Advances in Cancer Research, Volume 30

ADVANCES IN CANCER RESEARCH VOLUME 30 Contributors to This Volume Faye C. Austin Alexei J. Likhachev Joseph Ba16 ...

Author: Jesse Philip Greenstein

10 downloads 731 Views 18MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN CANCER RESEARCH VOLUME 30

Contributors to This Volume

Faye C. Austin

Alexei J. Likhachev

Joseph Ba16

Willie Mark

Charles W. Boone

Alpha Peled

E. H. Cooper

Kazyrnir M. Pozharisski

Necharna Haran-Ghera

Jacob D. Shaposhnikov

Christopher R. Kintner

V. S. Shapot

Valeri F. Klimashevski

Joan Stone

Hiroshi Kobayashi

Bill Sugden

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania

Volume 30-7979 ACADEMIC PRESS

New York

San Francisco London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT @ 1979, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM O R BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING F R O M THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER:5 2 - 13360 ISBN 0- 12-006630-0 PRINTED IN THE UNITED STATES OF AMERICA

79808182

9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS TO VOLUME 30

...............................................

ix

Acute Phase Reactant Proteins in Cancer E . H . COOPERA N D JOANSTONE I. I1 . 111 IV . V. VI . VII . VIII . IX . X XI . XI1. XI11. XIV

.

. .

Introduction .......................................................... Production. Half.Lives. and Destruction of Acute Phase Reactant Proteins . Prostate and Breast Cancers ........................................... Gastrointestinal Tract Cancers ......................................... Bladder and Gynecological Cancers ..................................... Lung Cancer ......................................................... Liver Cancer ......................................................... Lymphoma and Leukemia ............................................. Peritoneal and Pleural Effusions ........................................ Extrahepatic Synthesis and Concentration of Acute Phase Reactant Proteins Animal Tumor Systems ............................................... Haptoglobin Phenotypic Variation ...................................... The Biological Effects of Acute Phase Reactant Proteins in Cancer . . . . . . . . Mathematical Addendum .............................................. References ...........................................................

1 4

8 13 15

17 19 20 23 25 26 21 28 30 31

Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants NECHAMA HARAN-GHERA A N D ALPHAPELED I. I1 . I11. IV .

Introduction .......................................................... Radiation Leukemogenesis ............................................. Induction of Leukemia by the Radiation Leukemia Virus Variants ......... Concluding Remarks .................................................. References ...........................................................

45 48

62 81 83

On the Multiform Relationships between the Tumor and the Host V . S. SHAPOT

I . Introduction .......................................................... I1. Competitive Relationships between the Tumor and the Host . . . . . . . . . . . . . . . V

89 93

vi

CONTENTS

111. Effect of the Tumor on Biological Characteristics of the Host Tissue

.......

IV. Prospects for the Clinic ............................................... V. Conclusion ........................................................... References ...........................................................

115

137 139 143

Role of Hydrazine in Carcinogenesis JOSEPHB A L ~

.................................................

151

IV. Occurrence of Hydrazines in the Environment ........................... V. The Oncogenicity of Isonicotinylhydrazide .............................. VI . Experiments of H. Druckrey in the Production of Tumors with Hydrazine Compounds ........................................... VII. Production of Polyps and Tumors in the Intestinal VIII. Hydrazine-Caused Cancer ..................... IX. Does INH Produce Tumors in Humans? ........................... X. Methylhydrazine Derivatives, a New Class of Cyt XI. Hydrazine Therapy in Hodgkin’s Disease ................... ................................................ XII. Summary . . . References ........................................

153 I53

I. Introduction.

zine ........................................ 11. Toxic Effect 111. Hydrazine-Induced Alteration in Rat Liver .........................

155

161

Experimental Intestinal Cancer Research with Special Reference to Human Pathology KAZYMIR M. POZHARISSKI, ALEXEIJ. LIKHACHEV, VALERIF. KLIMASHEVSKI, A N D JACOBD. S H A P ~ S H N I K O V I. 11. 111. IV. V. VI , VII. VIII. IX.

X.

Introduction .......................................................... Experimental Models of Intestinal Tumors .............................. Morphology and Morphogenesis of Experimental Intestinal Tumors . . . . . . . . Factors Modifying Intestinal Carcinogenesis ............................. The Kinetics of Intestinal Epithelial Populations in Tumors and during Carcinogenesis ............................................. Biochemistry ......................................................... Immunology ......................................................... Metabolism of 1,2-Dimethylhydrazine and Related Substances . . . . . . . . . . . . . Interaction of 1,2-Dimethylhydrazine and Related Compounds with Cell Components ..................................................... Conclusion ........................................................... References ...........................................................

166 166 169 184

196 206 211 216 222 226 227

vii

CONTENTS

The Molecular Biology of Lyrnphotropic Herpesviruses BILLSUGDEN. CHRISTOPHER R . KINTNER. A N D WILLIEMARK I . Introduction .........................

..

. . . . . . . . . . . . 239

... ... IV . Experimental Tumor Studies ........................................ . . . V . Studies of MDV. HVS. and EBV in Tissue Culture . . . . . . . . . . . . . . . . . . . . . . V1. Identification and Properties of Virus-Related Products for MDV. HVS. and EBV ............ ..................... ... VII . Conclusion ................................................ ... I1 . A Brief Survey of Lymphotropic Herpes .................... I11. MDV, HVS. and EBV in Their Natural Hosts ........................

Addendum References

........................... ....................... ................................................

240 244 248 249

258 266 . . . 268 . . . 268

Viral Xenogenization of Intact Tumor Cells HIROSHIKOBAYASHI I. I1. 111. IV . V.

Introduction .......................................................... Acquisition of a Virus-Specific Antigen ................................. Increase in the Antigenicity of a Tumor-Specific Antigen . . . . . . . . . . . . . . . . . . Immune Responses against Xenogenized Tumor Cells .................... Summary ........................................................... References ..........................................................

279 280 287 292 295 297

Virus Augmentation of the Antigenicity of Tumor Cell Extracts FAYEC . AUSTINA N D CHARLES W . BOONS Introduction ......................................................... Virus Therapy of Cancer ............................................. Augmented Immunogenicity of Virus-Infected Tumor Cell Extracts ....... Mechanisms of Virus Augmentation of TATA Activity . . . . . . . . . . . . . . . . . . . Prospects for the Application of Virus-Augmented Tumor Antigens in Immunodiagnosis and Immunotherapy ............................... VI . Summary ........................................................... References .......................................................... I. I1. 111. IV . V.

SUBJECT INDEX ............................................................ CONTENTSOF PREVIOUS VOLUMES...........................................

301 303 308 329 338 339 340 347 351

This Page Intentionally Left Blank

CONTRIBUTORS TO VOLUME 30 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

FAYEC. AUSTIN,Cell Biology Section, Laboratory of Viral Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 (301) JOSEPH B A L ~Department , of Pathological Anatomy, Semmelweis Medical University, Budapest, Hungary (151) CHARLESW. BOONE,Cell Biology Section, Laboratory of Viral Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 (301) E . H. COOPER,The Unit for Cancer Research, University of Leeds, Leeds, England (1) NECHAMAHARAN-GHERA, Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel (45) CHRISTOPHER R. KINTNER,The McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 (239) VALERIF . KLIMASHEVSKI, Laboratory of Experimental Tumors, N.N. Petrov Research Institute of Oncology, USSR Ministry of Public Health, Pesochny-2, Leningrad 188646, USSR (165) HIROSHIKOBAYASHI, Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan (279) ALEXEIJ . LIKHACHEV, Laboratory of Experimental Tumors, N . N . Petrov Research Institute of Oncology, U S S R Ministry of Public Health, Pesochny-2, Leningrad 188646, USSR (165) WILLIEMARK,The McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 (239) ALPHAPELED,Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel (45) KAZYMIRM . POZHARISSKI, Laboratory of Experimental Tumors, N.N. Petrov Research Institute of Oncology, USSR Ministry of Public Health, Pesochny-2, Leningrud 188646, USSR (165) JACOB D. SHAPOSHNIKOV, Laboratory of Experimental Tumors, N.N. Petrov Research Institute of Oncology, USSR Ministry of Public Health, Pesochny-2, Leningrad 188646, USSR (165) ix

X

CONTRIBUTORS TO VOLUME

30

V. S. S H A P o r , Cancer Research Center, USSR Academy of Medical Sciences, Moscow, USSR (89) JOAN STONE, Department of Mathematics, University of Bradford, Bradford, England ( 1 ) BILLSUGDEN, The McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 (239)

ADVANCESINCANCERRESEARCH VOLUME 30


ADVANCES IN CANCER RESEARCH,

VOL. 30

ACUTE PHASE REACTANT PROTEINS IN CANCER

E.

H.Cooper and Joan Stone

The Unit for Cancer Research, University of Leeds. Leeds, England; and Department of Mathematics, University of Bradford, Bradford. England

I. Introduction .......................................................... 11. Production, Half-Lives, and Destruction of Acute Phase Reactant Proteins . . 111. Prostate and Breast Cancers ............................................ A. Prostate Cancer ..... ................................... B. Breast Cancer ..................................................... IV. Gastrointestinal Tract Cancers .......................................... V. Bladder and Gynecolog VI. Lung Cancer.. ....... VII. Liver Cancer ............................................. VIII. Lymphoma and Leukemia.. ............................................ IX. Peritoneal and Pleural Effusions ........................................ X. Extrahepatic Synthesis and Concentration of Acute Phase Reactant Proteins XI. Animal Tumor Systems ................................................ XII. Haptoglobin Phenotypic Variation ....................................... XIII. Biological Effects of Acute Phase Reactant Proteins in Cancer . . . . . . . . . . . . . XIV. Mathematical Addendum ............................................... Multivariate Methods .............................................

..........................

1

4 8 8 10 13 15 17 19 20 23 25 26 27 28 30 33 37

I. Introduction

The acute phase reactant proteins (APRPs) are mainly glycoproteins that alter their plasma concentration in response to stimuli produced by many forms of tissue injury, acute and chronic inflammation, connective tissue disorders, and cancer. Clearly as they respond to such a wide variety of stimuli it is self-evident that any changes in these proteins must be regarded as nonspecific. (For general review see Owen, 1967: Koj, 1974: Fisher and Gill, 1975.) Nevertheless, the quantitative and temporal responses of individual members of the APRPs can differ according to the nature of the stimulus and therefore may have diagnostic implications (Laurell, 1974 Hiramatsu et a / . , 1976; Fisher and Gill, 1975). The interest of APRPs in cancer can be considered from both a fundamental and an applied point of view. The underlying fundamental question is the reason for the alteration of the APRPs in response to I Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved ISBN 0-12-006630-0

2

E. H. COOPER A N D JOAN STONE

chronic degeneration and cancer and whether this has an advantage to the host or is an aberrant modification of various protective mechanisms that are a vital part of wound healing and the response to infection. The fundamental aspects concern the function of proteins such as a,-acid glycoprotein (alAGP), C-reactive protein (C-RP), and the C3 component of complement in the chronic tissue damage of cancer, as well as the local function of the antiproteases, a,-antitrypsin (a,AT) and a,-antichymotrypsin (alACT), in relation to the progression of an invading neoplasm as it infiltrates the normal host tissues. The main features of this family of proteins are shown in Table I. On the other hand, renewed interest is being taken in APRPs as components of a battery of biological tests for the monitoring of cancer. The history of investigation of APRPs in cancer shows how most of the earlier authors were content to produce a catalog of changes of a particular protein in a wide variety of cancers with very little attention to age, tumor load, or the patients' performance status. They were also handicapped by various technical problems which resulted in the proteins being quantitated indirectly as a measurement of their enzyme inhibitor activity (a,AT), binding capacity as in haptoglobin (HP), as indirect enzyme assays as in ceruloplasmin (CPL), or as partially purified serum fractions, e.g., seromucoid. The general availability of commercial specific antisera and the introduction of the simple single radial immuodiffusion technique of Mancini rf a / . (1965) has greatly enhanced its use in laboratory medicine both for research and routine. In patients in whom there is established cancer or a high probability of cancer, the tests may be required to help assess the prognosis, in particular to increase the accuracy of prediction of probable recurrence or TABLE I TO CLINICAL THEACUTEPHASE REACTANTPROTEINS WITH PARTICULAR RELEVANCE ONCOLOGY Protein a,-Acid glycoprotein a,-Antitrypsin a,-Antichymotrypsin Ceruloplasmin C-Reactive protein Haptoglobin Type 1-1 Type 2- I Type 2-2 Fibrinogen

Symbol aIAGP @,AT aIACT c PL C-RP HP HP 1 - 1 HP 2-1 HP 2-2

Molecular Weight 40,000 54,000 68,000 5 1,000

100,000 -200,000 =400,000 340,000

Amount in normal serum (gdliter) 0.55-1.4 2.0-4.0 0.3-0.6 0.15-0.6 < I 0 (mg/liter) 1 .o-2.2 1.6-3.0 I .2-2.6 2.0-4.5


3

metastases in an individual. The tests may be required to help monitor therapy, especially when the tumor is no longer clinically detectable. Finally in this context is the provision of long-term monitoring of the patient at risk, so that early warning of relapse or progression can be given. As it is quite improbable that APRPs could play any role in population screening for cancer, especially as none of the presently available tumorrelated markers can seriously be advocated for this purpose (Hobbs, 1974; Neville and Cooper, 1976; Schwartz, 1978), this topic will not be discussed further. A second general concept germane to this review is the variation in an individual's general reaction to cancer, as witnessed by such gross indicators as weight loss and a vast array of more subtle biochemical imbalances. In some tumors the metabolic disturbances in the host are gross and coupled to major defects in organ function or cachexia (Theologides, 1971; Bodansky, 1975); in others the abnormalities are often subclinical, but they can decline fairly rapidly from a metastable state to a severe life-threatening illness for what is a relatively small increase of tumor burden or additional burden on a defective system. If skin cancers are excluded, then only 5% of all cancer patients will survive (Seidman et af., 1976) and since the greater proportion of deaths are associated with the effects of metastatic disease, it can be seen that knowledge of the effect of locally recurrent or disseminated tumor on biochemical homeostasis is needed as part of the information for guiding therapy on patients with advanced disease. Within the context of identifying the cancer patient's biochemical status a matrix of levels of various serum proteins, especially those with short half-lives and small pool sizes including certain APRPs, is a promising area for study. The early studies confirmed that advanced cancer was usually accompanied by a rise of a-globulins (Winzler, 1953), and later this was attributed to APRPs but the lack of specificity tended to discourage clinical oncologists from the idea that APRPs could be of any practical value. This discouragement was strongly reinforced by the belief in the late sixties and earlier part of this decade that the age of specific tumor markers had arrived, the virtue of tests being cancer specific was then loudly proclaimed. Unfortunately, tests such as the measurement of plasma carcinoembryonic antigen (CEA), a-fetoprotein (AFP), and beta subunit of human chorionic gonadotropin (PHCG) are nowadays realized to either lack specificity or to have some-;Jhat limited circumstances in which they can be used as an optimal marker (see Neville and Cooper, 1976: Bagshawe and Searle, 1977; King, 1978: Coletta, 1978 Schwartz, 1978; Krebs et al., 1978). Indeed if the behavior of PHCG in

4

E. H . COOPER A N D JOAN STONE

choriocarcinoma is the paradigm (Bagshawe, 1969), few markers can attain such sensitivity, and certainly it leaves many of the common forms of cancer without reliable specific markers, especially at the earlier stages of disease. This experience has resulted in a reappraisal of the use of some of the available nonspecific markers and examination of the value of including them in a battery of markers to monitor cancer, especially in cancers such as the kidney, bladder, and lymphomas where tumorrelated products have little to offer as markers (Neville and Cooper, 1976). There are several studies in the literature that strongly reinforce the view that changes in certain fractions of the plasma proteins, especially the seromucoids (Harshman et ul., 1974); Randle et ul., 1974) and Hp (Jayle e t al., 1968, might be helpful in monitoring cancer, the choice of the particular protein or group being influenced by the techniques available. The advent of monospecific antisera to many human plasma proteins provided a new opportunity to examine the changes in the individual APRPs and their implications in cancer. However, before assessing in what way APRPs could contribute to such a battery, it is important to have a clear idea of the objectives of monitoring. Essentially, monitoring is the collection of biochemical intelligence about the patient which, when taken with the appropriate clinical information, can help the clinician in decision making. The roster of markers from which a battery may be chosen are given in Table 11: for a discussion of multiparametric screening in clinical chemistry see Wolf et al. (1973) and Galen (1975).

II. Production, Half-Lives, and Destruction of Acute Phase Reactant Proteins

The plasma half lives of several APRPs based on tracing iodinated proteins injected into healthy subjects were established in the 1960s. Thus the estimates were 5.2 days for (wlAGP(Weisman el a l . , 1961), 24 days for Hp (Freeman, 1964; Krauss et al., 1966), 4.2 days for CPL (Koskelo et al., 1967, and 3.2 days for fibrinogen (McFarlane et a / . , 1964): this method gives albumin a plasma half-life of 19 days (Peters, 1970). These data in conjunction with the blood levels cannot provide accurate information on the rates of synthesis, as the blood level is the resultant of synthesis, catabolism, and partition between the blood and tissue fluids. The intravenous pool is estimated to be 40% of the total pool of albumin (Peters, 19751, 40% of the total CPL pool (Koskelo et al., 1963, and about 80% of the total fibrinogen pool (McFarlane et al., 1964


5

TABLE I1 LIST OF SOMEPOTENTIAL TUMOR MARKER SUBSTANCES" 1. Tumor-Associated Antigens

Oncofetal wfetoprotein CEA Fetal sulfo-glycoprotein antigen B. Other tumor-associated antigens DNA Binding Proteins Hormones ACTH Calcitonin hCG and PHCG Prolactin Enzymes Acid phosphatase Alkaline phosphatases Aldolase Aminopeptidases Amylase Aryl sulfatase Aspartate arninotransferase Clycos yltransferases y-Glutarnyl transpeptidase Glutathione reductase Histaminase Metabolites and Degradation Products P-Aminoisobutyric acid Fibrinogen degradation products H ydroxyproline K-casein u-lactalburnin Catechol amines and metabolites A.

11.

111.

IV.

V.

Isocitrate dehydrogenase Lactate dehydrogenase Malate dehydrogenase Muramidase 5'-Nucleotidase Pepsinogen isoenzymes Phosphohexose isomerase Ri bonuclease Terminal deoxynucleotidyl transferase Tyrosinase Minor nucleosides Myeloma proteins Polyamines Protein-bound fucose p,-Microglobulin Acute phase reactant proteins

Modified from the list by Dr. R. W. Ruddon, Frederick Cancer Research Center, Frederick, Maryland 21701. (I

Takeda, 1966). The phenotype of Hp profoundly influences the balance between the intra- and extravascular pools, with Hp 1- 1 (mol. wt. 85,000) having an almost equal partition between the pools and Hp 2-2 (mol. wt. 200,000) with only 25% in the extravascular pool (Krauss, 1969). Clearly any extensive inflammatory process altering the permeability of the capillaries might influence this partition between the vascular and extravascular pool as is seen in inflammatory and noninflammatory effusions (Agostoni and Marasini, 1977). Koj (1974) has reviewed the evidence on the metabolism of APRPs in animals and humans during the course of acute and chronic elevations of the serum levels of these proteins. The

6


general consensus is that the fractional catabolic rate is not influenced by the level of the APRPs in the blood. In relation to cancer the fractional catabolic rate of Hp has been reported to be unchanged in Hodgkin’s disease, even when it was raised to a concentration of 7.0 gm/liter (Krauss, 1969). O’Hara et al., (1967) investigated the Hp half-life in three normal subjects and in three patients with cancer using radioiodinated human Hp, the t l l z was 1.9, 2.1, and 2.1 days in their controls and 2.4, 2.7, and 2.8 days in the cancer patients. But these results must be regarded with some reservation as there is an uncertainty as to what is the true range of normal plasma half-lives and the influence of phenotype on the distribution of Hp in disease (Putnam, 1975). A similar stability has been observed in fibrinogen catabolism in inflammation and cancer (see Koj, 1974). More recently Lyman et al., (1978) have investigated the half-lives of fibrinogen in 30 cancer patients; they found that f 1 / 2 was 3.89 L 1.38 days in controls and 3.01 1.09 in cancer patients, and the half-life was shortest in patients receiving chemotherapy. However, compared to the other APRPs, there is the added complication of various changes in local or generalized deposition of fibrin that may influence the rate of removal of this protein. A starting point for a new chapter in the biochemistry of glycoproteins was the observation that the activity of certain hormones appeared to be dependent on their sialic acid content. This was first demonstrated for follicle stimulating hormone (Gottschalk rt al., 1960) and HCG (Goverde c>t al., 1968: Mori, 1969). Later, the studies of Ashwell and his group (see Ashwell and Morell, 1974, for review) were to extend this observation and produce a general hypothesis that the partial or complete loss of sialic acid from a plasma glycoprotein causes it to be rapidly removed from the circulation (Fig. 1). Beginning with a study of CPL, they were able to show this also applied to alAGP, Hp, and fetuin. Radioisotopic labeling indicated that the liver was the organ responsible for removing the asialated glycoprotein where it undergoes enzymic hydrolysis. A typical result is the finding that the half-life of human a,AT in the circulation of the rat is 18 hours, while in asialic AT it is almost toally cleared in about 30 minutes (Yu and Gan, 1977). The effect of the desialation seems to depend on the exposure of galactose residues which then trigger the recognition processes in the liver: this effect can be inhibited by altering the galactose group (Van den Hamer rt al., 1970). The binding sites on the liver cells appear to involve sialic acid, as treatment of hepatic parenchymal cells with neuraminidase removes the binding activity for asialoglycoprotein, but sialic acid also blocks the binding of glycoproteins to these cells (Ashwell, 1974); this dual function of sialic acid is still an enigma.

*


7

FIG.1. Plasma survival of labeled asialoglycoproteins (from Ashwell and Morrell, 1974).

This mechanism could provide a way of distinguishing recently synthesized molecules from those that are “aged” and ready for removal, that is assuming the aging would involve loss of sialic residues. It is of interest that, in those proteins where a biological activity can be measured, desialation is not associated with a loss of function, for example asialo-a,AT, partially desialated alAT, and partially desialated alAT with oxidized galactose, maintain their trypsin inhibitory and chymotrypsin inhibitory potential (Yu and Gan, 1977). Hence the loss of hormone activity as the result of desialation is due to removal from the circulation rather than interference with the functional moiety. The clearance of alAT in subjects with alAT deficiencies has shown that the protein has a half-life of 6 days (Makino and Reed, 1970), while

8

E. H. COOPER

A N D JOAN STONE

iodinated alAT in recipients of the MM phenotype indicated the half-life was 4 days, and 5.5-6.5 days in the MM and MZphenotypes (Keuppers and Fallat, 1969). This is a somewhat unexpected result as these authors treated their preparation with neuraminidase. More recently, the behavior of the alAT isolated from PiM and PiZ subjects has been investigated by the simultaneous injection of the two labeled proteins into PiM subjects (Laurell et al., 1977). The mean fraction catabolic rates for M protein and Z protein were 0.26 and 0.40,respectively, which is too small a difference to explain why the alAT content of blood in PiZ phenotypes is only 15% of normal. Following injury or estrogen stimulation in M Z and ZZ patients there is a similar proportional increase of a,AT, but the absolute response is less in the ZZ patients. This points to either a small mRNA pool or a slower translation of the Z gene (Laurell or al., 1977). Ill. Prostate and Breast Cancers

It is convenient to consider these cancers together, as they are both hormone dependent and estrogens are frequently used to control their growth. This treatment produces a profound alteration in the plasma protein by its direct effects on their synthesis independent of any effect on the cancer (Musa or af., 1965; Laurell er al., 1968).

A. PROSTATE CANCER

Untreated carcinoma of the prostate has to be associated with raised serum a-globulins which are related to tumor stage and the treatment (Ablin c>t ul., 1973). An elevated pretreatment plasma fibrinogen in prostatic cancer has been significantly correlated with an increased proportion of deaths from all causes and from carcinoma of the prostate (Seal rr ul., 1976). More recently Seal rf ul. (1978) have confirmed that serum Hp levels are raised in prostatic cancer stages 111 and IV, stage I V being significantly higher than state 111. The pretreatment values were significantly correlated with death rates. Ward c’t af. (1977) examined the serum levels of Hp, alAGP, and CPL in benign prostatic hypertrophy (BPH), and untreated carcinoma of the prostate with and without bone metastases. The levels of this protein profile were not statistically different in BPH and the carcinoma without metastases, while carcinoma with bone metastases was accompanied by a significant increase in the four APRPs. They demonstrated that a mean discriminant function using the levels of the proteins in combination with the level of tartrate resistant serum acid

9


phosphatase (SAP) and prealbumin (PALB) could give a correct classification of cases with and without bone metastases 88.6% of the time. The evidence for the bone metastases was the uptake of technicium 99 into the bone as shown by scintigraphy. Figure 2 illustrates the separation achieved by this discriminant function. Subsequently, Houghton ( 1978) reexamined the discriminant on a further set of 25 prostatic cancer patients without bone metastases and 45 with bone metastases and examined the value of the levels of ACT and C-RP in addition to the variates used in the earlier study. A stepwise discrimination showed the importance of the variates in descending order was SAP, alACT, Hp, PALB, alAT, alAGP, and C-RP. He obtained a 90% overall correct assignment with his discriminant function. However, the recent advances in the isolation of specific prostatic acid phosphatase (Foti rt al., 1977) and the finding that serum ribonuclease levels may be a guide in monitoring prostatic cancer (Chu et al., 1977) may result in a more powerful discriminant which would be less liable to be influenced by the vagaries of the APRPs’ response to therapy (Chu, 1978). Several forms of estrogen therapy and estrogen-containing drugs such as estramustine phosphate, a nornitrogen mustard linked to 17p-estradiol phosphate (Jonsson et al., 1975), have major effects on the plasma protein profile in prostatic cancer. These effects include a rise of serum alAT and CPL levels (Ward rt al., 1978) and plasminogen (Seal et al., 1976) and a fall of aIAGP and Hp (Ward rt al., 1978) and fibrinogen (Seal rt af., 1976). Seal and his colleagues (1976) pointed out that the fall of fibrinogen was unexpected as it rises in pregnancy and a rise may be induced by several forms of estrogen-containing oral contraceptives in women. They were unable to account for the rise in patients with prostatic cancer, except to note its effect was maximal when a 1 mg/day dose of diethylstilbestrol was used, the effect being absent at 5 mg/day. Provera, another estrogen used for

I

I

I

I

FIG. 2. Discriminant analysis scores using prealbumin, a,-antitrypsin, a,-acid glycoprotein, haptoglobin, serum acid phosphatase with prior logarithmic transformation of the data. Closed triangles represent metastatic carcinoma of the prostate. Circles represent nonmetastatic carcinoma of the prostate. Discriminant (D)= - 0.638(log,.SAP) 0.767(log,, PALB) - 2.074(log, a,AT) + 0.605(log, AGP) - 0.91 I(log,. Hp) + 4.996. From Ward et (I/. (1977).

+

10


the management of prostatic cancer, had no effect on the APRPs as judged by changes in plasminogen or fibrinogen. The high a,AT to a,AGP ratio can provide an indication that the patient is receiving estrogens: this is sometimes a useful parameter to measure in the elderly on oral estrogens, as they do not always comply with the physician’s instructions (Table 111). Various other estrogen and steroid binding proteins are increased in the plasma in response to estrogen therapy: these include transcortin (Sandberg r t al., 1975), sex hormone binding globulin (Houghton r t a / . , 1977; Bartch et ul., 1977), and beta steroid binding protein (SP2) (Ward e t al., 1978). The last two proteins are probably the same (Bohn and Krantz, 1973; Bohn, 1974) though the former is assayed by competitive binding of labeled dihydrotestosterone and SP2 by immunodiffusion. B. BREASTCANCER Minton and Bianco (1974) reported serum electrophoresis has demonstrated a raised level of a,-globulins in 32 out of 39 advanced breast cancers with spread beyond local nodes, while in only 1 out of 1 1 with local disease. Pettingale and Tee (1977) examined the plasma protein profiles before and a year after excising an early breast cancer in 30 women, and in 30 women with benign breast disease. Only the levels of p,-glycoprotein preoperatively and CPL after one year were significantly higher in patients with breast cancer than in those with benign disease. An evaluation of APRPs as potential members of an array of markers to monitor breast cancer has been made by Cowen et al., (1978). In their study serum Hp, alAGP, CPL, and a l A T levels were evaluated in association with blood levels of CEA, K-casein, PHCG, and alkaline phosphatase. The values of the proteins in 501 women attending a breast clinic are shown in Table IV. The alAT levels were shown to be of no value as potential markers. They also made a limited study of serum CRP levels to identify the frequency of an abnormal value (>lo mdliter). The following results were obtained: Primary tumors Overt metastases after mastectomy without treatment Follow-up mastectomy clinically tumorfree Benign breast disease Controls

stages 1-111 stage IV

11/81 11/26 1124 3/102 0155 0126

(13.5%) (42%) (29%) (2.9%)

(0%) (0%)

Eighteen out of the 32 positive results (56%) had evidence of at least one

TABLE 111 alAT TO alAGP RATIOI N EACHGROUPI N PROSTATIC CANCER^

No estrogens Groupb

n Mean and standard deviation

1v I1 111 I 22 24 23 27 2.54 f 0.69 2.79 f 0.63 2.86 2 0.70 2.85 ? 0.77

Estrogens V 20 5.79

?

v1

VII

VIII

24 32 27 1.60 7 . 6 4 k 1.98 6.13 ? 2.12 6.50 ? 2.16

From Ward et al. (1977). I = benign prostatic hypertrophy, I1 = cancer prostate (CP) nonmetastatic untreated, 111 = CP metastatic untreated, IV = CP metastatic orchidectomy, V = CP nonrnetastatic-stilbestrol, 1 mg daily, V1 = CP nonmetastatic-stilbestrol, 3 mg daily, VII = CP rnetastaticstilbestrol, I rng daily, and VlII = CP metastatic-stilbestrol, 3 rng daily. a

TABLE IV INCIDENCE OF WOMENWITH BREASTCANCER A N D CONTROLS WITH RAISEDLEVELSOF TUMORASSOCIATED (TA) PRODUCTS AND THOSEWITH RAISEDLEVELSOF TUMOR ASSOCIATED AND/OR TUMORRELATED(TR) PRODUCTS

Control Benign breast disease Primary breast cancer stages I, 11, and I11 Postmastectomy clinically tumor-free Primary breast cancer stage IV and untreated metastatic cancer Metastatic breast cancer receiving treatment

Hpt >5.2 gdliter

AGP >1.4 gdliter

3/42

3/42

6/100

5/100

3/46

Alkaline C P L Phosphatase More than one >0.55 233 T A product gdliter IU/liter raised

Any one T A or TR marker raised"

Any two TA and one TR or Any two TA or any two TR and TR markers one TA markers raised" raised"

1/46

2/41 5/98 1/44

214 1 4/99 13/44

214 1 2/99 2145

141156

191156

11150

441155

14115s

48.1

16.0

5.1

14/34

11/34

5/34

23132

16/32

93.8

73.5

20.6

6/49

11/50

9/41

23/46

14/46

82.0

44.0

14.0

26.2% 29.0 39.1

OF

4.8% 5.0 8.6

0%

0 0

Expressed a s the minimum percentage. Some patients excluded as all the markers were not estimated. From Cowen et a/. (1978).


13

other acute phase reactant protein elevated above the discriminant level. Coombes et al., (1977, who examined a much wider panel of "markers" though in only 36 patients with breast lumps prior to diagnosis and 17 breast cancers with overt metastases, considered that serum alAGP and C-RP levels were potentially useful and included them with ferritin, CEA, sialyl-transferase, alkaline phosphatase, and urinary hydroxypro1ine:creatinine ratios as part of their final panel. However, as Mach et a / . (1977) have pointed out, there is a paucity of reliable markers in breast cancer. The APRPs tend to show a general pattern of elevation that depends on state and tumor load, but there is very considerable variation within this pattern. The long natural history of breast cancer, especially in younger women is well known, where there may be a disease-free interval of several years between the treatment of the primary tumor and the occurrence of metastases. This long time has deterred any longitudinal studies, and unfortunately the long-term adjuvant treatment programs in the United States (Bonadonna, 1977) and Italy (Fisher, 1977) have not included any studies of the evolution of markers in the patients at risk. IV. Gastrointestinal Tract Cancers

The confirmation that the levels of plasma CEA can be raised in many forms of gastrointestinal diseases other than cancer (see Neville and Cooper, 1976) has raised the question of whether the alterations of serum proteins in various neoplastic and inflammatory bowel disease might interfere with the assay of CEA. Crawley et af. (1974), examining malignant and benign bowel disease and liver disease, found that CEA and a,AGP, and CEA and alAT showed significant correlations (p = . Comparative studies of the properties of thymidine kinase in the epithelial cells of the intestines of rat embryos, rats of varying ages, and those of DMH-induced tumors suggested that the embryolike properties of tumor enzymes develop before the beginning of tumor formation (270 1. These authors also demonstrated that the activity of ornithine decarboxylase, another enzyme participating in cell proliferation, in the epithelial cells of rat colon increases 50-fold following DMH treatment, as compared with normal tissues. On the basis of a comparative study of the topography of proliferating epithelial cells in patients with solitary polyp, familial polyposis, and colonic carcinoma, and in experimental intestinal tumors induced in animals, Lipkin (160) could distinguish two stages of disturbances in intestinal epithelial proliferation during carcinogenesis. While in phase I , the cells of the grossly and microscopically unaltered mucosa continue to incorporate 3H-TdR as they move toward the gut lumen. The continued proliferation of cells even in superficial epithelium is due to the inadequate suppression of DNA synthesis. However, since the production and loss of epithelial cells are in equilibrium, the overall cell mass of the mucosa is not increased and, therefore, the appearance of the latter is not changed. At phase 2, highly proliferating cells are not shed into the gut lumen: they accumulate in the rnucosa, thus leading to the formation of intestinal tumors. However, the mechanisms responsible for the development of disturbances in cell reproduction are not clear yet. The dynamics of changes in the kinetics of populations of intestinal epithelial cells at the incipient stages of tumorigenesis has been studied in great detail by Pozharisski et ul. (240). The investigation was concerned with the microscopically unaltered mucosa of rat descending colon, examined 1, 2, 3, and 4 months after DMH treatment as well as such early malignant neoplasms arising in this segment of the intestines as in sitri carcinoma, superficial cancer, and slightly invasive, small adenocarcinoma. The evaluation of percentage labeled mitosis (PLM) curves undertaken in this study showed an increase in the duration of the short mitotic cycle from 11 hours under normal conditions to 15-16 hours in the cells of microscopically unaltered mucosa, in sitrf carcinoma, superficial cancers, and small adenocarcinomas. While f, and tG, 4 I,,,

+

200

KAZYMIR M . POZHARISSKI ET AL.

remain practically unchanged, the increase in mitotic cycle duration is due to the increase in GI-period to 3-6 hours, as compared with 2 hours in control. A comparison of PLM curves for microscopically unaltered mucosa and early malignant lesions revealed some changes-a shoulder-in the descending part of the first wave of PLM curve (240). This shoulder becomes increasingly pronounced by month 4 and is particularly well defined on PLM curves for in sit14 carcinoma, superficial cancer, and small adenocarcinoma with incipient invasion. Possible explanations of this shoulder on the descending part of PLM curves are discussed in the report of Pozharisski et a / . , (237,238). The most plausible seems to be the variations in cell population versus mean duration of S-period. The studies of changes in the fraction of labeled epithelial cells in different compartments of the crypt, following multiple injections of 3HTdR, and an evaluation of label cumulation (LC) curves showed that the compartment of maximum cell proliferation in the crypts of normal mucosa of rat descending colon is characterized by a varying mean length of the mitotic cycle of cell population. The latter, in turn, consists of three subpopulations of cells with different mean lengths of mitotic cycle. The bottom of the crypt was shown to comprise a four-component population of cells also made up of subpopulations with different mean lengths of mitotic cycle (237,238). The analysis of LC curves for the microscopically unaltered mucosa of the descending colon of rats, for different terms of DMH treatment, showed that the structure of the proliferating cell population both in the compartment of maximum proliferation and the bottom of the crypt becomes less complicated (240). After 4 months of DMH treatment, 95% of the cells in the cells in the compartment of maximum proliferation of the crypts of the microscopically normal mucosa of the descending colon proliferated through a short (15-16 hour) cycle, while about 5% of the remaining cells either had a long mitotic cycle or were able to enter the resting phase R,. These subpopulations occur in nearly the same ratio in in situ carcinoma, superficial cancer, and small adenocarcinomas. Another indication of neoplastic changes in the intestines is a shift in the ratio of mitotic phases, with the fraction of pathological forms of mitosis increasing (240). For example, prophases constituted 26% in the epithelium of the descending colon in control rats, and metaphases 57%; whereas after 4 weeks of DMH treatment, the fraction of prophases dropped to 6.396, while that of metaphases rose to 74%. There were no significant changes in this ratio at the later stages of the experiment. The epithelium of the descending colon of control rats was found to contain 4% of pathologic mitoses. But their fraction increased up to 51%

EXPERIMENTAL INTESTINAL CANCER RESEARCH

20 1

as early as 1 month after the beginning of the experiment. At later stages, it remained more or less stable within 57% to 60%. Cessation of DMH treatment was not followed by a decline in the number of abnormal mitoses in intestinal adenocarcinomas within the next 2 or 3 months. This observation suggests that the appearance of pathological mitoses was not due to the toxic effect of the carcinogenic substance. Diverse forms of pathologic mitoses (bridges, the lag of chromosomes and their fragments during divergence, chromosome scatter, hollow or three-group metaphases, monocentric and multipolar mitoses, etc.) with an apparent predomination of the lag of chromosomes and their fragments in metakinesis (12%-17%) and K-mitoses were observed for different terms of DMH treatment (in microscopically unaltered mucosa) and in developed adenocarcinomas, too. The iliac epithelium did not show any changes in the ratio of mitotic phases throughout the experiment, nor was the fraction of their pathological forms in excess of 3.5%. These changes in the kinetics of populations of intestinal epithelial cells seem to be manifestations of the malignant transformation of these cells (240 ) .

B. THEPECULIARITIES OF THE KINETICS OF TUMOR CELLS The analyses of tagged cell distribution after pulsed labeling with 3Hthymidine revealed that there is no clear distinction between the compartments of proliferating and nonproliferating cells in colonic neoplasm tissues and, therefore, DNA-synthesizing cells are distributed all over the tumor (139,239,338,348). Thus, the entire tumor is the proliferative zone. The analysis of percentage labeled mitosis curves showed that the mean length of cell cycle of the fastest proliferating subpopulation of cells of invasive tubular adenocarcinoma is 16 hours, as compared with 11 hours for the normal mucosa of the descending colon (239). This increase is due to the prolongation of GI-period, with the values oft, and tG, + f t , being practically the same. The slower rate of proliferation of adenocarcinoma cells, as compared with those in the proliferative compartment of the crypts of normal mucosa, was also reported by other authors (313,348). The evaluation of percentage labeled mitosis curves revealed a heterogeneity of the proliferating cells of adenocarcinoma with respect to the mean duration of S-period (239). Moreover, on the basis of the data showing that the maximum of the first wave of PLM curve remains short of loo%, the authors suggest this also to be an indication of the hetero-

202


geneity of tumor cell population with respect to the mean duration of G,period. It may as well indicate even the existence of an R,-subpopulation. The label cumulation curve obtained in this study also points to the increasing heterogeneity of adenocarcinoma cell population with respect to the mean length of mitotic cycle. This curve is identical to that obtained for the crypt bottom of normal descending colon, in which a complicated four-component structure of cell population was established and epithelial stem cells occur (237,238). These findings led these authors to suggest that the properties of tumor cells resemble those of the epithelial stem cells of the descending colon. The similar rates of cell proliferation in adenocarcinoma and the crypt bottoms in the normal mucosa of rat colon were also reported by other authors (313 1. While the determinations of cell cycle parameters from percentage labeled mitosis curves produce leveled indices for separate tumors, the application of the thymidine-colchicine method makes it possible to determine separate parameters of cell cycle for each tumor, both of the same and different histological types (235). Using the latter method, these authors demonstrated that, among such experimental tumors as adenocarcinoma, signet-ring cell, and mucinous cancer, signet-ring cell cancer and adenocarcinoma exhibit the highest proliferative activity (mitotic cycle length, T,, is 47 and 54 hours, respectively), while mucinous carcinoma exhibits the lowest one (T, = 124 hours). These authors give the following explanation for these differences: The cells of signet-ring cancer and glandular structures of adenocarcinoma are located in a highly vascularized stroma so that their requirements in nutrition and oxygen supply are fully satisfied. Mucinous cancer, on the other hand, consists of large lacunas of mucus, in which separate tumor cells or their aggregates are located. It is easy to understand that under such conditions, metabolic processes proceed on a reduced scale in tumor cells. This manifests itself in a varying rate of cell proliferation. The parameters of generation cycle in the tumors of the same histological structure, which formed in one and the same animal, were also found to vary. Marked variations in the magnitude of generation cycle parameters of the same histological type are believed to characterize the rate of cell proliferation, both in the entire tumor and its separate compartments. Variations in the proliferative rate within one and the same tumor were confirmed by the results of the subsequent studies of these authors (Pozharisski and Klimashevski, unpublished data). By means of the thymidine-colchicine method of determination of generation cycle parameters and analysis of the curves of labeled mitoses and those for changes in the fractions of labeled cells, following multiple injections of 3H-TdR into three regions (periphery, center, and zone of invasion) of


203

tubular adenocarcinoma, it was found that the cells of tumor periphery are characterized by the highest rate of proliferation. A lower rate of cell proliferation was observed in the central part of tumor and in its zone of invasion. The latter finding is rather strange. We should expect that the invasive zone would comprise cells capable of increased proliferation, indispensable for the infiltrative growth of tumor. Two maximum values of label index-near the surface and in the base of the mucosa, at an area of invasive growth into the submucosa-were established in the neoplasms considered to be adenomatous polyps with malignant transformation and incipient infiltrative growth (338). There are very few data available on studies on mitotic cycles in colonic and rectal tumors in humans. This seems to be due to the difficulties involved in taking multiple specimens for further autoradiographic investigations as well as moral considerations. Nevertheless, a few papers contain data on the quantitative parameters of the kinetics of cell populations in human intestinal tumors (2/,25,36,/59,302).Apart from autoradiography, the stathmokinetic method based on the use of the ability of colchicinelike preparations to block cell division at metaphase has been employed for the determination of cell proliferation rates (25.36 ). It is possible to infer from the results of these investigations that the rate of proliferation of tumor epithelial cells is generally lower than that of normal ones. The autoradiographic studies of the kinetic parameters of adenocarcinoma of human colon and rectum, involving the use of double label (3HTdr and 14C-Tdr),showed that DNA synthesis duration in tumor is about twice that of normal mucosa for both sites ( 2 1 ) . These authors attach much importance to this fact and suggest that any increase in the value oft, may be considered sufficient evidence to suspect carcinogenesis. Of similar importance is another finding that a rise in t , magnitude was observed in morphologically normal mucosa with an atypical arrangement of proliferating cells. At the same time, however, the authors are right in saying that more evidence is required to accept the rise in t , as a signal of malignant transformation or precancerous state development. Since the data are scarce, it is impossible to advance any theory on the peculiarities of cell population kinetics in human intestinal neoplasms. Such important factors as the proliferative pool and degree of cell loss were studied in few investigations only and the data obtained vary within a considerable range (36,302). In order to obviate these difficulties in studies of human tumors, the parameters of the mitotic cycle in human colonic and rectal adenocarcinoma were measured, following the transplantation of these tumors to mice, in which immunological reactivity had been suppressed (223). It was established that the lengths of mitotic

204

KAZYMIR M . POZHARISSKI ET A L .

cycle and its separate phases do not differ materially from those determined in relevant tumors of man. However, these authors withhold their judgment on the identity of the kinetic parameters in the cells of human colonic adenocarcinoma and its heterotransplants, owing to the scarcity of literature data on the peculiarities of proliferation in the primary tumors.

C.

STEMEPITHELIAL CELLSOF CARCINOGENESIS

THE

INTESTINES AND THEIRROLEI N

The role of the proliferating cells of organs and tissues in carcinogenesis may be considered well established at the present time (see 214). However, the proliferating cells of tissues may be part of different cell populations undergoing different stages of cell differentiation and, therefore, their contribution to carcinogenesis may be different. Meanwhile, the detection of a factor, responsible for tumorigenesis, on a cell level is vitally important for the elucidation of the nature of tumor growth. The following facts point to the leading role of enterocytes in carcinogenesis (241 ): 1. Intestinal epithelium is a quickly renewing tissue, and the first tumor lesions develop within 2 to 3 months after repeated injections of DMH (230). Since epithelial cells renew several times during this period, a source of carcinogenesis should be sought among more stable elements which can store carcinogenic effects. Stem epithelial cells are probably such elements, because they are located in the crypt bottom (41,238) and, therefore, do not enter the cell flow toward the superficial layers of the mucosa, where they are shed into the gut lumen. DMH treatment affects the hereditary mechanism of stem cells and they produce cells with an impaired heredity. It should be also mentioned that a single administration of DMH is sufficient to induce intestinal tumors. 2. The malignant transformation of enterocytes, as revealed by continued DNA synthesis in cells, which go on dividing outside the proliferative compartment in intestinal crypts, is a rather rare phenomenon. This means that it is actually brought about by a relatively small population of cells, i.e., stem enterocytes, rather than by the bulk of proliferating nonstem cells. 3. In the kinetic parameters of cell populations, the cells of large invasive adenocarcinomas resemble those in the bottom of colonic crypts, where stem cells are located (238). 4. A local stimulation of intestinal epithelium proliferation due to a chronic nonspecific injury of the mucosa results in a sharp rise in exper-


205

imental tumor incidence at the injured site. An enhanced proliferation of epithelial cells is not likely to be due to a shorter length of mitotic cycle of nonstem cells, because their rate of replication is very high under normal conditions. It is probably due to the entry of a greater portion of the stem cells of the intestinal epithelium into the mitotic cycle. Therefore, it may be inferred that stem enterocytes act as acceptors of carcinogenic influences (236). 5 . This conclusion is further confirmed by the correlation between the rate of tumor incidence in certain segments of the intestines, on the one hand, and the amount of stem cells, their proliferative pool, and the length of their life cycle in these segments, on the other. To illustrate, the descending colon, in which DMH treatment induces tumors in 100% of rats, reveals a greater fraction of stem cells, a higher proliferative pool, and shorter life cycle as compared with the ileum, which is practically insusceptible to tumors (236). 6. The conversion of adenocarcinoma cells into those of signet-ring cell cancer, and retransformation of the latter into the former, support the unitary theory of the origin of all types of epithelial cells (41) and demonstrate that, despite the cytological characteristics of cell differentiation, tumor cells retain stem-like properties. 7. A specific glycoprotein, similar to cancer embryonal antigen (CEA) observed in patients with carcinoma of the colon, was detected in experimental intestinal tumors and blood serum of tumor-bearing animals (216 ). However, this antigen is also found in the blood serum of patients with chronic nonspecific lesions of intestinal mucosa characterized by an intensified proliferation of the epithelium (243 ). As mentioned above, the increase in the fraction of proliferating epithelial cells is caused by the entry of a greater number of stem cells into mitotic cycle. Hence, it may be suggested that CEA is produced by stem cells passing through the mitotic cycle and that the stem cells of intestinal epithelium and the cells of an intestinal tumor possess common antigenic properties. It was suggested recently by Pierce et al. (224), who found the same ultrastructure in least-differentiated cells of normal colon of rats and intestinal tumor transplants in these animals, that stem cells serve as targets in carcinogenesis. On this basis, the mechanism of development of experimental tumors of the colon may be visualized as follows (240). As a result of DMH treatment, stem cells are transformed while in mitotic cycle and the descendants of the altered cells become capable of proliferating in those compartments of crypts in which differentiated epithelial cells are normally located. This means that transformed cells cease to differentiate. They are distributed throughout the entire length of the crypt and even

206


reach its mouth. Since stem epithelial cells are very few and their fraction passing through mitosis (i.e., when cells are susceptible to the transforming effect of the carcinogen) is exceedingly small, morphological means fail to detect any malignant alterations at initial stages. It is very likely that some altered epithelial cells are eliminated by immunological mechanisms. Repeated administrations of the carcinogenic substance result in the transformation of a greater amount of stem cells. Subsequently, local disturbances in the steady state of intestinal epithelium occur, which are manifested by the accumulation of atypical cells with altered proliferative properties and formation of in situ carcinoma in the superficial layers of the crypts. The formation of tumor lesions follows as a result of an upset equilibrium between cellular output and loss, because the latter diminishes. As these lesions continue to develop, the lamina propria of the mucosa becomes invaded, and superficial cancer is formed.

VI. Biochemistry

A. NUCLEIC ACIDS, NUCLEOTIDES, A N D ENZYMES PARTICIPATING IN THEIRSYNTHESIS A N D METABOLISM Although there are abundant data on the structure and function of nucleic acids in different experimental tumors, communications on studies of DNA in DMH-induced tumors of the intestines are very scarce (115). Equilibrium centrifugation of the DNA from the cells of mouse colonic tumor revealed only one peak of DNA, which points to the homogeneity of isolated DNA. Its buoyant density corresponded to that of liver DNA (1.700 gm/ml). Much attention is being given to studies of the so-called “second messengers” (cyclic nucleotides), which play an important role in the regulation of mitotic activity, alteration of membrane properties, control of the rates of metabolic reactions, etc. (52). For example, guanylate cyclase-guanosine 3’,5’-monophosphate in the mucosa of rat colon may be activated under the influence of N-methyl-N’-nitro-N-nitrosoguanidine ( 6 0 ) . I n contrast, the level of cyclic AMP in X-ray-induced adenocarcinoma of the jejunum is reduced (298). Similar results are reported for tumors of human colon (59). Adenylate cyclase activity in these tumors was much lower than in normal mucosa. These authors conclude that the low level of cyclic AMP in tumor should be accounted for by a decrease in its synthesis rather than an increase in its degradation. However, the mean level of cyclic AMP in human adenocarcinoma appeared to be lower than in normal tissues, which is consistent with the above experimental findings. In contrast with the data of DeRubertis et al. (5Y), the


207

results of Minton er al. (188) indicate that cyclic AMP level in colonic tumors does not differ from that of normal mucosa in man. Much more data are available on the role of enzymes participating in nucleic acid synthesis in normal and malignant intestinal tissues. Ball rt al. (14) conducted a study on precancerous lesions, an informative and the only investigation on the subject, in which two enzymes associated with cell division were examined. Ornithine decarboxylase activity is low in the rapidly dividing colonic cells, which are the targets for DMH carcinogenesis, whereas it is rather high in the nondividing cells of the small intestine. DMH administration resulted in an enhanced activity of this enzyme in colonic epithelium but did not affect it in the liver: this points to the specificity of this effect. The study of the other enzymethymidine kinase-revealed that the fetal and tumor enzymes are alike in possessing a high level of activity and an ability to be stimulated by phospholipase C. The properties of the colonic tissues of the rats exposed to DMH range between those of normal and tumor tissues. The activity and regulation of thymidine kinase in normal and malignant intestinal epithelium in humans were studied Pt the same laboratory (269). Unlike in rats, the difference in these characteristics between tumor and fetal intestinal tissues proved to be considerable: in carcinoma, the enzymic activity was higher and was not stimulated by phospholipase C, while in the fetus, it was 7-10 times as high, and in the normal mucosa of adult humans 2.5 times as high. It was inferred from these results that cell division regulation in gastrointestinal tumors is not identical to that of the rapidly dividing cells of the crypts and fetal intestine. The activity of thymidine kinase and thymidine phosphorylase appeared to be maximal in tumors and young proliferating cells, declining as the degree of cell differentiation increased and as cells migrated toward the tissue surface. The activities of adenine- and hypoxanthine phosphoribosyltransferases in tumor and young proliferating cells were minimal, and increased as cells became more differentiated and migrated toward the surface (307). The DMH-induced tumors of mouse colon show an enhanced activity of some enzymes, which play a key role in RNA synthesis: nuclear RNA polymerase, nucleolar RNA polymerase, ATP-tRNA-adenylyltransferase, L-methionine-tRNA-ligase, L-leucine-tRNA-ligase, and L-lysinetRNA-ligase (222 ).

B. NUCLEAR PROTEINS Some noteworthy results have been reported in papers dealing with the studies of nonhistone protein behavior during carcinogenesis and in

208


DMH-induced tumors of rat colon. The basic difference was found to consist in a sharp rise in the levels of the proteins with molecular weights of about 44,000 and 62,000 daltons in the course of carcinogenesis (24 ). Selective synthesis of proteins sets in long before the development of any morphological signs of neoplasms. It is likely that the accumulation of these acidic proteins is not simply due to the increase in the fraction of epithelial cells passing through the proliferative phase (22 1. Since the program of cell growth and differentiation under normal conditions and, therefore, that of cell dedifferentiation in tumor tissue are determined to a considerable degree by the interaction of DNA and firmly bound chromosomal proteins, which regulate gene activity, it is easy to appreciate the significance of the observation of a considerable selective accumulation of the same proteins in HT-29 cells, which originate from the adenocarcinoma cells of human colon ( 2 2 ) . Their level in human tumors of the colon is also raised ( 2 2 ) . These proteins appear in the adenocarcinomas which arise in patients suffering from familial polyposis long before any signs of malignant transformation develop (23). This is correlated with similar results in experimental tumors ( 2 4 ) and may be of diagnostic value. The protein with a molecular weight of 62,000 daltons, isolated from rat tumors, does not bind to DNA in vitro, while the other one (44,000 daltons) shows a great affinity for DNA, which points to its association with the active part of the genome (23 ). A two-dimensional electrophoresis showed a considerable difference in the isoelectric points and molecular weights of these proteins (23 1. It was also shown in this laboratory that sodium cyanate selectively inhibits 3H-labeled amino acid incorporation into the cytoplasmic and nuclear proteins of DMH-induced tumors. This effect was not observed in the HT-29 cell culture (5 ).

C. ENZYMES The cell membranes of mammals contain glycoproteins, glycolipids, and, sometimes, glycosoaminoglycans, and fucolipids in the microvilli of intestinal cells. All these components may be responsible for such functions of the cell as adhesion, recognition, “contact inhibition,” agglutination, growth regulation, etc. Hence, cell membranes and their constituents have aroused considerable interest. For instance, La Mont et (11. (149) demonstrated that the malignant transformation of rat intestinal cells is accompanied by changes both in their agglutination by lectins (cells of the tumors of small intestine are agglutinated by concanavalin


209

A and those of colonic tumors by wheat germ agglutinin) and the activity of glycosyltransferase of cell surface (activity in tumors of small intestine was higher and in colonic tumors lower than in normal homologous organs). Similar results for a number of ,glvcosyltransferases (sialyl-, galactosyl-, fucosyl- and polypeptidyl-N-acetyl galactosaminyltransferases) were obtained by Kim and Isaaks (140) who studied colonic tumors in humans. The same authors reported the absence of any changes in glycosidase activity and reduced levels of fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid in the membranes of tumor cells. These findings are at variance with the data of Bossmann and Hall (27) who discovered elevated levels of activity of glycosidases, some pro tease^,^ and sialyltransferase. Taking into consideration the use of the same techniques for enzymic activity determinations by the authors of the two latter publications, it is hard to find an explanation for this discrepancy. Unlike Kim and Isaaks (140), Mian and Cowen (184) found that the activity of glycosidases (N-acetyl-8-D-glucosaminidase, N-acetyl-p-Dgalactosaminidase, p-D-galactosidase, and a-L-fucosidase) in the DMHinduced colonic tumors of mice and rats is 2 to 4 times that of normal epithelium. Subsequently, Mian et al. (185) reported that K,'s for these enzymes in tumors are similar to those in embryonic tissue and differ from those in normal intestinal tissue. Moreover, Mian and Nutman (186) showed that the activity of phexosaminidase A in tumors induced by DMH in rats is lower than in normal intestinal epithelium. This observation is supported by the data of Brattain et ol. (29) on a relatively higher level of P-hexosaminidase B and a lowered level of isozyme A in tumors of human colon, while this ratio is reversed in normal tissue. Both isozymes have identical K,,, and V,,,,, although thermal stability and optimal pH are different. Schwartz (275) reported that the activity of enzymes which catalyze the incorporation of N-acetylneuraminic acid, galactose, fucose, and N acetylamine in the glycoproteins of colonic carcinoma in humans is relatively high: and that glycosidase activity in DMH-induced tumors of rats is elevated. These data point to changes occurring in the structure and function of cell membranes. They are further corroborated by the evidence on the altered permeability of the cell membranes of the DMH-induced tumors It should b e mentioned that in spite of this, the levels of inhibitors of proteases in human tumors are either identical to those in normal tissues (acid a,-glycoprotein, aPmacroglobulin, and antithrombin 111) (314 ) or even elevated (a,-antitrypsin, acid a,-glycoprotein, haptoglobin, and prealbumin) (320 ).

210


of rat colon (189). The permeability of cell membranes was assessed by the appearance of hexokinase-a key enzyme in glycolysis-in serum. This enzyme is not detected in the serum of intact animals, while, in DMH-treated rats, it was found 1 month after the beginning of carcinogen treatment and reached a maximum at month 5 . Simultaneously with the intensification of enzyme synthesis by tumor cells and its release into the blood, the activity of the isozyme to migrate fastest to the anode disappeared or pronouncedly decreased in experimental tumor. Only one of three isozymes, characteristic of normal intestinal tissue, remained in the metastases of signet-ring cell carcinoma. The K,, in tumors was 2-3 times less than in normal tissues. These results are consistent with the previous data on colonic tumors in humans (206). Changes in the isozyme composition of adenosine deaminase were reported recently by Trotta and Balk (308). The colonic tumors induced in rats by exposure to methylazoxymethanol acetate showed a rise rather than a decrease in the number of isozymes. Moreover, normal and tumor variants of enzymes differed in substrate specificity and responsiveness to the inhibition by 9-erythro(2-hydroxy-3-nonyl)adenine.In the opinion of these authors, the latter fact may suggest application of this preparation for chemotherapy. It is to be noted that, in normal mucosa of the jejunum, there were two enzymes, as in tumor of the colon with molecular weights of 37,000 and 33,500daltons, and PI 4.80 and 4.85, respectively. Pegg (221 ) and Pegg and Hawks (222) revealed an enhanced activity of tRNA-methylases in the DMH-induced tumors of rat colon. Of considerable interest are the results of Fiala et al. ( 9 7 ) who found an increased activity of y-glutamyltranspeptidase in the DMH-induced carcinoma of the colon and the intestines of newly born animals. Due to the simultaneous detection of CEA and an enhanced activity of this enzyme, it became possible to distinguish the metastases of colonic tumor in the liver from local recurrences and other metastases in human patients (2Y5), and the metastases of colonic carcinoma in the uveal tract from a genuine uveal melanoma (187). The mucosa of adenocarcinoma of the jejunum of rats treated with DMH shows a reduced concentration of retinyl ethers and contains no free retinal at all (300), unlike normal tissue of the small intestine. In summary, it may be concluded that the available results of the biochemical studies of experimental intestinal tumors still leave many gaps in the present-day knowledge of the subject. The situation is somewhat better in the field of biochemical peculiarities of human intestinal neoplasms. Experimental investigations in those biochemical parameters, which have been studied in man, seem to have considerable promise. They may, for instance, include the activity and isozymic spectrum of


211

lactate hydrogenase (301 ), which are generally different in the tumors of other sites, as well as p-glucuronidase and other glycosidases (73 ), important for the etiology of colonic tumors (see Sections IV,A,4 and VII1,A) and polyamines (putrescine, spermidine, spermine) (see 267 ), which play a role in nucleic acid and protein synthesis.

VII. immunology

In 1965, Gold and Freedman (107) found that human tumors of the colon produce an embryo-specific glycoprotein, which was designated as a cancer-embryonal antigen (CEA). Later on, the presence of CEA in patients with tumors of the gastrointestinal tract was confirmed by numerous reports (for references see the reviews 33,102,126,142). Today, CEA is the only biochemical marker for which the U.S. Food and Drug Administration granted a license to Hoffman-La Roche, Inc. for production and sale of kits for radioimmunoassay (179). However, no analog of CEA in experimental animals with intestinal tumors has been found so far; and this fact presents certain difficulties in conducting a detailed study of the pathogenic nature of this phenomenon.

A. ANTIGENS IN CHEMICAL-INDUCED TUMORS OF RAT COLON The earliest investigations involving the use of precipitation techniques detected an antigen in DMH-induced tumors, which was also present in embryonal tissues but was not detected in the intestinal tissues of adult rats. This antigen seems to circulate in tumor-bearing animals (103,104). Subsequently, the antigen was found in spleen extracts and, by immunohistochemical means, in marrow, macrophages, and polymorphonuclear leukocytes, but not in tumor cells; its presence was supposed to be due to tumor infiltration by leukocytes (168,f7 0 ) . The fact that Martin et nl. (170) failed to discover the antigen in perchloric acid extracts indicates that it differs from the one found by Abeyounis and Milgrom ( 2 ) and Okulov and Pozharisski (216) (see below). Moreover, experimental intestinal tumors generally do not reveal any pronounced infiltration by leukocytes, except necrotic foci (228,230). However, such neoplasms often develop in the regions of Peyer’s patches and lymphoid follicles (230,264),and it may be supposed that the antigen, which is not directly associated with tumor elements, was found only because specimens were taken from these areas.

2 12


The above investigation of Martin rt al. (170) revealed two antigens in tumor cells and, at a low concentration, in normal intestinal tissues. Finally, when rabbits were immunized by cultured rat adenocarcinoma cells and the antiserum was absorbed in vivo in syngeneic rats, there was revealed a membrane-associated antigen which was independent of the strain of rats, tumor site in the intestine, and carcinogens used for the induction of these tumors (169). These authors suspect the antigen to be tumor-specific and consider it to be a homolog of CEA in patients with colonic tumors. The sera of rats with transplanted intestinal tumors were shown to contain antibodies to this membrane-associated antigen (172). Okulov and Pozharisski (216) and Abeyounis and Milgrom (2) detected an antigen in the perchloric acid extracts of DMH-induced tumors of rats. It appeared to resemble human CEA in some physicochemical and biological properties (thermal stability, glycoprotein composition, immunoelectrophoretic migration, similar to p-globulins, and high level in embryonal and tumor intestinal tissues). The general consensus is that the antigens from experimental and human intestinal neoplasms do not cross-react (2,103,104,170).

B. ANTIGENS I N TUMORS OF

THE

SMALL INTESTINE

A recent investigation of X-ray-induced tumors of the ileum and jejunum revealed an antigen not detected in the tissue homogenates of the liver, kidney, colon, spleen, lungs, urine, and feces of tumor-bearing animals (297). At the same time, its immunological properties were identical to those of protein found in the 17- to 19-day-old rat embryos. This antigen, however, was not detected in irradiated rats, in which tumors failed to be induced (296). In such characteristics as thermal stability and mobility in immunoelectrophoresis similar to p-globulins, it resembles CEA. However, when extracted with perchloric acid, some of its antigenic determinants were hydrolized. They were completely hydrolized when extracted in 0.1 N HCI, but, 0.1 N NaOH did not affect the immunological activity of the antigen (296). The antigen was also observed in blood (83). A superficial tumor-associated antigen was detected in the cells of carcinoma of human small intestine by immunohistochemical means (210). For this purpose, an antiserum obtained by immunization of rabbits with the tissue of well-differentiated adenocarcinoma of human colon was used. This seems to point to the common nature of the antigenic composition of tumors of the large and small intestine.


213

C. ANTIGENS I N NONSPECIFIC LESIONSOF THE COLON An antigen, similar to the one described above in connection with DMH-induced tumors of the colon (216), was discovered in rat serum immunoprecipitation in agar, with nonspecific injury of the cecum (243 ). This means that the production of a CEA-like antigen in adult organism is not limited to neoplastic tissues only, and it is probably also synthesized in normal intestinal epithelium. This supposition is supported by the literature data showing that, although in small concentration (not more than 2.5 ng/ml), CEA was also found in the serum of healthy subjects (44,126,142). The appearance of the antigen in serum corresponds under experimental conditions to the period of the most enhanced reparative proliferation of intestinal epithelial cells. Since the increase in the proliferative activity of the epithelium is probably due, chiefly, to the entry of stem enterocytes into mitotic cycle (22Y), it may be suggested that CEA is produced precisely by these cells. On the other hand, immunohistochemical data show that CEA is located mainly in the glycocalyx of the most-differentiated cells of adenocarcinoma and normal intestinal mucosa in humans (130). But it seems that CEA is only absorbed on the surface of enterocytes (261), while it is actually synthesized in the cytoplasm of nondifferentiated cells (130). In general, the manner in which CEA is produced and released has very much in common with that of the mucopolysaccharides of intestinal goblet cells (207). The detection of a CEA-like substance in the experimental animals with nonspecific lesions of the intestine may account for the false positive results of the diagnosis of colonic carcinoma in man when CEA is used (44), as well as its high concentration in the blood serum of patients suffering from chronic ulcerative colitis of different etiologies (192,303). D. ANTIGENS I N INTESTINALCARCINOMA A N D ESTROGENS It has been shown recently that the administration of estrogens-propionate diethylstilbestrol-may cause an increased release of a CEA-like substance into blood serum in rats bearing DMH-induced tumors and in animals with chronic nonspecific injury of intestinal mucosa (215). This seems to be due to the presence of estrogen receptors in colonic neoplasms similar to those found in cancer patients (181 ). These data suggest the possibility of administration of estrogens to stimulate an increased release of CEA into blood serum, when it is not detectable in patients with intestinal tumors, for diagnostic purposes (215 ).

214

KAZYMIR M. POZHARISSKI ET AL.

E. CELL-MEDIATED IMMUNITY The cytotoxic action of immune lymphocytes on the cell cultures of colonic tumors induced in rats by N-methyl-N’-nitro-N-nitrosoguanidine, 3,2’-dimethyl-4-aminobiphenyl, and DMH was described by Steele and Sjogren (290). These authors claimed that chemical-induced tumors of rat colon have a common tumor-specific superficial antigen(s). At the same time Steele and Sjogren (291 ) revealed an antigenic cross-reaction between colonic tumors and fetal intestine, which points to the presence of a fetal organospecific antigen (or antigens) in carcinoma. A suggestion was made that tumor cells produced neoantigens which were the products of reexpressed fetal genes (291). Moreover, it was shown that lymphocytes in rats with X-ray-induced tumors of the small intestine exerted a cytotoxic effect on the cells of small bowel adenocarcinoma ( 8 2 ) . The cytotoxicity of the cells of lymph nodes obtained from rats with colonic tumors regarding target cells showed a sharp decrease on addition of a soluble fraction of cytoplasmic proteins and solubilized tumor membranes prepared by treatment of the cells with 3 M KCl or papain (293),as well as the serum of tumor-bearing animals (290,291,293,294). It is to be noted that the cytotoxic effect of lymphocytes and the blocking activity of the serum of rats with intestinal tumors become apparent before or at the earliest stage of tumor detection by doublecontrast X-ray examination (294). The blocking effect of serum was not observed immediately after the removal of tumors, unless any recurrences developed (294 ). Sjogren and Steele (281) give the following description of the tissue and embryonal specificities of antigens in the chemical-induced tumors of the colon: The former include the cytotoxic action of the cells of lymph nodes and blood lymphocytes in rats with intestinal tumors on the cells of intestinal carcinoma. They do not, however, exert this effect on the cells of other tumors or adult animals. The cytotoxic effect of lymph node cells in tumor-bearing animals on carcinoma cells was specifically inhibited by the serum of tumor-bearing animals. The cytotoxic effect of lymphocytes and the blocking activity of serum become manifest before or at the earliest stage of colorectal cancer detection. The blocking activity of serum ceases immediately on removal of primary tumors. The embryonal specific characteristics include the cytotoxic effect of the cells of lymph nodes of multiparous (more than three litters) pregnant rats on those of colonic carcinoma but not on the cells of adult animals. The lymphocytes of tumor-bearing rats are cytotoxic on the cells of fetal gut but not on those of fetal kidney, liver, lungs, or adult rats, intestinal mucosa included. The serum of pregnant rats can block the cytotoxic


215

action of lymphocytes on fetal intestine and intestinal carcinoma. Apart from the intestine-specific embryonal antigen, there are the so-called widely distributed embryonal antigens. Antigens expressed on the cells of fetal intestine are observed in a solubilized tumor material. In 1972, Hellstrom and Hellstrom (118) demonstrated that the lymphocytes of patients with colonic tumors are cytotoxic with respect to tumor cells in vitru but do not damage the normal epithelial cells of the intestines of the same patient. The lymphocytes from patients with other neoplasms as well as from healthy donors do not damage colonic tumor cells in vitro. However, both autochthonous and allogeneic lymphocytes of patients with colonic tumor can destroy tumor cells. On the basis of these data, it was concluded that tumors of human intestines have a common antigen. It was also shown that the lymphocytes of patients suffering from tumors of the colon can destroy the epithelial cells of fetal gut but not those of fetal kidneys or normal intestinal tissue of adult humans. The response of peripheral blood lymphocytes of patients with colonic carcinoma to phytohemagglutinin in vitro is markedly decreased (183 ). On removal of tumor the level of growth stimulation is restored. These authors suggest the following explanations: (1) the decline in the ratio of circulating T and B lymphocytes, which reflects the quantitative rather than qualitative anomaly of lymphocytes: (2) the production in patients of a factor, capable of interacting with “receptor sites for phytohemagglutinin” on lymphocyte surface: and (3) the rate and duration of the action of tumor-specific antigens on lymphocytes may change the ability of these cells to respond to other exogenous factors. However, each of these suppositions should be experimentally tested. In conclusion, owing to absolutely different approaches available at present, it is difficult to compare antigens which contribute to humoral and cell-mediated immunity developing in cases of colonic carcinoma, although, according to Martin et a / . (171), they may be identical. On the other hand, Hellstrom and Hellstrom (118) showed that lymphocytes obtained from patients with colonic tumors inhibit the growth of the cells of intestinal tumors but not those of the normal tissues of other organs or tumors of other sites. Therefore, the antigen (or antigens) which contributes to antitumor immunity seems to be strictly organspecific. However, CEA, nonspecific cross-reacting antigens and membrane-associated tissue antigens do not possess such a specificity. All these facts pose problems, the solution of which is of vital importance for tumor therapy: Why does colonic carcinoma progress quickly in v i w , though its cells may be killed by the lymphocytes of cancer patients in vitro? This may be due to the action of the above blocking elements of serum, the nature of which has not yet been identified.

216

KAZYMlR M. POZHARISSKI ET AL.

Therefore, a better immunotherapeutic effect in the management of tumors should be, probably, achieved by reducing the blocking activity of serum rather than by bolstering antitumor cell-mediated immunity (118).

VIII. Metabolism of 1,2-Dirnethylhydrazine and Related Substances

The fact that DMH and homologous substances induce tumors in the intestine only but never at the sites of administration suggests that these agents undergo certain metabolic changes before they exert their carcinogenic effect. On the basis of analogy with the metabolism of nitrosocompounds and some speculation, Druckrey and co-workers (75,247) suggested the following mechanism of DMH metabolism in a living organism: At first, it is oxidized, probably, without participation of enzymes, to azomethane (AM). This reaction is catalyzed by traces of heavy metals (247). Azomethane is further oxidized to azoxymethane (AOM). Then azoxymethane is subjected to enzymatic a-hydroxylation to form methylazoxymethanol (MAM). MAM may, in turn, be dealkylated by means of concerted reactions, forming an alkylating metabolite-methyldiazonium. Since the metabolic transformations of AOM and dimethylnitrosamine are similar, according to the above authors, it might be expected that they should produce similar carcinogenic effects. In actuality, they are characterized by a totally different organotropism, and the above mechanism does not account for these differences. Preussmann rr ul. (247) and Druckrey (75) believe that such differences in organ specificity are due to the presence of specific hydrolases in a target organ. In an attempt to explain the organ-specific carcinog :nic effect of DMH treatment, Weisburger (333) suggested that MAM is conjugated with glucuronic acid in the liver and then is transported in this form, together with bile, to the intestinal tract, where it is freed again by microbial enzymes and forms the final alkylating metabolite, which interacts with the macromolecules of enterocytes. The data available on the carcinogenicity of cycasin, a glycoside of MAM, which exhibits a similar organotropism, point to the likelihood of such a mechanism responsible for the organotropism of DMH. A. EXPERIMENTAL EVIDENCE ON DMH METABOLISM The experimental data supporting the above hypothesis on the metabolism of DMH and homologous compounds, capable of the induction of


217

intestinal tumors, may be divided into two groups. One of them includes the evidence on the detection of the postulated metabolites of DMH in experimental animals. Numerous studies of DMH metabolites involving the use of labeled carcinogenic agents have been carried out recently. For example, the air exhaled by l4C-DMH-tieated rats was shown to contain some AM, in addition to CO, (92,94,95):its level varied, depending on the dose. When 21 mg/kg of DMH were administered, exhaled air contained 14% of injected radioactivity, as AM (94,95).As DMH dosage was increased to 200 mg/kg, the level of AM in exhaled air rose considerably (up to 23% of injected radioactivity) (95 ) , Simultaneously, the fraction of exhaled CO, was found to decrease in such animals considerably-from 11%16% to 4% (95,116).This seems to show incomplete metabolism of DMH when it is administered in high doses. While AM is detected in exhaled air immediately after DMH administration and most of it is excreted within 4 to 5 hours, COPbegins to be exhaled at a later stage ( 9 5 ) . Such a delay in CO, formation is probably due to a complete oxidation of DMH metabolites or the induction of relevant enzymes ( 9 5 ) .The rate of DMH metabolism in mice seems to be higher, because the treatment with the same dosage of this substance results in CO, formation in an amount twice that in rats (116). Apart from AM and C 0 2 , the air exhaled by DMH-treated rats was found to contain monometh ylhydrazine and methylamine. A total of 45%-60% of radioactivity was shown to be excreted with air within 24 hours ( 9 6 ) . When 21 mg/kg of 3H-DMH was injected into rats, 3.7% of radioactivity was excreted in bile during 24 hours (245). However, when 14C-DMH was administered to rats, only 0.4%-0.9% of radioactivity went to bile, irrespective of carcinogen dosage (96,f16 ). All radioactivity was found in unmetabolized DMH when thin-layer chromatography was used (116 ). However, other investigators revealed different metabolites of DMH in rat bile. For example, chromatographic determinations on Sephadex LH-20 detected six different metabolites of DMH, including AOM, in bile ( 9 6 ) . Since AOM is eluted together with MAM in this chromatographic procedure, high-pressure liquid chromatography was carried out, which showed bile to contain AOM also. Unconjugated MAM was not detected. Apart from AOM this chromatographic assay showed some other peaks which probably represented MAM glucuronides and sulfates ( 9 2 ) . Another study was concerned with determinations of AOM, DMH, and AM in bile ( 9 4 ) . As much as 66% of radioactivity went to urine in the rats treated with 3H-DMH (245). Sephadex LH-20 chromatography detected DMH, MMH, and AOM in urine: 12% to 15% of radioactivity (14C-DMH)was excreted with these compounds (96 ). High-pressure liq-

2 18


uid chromatography revealed the presence of DMH, AM, AOM, and MAM in urine ( 9 4 ) . MAM was also found in the urine of the AOMtreated rats ( 9 3 ) . Hence, the results of these studies confirm the hypothesis of Druckrey, Preussmann, and Weisburger on the metabolic pathways of DMH, because all the substances supposed to be DMH metabolites were detected in the body after administration of this carcinogenic agent. The ability of the substances supposed to be DMH metabolites to induce intestinal tumors selectively is another point in support of the above hypotheses on metabolic pathways. For instance, tumors of this localization are invariably induced by AOM, MAM, and a related carcinogenic substance-cycasin (75,247). On the other hand, carcinogenic agents, which belong to hydrazo-, azo-, and azoxycompounds but are not metabolized to intermediary products identical to those of DMH, do not induce intestinal tumors. For example, 1,2-diethylhydrazine and its derivatives (azo- and azoxyethane) exhibit a specificity for other organs, inducing tumors of the nervous system, mammary gland, liver, and leukemia (75). Preussmann E t al. (247) claim that intestinal carcinogenesis is caused by those hydrazo compounds, which metabolize, forming a methylcarbonium ion. It is natural, therefore, that such substances as I-methyl-2ti-butylhydrazine and 1-methyl-2-benzylhydrazine should induce both neurinomas and intestinal neoplasms (247). On the basis of the above data on DMH metabolism and its reactivity, Pozharisski et al. (245) suggested a scheme of metabolism of this agent, which is intended to complement and detail the available concepts of Preussmann et al. (247), Weisburger (333), and Druckrey (75). According to this scheme, after DMH has found its way into the circulation, it is dehydrogenated to form AM. Subsequently, this product may undergo the three following changes: (I) homolytic degradation to nitrogen and methyl radical: (11) N-oxidation, forming N-oxyazomethane (AOM): or (111) a-C-hydroxylation (Fig. 3). There is some evidence which precludes the first possibility. For instance, the macromolecules of enterocytes fail to be labeled following total hepatectomy (234). It is significant that tumors do not arise at the sites of DMH administration and in the lungs, i.e., at the site where most of AM is excreted. Moreover, there are data demonstrating that the enzymic systems of the liver participate in the metabolism of DMH and its derivatives (93,247). The other two pathways of azomethane oxidation in biological systems are equally probable: Two maxima of label incorporation into the DNA and protein of different organs recorded at different times after injection

219


homolyaie

1,E-Dimethylhydrazine

Yethylazomethanol

II

.

N-oxidation1 in liver CH N=NCHJ trena- and cis-Nethylazoxymethane

3'

CH,,~NCH~OH trenm- end cia-NethylO"N-esoxymeth.no1

0

r(

C-hydroXY1etion W

h*temlyeis

-Hcwo

rCHjn.nOn

-pmo2

Yethyldiuo, hydroxide

uthylnitruine

Alk-1-CH2COOH I

ma3

1Jsthyl-3-lkyl-3cuboxymthyl-trlezene

FIG.3. Possible metabolic pathways of DMH. *Able to form glucuronides. From K . M. Pozharisski et ul. (245 ).

of 3H-DMH (234) suggest that DMH metabolism may occur by these two pathways simultaneously, though, probably, at different rates. a-C-hydroxylation of azomethane (Pathway 111) may result in the formation of methylazomethanol which may degrade to formaldehyde and methyldiimine, the latter, probably, forming a methyl radical, as a result of homolysis. When DMH is metabolized, forming methylazoxymethane with its subsequent hydroxylation (Pathway 11), unstable products-derivatives of MAM-are formed. The possibility of identification of the site at which this reaction takes place is confirmed by the results of in virro experiments which demonstrated hydroxylation of AOM, forming MAM, to occur due to hepatic homogenates and microsomes in the presence of NADPH. At the same time, this reaction failed to take place on addition of homogenates or microsomes of colonic mucosa (93,94). MAM derivatives are subsequently converted to methyldiazohydrate. Since these products form, as a result of DMH metabolism in the liver, while tumors arise mostly in the intestine, it was assumed that the liver is the site of the formation of DMH metabolites, which are released in target organ, as a result of the action of its specific factors. The most likely in this connection seems to be the formation of glucuronides, which synthesize a carcinogenic metabolite, probably, methylazoxymethanol, in the presence of bacterial P-glucuronidase. Recent investigations have revealed that tissue enzymes participate in MAM metabolism, because

220


the cytosols of the colon and cecum and, particularly, that of the liver reduce NAD to NADH in the presence of MAM acetate as a substrate. Meanwhile the cytosol of the jejunum and ileum, which are resistant to MAM, exhibits a low NAD-dependent alcohol dehydrogenase activity in the presence of MAM (109). These findings point to MAM being metabolized by alcohol dehydrogenase-like enzymes and suggest that NADdependent enzymic reactions, occurring in tissue cytolsol, may determine the organotropism of the carcinogenicity of MAM in rats also. These results further support the concepts of Preussmann et al. (247) and Druckrey (75) who think that MAM can be dealkylated spontaneously. Most of the labeled metabolites of DMH (96%)enter the intestine with bile (245),while as little as 4% of all radioactivity detected in the intestine is delivered there by blood flow. Nevertheless, tumors were also induced in isolated fragments of the intestine (227). Therefore, it is from the circulation that carcinogenic metabolites penetrate into the intestinal wall. As mentioned above (Y2), AM and AOM were found in bile, in addition to DMH, while other metabolites of this agent have not yet been identified. Pozharisski et al. (245) suggested that, apart from glucuronides, other synthesized in the methyl group-carrying substances-triazenes-are liver. These substances may be the products of a reaction of methyldiazonium and protein amino acids. These authors believe that triazenes are likely, by alkylation, to be responsible for tumor formation in germfree rats (233,257) as well as in the duodenum, in which bacterial flora are scare. It may as well be attributed to the effect of P-glucuronidase, which is contained in intestinal epithelium at high concentration. The metabolism of DMH, forming AOM (Pathway II), and a-C-hydroxylation of AM (Pathway 111) would result in different end productsa carbonium ion (CH,+) or a methyl radical (CH3.). They are different in chemical nature and reactivity. Since Pathway I11 of DMH metabolism produces a derivative of monomethyl hydrazine, which is a poor alkylator and does not induce intestinal tumors (116), it is most likely that the carcinogenic effect of DMH is due to its metabolism by Pathway 11, forming a carbonium ion. It was shown quite recently that, following a pretreatment with disulfiram which blocks N-oxidation of AM (94), the bases of DNA were not alkylated, while the label from 3H-DMH was incorporated into unmethylated purines ( 1 5 7 ~ )These . findings prove that DNA is methylated by the carbonium ion alone, rather than by the methyl radical. Thus, on the basis of the available data, Pozharisski et ul. (245) distinguish the following stages of DMH metabolism, leading to the initiation


22 1

of carci nogenesis : (a) formation of “active” metabolites (methylazoxymethanol or methyldiazohydrate) in the liver; (b) binding of the metabolites to glucuronic acid: (c) delivery of conjugates to the intestine via blood flow: (d) liberation of “active” metabolites due to enzymic activity of intestinal flora (P-glucuronidase): (e) formation of carbonium ion; and (f) specific methylation of enterocyte macromolecules.

B . THEEFFECTOF CERTAIN MODIFYING FACTORS ON DMH METABOLISM The suppression of the carcinogenic effect of DMH treatment in mice by administration of disulfiram (331) has stimulated studies of the mechanism of this phenomenon. A pretreatment with disulfiram (94) or some of its metabolites-diethyldithiocarbamate, bis(ethylxanthogen), and carbon disulfide-to rats before injection of 14C-DMH(94 ) appeared to result in a trebled level of azomethane in exhaled air, while carbon dioxide concentration fell by 65%-80%, as compared with the animals treated with DMH alone. AOM and MAM concentrations in the urine of these rats either dropped considerably or were not detected at all. The levels of 14C incorporation into the liver, kidneys, lungs, spleen, and colonic mucosa decreased by 43%-70%. As a result, Fiala rt a / . (94 ) concluded that these substances block the N-oxidation of AM and, therefore, prevent its conversion to AOM. However, the pretreatment with disulfiram or carbon disulfide also blocked the oxidation of the AOM administered to rats, which manifested itself in a complete suppression of carbon dioxide formation within 6-8 hours. The concentration of AOM in exhaled air, however, was rather high. Moreover, disulfiram and carbon disulfide reduced the level of MAM and raised that of AOM in the urine of these rats (93). The decrease in the carbon dioxide level following the administration of disulfiram and its metabolites was probably due to the diminished formation of formaldehyde, a decline in its oxidation, or a combination of both these processes ( 9 4 ) . Therefore, disulfiram and its derivatives not only block the N-oxidation of AM but inhibit further metabolism of AOM. It is most likely that DMH metabolism becomes blocked by disulfiram and homologous compounds as a result of the inhibition of the activity of the microsomal cytochrome P-450-dependent oxygenases.

222

KAZYMlR M . POZHARlSSKl ET AL.

Unlike disulfiram, a preliminary administration of phenobarbital to rats, followed by subcutaneous injection of AOM, resulted in a 1.5-fold increase in the level of I4CO2exhaled, while the level of MAM excreted with urine increased tens of times. At the same time, the amount of unmetabolized AOM in urine decreased ( 9 3 ) . All these data point to an intensified metabolism of the carcinogenic substance, following the administration of phenobarbital. Chrysene exerted a still more pronounced effect (93 1. IX. Interaction of 1,2-DimethyIhydrazine and Related Compounds with Cell Components

On the basis of the above data on the pathways of DMH metabolism, leading to the formation of an alkylating metabolite, one might expect a priori that DMH treatment of animals would result in the alkylation of different macromolecules. The alkylating potential of DMH was confirmed by Hawks et al. (f 1 7 ) , who by Dowex-50 chromatography of hydrolyzates of liver and colon DNA and RNA of mice treated with DMH 6 or 24 hours before, detected 7-methylguanine in all samples studied. Hawks and Magee (116) found that within 6 hours after injecting 15 mg/kg of 14C-DMH into mice, 7methylguanine was present in the nucleic acids of all organs studied. Its level ranged from 1.5% (small intestine and lungs) to 4%-5% (large intestine, kidneys, and spleen) of that in liver DNA. In the RNA from the same tissues, the levels were similar. The distribution pattern in the rats sacrificed 6 hours after an injection of 200 mg/kg of the carcinogenic substance was somewhat different: 7-methylguanine in intestinal DNA and RNA was more than 50% and, in the nucleic acids of kidney tissue, about 12% of that of liver. The rate of alkylation of nucleic acids in mice appeared to be higher than in rats, and like nitrosamines, DMH methylated more RNA than DNA. No alkylated products other than 7-methylguanine were found in this study. Although these investigations confirm the alkylating potential of DMH, they do not account for the organotropism of DMH. Both in rats and mice, maximal alkylation was observed in liver nucleic acids, while tumors are induced by this agent mostly in the colon. To establish possible correlations between the organotropism of DMH and its interactions with DNA, RNA, and proteins, a time-course study was conducted (234).In all tissues under study, the specific radioactivity of DNA appeared to have two maxima: the first peak of different segments of the intestines (duodenum, small intestine, ascending and de-


223

scending colon) was observed within 1 hour and that of liver and kidney DNA 3 hours after carcinogen treatment. Subsequently, the radioactivity level decreased followed by a rise by hour 9. By hour 12, there was a sharp fall in specific radioactivity level in the DNA of all tissues, followed by a gradual decline until day 3 after exposure. By day 7, the radioactivity rose again, but by four weeks after 3H-DMH treatment, no tissues contained any radioactivity. It was suggested that the rise in radioactivity of DNA between days 3 and 7 was due to the incorporation of methylated precursors from the cell pool into the newly synthesized DNA, which is consistent with other observations. In all RNA samples, radioactivity increased within the first 6-9 hours. By hour 12, there was a sharp fall, followed by a gradual decrease. Between days 3 and 7, radioactivity, except for the liver and small intestine, remained practically unchanged. Like DNA, this may be due, probably, to incorporation of labeled precursors. Radioactivity was negligible by week 4. Throughout the entire experiment, radioactivity levels in the DNA and RNA from the liver and kidneys were higher than those of different intestinal segments. The radioactivity in total protein was maximal within 1 hour after treatment with the carcinogen and its levels in all segments of the intestine were much higher than in parenchymatous organs. By hour 3, the protein radioactivity fell in all organs, but at hour 9, it increased in protein of the intestines. Within the period from hour 12 to day 7 , protein radioactivity remained practically unchanged and it could not be detected 4 weeks after treatment. In control rats injected with a substance homologous to DMH-3H-1,2diethylhydrazine (3H-DEH), no label was detected in either DNA, RNA, and protein of the intestine, liver, or kidneys. However, radioactivity levels in all fractions of tissues of DEH-target organs (thymus, spleen, and brain) were high. Thus, the experiments of Pozharisski et al. (234) did not reveal any correlation between the radioactivity level of the macromolecules of different organs of rats and their susceptibility to the carcinogenic effect of DMH. These results as well as those of Hawks et al. (117), Hawks and Magee (116), and Shimitzu and Toth (276), who autoradiographically revealed a maximum incorporation of label from 14C-DMH by mouse hepatocytes rather than enterocytes, fail to explain why DMH-induced tumors arise predominantly in the colon. Pozharisski et al. (234) suggested that in those organs in which DMH treatment fails to induce tumors a “nonspecific” methylation or participation of the methyl groups of 3H-DMH in the normal metabolism of nucleic acids occurs. “Specific”

224


methylaction probably occurs in the tissues in which DMH induces tumors predominantly (colon). It has recently become obvious that the level of alkylation of DNA guanine in the 7 position is not correlated with the organotropism of the carcinogenicity of many alkylating agents. On the other hand, there is much evidence that the carcinogenicity of alkylating agents may be caused by 0 6-methylguanine formation in DNA. It may be the persistence of 06-methylguanine in DNA rather than the initial level that is actually responsible for its carcinogenic effect (153,165 ). Loveless (163 ) suggested that, being a potential mutagenic base, 0 6-methylguaninecan block base pairing with cytidine and cause the misincorporation of thymine during DNA replication (see 153). This effect was confirmed in in vifro experiments, using the RNA-dependent RNA polymerase and an 0 %nethylguanine-containing template. The product formed in this system incorporated uracil, an analog of thymine but not cytosine, whereas 7methylguanine formed a normal pair with cytosine. As a result, 0 6 methylguanine may cause a mutation (see 153). Likhachev et al. (157) carried out a detailed study of the alkylated products of the DNA of different tissues of rats killed 3 hours after 3HDMH injection. Similarly to the results of Hawks and Magee ( 1 1 6 ) , the highest level of 7-methylguanine was found in liver DNA, a lower level in colon DNA (14% of that in the liver), and levels in the DNA of kidneys, testes, lungs, and small intestine of 1.7%-3.2% of that in liver. At the same time, the DNA of all organs incorporated radioactive products of DMH breakdown, as a result of de nova synthesis of its precursors. The uptake of radioactivity in adenine varied greatly and the highest level was detected in the adenine of the DNA of the duodenum, in which alkylated purines were not found at all. Therefore, the specific radioactivity of total DNA cannot be used as an indication of the degree of alkylation. Besides 7-methylguanine, the DNAs of the liver and colon had 0 6 methylguanine, which points to a high potency of DMH as an alkylator, like MNU and DMNA (153). Likhachev et ul. (157) showed that the 0 6 methylguanine to 7-methylguanine ratio in the DNA of the target organthe colon-to be more than four times that in the DNA of the liver, in which tumors do not arise after a single exposure to DMH. Unlike in other organs, the level of 06-methylguanine of the colon DNA did not vary within 3 days (190), and O6-rnethylguanine was not detected at all in the mucosa of the ileum, where practically no tumors arise. Since the initial 0 6-methylguanine to 7-methylguanine ratio of DNA is determined by the type of the carcinogen used but not by the target organ (153), this


225

evidence demonstrates that the colon’s ability to eliminate 0 fi-methylguanine from DNA is much lower than that of the liver. Hence, the results support the hypothesis of Pozharisski et ul. (245) that although the levels of total radioactivity of DNA in different segments of the intestine are similar, the “specific” alkylation of DNA occurs in the colon only. The findings of Likhachev et al. (157) were confirmed by experiments of Rogers and Pegg (266 ), who studied the alkylation of rat liver, kidney, and colon DNA at intervals after intraperitoneal or subcutaneous administration of 14C-DMH.After intraperitoneal administration of the carcinogenic agent, the formation of 7-methylguanine and 0 6-methylguanine was observed in the DNA of all organs under study. Moreover, liver DNA contained small amounts of 1-methyladenine, 6-methyladenine, 3methyladenine, and 3-methylguanine. The highest levels of alkylated bases were detected in all three organs 6 hours after DMH treatment. At hour 48, 0 fi-methylguaninelevel in liver DNA dropped by nearly twothirds, while that of 7-methylguanine by half. 7-Methylguanine was eliminated from kidney DNA at the same rate as from liver DNA, while Ofimethylguanine level dropped by only 40% within the same period. The level of 7-methylguanine in colon DNA decreased fourfold, while that of 0 s-methylguanine threefold. Thus, within 48 hours after intraperitoneal injection of DMH, the 0 s-methylguanine to 7-methylguanine ratio in liver DNA decreased, whereas it grew in the DNA of the kidneys and colon. A somewhat different situation developed after subcutaneous injection of I4C-DMH. While the 7-methylguanine level in rat liver DNA was identical to that after intraperitoneal administration, it was twice as high in kidney DNA, and somewhat lower in colon DNA. During a 48-hour interval after subcutaneous injection of DMH, there was a rise in Ofimethylguanine level in kidney and colon DNA, indicating that DNA methylation proceeded throughout the experiment, although exhalation of radioactivity and, therefore, DMH metabolism, actually ceased in these rats by hour 24. The 0 fi-methylguanineto 7-methylguanine ratio increased in the DNA of all three organs within 48 hours. However, it rose only 1.3-fold in liver DNA, but 3.4-fold in kidney DNA and at least 9-fold in colon DNA. After DMH administration by both routes, 7-methylguanine and 0 fimethylguanine levels in liver DNA were about 10-fold higher than in the DNA of the kidneys and colon. Therefore, the 0 fi-methylguanine to 7-methylguanine ratio in the DNA of the target organ (colon) was higher than those of other organs. The fact that the 0 6-methylguanine level in kidney and colonDNA was

226


increasing during the whole 48-hour period after exposure to DMH, when the latter had been excreted, is interpreted by us as a manifestation of either a continued liberation of an alkylating metabolite from the corresponding glucuronide or an incorporation of alkylated precursors into the newly synthesized DNA. Unfortunately, Rogers and Pegg (266 ) were not concerned with a comparative study of DNA methylation in the small intestine, in which tumors fail to be induced by DMH. As a consequence, it is impossible to adequately appraise the role of DNA alkylation in the tropism of carcinogenesis of DMH in different parts of the intestine. Unlike the data reported by Likhachev et al. (157), Rogers and Pegg (266) found that the level of alkylation of colon DNA and, particularly, that of 06-methylguanine was one order higher than methylpurine level in liver DNA. This discrepancy may be due to the fact that Rogers and Pegg (266 ) determined the alkylation of purines of the DNA isolated from the entire mass of the colon, but not from its mucosa, which is the target tissue for DMH and constitutes only a small portion of the whole mass of the intestinal wall. Some recent reports demonstrate that the alkylation by DMH may be studied in cultured tissues of humans and animals. For instance, after incubation of cultured human colon with 14C-DMH,the labeling of DNA and protein was much higher than with 14C-dimethylnitrosamine and, particularly, 14C-benzo[a]pyrene. After a 24-hour incubation with DMH, 3-methyladenine, 7-methylguanine, and 0 6-methylguanine were found in colon DNA (11 ) and also in DNA after a 24-hour incubation of slices of human bronchi in the presence of DMH. 0 s-Methylguanine level was the highest-three times that of 7-methylguanine (114). DMH metabolites also bind to macromolecules of the epithelium of rat intestine cultured i n vitro (10). Thus these tissues of human and animal intestines and bronchi contain enzymes indispensable for the synthesis of an alkylating metabolite of DMH, which methylates the DNA of these organs. In conclusion, it should be mentioned that apart from 0 6-alkylguanine formation, alkylation of other positions in the purine and pyrimidine bases of DNA may also play a role in carcinogenesis (153,279). For instance, 0 4-methylthyminewas also shown to form a mispair with guanine, instead of adenine, during DNA synthesis in vitro (I ). X. Conclusion

The study of various aspects of intestinal carcinogenesis has become possible owing to the development of relevant experimental models. Considerable advances have occurred in the study of morphology and


227

morphogenesis of intestinal cancer, which contribute to the elucidation of the mechanisms of human colon carcinogenesis. It has been shown that the malignant potency of adenomatous polyps was exaggerated; however, local lesions of intestinal mucosa, which promote carcinogenesis, though not determined genetically, have been detected. Experimental intestinal tumors are finding a wide application in the studies of the immunology and kinetics of tumor cell populations, opening up new vistas in the explorations of the nature of tumor growth. Such animal models are being used for the improvement of X-ray (32,260) and clinical (70) diagnosis of intestinal tumors. The available data show that the pathogenesis of experimental intestinal tumors is a complex of processes, taking place at all levels-from the molecule to the body, including multistage metabolism of the carcinogen involved. This metabolism is determined, to a considerable degree, by the enzymic function of the liver, specific transport of carcinogenic metabolites to the target organ, and enzymic activity of intestinal bacterial flora, terminating in the alkylation of DNA bases, which persist for a long time. Of vital importance are the cell types, which interact with the end metabolite, and the phase of their life cycle. Moreover, such factors as genetic characteristics, age and hormonal status of the organism, and diet also contribute.

ACKNOWLEDGMENT The authors consider it their pleasant duty to thank Prof. N . P. Napalkov for his unflagging interest to this study and valuable advice, Prof. H. Druckrey for discussions on certain aspects of the problem, and Mr. I. K. Arkhipov for assistance in the preparation of the manuscript for publication.

REFERENCES 1. Abbott, P. J., and Saffhill, R. (1977). N!4ct. Acids Res. 4, 761-769. 2. Abeyounis, C. J., and Milgrom, F. (1976). J. Irnrnunol. 116, 30-34. 3 . Abrams, G. D., Bauer, H., and Sprinz, H. (1963). Lab. Invest. 12, 355-364. 4 . Ackerman, L. V., and Spratt, J. S., Jr. (1963). Gastroenterology 44, 905-909. 5 . Allfrey, V. G., Boffa, L. C., and Vidali, G. (1977). Cancer 40, 2692-2698. 6 . Andreassen, J. C. (1%9). Actu Chir. Scand. Suppt. 396, 123-169. 7. Aries, V . , Crowther, J. S.,and Drasar, B. S. (1969). Gut 10, 334-335. 8 . Armstrong, B., and Doll, R. (1975). In?. J . Cancer 15, 617-63 I . 9. Asano, T., Pollard, M., and Madsen, D. C. (1975). Proc. SOC. Exp. B i d . Med. 150, 780-785. 10. Autrup, H., Hanis, C. C., Stoner, G. D., and Fugaro, S. (1977). In vitro 13, 192. 11. Autrup, H . , Hams, C. C., Stoner, G . D., Jesudason, M. L., and Trump, B. F. (1977). J . Nail. Cancer Inst. 59, 351-354.

228


l 2 . Baker, R. K. (1953). Cancer Res. 13, 137-140. 13. Balish, E., Shih, C. N., Croft, W. A., Pamukcu, A. M., Lower, G., Bryan, G . T., and Yale, C. E. (1977). J . Natl. Crrncer fnst. 58, 1103-1 106. 14. Ball, W. J., Jr., Salser, J. S . , and Balis, M. E. (1976). Cancer Res. 36, 2686-2689. I S . Bansal, S. C., Mark, R., Bansal, B., and Rhoads, J. E. (1978). J . Natl. Crrncer I n ~ t . 60, 667-675. 16. Barbolt, T., Ringwood, N., and Abraham, R. (1977). Tuxirol. Appl. Phannrrcol. 41, 157. 17. Barkla, D. H., and Tutton, P. J. M. (1977). C m c e r Res. 37, 262-271. 18. Barthold, S. W., and Jonas, A. M. (1977). Cancer Res. 37, 4352-4360. 19. Bielschowsky, F. (1946). Br. J . E x p . Path. 27, 135-139. 20. Birbeck, M. S . C., and Dukes, C. E. (1963). Proc. R. Soc. Med. 56, 793-797. 21. Bleiberg, H., and Galand, P. (1976). Ccrncer Res. 36, 325-328. 22. Boffa, L. C., and Allfrey, V. G. (1976). Cancer Re.?. 36, 2678-2685. 23. Boffa, L. C., and Allfrey, V. G. (1977). Cuncer 40, 2584-2591. 24. Boffa, L. C., Vidali, G., and Allfrey, V. G. (1976). Exp. Cell Res. 98, 396-410. 25. Bone, G., Camplejohn, R. S. (1975). Am. J . Procrol. 26, 65-76. 26. Bonser, G. M., Clayson, D. B., and Jull, J. W. (1956). Br. J . Cajicer 10, 653-667. 27. Bosmann, H. B., and Hall, T. C. (1974). Proc. Natl. Acad. Sci. U . S . A . 71, 18331837. 28. Bralow, S. P., Gruenstein, M., Meranze, D. R., Bonakdarpour, A., and Shimkin, M. B. (1970). Cancer Re.\. 30, 1215-1222. 29. Brattain, M. G., Kimball, P. M., and Pretlow, T. G., 11. (1977). Crrncer Res. 37, 731735. 30. Broitman, S. A., Vitale, J. J., Vavrousek-Jakuba, E., and Gottlieb, L. S. (1977). Clrncer 40, 2455-2463. 31. Burkitt, D. P., (1974). Dis. Colon Rectrun 17, 456-461. 32. Burkle, G . , von. (1975). Fortschr. Geh. Riientgemtr. Nrikleurmrd. 122, 352-364. 33. Burtin, P. (1974). Cancer 34, 829-834. 34. Calman, K. C. (1974). Gut 15, 552-554. 35. Campbell, R. L., Singh, D. V., and Nigro, N. D. (1975). Cancer Res. 35, 1369-1371. 36. Camplejohn, R. S . , Bone, G., and Aherne, W. (1973). B r r . J . Cuncer 9, 577-582. 37. Carter, S. (1976). Semin. Oncol. 3, 433-443. 38. Castleden, W. M. (1977). Br. J . Cuncer 35, 491-495. 39. Castleman, B., and Krickstein, H. I. (1962). N. Engl. J . Med. 267, 469-475. 40. Castleman, B., and Krickstein, H. I. (1966). Ga.strcietitero/og~51, 108-1 12. 41. Cheng, H., and Leblond, C. P. (1974). Am. J. Ancrf. 141, 537-562. 42. Chlopin, N. G. (1946). “Biological and Experimental Fundamentals of Histology.” Publ. House of the U.S.S.R. Academy of Sciences. 43. Chomchai, C., Bhadrachari, N., and Nigro, N. D. (1974). Dis. Colon Rectum 17,3103 12. 44. Chu, T. M., Reynoso, G., and Hansen, H. J. (1972). Ncrtrire (London) 238, 152-153. 45. Cleveland, J. C., and Cole, J. W. (1969). Cancer 23, 1200-1203. 46. Cleveland, J. C., Litvak, S. F., and Cole, J. W. (1967). Cancer Res. 27, 708-714. 47. Cole, J. Quoted by Wynder, E. L., Kajitani, T., Ishikawa, S . , and Takano, A. (1969). Cancer 23, 1210-1220. 48. Cole, J . W., and McKalen, A. (1963). Crrncer 16, 998-1002. 49. Colvert, J. R.. and Brown, C. H. (1948). Am. J . Med. Sci. 215, 24-32. 50. Corbett, T. H., Griswold, D. P., Jr., Roberts, B. J., Peckham, J. C., and Schabel, F. M., Jr. (1975). Cancer Res. 35, 2434-2439.


229

51. Cox, A. J., Wilson, R. H., and DeEds, F. (1947). CuIrcer Res. 7, 647-657. 52. Criss, W. (1974). O / i c d o g y 30, 43-80. 53. Cuatico, W., Cho, J.-R., and Spiegelman, S. (1974). Proc. N o t / . A c d . Sci. U . S . A . 71, 3304-3308. 54. Cunningham, M. P., and Wilhoite, R. (1973). Dis. Co/o,7 Recrrrni 16, 145-148. 55. Czernobilsky, B., and Tsou, K-C. (1968). C o w e r 21, 165-177. 56. Daland, E. (1954). N . &a/. J. Med. 251, 664-669. 57. Deger, G. E. (1976). Anu. Intc~rn.Mid. 85, 204. 58. Denk, H., Holzner, J . H., and Obiditsch-Mayer. I. (1975). Verli. Dtsch. Ccs. Ptrthol. 59. Tug. Stuftgurt 490. 59. DeRubertis, F. R., Chayoth, R., and Field, J . B. (1976). J. C/i/r./ / w e s t . 57, 641-649. 60. DeRubertis, F. R., and Craven, P. A. (1977). Cancer 40, 2600-2608. 61. Deschner, E. E. (1974). Cuncer 34, 824-828. 62. Deschner, E. E., and Lipkin, M. (1970). J . N u t / . Cancer I n s t . 44, 175-187. 63. Deschner, E. E., and Lipkin, M. (1976). C h . Gus/roe/r/ero/.5, 543-561. 64. Deschner, E. E., Lipkin, M., and Solomon, C. (1966). J. N u t / . Cu~icc~v I r r s t . 36, 849858. 65. Deschner, E. E., Long, F. C., and Katz, S. (1975). Am. J. D i g . Dis. 20, 418-424. 66. Deschner, E. E., Winawer, S. J., Long, F. C., and Boyle, C. C. (1977). Crrrrwr 40, 2625-263 1. 67. Dilman, V. M. (1971). Luncer 1, 1211-1219. 68. Dilman, V. M.. Sofronov, B. N., Anisimov, V. N., Nasarov, P. G., Lvovich, E. G . , and Polushina, L. I. (1977). Vopr. Oticol. 23, 50-54. 69. Diwan, B. A., Meier, H., and Blackman, K. E. (1977).J . N u t / . CUMPT/ m i . 59, 455458. 70. Dixon, M. F., Cowen, D. M., and Cooper, E. H. (1975). Bionirdicine 23, 247-252. 71. Doll, R., Muir, C., and Waterhouse, J . (eds.) (1970). "Cancer Incidence in Five Continents." UICC, Geneva. 72. Double, J. A., and Ball, C. R. (1975). Cuncczr C h ~ m o f h ~Rrc.p . 59, 1083-1089. 73. Double, J. A., Cooper, E. H., and Goligher, J. C. (1977). Biomcdici/rc. 27, 11-13, 74. Drasar, B. S . , and Jenkins, D. J. A. (1976). Am. J. C/;/t.Nritr. 29, 1410-1416. 75. Druckrey, H. (1972). 111 "Topics in Chemical Carcinogenesis. 2nd International Symposium of the Princess Takamatsu." Univ. of Tokyo Press, Tokyo. 76. Druckrey, H., and Lange, A. (1972). Fed. Proc. 31, 1482-1484. 77. Druckrey, H., Preussmann, R., Ivankovic, S., and Schmahl, D. (1967). Z. Krebs,forsch. 69, 103-201. 78. Druckrey, H., Preussmann, R., Matzkies, F., and Ivankovic, S. (1967). Nrrtrir~~,is.senschuften 54, 285-286. 79. Druckrey, H . , Steinhoff, D., Preussmann, R., and Ivankovic, S . (1964). Z. Krrbsjbrsch. 66, 1-10. 80. Dunn, T. B. (1965). I n "Carcinoma of the Alimentary Tract" (W. J . Burdette, ed.), pp. 45-54. Univ. of Utah Press, Salt Lake City, Utah. 81. Dunphy, J. E., Patterson, W. B., and Legg, M. A. (1959). Ann. Srirg. 150, 488-498. 82. Englund, C. W., Stevens, R. H., Chan, S. W., and Osborne, J. W. (1976) Radicrt. Res. 67, 568. 83. Englund, C . W . , Stevens, R. H., Cheng, H. F., and Osborne, J. W. (1975). Rudiut. Res. 62, 603. 84. Enker, W. E., and Jacobitz, J. L. (1976). Surgery 80, 164-170. 85. Evans, I. A., and Mason, J. (1965). Nature (London) 208, 913-914.

230


86. Evans, I. A,, Widdop, B., Jones, R. S., Barber, G. D., Leach, H., Jones, D. L., and Mainwaring-Burton, R. (1971). Biochem. J. 124, 28P. 87. Evans, J. T., Hauschka, T. S., and Mittelman, A. (1974). J . Nail. Cancer Inst. 52, 999- 1000. 88. Evans, J. T., Lutman, G., and Mittelman, A. (1972). J. M e d . 3, 212-215. 89. Evans, J. T., Mittelman, A., and Hauschka, T. S. (1975). Proc. Am. Assoc. C u ~ c e r Res. A m . Soc. Cliii. Oncol. 16, 160. YO. Evans, J . T., Shows, T. B., Sproul, E. E., Paolini, N. S., Mittelman, A., and Hauschka, T. S. (1977). Cancer Res. 37, 134-136. 91. Farmer, R. G., Hawk, W. A., and Turnbull, R. B., Jr. (1971). Cancer 28, 289-292. 92. Fiala, E. S. (1975). Cancer 36, 2407-2412. 93. Fiala, E. S. (1977). Cuncer 40, 2436-2445. 94. Fiala, E. S . , Bobotas, G., Kulakis, C., Wattenberg, L. W., and Weisburger, J. H. (1977). Biochem. Phurmtrcol. 26, 1763-1768. 95. Fiala, E. S., Kulakis, C., Bobotas, G., and Weisburger, J. H. (1976). J . Natl. Cuncer Inst. 56, 1271-1273. 96. Fiala, E. S., and Weisburger, J. H . (1975). Toxicol. Appl. Pharnracol. 33, 178. 97. Fiala, S., Fiala, A . E . , Keller, R. W., and Fiala, E. S. (1977).Arch. Grsc1iwrtlsrfor.sch. 47, 117-122. 98. Filipe, M. I. (1975).Br. J . Cuncer 32, 60-77. 99. Filipe, M. I., and Branfoot, A. C. (1974). Cancer 34,282-290. 100. Fischer, E. R., and Sharkey, D. A. (1962). Cancer 15, 160-170. 101. Frank, J. D., and Shorey, B. A. (1973).Gut 14, 120-124. 102. Freedman, S. 0. (1972).J . Allergy C1iIi. Immrrnol. SO, 348-357. 103. Garmaise, A.B-K., Rogers, A., Newberne, P. M., Saravis, C. A., Kupchik, H . 2.. and Zamcheck, N. (1974). Nurure (London) 248, 706-707. 104. Garmaise, A.B-K., Rogers, A. E., Saravis, C. A., Zamcheck, N., and Newberne, P. M. (1975).J. Natl. Cancer I / i s t . 54, 1231-1235. 105. Gennaro, A. R., Villanueva, R., Sukonthaman, Y., Vathanophas, V., and Rosernond, G. P. (1973). Crrncer Ros. 33, 536-541. 106. Georgsson, G., and Vigfusson, H. (1973).Actu Vet. Scaird. 14, 392-409. 107. Gold, P., and Freedman, S. 0. (1965).J. Exp. Med. 122, 467-481. 108. Goldin, B. R., and Gorbach, S. L. (1976).J. Nut/. Cuncer Inst. 57, 371-375. 109. Grab, D. J., and Zedeck, M. S. (1977). Fed. Proc. Fed. Am. Soc. Exp. B i d . 36, 348. 110. Grinnel, R. S . , and Lane, N. (1958). Irrt. Ahstr. Srirg. 106, 519-538. 1 1 1 . Haase, P., Cowen, D. M., Knowles, J. C., and Cooper, E . H. (1973). Br. J . Cunccv 28, 530-543. 112. Haenszel, W., and Correa, P. (1973). [ A s . Colon Rectum 16, 371-377. 113. Harber, P. S. (1975). Schweb. Mcd. Wochenschr. 105, 564-569. 114. Harris, C. C., Autrup, H . , Stoner, G. D., McDowell, E. M., and Trump, B. F. (1977). Cancer Res. 37. 2309-23 11. 115. Hawks, A., Farber, E., and Magee, P. N . (197U1972). Chem.-Bio/. Ititeruct. 4, 144148. 116. Hawks, A., and Magee, P. N. (1974). Br. J. Cuncw 30,440-447. 117. Hawks, A., Swann, P. F., and Magee, P. N . (1972). Biocheni. Pliurmucol. 21, 432433. 118. Hellstrom, I., and Hellstrom, K. E. (1972). Dis. Colotr Rectum 15, 100-105. 119. Higginson, J . (1967). 111 "Natl. Cancer Inst. Monograph," Vol. 25, pp. 191-198. Washington, D.C. 120. Hill, M. J. (1975). Crrnwr Ros. 35, 3398-3402.


23 1

121. Hill, M. J., and Aries, V. C. (1971). J . Pathol. 104, 129-139. 122. Hirono, I., Fushimi, K., Mori, H., Miwa, T., and Haga, M. (1973). J. Nail. C ~ n c e r Inst. 50, 1367-1371. 123. Hirono, I., Sasaoka, I., Shibuya, C., Shimizu, M., Fushimi, K., Mori, H., Kato, K.. and Haga, M. (1975). Gann Monogr. Cancer Res. 17, 205-217. 124. Hirose, F., Fukazawa, K., Watanabe, H., Terada, Y., Fujii, I., and Otsuka, S. (1977). Gunn 68, 669-680. 125. Hollmann, K. H. (1964). Arch. Ma/. Appiir. Dig. 53, 975-994. 126. Holyoke, D., Reynoso, G., and Chu, T. M. (1972). Ann. Siirg. 176, 559-563. 127. Hornburger, F., Kerr, C. S., and Hsueh, S. S. (1971). Narirre (London) New Biol. 234, 28-29. 128. Horava, A., and Haam, E., von. (1958). Cancer Res. 18, 764-767. 129. Horn, R. C. (1971). Cuncer 28, 146-152. 130. Huitric, E., Laumonier, R., Burtin, P., Kleist, S., von, and Chanavel, G., (1976). Lab. Invest. 34, 97-107. 131. Hultborn, K. A. (1954). Acia Radio/. (Stockholm) Siippl. 113, 1-71. 132. Iatropoulos, M. J., Golberg, L., and Coulston, F. (1975). Exp. M o l . Pathol. 23, 386401. 133. Imai, H., Saito, S., and Stein, A. A. (1965). Gustroenterology 48, 188-197. 134. Ioachim, N.-J., Delaney, W. E., and Madrazo, A. (1974). Cancer 34, 586-596. 135. Jacobs, M. M., Jansson, B., and Griffin, A. C. (1977). Cuncer Lerr. 2, 133-138. 136. Jimenez, C. J. M., Diener, K. A . , Alvarez, E. E . , Elizabeth, Q., and Maldonado, M. (1971). Ani. J . Proctol. 22, 301-307. 137. Joshi, S . R., Rice, J . M., Wenk, M. L., Roller, P. P., and Keefer, L. K. (1977). J . Nail. Cancer Insf. 58, 1531-1535. 138. Kanazawa, K., Yarnamoto, T., and Sato, S. (1975).Jupun. J . Exp. Med. 45,439-456. 139. Kikkawa, N . (1974). Med. J . Osaha Univ. 24, 293-314. 140. Kim. Y. S . , and Isaaks, R. (1975). Cancer Res. 35, 2092-2097. 141. King, E. S . J . , and Varasdi, G. (1959). Aiist. N . 2. J . Surg. 29, 38-53. 142. Kleist, S., von. (1977). Z. Krehsjbrsch. 90, 1-11. 143. Knudson, A. G . , Jr. (1973). A d v . Cancer Res. 17, 317-352. 144. KorpBssy, B., and Tiboldi, T. (1957). Oncologici 10, 330-335. 145. Koudstaal, J., Makkink, B., and Overdiep, S. H. (1975). Eirr. J. Cancer 11, 105-109. 146. Koudstaal, J., Makkink, B., and Overdiep, S. H. (1975). Eirr. J . Cunccv 11, 111-115. 147. Kroes, R., Berkvens, J. M., and Weisburger, J. H. (1975). Cancer Res. 35, 26512656. 148. Kronman, B. (1971). Cancer 28, 82-88. 149. L a Mont, J . T., Weiser, M. M., and Isselbacher, K. J. (1974). Cuncer Res. 34, 32253228. 150. Laqueur, G. L. (1965). Virchows Arch. 340, 151-163. 151. Laqueur, G. L., and Spatz, M. (1975). Cann Monogr. 17, 189-204. 152. Laumonier, R. (1975). Biomedicine 22, 358-367. 153. Lawley, P. D. (1974). Miifui. Re.\. 23, 283-295. 154. Leaver, D. D., Swann, P. F., and Magee, P. N. (1969). Br. J . Ciinci,r 23, 177-187. 155. Lee, J. A. H . (1976). I n f . J. Epidemol. 5, 187-194. 156. Lieb, L. M., and Lisco, H. (1966). Cancer Res. 26, 733-740. 157. Likhachev, A. J . , Margison, G. P., and Montesano, R. (1977). Chem. Biol. Interoct. 18, 235-240. 1 5 7 ~ .Likhachev, A. J., Petrov, A. S., Prvanova, L. G., and Pozharisski, K. M. (1978). But/. Exp. B i d . Mrif. 86 (121, 679-681.

232 158. 15Y. 160. 161.

162. 163. 164. 165. 166. 167. 168. 16Y. 170.


Lingeman, C . H., and Garner, F. M. (1972). J. Noil. C o w e r I n s / . 48, 325-346. Lipkin, M. (1971). Cancer 28, 38-40. Lipkin, M. (1974). Cancer Srtppl. 34, 878-888. Lipkin, M., Bell, B., Stalder, G., and Troncale, F. (1971). In "Carcinoma ofthe Colon and Antecedent Epithelium" (W. J. Burdette, ed.), pp. 213-221. Thomas, Springfield, Massachusetts. Lisco, H., Brues, A. M., Finkel, M. P., and Grundhauser, W. (1947). Ctiticer R e s . 7, 721. Loveless, A. (1969). Ntrrrrrc. (London) 223, 206-207. Lowenfels, A. B., and Sonni, A. (1977). C a m w L e t / . 3, 83-86. Magee, P. N . , Montesano, R., and Preussmann, R. ( 1976).A m . Coric.er Soc. Moriogr.. S e r . 173, 491-625. Marks, S., and Sullivan, M. F. (1960). Noirrrc (Lomfon)188, 953. Marsden, J. R., and Dawson, J. M. P. (1974). Giii 15, 783-787. Martin, F. (1976). J. Nrril. Cuwcer Insi. 56, 212. Martin, F., Knobel, S . , Martin, M., and Bordes, M. (1975). Ctrticcv R e s . 35, 333-336. Martin, F., Martin, M. S., Bordes, M., and Knobel, S . (1975). / t i t . J . Crrncer 15, 144-

151. 171. Martin, F., Martin, M. S. , and Knobel, S . (1975). Proc. I n / . Ctrnccr C o ~ t g r ./ / / / I , F lo r i~n c1~~ Y74 I , 300-303. 172. Martin, F., Martin, M., Lagneau, A., Bordes, M., and Knobel, S. (1976). Crtncc,r R P ~ .

36, 3039-3042. 173. Martin, M. S., Bastien, H., Martin, F., Michiels, R., Martin, M. R., and Justrabo, E. (1973). BionitdiciniJ 19, 555-558. 174. Martin, M. S., Martin, F., Justrabo, E., Michiels, R., Bastein, H., and Knobel, S .

(1974).J . N a i l . Ctrncer I n s t . 53, 837-840. 175. Martin, M. S., Martin, F., Justrabo, E., Michel. M. F., and Lagneau, A. (1977). Git/

18, 232-235. 176. Maskens, A. P. (1976). C a ~ t c e rR e s . 36, 1585-1592. 177. Matsuyama, M., [to, M., Yamada, S., Nakamura, T., and Nagayo, T. (1976).J. Ntrrl. Ct//iccJrI / i s t . 56, 791-795. 178. Matsuyama. M., Nakamura, T., Suzuki, H., and Nagayo, T. (1975). Ctrriii Moriogr.

17, 269-281. 179. Maugh, T. H. (1977). StYcvice 197, 544. 180. McCall, D. C . , and Cole, J . W. (1974). Cancer, 33, 1021-1026. 181. McClendon. J. E., Applely, D., Clandon, D. B., Donegan, W. L., and DeCosse, J . J. (1977). Arch. Srrrg. 112, 240-241. 182. McGinty, F., Deliges, G., and Harrison, D. (1973). G I / / 14, 502-505. 183. Mclllmurrey, M. B., Gray, M., and Langman, M. J . S. (1973). Girt 14, 541-544. 184. Mian, N., and Cowen, D. M. (1974). Br. J . Cancer 29, 438-446. 185. Mian, N., Herries, D. G., Cowen, D. M., and Batte, E. A. (1975). Biochim. Biop1t.v.s. A c / a 391, 179-188. 186. Mian, N., and Nutman, C. A. (1975). Br. J. Ctrncer 32, 252-253. 187. Michelson, J . B., Felberg, N. T., and Shields, J . A. (1977). Arch. Oph//ictlnrol. 95,

692-694.

P.,Matthews, R. H.. and Wisenbaugh, T . W. (1976). J. N u t / . C t r m w / U S / . 57, 39-4 I . 189. Monakhov, N . K., Pozharisski, K. M., and Schwartzman, A. L. (1975). Bull. E,vp. B i d . M e t l . 79, 53-56. /YO. Montesano, R., Margison, G. P., and Likhachev, A. J. (1977). //7 "Pathophysiology 188. Minton, J.


233

of Carcinogenesis in Digestive Organs” (E. Farber et u I . , eds.), pp. 221-231. Univ. of Tokyo Press and Univ. Park Press, Tokyo and Baltimore. 1Y1. Moon, R. C., and Fricks, C. M. (1977). Cancer 40, 2502-2508. 192. Moore, T., Kantrowitz, P. A . , and Zamcheck, N. (1972). J . AMA 222, 944-947. 193. Mori, H., and Hirono, I. (1977). Br. J. Cuncer 35, 369-371. 194. Mori, K., Ichii, S., and Shigeta, Y. (1955). Cunn 46, 631-634. 195. Moms, H. P., Wagner, B. P., Ray, F. E., Snell, K. C., and Stewart, H. L. (1961). N u t / . Cancer Inst. Morrogr. 5, 1-53. 196. Morris, H. P., Wagner, B. P., Ray, F. E., Stewart, H. L., and Snell, K. C. (1963). J . N u t / . Cancer Inst. 30, 143-161. 1’97. Morson, B. C. (1976). ”Histological Typing of Intestinal Tumours.” International Histological Classification of Tumours. No. 15, World Health Organization, Geneva. 198. Nachlas, M. M., and Hannibal, M. J. (1961). Siwg. Gynec. Obstet. 112, 534-542. I Y Y . Narisawa, T., Magadia, N. E., Weisburger, J. H., and Wynder, E. L . (1974). J . N u t / . Cuncer I n s f . 53, 1093-1097. 200. Narisawa, T., Reddy, B. S . , Wong C.-Q., and Weisburger, J. H . (1976). Cancer Res. 36, 1379-1383. 201. Narisawa, T., Sato, T., Haykawa, M., Sakuma, A., and Nakano, H. (1971). Gunn 62, 231-234. 202. Narisawa, T . , and Weisburger, J. H. (1975). Proc. Soc. Exp. Biol. Mrd. 148, 166-169. 2 W . Narisawa, T., Wong, C.-Q., Maronpot, R. R., and Weisburger, J. H. (1976). Cancer Res. 36, 505-510. 204. Narisawa, T . , Wong, C.-Q., and Weisburger, J. H . (1976). Gunn 67, 41-46. 205. Navarrete, A., and Spjut, H. J. (1967). Cnncer 20, 1466-1472. 206. Neifakh, S. A., Monakhov, N. K., and Schwartzman, A. L. (1972). Dokl. AN U.S.S.R. 206, 743-746. 207. Neutra, M., and Leblond, C. P. (1966). J . C d Biol. 30, 119-136. 208. Newberne, P. M., and Rogers, A. E. (1973). J. N u t / . Cancer I n s t . SO, 439-448. 20Y. Newberne, P. M., and Suphakarn, V. (1977). Cuncer 40, 2553-2556. 210. Newman, R., Bennett, S. J., and Pascal, R. R. (1975). C a n c i v 36, 2016-2019. 211. Nigro, N. D., Bhadrachari, N., and Chomchai, C. (1973). Dis. Colon Rectum 16,438443. 212. Nigro, N. D., Campbell, R. L., Gantt, J . S., Lin, Y. N., and Singh, D. V . (1977). Cuncer Res. 37, 3198-3203. 213. Nigro, N. D., Singh, D. V., Campbell, R. L., and Pak, M. S. (1975). J. N u t / . Cuncer Inst. 54, 439-442. 214. Oehlert, W. (1973). Cell Tissue Kinet. 6, 325-335. 215. Okulov, V. B., Anisimov, V. N., and Pozharisski, K . M. (1977). Bull. E.rp. B i d . M d . 83 (5), 582-583. 216. Okulov, V. B., and Pozharisski, K. M. (1975). B i d / . Exp. B i d . Mid. 80 (11). 78-81. 217. Osswald, H., und Kriiger, F. W. (1969). Arineiwr. Forsc/i. 19, 1891-1892. 218. Otto, H. F., Winkler, R., and Heitmann, C. (1976). Z . K r c h f o r s c l r . 87, 333-342. 21Y. Pamukcu, A. M., and Price, J . M. (1969). J . Nut/ Cunccr Inst. 43, 275-281. 220. Pamukcu, A. M., Yalqiner, S., and Bryan, G. T. (1977). Crincer 40, 2450-2454. 221. Pegg, A. E. (1974). Biockcm. J. 137, 239-248. 222. Pegg, A. E., and Hawks, A. M. (1974). Bioc/rc,m. J. 137, 229-238. 223. Pickard, R. G., Cobb, L. M., and Steel, G. G. (1975). Br. J. C n n c ~ r31, 36-45. 224. Pierce, G. B., Nakane, P. K., Martinez-Hernandez, A., and Ward, J . M. (1977). J. Nntl. C i i m ~ i I~nrs t . 58, 1329- 1345. 225. Potet, F., and Soullard, J . (1971). Girt 12, 468-482.

234


226. Pozharisski, K. M. (1972). V o p r . Oticol. I8 ( I ) , 64-71. 227. Pozharisski, K. M. (1973). V r s t n . Chit-. 111 (8), 52-57. 228. Pozharisski, K. M. (1973).In ”Pathology of Tumours in Laboratory Animals” (V. S. 22Y. 230. 231. 232. 233. 234. 235. 236. 237. 238. 23Y. 240. 241.

242. 243. 244. 245. 246. 247. 248. 249. 250. 25l. 252. 253. 254. 255.

Turusov, ed.), Vol. I, pt. 1 , pp. 119-140. IARC Scientific Publication N o 5, Lyon. Pozharisski, K. M. (1975). Cnt~crrRrs. 35, 3824-3830. Pozharisski, K. M. (1975). J. Natl. Cuncrr Inst. 54, 1115-1 135. Pozharisski, K. M. (1978).Arch. Pathol. 40 ( 5 ) . 76-84. Pozharisski, K. M . , and Anisirnov, V. N. (1975). Pathol. Physiol. I , 47-50. Pozharisski, K. M., Dushkin, V. A., and Podoprigora, G. I. (1974). Bull. E x p . Biol. Med. 78 (lo), 81-84. Pozharisski, K. M., Kapustin, Yu. M., Likhachev, A. J., and Shaposhnikov, J. D. (1975). Int. J . Cuticer IS, 673-683. Pozharisski, K . M., and Klimashevski, V. F. (1974). Exp. Puthol. 9, 88-98. Pozharisski, K. M., Klimashevski, V. F., and Gushchin, V. A. (1975). Do/,/. AN U . S . S . R . 220 ( l ) , 216-219. Pozharisski. K. M., Klimashevski, V. F., and Gushchin, V. A. (1977). Tsitologiiu 19, 303-317. Pozharisski, K. M., Klimashevski, V. F., and Gushchin, V. A. (1977). Tsitologiirr 19, 318-328. Pozharisski, K. M., Klimashevski, V. F., and Gushchin, V. A. (1977). Tsitologiiu 19, 537-544. Pozharisski, K. M., Klimashevski, V. F., and Gushchin, V. A. (1977). Tsitologiiu 19, 768-780. Pozharisski. K. M., Klimashevski, V. F., Gushchin, V. A., and Okulov, V. B. (1977). I n “The Role of Stem Cells in Leucoso- and Carcinogenesis” (P. E. Kavetsky, ed.), pp. 21-24. Naukova Dumka, Kiev. Pozharisski, K. M., and Kolodin, V . I. (1975). Vopr. Oncol. 21 ( 5 ) . 80-86. Pozharisski, K. M., and Okulov, V. B. (1976). B d l . Exp. B i d . Med. 81, 467-470. Pozharisski, K. M., and Savostjanov, G. A. (1976). Vopr. Oncol. 22 ( I ) , 59-68. Pozharisski, K. M., Shaposhnikov, J. D., Petrov, A. S., and Likhachev, A. J. (1976). 2. Krehs:forsch. 87, 67-80. Pretlow, T. P., Glover, G. L., and Pretlow, T. G., 11. (1977). J . N u t / . Cuncer I n s t . 59, 98 1-987. Preussmann, R., Druckrey, H., Ivankovic, S., and Hodenberg, A., von. (1969). Ann. N . Y . A C N ~Sci. . 163, 697-716. Qizilbash, A. H. (1974). Arch. Puthol. 98, 118-121. Qizilbash, A. H. (1974). Uis. Colon Rectum 17, 540-549. Reddy, B. S., Narisawa, T., Maronpot, R., Weisburger, J. H., and Wynder, E. L. (1975). Cancer Res. 35, 3421-3426. Reddy, B. S., Narisawa, T., Vukusich, D., Weisburger, J . H., and Wynder, E. L. (1976). Proc. Soc. Exp. Med. 151,237-239. Reddy, B. S., Narisawa, T., and Weisburger, J. H. (1976). Cancer Res. 36, 28742876. Reddy, B. S.. Narisawa, T., and Weisburger, J. H. (1976). J . N u t / . Cuncer Inst. 57, 567-569. Reddy, B. S., Narisawa, T., Weisburger, J. H., and Wynder, E. L. (1976). J . Natl. Cancer Inst. 56, 441-442. Reddy, B. S., Narisawa, T., Wright, P., Vukusich, D., Weisburger, J. H., and Wynder, E. L. (1975). Cancer Res. 35, 287-290.


235

256. Reddy, B. S . , Watanabe, K., Weisburger, J. H., and Wynder, E . L. (1977). Cancer Res. 37, 3238-3242. 257. Reddy, B. S., Weisburger, J . H., Narisawa, T., and Wynder, E. L . (1974). Cancer Rrs. 34, 2368-2372. 258. Reddy, B. S . , Weisburger, J . H., and Wynder, E . L. (1974). J. Narl. Cancerlnst. 52, 507-51 1 . 259. Reuber, M. D., and Thomas, A. (1973). Proc. A m . Assoc. Cancer Res. 14, 5 5 . 260. Reznik, G., Eckel, H., Green, U . , Rippel, W., and Mohr, U. (1976). 2. Krebsforsch. 87, 321-326. 261. Rogalsky, V. J . (1975). J. Nutl. Cancer Inst. 54, 1061-1071. 262. Rogalsky, V. J., Konovich, E. A., Rivkin, V. L., and Kapuller, L. L. (1972). Vopr. Oncol. 18 (4) 58-63. 263. Rogers, A. E., and Gildin, J . (1975). J. N d . Cancer Inst. 55, 385-391. 264. Rogers, A. E., Herndon, B. J., and Newberne, P. M. (1973). Cancer Res. 33, 10031009. 265. Rogers, A. E., and Newberne, P. M. (1973). Nutiirt (London) 246, 491-492. 266. Rogers, K . J., and Pegg, A. E. (1977). Cancer Res. 37, 4082-4087. 267. Russel, D. H. (1977). Clin. Chem. 23, 22-27. 268. Salser, J. S., and Balis, M. E. (1973). Cancer Res. 33, 1889-1897. 269. Salser, J. S . , and Balis, M. E. (1974). Cancer 34, 889-895. 270. Salser, J. S . , Ball, W. J., and Balis, M. E. (1976). Cancer Res. 36, 3495-3498. 271. Schauer, A., and Kunze, E. (1971). Sci. Tech. Inf. 11, 45-49. 272. Schauer, A., Vollnagel, Th., and Wildanger, F. (1969). Z. Gesamfe Exp. M t d . 150, 87-93. 273. Schmahl, D., Danisman, A., Habs, M., and Diehl, B. (1976). Z. Krehsforsch. 86, 8994. 274. Schoental, R., and Bensted, J . P. M. (1968). Br. 1. Cancer 22, 316-323. 275. Schwartz, M. K. (1977). Cancer 40, 2620-2624. 276. Shimizu, H . , and Toth, B. (1975). Gann 66, 589-601. 277. Shockman, A. T. (1973). Am. J . Gastroentrrol. 60, 178-184. 278. Simpson, B. H., and Jolly, R. D. (1974). J . Pathol. 112, 83-92. 279. Singer, B. (1976). Nature (London) 264, 333-339. 280. Singh, D. V., Campbell, R. L., Lin, Y. N., and Nigro, N. D. (1977). Experientia 33, 1516-1518. 281. Sjogren, H.O., and Steele, G. (1975). Cancer 36, 2469-2471. 282. So, B. T., Magadia, N. E., and Wynder, E. L. (1973). J. Narl. Cancer Inst. 50, 927932. 283. Spitz, S., Maguigan, W. H., and Dobriner, I(.(1950). Cancer 3,789-804. 284. Spjut, H. J. (1972). Dis. Colon Recium 15, 94-99. 285. Spjut, H. J., and Noall, M. W. (1971). Cancer 28, 29-37. 286. Spjut, H. J., and Smith, M. N. (1967). Exp. Mol. Pathol. 6, 11-24. 287. Spjut, H . J., and Spratt, J . S. (1965). Ann. Surg. 161, 309-324. 288. Spratt, J. S . , Ackerman, L. V., and Moyer, C. A. (1958). Ann. Surg. 148, 682-698. 289. Springer, P . , Springer, J., and Oehlert, W. (1970). Z. Krebsforsch. 74, 236-240. 290. Steele, G., Jr., and Sjogren, H. 0. (1974). Cancer Res. 34, 1801-1807. 291. Steele, G . , Jr., and Sjogren, H. 0 . (1974). In?. J . Cancer 14, 435-444. 292. Steele, G., Jr., and Sjogren, H. 0. (1977). Surgery 82, 164-169. 293. Steele, G . , Jr., Sjogren, H. O., and Price, M. P. (1975). Inr. J. Cancer 16, 33-51. 294. Steele, G . , Jr., Sjogren, H. O., Rosengren, J. E., Lindstrom, C., Larsson, A., and Leandoer, L. (1975). J. Narl. Cancer Insr. 54, 959-967.

236


2Y5. Steele, L., Cooper, E. H., MacKay, A. M., Losowsky, M. S., and Goligher, J . C. (1974). Br. J. Ccr/iccr 30, 319-324. 2Y6. Stevens, R. H . , Englund, C. W., Osborne, J. W., Cheng, H. F., and Hoffman, K . L. (1976). Cuticrr Res. 36, 3260-3264. 2Y7. Stevens, R. H., Englund, C. W., Osborne, J. W., Cheng, H. F . , and Richerson, H. B. (1975). J. N u / / . Ccrncrr Irisf. 55, 1011-1013. 2Y8. Stevens, R. H . , Smith, D. D., Osborne, J. W., and Oberley, L. W. (1976). J . Natl. Cuncrr I r i s / . 57, 43-45. 299. Stewart, H. L. (1953). I n “The Physiopathology of Cancer” (F. Homburger and W. H . Fishman, eds.), pp. 3-45. Cassell, London. 300. Sundaresan, P. R., and De Luca, L. M. (1977). J. Natl. C ~ n c e Ir/ i s t . 58, 1643-1645. 301. Tayler, R., Cumberland, V. H., and Piper, D. W. (1977). Grit 18, 45-47. 302. Terz, J. J., Curutchet, H. P., and Lawrence, W., Jr. (1971). Crrncrr 28, 1100-1 110. 303. Thurnher, N., Deschner, E. E., Stonehill, E. H., and Lipkin, M. (1973). Cancw R i v . 33, 940-945. 304. Topping, D. C., and Visek, W. J. (1976). J. Nirtr. 106, 1583-1590. 305. Toth, B. (1977). Cancer 40, 2427-2431. 306. Toth, B., Malick, L., and Shimizu, H. (1976). Am. J . Pufli01. 84, 69-86. 307. Troncale. F., Hertz, R., and Lipkin, M. (1971). Ccrticer R r s . 31, 463-467. 308. Trotta, P. P.. and Balis, M. E. (1977). Crrncer 40, 2592-2599. 30Y. Turell, R. (1973). Cu 23, 91-93. 310. Turkevitch, N. M., and Samundjan, E. M. (1975). “Hormones and Carcinogenesis.” Naukova Dumka, Kiev. 311. Turner, M. D., Kleinman, M. S., and Thayer, W. (1973). Digestiori 9. 116-123. 312. Turusov, V . S . , Lanko, N . C., and Bazlova, L. S. (1977). V o p r . Onkol. 23 (7). 39-42. 313. Tutton, P. J. M., and Barkla, D. H. (1976). VirchoMBs Arch. B: Cell Pntliol. 21, 147160. 314. Twining, S. S . , and Brecher, A. S. (1977). Clin. Chim. Acfri 75, 143-148. 315. Varghese, A. J., Land, P., Furrer, R., and Bruce, W. R. (1977). Proc. A m . Assoc. Criiicrr Res. IS, 80. 316. Vollnagel, T., Wildanger, F., and Schauer, A. (1969). Z. Giwimtc, Exp. Met!. 151, 208-2 13. 317. Walker, E. A., Bogovski, P., and Griciute, L. (eds.) (1976). ”Environmental N-nitroso Compounds. Analysis and Formation.” IARC Scientific Publications No. 14, Lyon. 318. Walpole, A. L., Williams, M. H., and Roberts, D. C. (1952). Br. J. Ind. Med. 9, 255263. 319. Walpole, A. L., Williams, M. H., and Roberts, D. C. (1955). Br. J . Cancer 9, 170176. 320. Ward, A. M., Cooper, E. H., Turner, R., Anderson, J . A., and Neville, A. M. (1977). Br. J. Concer 35, 170-1723, 321. Ward, J. M. (1974). Lab. Invest. 30, 505-513. 322. Ward, J. M., Rice, J. M., Roller, P. P., and Wenk, M. L. (1977). Cancer Res. 37, 3046-3052. 323. Ward, J. M., Sontag, J. M.,Weisburger, E. K., and Brown, C. (1975).J. N u / / .Cancer I m t . 55, 107-113. 324. Ward, J. M., and Weisburger, E. K. (1975). Cancer Res. 35, 1938-1943. 325. Ward, J. M., Yamamoto, R. S., and Weisburger, J. H. (1973). J. Nritl. Crrncrr I/i.st. 51, 713-715. 326. Ward, J. M.,Yamamoto, R. S., Weisburger, J. H., and Benjamin, T . (1973). J. Nut/. Cancer I n s t . 51, 1997-1999.


237

327. Watanabe, K., Yoshii, H., Iwashita, H., Muta, K., Hamada, K., Isaka, H., and Nishi. M. (1975). Gutin 66, 449-453. 328. Watson, D. W. (1974). C(117cer34, 867-871. 329. Wattenberg, L. W. (1959). Corrcer Res. 19, 1118-1 123. 330. Wattenberg, L. W. (1974). Am. J . Dig. Die. 19, 947-953. 331. Wattenberg, L. W. (1975). J. NNil. Ctrticcv Inst. 54, 1005-1006. 332. Wattenberg, L. W., Lam, L. K. T., Fladmoe, A. V., and Borchert, P. (1977). C'mcc,r40, 2432-2435. 333. Weisburger, J . H. (1971). Ctr/icer 28, 60-70. 334. Weisburger, J . H., Reddy, B. S., Narisawa, T., and Wynder, E. L. (1975). Proc. SOC. Exp. B i d . Mad. 148, 1119-1121. 335. Wellin, S., Yonker, J., and Spratt, J. S. , Jr. (1963). Am. J . R O O / l f g ~ ~ 090, / . 673-687. 336. Werner, B . , de Heer, K., and Mitschke, H. (1977). Z . Kwbsfbrsc/i. 88, 223-230. 337. Werner, B., de Heer, K., Mitschke, H., Knipper, A,, Thoran, P.. and Dahm, K. (1977). Z. Krehsforsch. 89, 53-60. 338. Wiebecke, B., Krey, U., Lohrs, U., and Eder, M. (1973). V i r c ~ l i o ~Arch. i ~ s A: PNIIIOI. Anni. 360, 179-193. 339. Wiebecke, B., Lohrs, U., Gimmy, J., and Eder, M. (1969). Z. Grscwiic, Exp. M r d . 149, 277-278. 340. Williams, R. D., and Fish, J. C. (1966). Am. J . Surg. 112, 846-849. 341. Williamson, R. C. N., Bauer, F. L. R., and Malt, R. A. (1977). Br. J. Cruicer- 36,401. 342. Wilson, R. B., Hutcheson, D. P., and Wideman, L. (1977). Ani. J . Clin. Nictr-. 30, 176- 18 1. 343. Winkler, R., Heitmann, C., and Otto, H. F. (1977). Chir. Gn,stroe/iterol. 11, 105-108. 344. Winneker, R. C., Tompkins, M., Westenberger, P., and Harris, J . (1977). Exp. Mol. Pothol. 27, 19-34. 345. Wittig, G., von, Wildner, G. P., and Zielbarth, D. (1971). Arch. G~~.sc/rii~/il.sffbr.sc~h. 37, 105-115.

346. Wood, D. A. (1967). "Tumors of the Intestines. Atlas of Tumor Pathology," Sect. 6, Fasc. 22. U.S. Armed Forces Institute of Pathology, Washington, D. C. 347. Wynder, E. L., Reddy, B. S., McCoy, G . D., Weisburger, J. H., and Williams, G. M . (1976). Clin. Gustrornterol. 5, 463-482. 348. Yamada, S., Ito, M., and Nagayo, T. (1971). Gaiiri 62, 471-478. 34Y. Yamamoto, T., Kato, H., and Smith, G . S. (1975). Gomi 66, 623-630.


ADVANCES IN CANCER RESEARCH, VOL. 30

THE MOLECULAR BIOLOGY OF LYMPHOTROPIC HERPESVIRUSES*

Bill Sugden, Christopher R. Kintner, and Willie Mark The McArdle Laboratory tor Cancer Research, University of Wisconsin, Madison, Wisconsin

I. Introduction ........................................................... 11. A Brief Survey of Lymphotropic Herpesviruses . B. Persistent and Latent Infections ......................................

EBV ............................................. A . Proteins in the Virion ...............................................

References

............................................................

239 240 244 244 246 248 249 249 25 I 25 1 258 259 259 262 265 266 268 268

I. Introduction

Lymphotropic herpesviruses are those members of the Herpesvirus family that infect lymphoid cells of their natural hosts. These herpesviruses frequently cause lytic infections as well as an infection that leads to a self-limiting lymphoproliferative disease or a malignant lymphoma. The ability of these viruses to cause these diseases is probably derived, at least in part, from their capacity to induce and maintain cell division in the infected cell. A major goal of research into the molecular biology of lymphotropic herpesviruses is to learn how they induce these growth changes in their target cells. If we can achieve this goal, we may gain insight into mechanisms of both oncogenesis and the control of normal development.

* We dedicate this paper to Dr. Harold Rusch, under whose leadership McArdle Laboratory has become a communal research laboratory where our science profits immensely from our environment. 239 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved ISBN 0-I?-006630-0

240

BILL SUGDEN ET AL.

The bulk of studies on the lymphotropic herpesviruses to date has focused on three viruses: Marek’s disease virus (MDV), found in domestic chickens; H e r p e s v i r ~ ssaimiri (HVS), found in squirrel monkeys; and Epstein-Barr virus (EBV), found in human beings. MDV causes a lymphoma in chickens which progresses rapidly to kill the animals (Witter, 1971; Nazerian, 1973). MDV is transmitted horizontally, and its DNA contains no detectable nucleic acid homology with chicken cell DNA (Lee et al., 1971; Tanaka et al., 1978). These findings distinguish MDV mechanistically from the transformation-defective Rous-associated viruses, which probably induce lymphoid leukosis by a process involving exchange of information with cell DNA with which they share sequence homology (Temin, 1971). We conclude that MDV and, perhaps, other lymphotropic herpesviruses induce tumors by themselves introducing the genetic information needed to render an infected cell malignant. EBV is necessary and sufficient to transform fetal and adult human lymphocytes into permanently dividing cultures of lymphoblasts (Pope et al., 1968). If we can understand how EBV changes an apparently differentiated end cell into a permanent blast cell, we may also learn how some cellular decisions are made in normal development. In this review we shall first present a general survey of the lymphotropic herpesviruses. We shall then describe our current knowledge of the molecular biology of the three prototypic viruses: MDV, HVS, and EBV. We shall also present an overview of the relationship of these viruses to their hosts it7 virw. It is these relationships that we hope eventually to understand at the molecular level. II. A Brief Survey of Lymphotropic Herpesviruses

A survey of the salient (for the purpose of this review) features of the identified lymphotropic herpesviruses is presented in Table I . MDV, HVS, and EBV will be discussed in detail later. We will now briefly describe the other lymphotropic herpesviruses. Herpesvirus s y l v i l a g ~ swas isolated from naturally infected, wild cottontail rabbits (Hinze, 1971a). This virus can be grown in tissue culture, but it has not been well characterized. When rabbits are infected experimentally with this virus, they may develop a lymphoproliferative disease (Hinze, 1971b). Herpesvirus of turkeys (HVT) is antigenically related to MDV (Witter et al., 1970). It is avirulent in chickens and has been used for vaccination of chickens against Marek’s disease (Okazaki et al., 1970; Eidson and Anderson, 1971). The virus is less frequently bound to cell debris than MDV when grown in tissue culture. This property of HVT permits the

MOLECULAR BIOLOGY OF LY MPHOTROPIC HERPESVIRUSES

24 1

relative ease of purifying cell-free virus needed for vaccine preparation. Little is known about the molecular biology of this virus. Most of the lymphotropic herpesviruses so far identified have been isolated from nonhuman primates, and more are likely to be found among them. Four species of New World monkeys have been found to have antibodies to HVS-associated antigens (Deinhardt et al., 1974). Eight species of Old World monkeys and four species of the great apes can carry antibodies that cross-react with EBV-induced antigens (Frank et al., 1976). These findings indicate that monkeys in the wild are exposed to herpesviruses that are related to EBV and HVS. It seems likely that these species of primates harbor the viral agent(s) and act as reservoirs for their horizontal transmission. The age-dependent acquisition of antibodies by these animals seems to support this conclusion (Landon and Malan, 1971; Deinhardt et al., 1974). In addition, when monkeys are housed together with seropositive animals, they develop antibodies that cross-react with EBV- or HVS-induced antigens (Deinhardt et al., 1974). Two of the most studied simian lymphotropic herpesviruses are HVS and Herpesvirus ateles (HVA). Melendez and colleagues first isolated HVS and HVA from fibroblast cultures derived from clinically healthy host animals (Melendez et al., 1968: Melendez et al., 1972). Subsequently, HVS and HVA have been isolated from peripheral blood leukocytes of their natural hosts by cocultivation with permissive cells (Falk et a/., 1973; Falk et al., 1974b). Both viruses grow well in tissue culture and can be propagated in several species of primate fibroblast cell lines. The occurrence and isolation of these two herpesviruses have been reviewed by Deinhardt et al. (1974). In this review, HVS will be used as a prototype virus for discussion. There is no proof yet that the more recent isolates of herpesviruses from other primates are distinct viruses. Herpesviruses have been identified in spontaneously transformed lymphoblastoid cell lines of chimpanzees (Landon et al., 1968) and baboons (Gerber e[ al., 1977). Antigenically, these two herpesviruses are related to EBV, since cell lines harboring the virus are stained by antisera that are specific for EBVinduced antigens (Gerber et al., 1976: Goldman et al., 1968). The viral DNAs present in these cell lines share sequence homology with EBV DNA (Gerber et al., 1976: Falk et al., 1977). Neither virus has been well characterized as a result of the difficulty in obtaining cell-free virus from the spontaneously transformed cell lines. Several attempts have been made to rescue the virus by transforming one species’ leukocytes with cell-free virus from the other (Gerber ef al., 1977, Falk et al., 1976). Transformed cell lines were established in these experiments. Under these circumstances, it is essential to demonstrate which virus is harbored in the established cell line. If the animal that donated the target cells for

242

BILL SUGDEN ET AL.

TABLE I SURVEY OF LYMPHOTROPIC

Virus Herpesvirus sylvilagus

Natural host (reference)

Related herpesviruses" (reference)

Cottontail rabbit (Hinze. 1971a)

Marek's disease virus (MDV)

Chicken (Churchill and Biggs, 1967: Biggs ei a l . , 1968)

Herpesvirus of turkeys (HVT

Turkey (Kawamura ei a / . , 1969)

Herpesvirus saimiri (HVS)

Squirrel monkey (Melendez ei a / . . 1968)

Herpesvirus ateles (HVA)

Spider monkey (Melendez ef a/.,1972)

Epstein-Barr virus (EBV)

Human (Epstein and Barr,

MDV (Kamamura ef a / . , 1969)

HVS (25%) (Fleckenstein r i a/., 1978)

1964)

Herpesvirus papio

Baboon (Falk ei a/. , 1976)

EBV (Falk et a l . , 1976)

Herpesvirus of chimpanzee

Chimpanzee (Landon ei a / . , 1968)

EBV (35-45%) (Gerber ei a/., 1976)

Herpesvirus pongo

Orangutan (Rasheed e / al., 1977)

EBV (30-40%) (Rabin ei al., 1978)

a As measured antigenically or by DNA sequence homology. Only one of each homolpairs.

MOLECULAR BIOLOGY OF LYMPHOTROPIC HERPESVIRUSES

243

HERPESVIRUSES Host of leukocytes for transformation in vifra

(reference)

Host for induction of lymphoid tumors (reference) Cottontail rabbit (Hinze, 1971b) Chicken (Biggs et a / ., 1968: Calneck et a / . , 1970)

Cotton-topped marmoset (Hunt et a / . , 1970) Owl monkey (Deinhardt ef a / . , 1974) New Zealand rabbits (Daniel et al., 1975) Cotton-topped marmoset (Falk et a l . , 1974a, 1978)

Cotton-topped marmoset (Melendez et al., 1972)

Human (Pope ef al., 1968) Cotton-topped marmoset (Miller and Lipman, 1973) Other primate monkey (Frank et a / ., I976

Cotton-topped marmoset (Frank et a / . , 1976: Miller et a / ., 1977a)

Human (Gerber et a / ., 1977) Cotton topped marmoset (Rabin et al., 1977) Some other primates (Falk et a / ., 1977) Human, baboon, and some other primates (Gerber et a/. , 1977) Gibbon (Rabin et a / . , 1978) ogous set is noted: parentheses indicate percent of DNA sequence homology between

244

BILL SUGDEN ET

AL.

the transformation experiments harbored cells already transformed in vivo by its herpesvirus, then those cells could also grow out as lines. Recently, a lymphoblastoid cell line derived from a leukemic orangutan was shown to contain a herpesvirus (Rasheed et al., 1977). This virus is related serologically and by DNA sequence homology to EBV (Rabin et al., 1978). However, the orangutan had been in captivity for ten years in a city zoo. It is possible that it had been in contact with human EBV carriers or other naturally infected primates during its captive life. Again it is important to determine the distinguishing properties of new isolates of viruses before we classify them as distinct lymphotropic herpesviruses.

Ill. MDV, HVS, AND EBV in Their Natural Hosts

It is apparent that infection by a lymphotropic herpesvirus can affect the host variously. We know that some strains of MDV are able to cause Marek's disease (MD), a malignant lymphoma, at a high efficiency in genetically susceptible chicken. In other conditions MDV causes a persistent infection. We know EBV in some cases causes one form of infectious mononucleosis in people and that it is possible but not proven that EBV is necessary to cause most cases of Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma. Finally, EBV infects so many people without causing overt disease that most human beings harbor it in a latent state. In order to identify the possible role that these viruses can play in causing disease, we must ascertain both if the virus is required causally and what modifying factors (for example, virus strains, genetic make-up, and immunological status of host) participate in determining the outcome of infection. A. VIRALDISEASES Marek's disease, in its acute form, is a lymphoproliferative disease that results in the development of lymphoid tumors in chickens. Animals are usually exposed to the virus early in life and become infected by 46 weeks ofage (Biggset al., 1968; Witter, 1971, 1972). The virus replicates efficiently in the feather follicle epithelium of the bird (Calnek and Hitchner, 1969: Nazerian and Witter, 1970): this probably accounts for the infectiousness of the disease. In some fashion lymphoid cells are infected by the virus, proliferate, and infiltrate many organs of the animal to give rise to the neoplastic disease. Several lymphoblastoid cell lines have been established from Marek's disease tumor tissue, all carry T-cell markers (Payne et a / . , 1974; Nazerian and Sharma, 1975: Matsuda et al., 1976),


245

and two have been shown to contain multiple copies of MDV DNA (Nazerian and Lee, 1974; Tanaka et al., 1978). These findings are consistent with the notion that the tumor cell in Marek's disease is t h e MDVharboring T lymphoblast. Not all chickens are susceptible to MD when exposed to the virus. There exist strains of inbred chickens that are genetically resistant to MDV-induced lymphoma (Cole, 1972). Longenecker and co-workers have found that resistance is associated with the B locus of the major histocompatibility complex (MHC) of the chicken (Longenecker et al., 1976). Briles et aI. (1977) have since confirmed this finding. Recently, Longenecker and his colleagues have discovered a second locus in the MHC that may also help to confer resistance to MD (Fredericksen et a l . , 1977). The mechanism of resistance is not known. Chickens that are not susceptible to neoplasia can harbor the virus in a latent form. In addition to host-related resistance to MD, there are strains of MDV that are not pathogenic in susceptible chickens. Some of these strains have been used to vaccinate birds against subsequent challenge with virulent strains of MDV (Churchill et al., 1969a). The mechanism of protection against disease by vaccination with avirulent MDV is not yet understood. However, this acquired immunity does not protect the host from persistent infections by MDV; that is, MDV can be isolated from these birds throughout life. Whereas HVS is not known to cause overt disease in its natural host, EBV does. The relationship between EBV and human disease cannot be as directly studied as can that of MDV in chickens. However, we accept the notion that EBV causes the heterophile antibody-positive form of infectious mononucleosis. The evidence for this etiologic role of EBV is necessarily indirect but it is still compelling. The varied evidence runs the gamut from an early demonstration of transmission of infectious mononucleosis by transfusion (Wising, 1942) to careful seroepidemiologic studies (Neiderman et al., 1968), which found a primary induction of antibodies to EBV-associated antigens soon after clinical recognition of the disease. Additional evidence includes the demonstration that a population of killer cells specific for cells harboring EBV information is induced i n vivo and remains detectable only during the acute stage of infectious mononucleosis (Svedmyr and Jondal, 1975)'; that about 1 per 1000 of the peripheral leukocytes of patients in this acute stage is infected This study used EBV-harboring lymphoblastoid cell lines as targets for the killer cells isolated from patients' blood. In most if not all experiments, therefore, the target and killer cells did not share histocompatibility antigens. The observed killing apparently does not require identity of some histocompatibility antigens, although such a requirement has been found for specific killing of murine cells lytically infected with several different viruses (Doherty et ol.. 1976).

246

BILL SUGDEN ET

AL.

by EBV (Klein et al., 1976); and that this number drops dramatically in the convalescent patient (Hinuma and Katsuki, 1978). Most patients early in the course of infectious mononucleosis secrete EBV orally and can continue to do so for many months thereafter (Chang and Golden, 1971; Niederman et al., 1976). These findings are consistent with the idea that the heterophile antibody-positive form of infectious mononucleosis results from the oral exposure of a susceptible person (usually a EBV seronegative adolescent or young adult) to EBV. The ensuing infection and proliferation of EBV-carrying B lymphoblasts lead to the stimulation of killer cells that eventually limit the B-lymphoblast proliferation and then decline in number as a result of the lack of further stimulation. This satisfying (and, inevitably, oversimplified) description of the role played by EBV in causing one form of infectious mononucleosis does not yet have its equally satisfying counterpart in relating EBV to Burkitt’s lymphoma and nasopharyngeal carcinoma. Several reviews describing the evidence that implicates EBV as being necessary to cause these two human tumors have been published (Klein, 1973; Miller, 1974; zur Hausen, 1975). We have presented major pieces of the evidence in Table 11. In general, if a tumor is monoclonal and the tumor cells all contain viral information, then that virus had to be present at the onset of tumor formation and might have played an active role in that formation event. There are sufficient uncertainties in the data for EBV to force us to regard this logical argument as speculative and in need of further evidence for the case of EBV and Burkitt’s lymphoma. Other kinds of evidence for the etiologic role of EBV are less direct than that presented in Table 11. EBV can induce malignant lymphomas in some New World monkeys (Shope et al., 1973) and transforms human B lymphocytes into “immortalized” blast-cell lines (Pope et al., 1968). These facts must be reconciled with the observations that both Burkitt’s lymphoma and nasopharyngeal carcinoma are rare diseases on a world-wide basis but occur frequently in restricted regions of the world, whereas infection with EBV occurs in 80% or more of people throughout the world. If EBV is necessary for these two human neoplasms, it is not alone sufficient to cause them.

A N D LATENTVIRALINFECTIONS B. PERSISTENT

MDV, HVS, and EBV can give rise to persistent or latent infections in their respective hosts. Vaccine strains of avirulent MDV yield life-long persistent infections (Jackson et al., 1974). More than 8% of clinically healthy squirrel monkeys exhibit antibodies to HVS antigens, and the virus can usually be isolated from these animals by cocultivating their

TABLE I1 EVIDENCE RELATINGEBV TO Two HUMANTUMORS ~~

Evidence

Burkitt's lymphoma

References

Nasopharyngeal carcinoma

~

References

I . Seroepidemiologic studies find higher titers of antibodies against virus-associated antigens in patients as compared with controls 2. Multiple copies of viral DNA are found in the tumor cell

+

Henle et a / . (1969)

+

Henle er a/. (1970a)

+

+

zur Hausen et a/. (1970):

3. The tumor is monoclonal in origin" 4. In some genetically immunodeficient people the viral infection appears to lead to tumor formation in several weeksb 5 . A prospective seroepidemiologic survey finds that children in Uganda with abnormally high titers of antibodies to some EBV-associated antigens have a 30-fold increased risk of contracting the neoplasm

+ +

zur Hausen et al. (1970); Nonoyama and Pagano (1973) Fialkow et a/. (1970) Bar ef a/. (1974): Purtilo et a / . (1978)

+

Anderson-Anvret et a / . (1977)

de-The et a/. (1978)

It has t o be noted that rapid growth of a single clone of a multiclonal tumor early in the history of the tumor could yield a tumor that by isozyme markers appears to be monoclonal in origin. It is not proven in the few examples of these patients if EBV is necessary for tumor formation. (1

248

BILL SUGDEN ET AL.

peripheral leukocytes with susceptible fibroblasts (Deinhardt et al., 1974). Infection by EBV appears to persist after infectious mononucleosis, because virus can frequently be isolated from throat washings 6 to 12 months after the clinical symptoms have passed (Niederman et al., 1976). After that time the virus remains in a latent state in that a small number of EBV-harboring B lymphocytes can be detected by growing them in tissue culture, whereas the large mass of lymphoid cells not carrying EBV fail to grow (Moore et al., 1967; Gerber and Monroe, 1968). In the majority of people infected by EBV the entire infection remains asymptomatic and the virus is generally detected only by the specific antibodies it stimulates or by cultivating virus-harboring cells in tissue culture. Additional evidence for the latent infection of seropositive people by EBV is found in studies on immunosuppressed adults, who usually begin to secrete detectable quantities of EBV in their saliva within several weeks of administration of immunosuppressive drugs (Strauch et d., 1974).

IV. Experimental Tumor Studies

MDV, HVS, and EBV can each induce lymphomas in experimental animals. Some of the accumulated data for tumor induction by these viruses are presented in Table I . These data indicate that MDV induces tumors in the chicken, its natural host, and that HVS can induce tumors in two kinds of marmosets as well as in rabbits but not in its natural host, squirrel monkeys. It should be noted, however, that the number of squirrel monkeys studied in captivity is tiny compared with the number of people observed medically. If tumors induced by HVS occurred as rarely in its natural host as Burkitt’s lymphoma and nasopharyngeal carcinoma occur in people, then it is statistically likely that these rare tumors in squirrel monkeys probably would not have been identified. The ability of EBV to induce tumors in cotton-topped marmosets is important but may not be relevant to its potential etiologic role in human tumors for two reasons. First, as expected, there are significant differences in the virusassociated tumors in the different hosts. For example, while Burkitt’s lymphoma appears to be monoclonal in origin (Fialkow et al., 1970). the EBV-induced lymphoma in marmosets appears to be polyclonal in origin (Deinhardt et al., 1975). Second, there are several precedents of viruses that apparently do not induce tumors in their natural hosts (human adenoviruses and simian virus 40, for example), but can induce tumors in experimental animals. A second mode of tumor formation has been used in the study of EBV-


249

related tumors. Biopsies or cell lines of Burkitt’s lymphoma and nasopharyngeal carcinoma biopsies grow as tumors following xenotransplantation into homozygous nu/nu mice (Nilsson et al., 1977; Klein et al., 1974). This foreign graft experiment provides a way of measuring tumorigenicity. It permits distinction among four kinds of cells harboring EBV: ( I ) those recently transformed in vitro, (2) those recently established from normal donors, (3) those passaged in culture for long times, and (4) those freshly established from tumors. The latter two yield tumors upon subcutaneous transplantation; the former two do not (Nilsson et al., 1977). V. Studies of MDV, HVS, and EBV in Tissue Culture

A. TYPESOF CELLSSUSCEPTIBLE TO INFECTION Types of cells that are susceptible to infection by MDV, HVS, and EBV in vivo and in vitvo are presented in Table 111. Although we know that M D V replicates efficiently in the feather follicle epithelium of the chicken, we do not know the sites for replication of HVS and EBV in their natural hosts. It is obviously important to identify sites of replication of EBV in vivo because in so doing we may be able to establish in culture a productive host cell for that virus. Detection of EBV in throat washings of patients with infectious mononucleosis (Chang and Golden, 197 I ; Niederman et al., 1976) and of EBV-associated antigens in a high percentage of B lymphocytes carrying complement receptors in tonsils (Veltri et al., 1977) indicates that the throat probably harbors a site for viral replication, but it has not yet been identified. As determined by xenotransplantation of nasopharyngeal carcinoma tissue into nude mice, epithelial tumor cells harbor viral DNA (Klein et al., 1974). Cells with epithelial morphology isolated from throat scrapings of patients with infectious mononucleosis harbor EBV DNA as detected by in sitrr hybridization (Lemon et al., 1977). However, it is not known if the virus can mature in and be released from these cells. MDV and HVS lytically infect the appropriate fibroblast cultures in vitro, but no such productive host for EBV has been identified. The lack of a productive host cell has severely restricted research on EBV in two ways. First, MDV and HVS can be genetically purified by repeated plaque formation on susceptible target cells, whereas EBV cannot. The potential heterogeneity of all stocks of EBV precludes some experiments and renders others difficult to interpret. Second, large-scale production and isotopic labeling of the virions in tissue culture are practicable for HVS but probably not for MDV and certainly not for EBV. The release

250

BILL SUGDEN ET AL.

TABLE 111 TYPESOF CELLS OBSERVED TO

INFECTION BY LYMPHOTROPIC HERPESVIRUSES

BE SUSCEPTIBLE TO

I n Viiw

Virus MDV

HVS

EBV

Lytic Infection (references)

Transformation (references)

Chicken feather Chicken T follicle lymphocytes (Calneck and (Payne el a / . , Hitchner, 1969: 1974: Nazerian Nazerian and and Sharrna, Witter, 1970) 1975: Matsuda P / ul. , 1976) Owl monkey T None lymphocytes (Wallen PI ul. , 1973) Marmoset and owl monkey lymphocytes (Deinhardt rt d.,1974) Human B None lymphocytes (Jondal and Klein, 1973: Huber et ul., 1976) Human epithelial cells of the nasopharynx and throat (Klein C I a l . , 1974: Lemon rt ul., 1977) Cot ton-topped marmoset lymphocytes (Frank ~t a / . , 1976)

Ill

Lytic infection (references)

Vi1r.o

Transformation (references)

Chick and duck embryo fibroblasts (Witter P I ul., 1969)

None

Green monkey kidney and owl monkey kidney cell lines (Deinhardt C I 01.. 1974)

None

None

Human B lymphocytes (Pattengale C I ul.? 1973: Mizuno ('1 n l . , 1974) Lymphocytes of 7 nonhuman primates (Frank PI ul., 1976)


25 1

of MDV into the extracellular fluid is too inefficient for large-scale virus production, presumably because the virus remains cell-associated. Therefore, many experiments on the molecular biology of MDV and EBV are not feasible. Neither MDV nor HVS has been shown to transform lymphocytes in culture. This lack may result from our inexperience in working with uninfected avian and primate lymphocytes in culture, or it may result from the fact that those cells that are susceptible to transformation in vivo may not be present in adequate concentrations in peripheral blood to be transformed in vitro. EBV does transform human B lymphocytes in culture, and clonal transformation assays for EBV have been developed with fetal (Yamamoto and Hinuma, 1976) and adult (Sugden and Mark, 1977) lymphocytes. These assays should permit quantitative studies of the transformation of target cells by EBV.

B. LYTICINFECTIONS A s shown in Table 111, MDV and HVS infect some fibroblastic cultures in vitro, and HVS grows productively in them. N o lytic host has as yet been identified for EBV. The molecular events that occur in the lytically infected cell, including uncoating, transcription and translation of viral products, viral DNA synthesis, and virion maturation, have not been elucidated for MDV or HVS. We do know that the DNA of HVS is infectious in tissue culture (Fleckenstein er al., 1975) as is herpes simplex DNA (Graham et a/., 1973; Sheldrick et al., 1973). This finding indicates that HVS requires no virion proteins for complete infection of cells and, by analogy only, that MDV and EBV probably do not require virion proteins for their successful infection of their target cells. Although a productive host cell in tissue culture has not been found for EBV, a number of cell strains and lines have been tested to determine whether they could serve in that capacity. A list of these negative results is given in Table IV, not to discourage but to guide investigators in testing other cells in culture.

C. TRANSFORMATION MDV and HVS have not yet been shown to transform cells in tissue culture. EBV, however, does transform both human and nonhuman lymphocytes in culture. This transformation of lymphocytes by EBV is distinct from transformation of fibroblasts by other animal DNA viruses (see Tooze, 1973, for a review). Transformation by EBV does not result in altered growth morphology, reduced nutrient requirements, or loss of

252

BILL SUGDEN ET AL.

TABLE IV CELLSTESTEDA N D FOUND INCAPABLE OF SERVING AS LYTICHOSTSFOR EBV Primary cells

Human embryonic tissues" Human placenta" Human epithelial cells from adenoids and tonsilsu Human macrophages' Human lymphocytes from patients with chronic and acute lymphocytic leukemia" Marmoset kidney, skin, and muscle" Squirrel monkey kidney, lung, and heart" Rhesus monkey kidney" Callothrix skin and muscle" Hamster embryo fibroblasts" Chicken embryo fibroblasts"*b Turkey embryo fibroblastsb Pheasant embryo fibroblastsb Duck embryo fibroblastsb Lymphoid cell cultures" of cotton-topped and white-lipped marmosets, squirrel monkey, woolly monkey, callothrix, slow loris, hamster, and guinea pig. Cell 1inc.s or cell strains

Human fibroblast and epithelial:

W13XR,HeLa", Hep2"ab, KBb, RPMI-26SOb~', A-2S3b*P, A-38Sb.' Human lymphoid: NC37". F26Sa, WILZ", DEMO, MOLT4cr,BJAB" Green monkey kidney: VERO"sb, BSC-I"*b.JR", CV-Ib Squirrel monkey lung" Owl monkey lungU Woolly monkey lung" Hamster: Iotau, BHK-21b Mouse: Lb, Friend virus erythroleukemias, GM-X6b and T3-CI Mink: normal fibroblastsb Cells tested by Dr. George Miller and his colleagues of Yale University who kindly provided us with their unpublished results. They screened EBV-exposed cultures for cytopathic effects, transformation, and expression of viral capsid antigens. Cells we tested. We screened EBV-exposed cultures for cytopathic effects, for expression of viral capsid antigens, and (after several passages) for the presence of EBV DNA by renaturation kinetics. Human epithelial tumor cell lines originating from the nasopharynx kindly provided us by Dr. Jorgen Fogh of Sloan-Kettering. (I

"topoinhibition," because these changes can affect only dividing cells. EBV infects and transforms a nondividing lymphocyte and converts it into a permanently dividing lymphoblast. Several kinds of assays have been established to monitor transformation by EBV: transformation in mass culture (Pope r t al., 1968), by end-


253

point dilution (Moss and Pope, 1972; Henderson et al., 1977), and by colony formation in a semisolid medium (Mizuno er al., 1976: Yamamoto and Hinuma, 1976: Sugden and Mark, 1977). Each of these assays has advantages and disadvantages. One disadvantage of transformation of total peripheral leukocytes in mass culture and in end-point dilution experiments is that many cells that are destined not to be transformed are close to and can interact with cells that will be transformed. This problem may be only academic for fetal cells, but is acute when adult leukocytes are used as targets, because adult T cells inhibit transformation of autochthonous B cells (Thorley-Lawson et ul., 1977). The problem can be avoided by first separating lymphocyte populations and using only B cells as targets. Clonal transformation techniques also avoid this problem but require that the transformed cells form colonies in a semisolid medium. For maximum efficiency both end-point dilution assays (Henderson et al., 1977) and clonal transformation assays (Sugden and Mark, 1977) require feeder layers of fibroblasts but are not affected by mercaptoethanol. Several quantitative characteristics for transformation of fetal lymphocytes as measured by end-point dilution (Henderson er al., 1977) and for adult lymphocytes as measured by clonal transformation (Sugden and Mark, 1977) have been determined and agree remarkably well: ( I ) one particle of EBV is sufficient to transform a cell: (2) between 3% and 10% of the total leukocyte population is susceptible to transformation by EBV; (3) the plating or cloning efficiency of the transformed cells ranges between 1% and 1%. We have stated that the human target cell for transformation by EBV is the B lymphocyte (Pattengale et al., 1973; Mizuno et al., 1974: Robinson et al., 1977; Katsuki et ul., 1977; and Thorley-Lawson et al., 1977). Hinuma and his colleagues have tried to determine whether only a fraction of human B lymphocytes are susceptible to transformation by EBV (Katsuki et al., 1977) and have found that the number of cells in a target population that are IgM-positive correlates best with the number of cells that become transformed (Katsuki er af., 1977). They find only '/a to '/z of the percentage of the total lymphocytes that bear IgM found by other investigators (Rowe er al., 1973; Kumagai et al., 19751, but they find that all cell lines established by transformation with EBV in vitro carry IgM (Katsuki and Hinuma, 1975). We find the suggestion exciting that peripheral leukocytes that express IgM on their surface are the necessary targets for EBV transformation. The majority of IgM-positive cells also carry IgD (Rowe et al., 1973), and it has been suggested that surface IgD serves as a trigger that permits

254

BILL SUGDEN ET AL.

maturation of the lymphocyte to a memory or plasma cell after encountering antigen (Vitetta and Uhr, 1975). It would be desirable to know if EBV preferentially transforms IgD-positive cells and if the transformed cell lines continue to express IgD. This information might help us to understand if EBV’s transforming a lymphocyte into a blast cell is a carefully regulated change that in part follows the normal ontogeny of lymphocyte maturation or represents a grosser level of virus-imposed regulation that overrides normal developmental controls. Miller and his colleagues have studied the surface characteristics of cell lines established by transforming nonhuman primate leukocytes with EBV (Robinson et ul., 1977; Andiman and Miller, 1978). In addition, they have separated populations of lymphocytes from cotton-topped marmosets and found that, although the population of cells enriched for those bearing complement receptors is most susceptible to transformation, the transformed cell lines that grow out lack complement receptors (Robinson et al., 1977). They favor the hypothesis that transformation of marmoset cells by EBV leads to alteration of specific surface properties of the target cells. An alternative explanation of these findings comes from the work of Thorley-Lawson et al. (1977), who found that adult human T cells inhibit transformation of autochthonous B cells by EBV. I n the experiments of Robinson et al. (1977) in which different fractions of marmoset leukocytes were tested for their susceptibility to being transformed, the T cells would largely copurify with those cells that lacked complement receptors. It is possible that these T cells would prevent transformation of the bulk of complement receptor negative cells with which they copurify . Those cells that lack complement receptors but contaminate the population both enriched with cells bearing that receptor and depleted of T cells could still be the targets for transformation by EBV. The above discussion illustrates a difficulty of transformation studies with EBV. Not only are all virus stocks potentially heterogeneous, but also all target cell populations are heterogeneous and the multiplicity of potential interactions that may take place makes it difficult to interpret results. Epstein and his colleagues use heterogeneous cell populations while studying whether cells harboring EBV in vivo are capable of growing in vitro and, if not, whether they release EBV, which then infects other cells that do grow in vitro (Rickinson et al., 1975; Rickinson et d., 1977). The authors favor the second explanation, which would require two steps to establish EBV-transformed cell lines from the peripheral blood of patients with infectious mononucleosis. The experiments involve cocultivating fetal cells with peripheral leukocytes of patients with infectious mononucleosis, and these investigators find that the transformed


255

cells that grow out are usually of fetal origin (Rickinson et al., 1975, 1977). Again the findings of Thorley-Lawson et al. (1977) provide a different possible explanation for the observed prevalence of this twostep transformation. Adult T cells from the infectious mononucleosis patient will inhibit the outgrowth of autochthonous EBV-transformed cells but do not influence the transformation of those fetal leukocytes that have been infected with virus released from the adult transformed cells (in general, the fetal and adult cells will not share histocompatibility antigens). In all events, although the work of Epstein and his colleagues shows that transformation can take place in two steps (that is, that some peripheral leukocytes of infectious mononucleosis patients do release EBV), the work of Hinuma and Katsuki (1978) demonstrates that two steps are not required. They found that at least 1 in lo5 peripheral leukocytes from patients with infectious mononucleosis can form colonies when plated directly into agarose (Hinuma and Katsuki, 1978). The early events that take place during transformation of human lymphocytes by EBV are difficult to study and to interpret not only because of heterogeneities of virus and target cells, but also because of the potential temporal asynchrony of those events. Nevertheless, several investigators are trying to define those early events. Using fetal cells infected with EBV, labeling them with tritiated thymidine, and determining radioactive uptake by autoradiography , two groups have found that one EBV-associated nuclear antigen, EBNA, is expressed about 12 to 16 hours after infection, although cell DNA synthesis is not detected in those cells within 30 to 36 hours after infection (Einhorn and Ernberg, 1978; Takada and Osato, in press). On the assumption (as yet unsupported) that EBNA is coded at least in part by EBV, these findings indicate that expression of some viral genes precedes both the stimulation of cell DNA synthesis and blast formation (Einhorn and Ernberg, 1978; Takada and Osato, in press). Infection with EBV stimulates cell DNA synthesis in a biphasic fashion with time, and UV-irradiation of the virus abolishes this stimulation (Thorley-Lawson and Strominger, 1978). The first stimulatory phase is not inhibited by the addition of phosphonoacetic acid (PAA), but the second is (Thorley-Lawson and Strominger, 1978). Growth of potentially transformed cells is inhibited by PAA (Thorley-Lawson and Strominger, 1976). PAA inhibits some DNA polymerases by binding to the site where pyrophosphate is released (Leinbach et al., 1976). In particular, PAA inhibits DNA polymerases specifically induced by herpes simplex virus (Mao et al., 1975) and MDV (Lee et al., 1976). Investigators who have used PAA to study transformation by EBV have tended to interpret observed inhibition by PAA to be the result of the drug’s inhibiting an

256

BILL SUGDEN ET AL.

EBV-induced DNA polymerase that performs a necessary role during transformation. This interpretation may or may not be correct because PAA also inhibits one DNA polymerase of uninfected cells (Lee er al., 1976), and that cell enzyme is necessary to synthesize the DNA molecules of other animal viruses (Edenberg et a l . , 1978; Otto and Fanning, 1978). Therefore, PAA may serve as a rather blunt scalpel to dissect early events during transformation by EBV. However, these experiments (Einhorn and Ernberg, 1978; Takada and Osato, in press; Thorley-Lawson and Strominger, 1978) permit us to reconstruct a likely sequence of events leading to the first stirnulatory phase early in the transformation of B lymphocytes by EBV: first, penetration and uncoating of the virus particle takes place; then transcription by host enzymes of some viral messages occurs; finally those messages are translated into viral proteins that act directly or that induce cell proteins to stimulate the initiation of cell DNA synthesis by a PAA-insensitive host DNA polymerase ( p DNA polymerase?). Further experiments are required to support or to refute this putative sequence of early events during transformation of cells by EBV. The amount of viral information required for the initiation and maintenance of the transformed state has been studied by inactivation experiments (Henderson et al., 1978; W. Mark and B . Sugden, unpublished). The slope of the curve of inactivation of the transforming capacity of EBV with doses of X rays over one decade (Henderson rr al., 1978) or with y rays over two decades (W. Mark and B. Sugden, unpublished) is the same as that for similar inactivations of plaque formation by herpes simplex virus type I (HSV-I). These findings indicate that the target size for transformation by EBV is the same as the target size for plaque formation by HSV-I when measured by inactivation with X or y rays. The findings differ strikingly from those resulting from similar studies performed with other DNA-transforming viruses. By X ray inactivation experiments, Benjamin (1965) found that only one-half of the polyoma genome was required to transform cells, and Graham el ul. (1974), by fractionating sheared viral DNA, found that less than 10% of Adenovirus 2 information was required to transform cells. There are two possible interpretations for these findings from inactivation studies of EBV. One is that all of the viral information must be expressed in order for EBV to transform a cell. The second interpretation results from the recognition that the major unrepaired lesion introduced by X and y rays into DNA is a double-stranded break. Breaking the linear viral DNA molecule into pieces may make it impossible for the virus to maintain its genome in the transformed cell. One may be able to distinguish between these two interpretations by comparing the slope of inactivation of transformation


257

by EBV with that for inactivation of plaque formation by HSV-1 when point mutagens are used as the inactivating agents. The concentration of viral DNA and release of mature virions have been studied in transformed cells after the first 20 to 30 cell divisions following exposure of cells to EBV. It appears certain that viral DNA undergoes disproportionate replication relative to cell DNA during this time (Sugden et al., J . Virol., in press). This conclusion was reached by infecting adult leukocytes with 0.01 to 0.1 viral particles per cell and cloning the infected cells in agarose immediately. When the clones had grown enough to have their viral DNA content determined by renaturation kinetics, the average number of copies of viral DNA per cell varied between 10 and 1000 among the different clones (Sugden et al., J . Virol. in press). This finding indicates that early in the life of the EBV-transformed cell, viral DNA synthesis is not tightly coupled with cell DNA synthesis: however, later in the history of the cell some kind of equilibrium is reached, because those clones with an average number of copies of viral DNA per cell between 100 and 1000 stabilize at their respective numbers. Most clones of transformed cells harbor small populations of cells that release infectious virus. This phenomenon is detectable several cell divisions after exposure of the target cells to the virus. I n those clones described above, the number of virus-producing cells in each clone does not correlate with the average number of copies of viral DNA per cell, and for most of the clones about I cell per 2000 to 1 per 10,000 releases virus (Sugden et al., J . Virol., in press). When cells other than adult human leukocytes are transformed by EBV, different percentages of their populations release virus. In particular, cotton-topped marmoset cell lines transformed by EBV in vitro uniformly release more virus than do their human counterparts (Miller and Lipman, 1973). U p to 10% of the marmoset cells may contain viral capsid antigens and, presumably, most of those cells will release virus. This difference between adult human and marmoset transformed cells indicates that the regulation of expression of viral functions in the cell is at least in part determined by that cell. The expression of viral functions in some EBV-transformed cells can be modulated exogenously in three ways: ( I ) infecting the cell with a nontransforming strain of EBV (Henle et ul., 1970b: Traul et al., 1977: Yajima et a / . , 1978): (2) exposing the cells to halogenated pyrimidines (Gerber, 1972: Hampar et a / . , 1972): (3) exposing the cells to the tumorpromoting agent, tetradecanoyl phorbol acetate (TPA) (zur Hausen et al., 1978). All three methods lead to the new expression of EBV-associated an-

258

BILL SUGDEN ET AL.

tigens in some of the affected cells, and all can lead to new or increased release of some infectious virus. The addition of TPA to EBV-transformed cotton-topped marmoset cell lines is particularly useful in stimulating many of the cells to release infectious virus (zur Hausen ef a l . , 1978). This finding permits sufficient EBV to be harvested so that some formerly impractical molecular biological experiments now become feasible. The mechanisms of modulating viral expression in EBV-transformed cells are not now understood.

VI. Identification and Properties of Virus-Related Products for MDV, HVS, and EBV

Many viral-induced antigens in infected cells have been detected. Some of these antigens are likely to be coded for by their respective viruses, but which ones are virus-coded has not been established. Few of the antigens have been purified, so that the antisera used to define most antigens come from infected hosts: for EBV this means that most of the antiserum comes from tumor patients who may be immunosuppressed and will be infected with many viruses. Much of this work has been reviewed recently (Nazerian, 1973; Klein, 1973, zur Hausen, 19751, so that we shall focus on known viral components, on those antigens that our current biases lead us to think are most likely to be coded for by their respective viruses, and on those virus-associated antigens that have recently been identified or purified. Most studies on defined viral products done on HVS and EBV have focused on the structure of the viral DNA that has been extracted from virions or from transformed cells. For HVS and EBV the DNA is the one viral product that can be unambiguously identified and routinely purified. The viral DNA of MDV is difficult to purify because its density does not permit ready separation from host cell DNA. Some work cataloging the number and map positions of viral RNA sequences in EBV-infected cells has been undertaken (Hayward and Kieff, 1976: Orellana and Kieff, 1977; Powell et ul., in press). These workers have found that the complexity of viral RNA expressed in cells that do not release virus ranges between 25% and 50% of that found in the cells from which they harvest virus. This work is obviously difficult both because of the complexity of EBV DNA and because so little of the DNA is available. Our recognition that animal viruses construct their messenger RNA molecules by splicing together sequences transcribed from distant regions of the DNA template adds yet another level of difficulty to the study of viral RNA in cells infected with lymphotropic herpesviruses (Berget et ul., 1977; Chow et al., 1977; Klessig, 1977).


259

A. PROTEINS I N THE VIRION Some work following a similar design has been done on the virion proteins of both MDV and EBV. Infected cells have been labeled with radioactive amino acids or sugars, and extracellular virions have been partially purified by various methods of differential centrifugation. The recovered virions were disrupted in mercaptoethanol and sodium dodecylsulfate, and labeled polypeptides were separated by electrophoresis in polyacrylamide gels and detected by cutting and counting the gels or by autoradiography. Chen et al. (1972) found eight polypeptides, two of which were glycosylated, in their preparations of MDV. Kieff and his colleagues (Dolyniuk et al., 1976a, b) found 33 polypeptides, 12 of which were glycosylated, in their preparations of EBV. These experiments serve as the foundation for further work to elucidate which of the identified polypeptides are constituents of the virions. It is likely that there are more polypeptides (particularly in the case of MDV) that are also components of the virions. Because herpesviruses are notoriously difficult to purify, further work must incorporate several independent criteria for identifying any polypeptide as a constituent of either an MDV or EBV viral particle.

B. DNA

I N THE

VIRION

The density of MDV DNA (and that of HVT DNA) is 1.706 gm/cm3 and its molecular weight 1 x lo8. In conjunction with sedimentation studies in alkaline sucrose, these findings indicate that the viral DNA is a double-stranded, linear molecule (Lee et al., 1971; Lee, 1972), containing 46% guanine plus cytosine residues. This value is quite close to the 42% G + C content of chicken and duck DNA. This similarity in density provides a major difficulty in purifying MDV DNA free from contaminating host sequences by equilibrium sedimentation, which is a common method for purifying the viral DNA of different herpesviruses. HVS DNA is the most thoroughly characterized of the three prototypic lymphotropic herpesviruses. Fleckenstein and Wolf (1974) found that the density of HVS DNA was 1.709 gm/cm3 and its molecular weight was 0.91 f 0.05 x lo8 daltons. They also observed that, after shearing, two populations of viral molecules were generated with remarkably different densities: a heavy moiety with a density of 1.729 gm/cm3, and a light one of 1.694 gm/cm3. Later work has shown that only the full-length molecule of intermediate density, containing both covalently attached light and heavy moieties, is infectious in tissue culture (Fleckenstein et al., 1975). In addition, the moiety of higher density was found to contain repetitious DNA (Fleckenstein et al., 1975). A detailed study of the structure of

260

BILL SUGDEN ET A L .

HVS DNA isolated from virions has been carried out with denaturation mapping by electron microscopy and separation of restriction endonuclease digestion products by gel electrophoresis (Bornkamm et al., 1976). The full-length, infectious molecule is a linear, double-stranded DNA of 1.01-1.05 x lo8 daltons. This molecule is composed of a low-density moiety, which has a mass of 0.71 x lo8 daltons and is flanked on both sides by differing lengths of the high-density moiety such that the sum of the masses of both high-density moieties on each molecule varies between 0.3 and 0.35 x lo8 daltons (see Fig. I ) . This means 30% of the mass of HVS DNA is devoted to its terminal repetitions. This more recent study (Bornkamm et al., 1976) has found the repeat length of the high-density moiety DNA to be 0.83 X los daltons. Some preliminary results have shown that treatment of the viral DNA with an exonuclease can lead to its intramolecular circularization (Bornkamm et al., 1976). It is not known if this repeated DNA is transcribed (it could code for up to 3 x lo4 daltons of protein) or whether it performs only a structural role in the life cycle of HVS. Purified populations of HVS DNA contain both infectious and noninfectious molecules. Among the noninfectious molecules there is a population of defective molecules that consists solely of the repetitive se-

HVS

UNIOUE DNA

EBV

TR t

l-RDl-1 .----I-

TR i

FIG. I . These drawings represent the structures of the duplex DNAs isolated from virions of HVS and EBV. Sources of the data used to construct these representations are cited in the text. Both viral DNAs are approximately IOR daltons in mass. HVS: The density of repetitious DNA (RD,,, RDI.) is 1.729 gm/cm3. The density of unique DNA is 1.6V4gm/cmD. RDI and RD,, vary in length. The sum of RD,, and RD,. is constant and represents 3W of the H V S genome. E B V The density of EBV DNA is 1.717 gm/cm3. Repetitious DNA (RDJ represents 21% of the EBV genome. The terminal repeat (TR) is about 200- 1000 base pairs long.


26 1

quences of high density normally found at the ends of the infectious HVS molecules. The generation of HVS defective molecules differs from that of defective molecules found in other animal viruses, because the HVS defective molecules arise at high frequency and independently of the multiplicity of infection. It has been proposed that some structural feature of the repetitive DNA is responsible for the ease of generation of defective molecules (Fleckenstein et al., 1978). Much work has been done to determine the structure of EBV DNA, but progress has been slow. This slow progress surely results from the small quantities of viral DNA available and from our inability to label it isotopically in vivo to a high specific activity. These problems again are consequences of our lack of a productive host cell for EBV. The virion DNA of two strains of EBV, P3HRl (Hinuma ef al., 1967) and B95-8 (Miller and Lipman, 1973) has been studied. The DNA isolated from a nontransforming strain of EBV, P3HR1, has been analyzed by means of renaturation kinetics, digestion with restriction endonuclease, and denaturation mapping (Pritchett et a / . , 1975: Sugden et al., 1976: Hayward and Kieff, 1977: Rymo and Forsblom, 1978: Delius and Bornkamm, 1978). This work in sum indicates that the DNA of the P3HRI strain is heterogeneous. Molecules with different sequence arrangements are found in each preparation of P3HRI DNA. In addition, biological experiments indicate that pools of this strain of EBV are heterogeneous in that exposure of homogeneous target cells to a highly diluted inoculum leads to expression of different virus-associated antigens in different individual cells (Fresen et al., 1977; zur Hausen and Fresen, 1977). We shall review only the work carried out on the B95-8 strain of EBV because that strain transforms cells, has a high infectivity-to-particle ratio (Henderson et al., 1977: Sugden and Mark, 1977), and its DNA is homogeneous as determined by partial denaturation mapping (Delius and Bornkamm, 1978). EBV DNA extracted from B95-8 virions is a double-stranded, linear molecule with a density of 1.716-1.718 gm/cm3 [as was first determined for the P3HRl strain (Schulte-Holthausen and zur Hausen, 1970)] and has a molecular weight of 1.01-1.05 x lo8 daltons as determined by length measurements in the electron microscope (Pritchett et a / . , 1975). The linear molecule is terminally repetitious. The repetitions have been demonstrated by digesting the molecules with A-exonuclease, renaturing, and observing circular molecules in the electron microscope (Kintner and Sugden, Cell, in press). When an average of 0.75% of each molecule is digested with A-exonuclease, up to 40% of the molecules observed are circles. This observation indicates that the length of the terminal repeat is approximately 1000 base pairs on each end of the molecule. Measurement of the joint region where the reannealed ends of extensively digested

262

BILL SUGDEN ET AL.

molecules overlap indicates that the length of the terminal redundancy is on the order of 200-1000 base pairs. Sugden (1977) cleaved viral DNA with the Bam restriction endonuclease and separated the products electrophoretically in agarose gels. The results indicated that one fragment of 1.8 x lo6 daltons was present in several copies per molecule. Rymo and Forsblom (1978), using a sensitive method of quantification, found I I copies of that fragment per molecule. Therefore, 20% of EBV DNA is repetitious with a complexity of 1.8 x lo6 daltons or less. I n addition, Rymo and Forsblom (1978) found that the repetitious DNA sequences all lay within one DNA fragment generated by digestion with the Eco RI restriction endonuclease, and that fragment has subsequently been mapped to lie between 8% and 32% from one end of the molecule (Given and Kieff, 1978). All of these findings are roughly consistent with a denaturation map of the B95-8 strain of EBV DNA produced by Delius and Bornkamm (1978). In sum, the data indicate that the DNA of the B95-8 strain of EBV is a linear molecule with terminal repetitions of approximately 200-1000 base pairs at each end and with a tract of internal repeated sequences that begins 8% from one end of the molecule and proceeds to about 28% from that end. It is important to recognize that the structure of EBV DNA is similar to that of HVS DNA in that it contains a significant amount of repetitious sequences (20% of EBV versus 30% of HVS), but is strikingly different in that its repetitious DNA lies as a single tract bounded at both ends by nonrepeated sequences. A schematic drawing comparing HVS and EBV DNA is presented in Fig. I . Recent findings indicate that a portion of the repeated DNA sequences in EBV are transcribed (Powell er af., submitted) but, as with HVS, it is not known if they are translated.

C. VIRUS-INDUCED ANTIGENS A N D PROTEINS I N INFECTED CELLS Virus-associated antigens were detected in MDV-infected cells soon after tissue culture methods were developed for this virus (Churchill et af., 1969b). One of these, the A antigen, was present in extracellular fluid and was lost from the infected cultures during prolonged passage in vitro (Churchill er al., 1969b). This passaging in vitro also led to attenuation of the virus. It was later found that some strains of MDV can be pathogenic and not express the A antigen (Purchase el af., 1971; von Biilow, 1971). Therefore, it appears that the A antigen is not required for the pathogenicity or for the infectivity of MDV. This antigen was found to be a glycoprotein (Ross er al., 1973), and subsequent work has found its molecular weight to be about 45,000 (Long er al., 1975a; Long et af.,


263

1975b). The B antigen has been partially purified from infected cells and also found to be a glycoprotein with a molecular weight of 55,000 (Velicer et al., 1978). The function of these antigens and their relationship to MDV are not known. A Marek’s disease tumor-associated surface antigen (MATSA) has been identified by antisera raised in rabbits or chickens against established MDV tumor cell lines (Witter et af., 1975; Matsuda et al., 1976). After adsorption these sera stain MDV tumor cells and tumor cell lines in immunofluorescence assays but do not stain normal tissue or fibroblast cultures lytically infected with MDV (Witter et al., 1975; Matsuda ef al., 1976). The antigen, therefore, is not necessarily associated with the presence of MDV but rather with MDV-induced tumor cells. Accordingly, MATSA is not found on other types of lymphoid tumors of the chicken. For example, it is not present on cells of a transplantable lymphoid tumor induced by an RNA leukosis virus (Witter er al., 1975). The variety of MDV tumor cells on which MATSA has been found indicates that it is not likely to be a histocompatibility antigen. The MATSA antigen may be an alloantigen that is specific to the differentiated cells infected by MDV. Tumor virus-associated alloantigens have been defined in the mouse (Stockert et af., 1971). An MDV-induced DNA polymerase activity has been identified and partially purified from infected duck embryo fibroblast cultures (Boezi et al., 1974). The enzyme has an apparent molecular weight of 100,000 and prefers activated DNA as a template. These two characteristics, in addition to the enzyme’s chromatographic behavior on phosphocellulose columns, distinguish it from the DNA polymerase activities present in uninfected cells. All herpesviruses studied have been shown to induce a new DNA polymerase activity in infected cells. These observations have conditioned virologists to view favorably the notion that herpesviruses code for a DNA polymerase. This idea, however, has so far received strong support only from studies of herpes simplex virus that have employed temperature-sensitive mutants (Aron et al., 1975). Of the three prototypes of lymphotropic herpesviruses, it would seem that HVS lends itself best to the study of its associated proteins and antigens. It infects established cell lines efficiently, has a reasonably short growth cycle, and is released into the extracellular fluid. However, of the three, it is the most recent to be studied and, consequently, little is known about its associated proteins. On the other hand, a great many observations have been made in EBVinfected cells on virus-associated antigens that are detected by patients’ antisera. These data have been reviewed by Klein (1975) and zur Hausen (1975). Recently work has progressed on characterizing the EBV nuclear

264

BILL SUGDEN ET AL.

antigen (EBNA) that was first identified by anticomplement immunofluorescence techniques (Reedman and Klein, 1973). This antigen is particularly useful in that it appears to be uniformly associated with all cells infected by EBV (Lindahl et al., 1974). EBNA has been purified to apparent homogeneity (Luka et al., 1978) and has been partially characterized (Lenoir et d . , 1976; Matsuo et d . , 1977; Luka et d . , 1977, 1978; Baron and Strominger, 1978). The molecular weight of the native antigen is about 200,000, whereas in denaturing gels the most purified preparations contain only one polypeptide with a mobility that corresponds to a molecular weight of 48,000 (Luka et al., 1978). The active species, therefore, appears to be a tetramer. It binds to double-stranded DNA cellulose columns in the presence of 0.2 M NaCl but is eluted at 0.4 M NaCl at a pH of 7.4. In two of its characteristics EBNA is reminiscent of the simian virus 40 (SV40) large T antigen. Both antigens are found in the nucleus of all virally infected cells, and both bind to double-stranded DNA. The SV40 large T antigen binds preferentially to a specific sequence of DNA near or at the site for initiation of D N A synthesis on the SV40 DNA molecule (Tijan, 1977). It is not now known if EBNA shows any sequence specificity of binding to double-stranded DNA. An EBV-associated cell surface antigen has been identified with rabbit antisera raised against two Burkitt’s lymphoma cell lines (Sakamoto and Hinuma, 1978). After the sera were adsorbed with normal cells, at least some of the cells of most lines infected by EBV were stained in immunofluorescent assays (Sakamoto and Hinuma, 1978). In this respect the EBV-associated cell surface antigen apparently differs from MATSA, which is found only on tumor cells. However, only EBV-infected lymphoid cells have been studied, so that the virus-associated surface antigen may also be a lymphoid differentiation antigen or, perhaps, a candidate for the target recognized by the immune killer T cells found in patients during the acute stage of infectious mononucleosis (Svedmyr and Jondal, 1975)* Three enzymic activities have been studied in cells that have been induced to express EBV-associated antigens by treating them with halogenated pyrimidines or by superinfecting them with the nontransforming strain of EBV. A ribonucleotide reductase activity resistant to hydroxyurea (Henry et al., in press), a thymidine kinase activity with a distinct electrophoretic mobility (Chen et al., 1978), and a DNA polymerase activity active in 50 mM ammonium sulfate (Miller et a / . , 1977b) have been shown to differ in some way from the corresponding activities in uninduced cells. These enzymic activities, found in the induced cells, have their counterparts in cells infected by other herpesviruses and, therefore, may be host enzymes that are generally induced by different herpesviruses or may be encoded by the viruses themselves.


D. VIRAL DNA

IN

265

TRANSFORMED CELLS

Viral DNA has been detected in tumor cells, in lines established from tumor biopsies, and in cells transformed in vitro by MDV, HVS, and EBV. Nazerian and Lee (1974) found one MDV tumor cell line to harbor 60-90 genome equivalents of MDV DNA by using the technique of hybridizing complementary viral RNA to cell DNA immobilized on nitrocellulose filters. Nonoyama and his colleagues have studied the structure of this intracellular viral DNA. Using velocity sedimentation analyses in neutral and alkaline glycerol gradients and equilibrium sedimentation in gradients of CsCl plus ethidium bromide, they have shown that at least 80%-90% of the viral DNA is extrachromosomal and behaves as a covalently closed, supercoiled molecule of full length (Tanaka et a/., 1978). It is not yet known if all of the nucleotide sequences of the virion DNA are present in these extrachromosomal molecules. Multiple copies of HVS DNA have been found in tumor biopsy cells and in nonproducer tumor cell lines with the technique of renaturation kinetics (Fleckenstein et a / . , 1976, 1977). The structure of HVS DNA in one nonproducer cell line has been carefully studied by means of denaturation mapping by electron microscopy and analysis of cleavage products generated by digestion with restriction endonucleases (Werner et a / . , 1977, in press). The viral DNA again has been found to be present as an extrachromosomal, covalently closed, supercoiled molecule. However, it has a molecular weight of l .3 x lo8, which is 30% larger than the virion’s molecule, and is composed of two low-density and two highdensity moieties with a portion of each of the low-density moieties being deleted from the intracellular viral DNA (Werner et al., 1977, in press). The study of intracellular viral DNA of lymphotropic herpesviruses was first begun with EBV when zur Hausen and Schulte-Holthausen (1970) detected viral nucleotide sequences in a cell line established from a Burkitt’s lymphoma biopsy. Multiple copies of EBV DNA were detected in both Burkitt’s lymphoma and nasopharyngeal carcinoma biopsies (zur Hausen el a/., 1972: Nonoyama and Pagano, 1973). Nonoyama and Pagano (1973) introduced the technique of isotopically labeling EBV DNA to high specific activity by “nick translation,” using DNA polymerase I from E . coli (Kelly et a / . , 1970). This technique is particularly important for the detection of EBV (and MDV) DNA because it is not practicable to label the viral DNA in vivo, and complementary RNA synthesized in vitro with DNA-dependent RNA polymerase is probably not homologous to all viral DNA sequences and is certainly not a uniform distribution of those sequences (B. Sugden, unpublished results). The location of the bulk of intracellular EBV DNA is extrachromosomal (Nonoyama and Pagano, 1972: Tanaka and Nonoyama, 1974) and consists

266

BILL SUGDEN ET AL.

of covalently closed, supercoiled molecules of about full length (Lindahl 1976). Within the limit of analysis of DNA by electrophoretic separation of cleavage products of restriction endonucleases, intracellular viral DNA in Burkitt’s lymphoma biopsies and in vitro transformed cell clones contains all the nucleotide sequences present in virion DNA (Sugden, 1977). This finding is strengthened by the observation that most clones of cells transformed in vitro by EBV release infectious particles, albeit at a very low level (Sugden et af.,J . Virol., in press), In some cell lines transformed by EBV a small portion of viral DNA is integrated into the cell genome (Lindahl et al., 1976). This conclusion is based on experiments that separate free viral from cell DNA by centrifugation in equilibrium sedimentation gradients. After three consecutive centrifugations about 1% of the viral nucleotide sequences are found in the position of cell DNA (Kaschka-Dierich et al., 1977). Upon shearing and recentrifugation these viral sequences shift their position to the density characteristic of free viral DNA. With similar techniques, tumor biopsy cells, tumor cell lines, and cell lines established from EBV-seropositive donors were found to harbor a small percentage of their viral DNA as integrated sequences (Lindahl et al., 1976; Adams et al., 1977; Kaschka-Dierich et al., 1977). Lindahl and his colleagues have identified one in vitro transformed cell line that lacked detectable integrated viral DNA (Kaschka-Dierich el al., 1977). Because so little DNA is integrated, it is technically difficult to demonstrate directly the presence or absence of integrated viral nucleotide sequences in a given cell line. Without direct experiments to measure the complexity of integrated viral DNA, it remains difficult to evaluate the role this information might play in transformation by EBV.

et al.,

VII. Conclusion

Are there reasons other than convenience to discuss lymphotropic herpesviruses separately from other members of the group? Two reasons are immediately apparent: ( I ) only the lymphotropic herpesviruses infect and transform lymphoid cells in their natural host; (2) only the lymphotropic herpesviruses induce lymphoid tumors in natural or experimental hosts. [With the possible exception of the Luck6 frog virus (Granoff, 19731, the lymphotropic herpesviruses are the only ones of this family known to induce tumors directly in any hosts.] These distinct features may be a function only of the tissue tropism of MDV, HVS, EBV, and their close relatives or may reflect genetic information peculiar to them. MDV, HVS, and EBV permit us to establish lymphoid cell lines of


267

chickens, cotton-topped marmosets, and human beings. Most established chicken cells of any kind are MDV-transformed T cells; most cottontopped marmoset lymphoid cell lines are transformed by HVS o r EBV; all B-lymphoid cell lines established from normal human donors and most lymphoid lines established from human tumor patients are transformed by EBV. Transformation by EBV in vitro is functionally equivalent t o the establishment of B-lymphoblast cell lines. It bears repeating that transformation by EBV is qualitatively distinct from transformation by papova-, adeno-, and other herpesviruses. In transformation by EBV, a B lymphocyte is converted into a permanently dividing B lymphoblast. The state of the viral DNA in cells transformed by MDV, HVS, and EBV is strikingly different from that usually found in cells transformed by papova-, adeno-, and other herpesviruses. In the latter cases, one o r a few copies of the viral DNA are usually found to be integrated into the host cell DNA (Sambrook et al., 1968), and only fragments of the viral DNA with as small a complexity as 1-2 x lo6 daltons may be present (Sambrook et al., 1974; Graham et al., 1974; Kraiselbard et al., 1975). Thus only 50% of the papovaviruses, 5%-1% of the adenoviruses, and 1%-2% of herpes simplex virus at most are required to maintain the transformed state. This information is maintained in the cell, presumably by dint of its being covalently linked to cell DNA. With MDV and EBV, copies of all or nearly all of the viral DNA are extrachromosomal; with HVS, in two cases, the majority but not all of the viral DNA is present extrachromosomally in multiple copies. The multiple copies of viral DNA in MDV-, HVS-, and EBV-transformed cells may result from a mechanism evolved to guarantee that both daughter cells would receive at least one copy. Obviously, in view of the limitations of our knowledge, other hypotheses involving gene dosage might also explain the observed features of viral DNA in cells transformed by lymphotropic herpesviruses. The viral DNAs of MDV, HVS, and EBV, within the limits of detection (1%-5% for HVS and EBV, more for MDV), share no sequence homology with their hosts’ DNA. In addition, within our ability to discern it, the virus and viral DNA in EBV tumor cells from human patients are not different from the virus and its DNA in cells cultured in the laboratory. We conclude, therefore, that EBV and, by analogy, MDV carry sufficient information to transform lymphocytes, that is, to infect some precursor cell in vivo and alter it such that it can replicate permanently in vitro. In addition, from among the cells transformed by MDV, neoplastic progeny are generated efficiently in genetically susceptible animals. Prominent goals in working with MDV, HVS, and EBV are to identify those gene products required for the initiation and maintenance of the transformed state, t o elucidate their mechanisms of action, and to deter-

268

BILL S U G D E N E T AL.

mine what role they play in contributing to the neoplastic phenotype. We need to overcome several major problems in order to achieve these goals. We need much information on normal lymphoid cells in vivo and in vitro, including how to identify different classes of leukocytes and their precursors and how to culture their precursors in vitro. We need in vitro transformation assays for MDV and HVS. Finally, we need to grow MDV in large quantities in a cell-free form and to be able to plaquepurify EBV and to propagate it efficiently in a productive host. These needs will be difficult to fulfill, but without them progress toward our goals will be painfully slow and confusingly indirect.

ACKNOWLEDGMENTS We thank our many colleagues who kindly sent us their papers prior to publication so that we might include their findings in this review. We thank Dr. George Miller of Yale University in particular for providing us with his unpublished findings which constitute the bulk of Table IV. Finally, we thank Drs. Ilse Riegel and Jeff Ross of the McArdle Laboratory for critically helping us with this manuscript. We were supported by National Institutes of Health Grants CA 07175, CA 22443, and CA 09135.

ADDENDUM In our discussion of transformation by EBV we referred to the work of Thorley-Lawson et ul. (1977), who found that the presence of adult T-cells inhibited transformation of

peripheral leukocytes by EBV. Those authors used target cells from both EBV seronegative and seropositive donors and did not distinguish between them in their protocols nor in their results. In a recent similar study by Moss et ul. (1978). these workers found that T-cells inhibited transformation of autochthonous cells only from donors who were seropositive for antibodies to EBV. These new findings obviate our alternative interpretation of the work of Robinson et a / . ( 1977) with cells of seronegative marmosets but do not change our reflections on the work of Epstein and his colleagues, who worked with cells of infectious mononucleosis patients [Rickinson rt a / . (1975): Rickinson (11. ( 1977)l.

ADDENDUM REFERENCE Moss, D. .I.Rickinson, A. B., and Pope, J. H (1978), Long-term T-cell mediated immunity to Epstein-Barr virus in man. I. Complete regression of virus-induced transformation in cultures of sero-positive donor leukocytes. I u t . J . Cuncer 22, 662-668

REFERENCES Adams, A., Bjursell, G., Kaschka-Dierich. C., and Lindahl, T. (1977). Circular EpsteinBarr virus genomes of reduced size in a human lymphoid cell line of infectious mononucleosis origin. J . Virol. 22, 373-380.


269

Andersson-Anvert, M., Forsby, N., Klein, G., and Henle, W. (1977). Relationship between the Epstein-Barr virus and undifferentiated nasopharyngeal carcinoma: Correlated nucleic acid hybridization and histopathological examination. Int. J. Cuncer 20, 486494. Andiman, W. A., and Miller, G. (1978). Properties of Epstein-Barr virus transformed woolly monkey lymphoblastoid cell lines. Proc. Soc. Exp. B i d . Med. 157, 489-493. Aron, G . M., Purifoy, D. J . M., and Schaffer, P. A. (1975). DNA synthesis and DNA polymerase activity of herpes simplex virus type I temperature-sensitive mutants. J . Virol. 16, 498-507. Bar, R. S., DeLor, C. J., Clausen, K. P., Hurtubise, P., Henle, W., and Hewetson, J . F. (1974). Fatal infectious mononucleosis in a family. N . Engl. J. Met/. 290, 363-367. Baron, D., and Strominger, J . L. (1978). Partial purification and properties of the EpsteinBarr virus-associated nuclear antigen. J. B i d . Chem. 253, 2875-288 I . Benjamin, T. L. (1965). Relative target sizes for the inactivation of the transforming and es of polyoma virus. Proc. Nutl. Acrid. Sci. U . S . A . 54, 121-124. Berget, S. M., Moore, C . , and Sharp, P. A. (1977). Spliced segments at the 5 ‘ terminus of adenovirus 2 late mRNA. Proc. Natl. Acud. Sci. U . S . A . 74, 3171-3175. Biggs, P. M., Churchill, A. E., Rootes, D. G., and Chubb, R. C. (1968). The etiology of Marek’s disease-an oncogenic herpes-type virus. In ”Perspectives in Virology” ( M . Pollard, ed.), pp. 21 1-237. Academic Press, New York. Boezi, J . A,, Lee, L. F., Blakesley, R. W., Koenig, M., and Towle, H. C. (1974). Marek’s disease herpesvirus-induced DNA polymerase. J. Virol. 14, 1209- 12 19. Bornkamm, G. W., Delius, H., Fleckenstein, B., Werner, F.-J., and Mulder, C. (1976). Structure of Herpesviriis suimiri genomes: Arrangement of heavy and light sequences in the M genome. J. Virol. 19, 154-161. Briles, W. E., Stone, H . A., and Cole, R. K. (1977). Marek’s disease: Effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 195, 193- 195. Calnek, B. W., and Hitchner, S. B. (1969). Localization of viral antigen in chickens infected with Marek’s disease herpesvirus. J . Natl. Cancer Inst. 43, 935-949. Calnek, B. W., Adldinger, H. C., and Kahn, D. E. (1970). Feather follicle epithelium: A source of enveloped and infectious cell-free herpesviruses from Marek’s disease. AiYrrn Dis. 14, 219-233. Chang, R. S., and Golden, H. D. (1971). Transformation of human leucocytes by throat washing from infectious mononucleosis patients. Nutitre (London)234, 359-360. Chen, J . H., Lee, L. F., Nazerian, K., and Burmester, B. R. (1972). Structural proteins of Marek’s disease virus. Virology 47, 434-443. Chen, S.-T., Estes, J . E., Huang, E.-S., and Pagano, J . S . (1978). Epstein-Barr virusassociated thymidine kinase. J. Virol. 26, 203-208. Chow, L. T., Gelinas, R. E., Broker, T . R., and Roberts, R. J . (1977). A n amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 12, 1-8. Churchill, A. E., and Biggs, P. M. (1967). Agent of Marek’s disease in tissue culture. Nutitre (London) 215, 528-530. Churchill, A. E., Payne, L. N., and Chubb, R. C. (1969a). Immunization against Marek’s disease using a live attentuated virus. Natirre (London) 221, 744-747. Churchill, A. E., Chubb, R. C., and Baxendale, W. (1969b). The attentuation, with loss oncogenicity, of the herpes-type virus of Marek’s disease (strain HPRS-16) on passage in cell culture. J. Gen. Virol. 4, 557-564. Cole, R. K. (1972). The genetics of resistance to Marek’s disease. I n “Oncogenesis and Herpesviruses” (P. M. Biggs, G . de-The, and L. N. Payne, eds.), pp. 123-128. International Agency for Research o n Cancer, Lyon.

270

BILL SUGDEN ET AL.

Daniel, M. D., Hunt, R. D.. Dubose, D., Silva, D., and Melendez, L . V. (1975). Induction of Herpesvirus saimiri Lymphoma in New Zealand white rabbits inoculated intravenously. I n "Herpesviruses and Oncogenesis, 11" (G. de-The, M . A. Epstein, and H. zur Hausen. eds.), pp. 205-208. International Agency for Research on Cancer, Lyon. Deinhardt, F. W., Falk, L. A., and Wolfe, L. G . (1974). Simian herpesviruses and neoplasia. Adv. Cciiicer Res. 19, 167-205. Deinhardt, F., Falk. L . , Wolfe, L. G., Paciga, J . , and Johnson, D. (1975). Response of marmosets to experimental infection with Epstein-Barr virus. I n "Oncogenesis and Herpesviruses 11'' (G. de-The. M. A. Epstein, and H. zur Hausen. eds.), pp. 161-168. International Agency for Research on Cancer, Lyon. Delius, H., and Bornkamm, G. W. (1978). Heterogeneity of Epstein-Barr virus. 111. Comparison of a transforming and a nontransforming virus by partial denaturation mapping of their DNAs. J . Virol. 27, 81-89. de-ThC, G., Geser, A., Day, N . E., Tukei, P. M., Williams, E . H., Beri, D. P., Smith, P. G . . Dean. A. G.. Bornkamm, G. W., Feorino, P., and Henle, W. (1978). Epidemiological evidence for causal relationship between Epstein-Barr virus and Burkitt's lymphoma: Results of the Ugandan prospective study. Nutrrrc, (London)274, 756-761, Doherty, P. C., Blanden, R. V., and Zinkernagel, R. M. (1976). Specificity of virus-immune effector T cells for H-2K and H-2D compatible interactions: Implications for H-antigen diversity. Trcoisplan/. Rei,. 29, 89-124. Dolyniuk, M.. Pritchett, R., and Kieff, E. (1976a). Proteins of Epstein-Barr virus I . Analysis of the polypeptides of purified enveloped Epstein-Barr virus. J . Virol. 17, 935-949. Dolyniuk, M., Wolff. E., and Kieff, E. (1976b). Proteins of Epstein-Barr virus 11. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine- and polysaccharide-containingcomponents of enveloped virus. J . Virol. 18, 289-297. Edenberg. H. J., Anderson, S . , and De Pamphilis, M. L. (1978). Involvement of DNA polymerase (I in simian virus 40 DNA replication. J . Biol. Chern. 253, 3273-3280. Eidson, C. S . , and Anderson, D. P. (1971). Immunization against Marek's disease. Avirrrr Dis. 15, 68-81. Einhorn, L., and Ernberg, I . (1978). Induction of EBNA precedes the first cellular S-phase after EBV-infection of human lymphocytes. I n t . J . C a n w r 21, 157-160. Epstein, M. A., and Barr, Y. M. (1964). Cultivation in iitro of human lymphoblasts from Burkitt's malignant lymphoma. Lancet 1, 252. Falk, L. A., Nigida, S . . Deinhardt, F., Cooper, R. W., and Hernandez-Camacho, J. I . ( 1973). Oral excretion of herpesvirus saimiri in captive squirrel monkeys and incidence of infection in feral squirrel monkeys. J . N u t / . Cancer Inst. 51, 1987-1989. Falk, L.. Wright, J., Wolfe, L., and Deinhardt. F. (1974a). Herpesvirrrs arrlrs: transformation in vitro of marmoset splenic lymphocytes. I n / . J . Cuncer 14, 244-25 I. Falk, L . A., Nigida, S. M., Deinhardt, F., Wolfe. L. G., Cooper, R. W., and HernandezCamacho, J. I . ( l974b). Herpesvirus atrles: Properties of an oncogenic herpesvirus isolated from circulating lymphocytes of spider monkeys (ateles sp.). I n t . J . C'crricer 14, 473-482. Falk. L., Deinhardt, F.. Nonoyama. M., Wolfe, L. G.. Bergholz, C . , Lapin, B., Yakovleva, L., Agrba, V., Henle, G., and Henle, W. (1976). Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus. I n / . J . Cancer 18, 798-807. Falk, L. A., Henle. G., Henle, W., Deinhardt, F., and Schudel, A. (1977). Transformation of lymphocytes of Herpesvirris papio. Int. J . Cancer 20, 219-226. Falk, L. A., Johnson, D., and Deinhardt, F. (1978). Transformation of marmoset lymphocytes in iti/ro with Hrrpc,svirns uteles. I n t . J . Cancer 21, 652-657.


27 1

Fialkow, P. J., Klein, G., Gartler, S. M., and Clifford, P. (1970). Clonal origins of individual Burkitt tumors. Lanccjt 1, 384-386. Fleckenstein, B., and Wolf, H. (1974).Purification and properties of Hivpesiiirus saimiri DNA. Virology 58, 55-64. Fleckenstein, B., Bornkamm, G . W., and Ludwig, H . (1975). Repetitive sequences in complete and defective genomes of Herpesvirus saimiri. J. Virol. 15, 398-406. Fleckenstein, B., Bornkamm, G. W., and Werner, F. J. (1976). The role of Herpesiirus saimiri genomes in oncogenic transformation of primate cells. I n “Comparative Leukemia Research 1975, Bibl. Haemat., No. 43” (J. Clemmesen and D. S. Yohn, eds.), pp. 308-312, Karger, Basel. Fleckenstein, B., Miiller, I., and Werner, J. (1977). The presence of Herpesvirus saimiri genomes in virus-transformed cells. Int. J. Cancer 19, 546-554. Fleckenstein, B., Bornkamm, G. W., Mulder, C., Werner, F.-J., Daniel, M. D., Falk, L. A., and Delius, H. (1978). Herpesvirus ateles DNA and its homology with Herpesvirus saimiri nucleic acid. J . Viorl. 25, 361-373. Frank, A., Andiman, W. A,, and Miller, G. (1976). Epstein-Barr virus and nonhuman primates: Natural and experimental infection. Adv. Cancer Res. 23, 171-201. Frederickson, T. L., Longenecker, B. M., Pazderka, F., Gilmour, D. G., and Ruth, R. F. (1977). A T-cell antigen system of chickens: Ly-4 and Marek’s disease. Inimunogcnetics 5, 535-552. Fresen, K.-0.. Merkt, B., Bornkamm, G.. and zur Hausen, H. (1977). Heterogeneity of Epstein-Barr virus originating from P3HR-I cells. I. Studies on EBNA induction. I n t . J. Cancer 19, 317-323. Gerber, P. ( 1972). Activation of Epstein-Barr virus by 5-bromodeoxyuridine in “virusfree” human cells. Proc. Natl. Acad. Sci. U . S . A . 69, 83-85. Gerber, P.. and Monroe, J. H. (1968). Studies on leukocytes growing in continuous culture derived from normal human donors. J . Natl. Cancer Inst. 40, 855-866. Gerber, P., Pritchett, R. F., and Kieff, E. D. (1976). Antigens and DNA of a chimpanzee agent related to Epstein-Barr virus: J . Virol. 19, 1090-1099. Gerber, P., Kalter, S. S., Schidlovsky, G., Peterson, W. D., Jr., and Daniel, M. D. (1977). Biologic and antigenic characteristics of Epstein-Barr virus-related herpesviruses of chimpanzees and baboons. fnr. J . Cancer 20,448-459. Given, D., and Kieff, E. (1979). DNA of Epstein-Barr virus I V . Linkage map of restriction enzyme fragments of 895-8 and W91 strains of EBV. J. Virol. 28, 524-542. Goldman, M., Landon, J. C., and Reisher, J. I. (1968). Fluorescent antibody and gel diffusion reactions of human and chimpanzee sera with cells cultured from Burkitt tumors and normal chimpanzee blood. Cancer Res. 28, 2489-2495. Graham, F. L., Veldhuisen. G., and Wilkie, N. M. (1973). Infectious herpesvirus DNA. Nature (London) New B i d . 245, 265-266. Graham, F. L., A b r a h a m , P. J., Mulder, C.. Heijneker, H. L., Warnaar, S. O., devries, F. A. J., Fiers. W., and Van der Eb, A. J. (1974). Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and Simian virus 40. Cold Spring Harbor Svmp. Qctanr. B i d . 39, 637-650. Granoff, A. (1973). The Luck6 renal carcinoma of the frog. In “The Herpesviruses” (A. Kaplan, ed.). pp. 627-640. Academic Press, New York. Hampar, B., Derge, J. G., Martos, L. M., and Walker, J. L. (1972). Synthesis of EpsteinBarr virus after activation of the viral genome in a ”virus-negative” human lymphoblastoid cell (Raji, made resistant to 5-bromodeoxyuridine. Proc. Nafl. Acail. Sci. U . S . A . 69, 78-82. Hayward, S. D., and Kieff, E. (1976). Epstein-Barr virus-specific RNA. I . Analysis of viral

272

BILL SUGDEN ET AL.

RNA in cellular extracts and in the polyribosomal fraction of permissive and nonpermissive lymphoblastoid cell lines. J. V i d . 18, 518-525. Hayward, S. D., and Kieff, E. (1977). The DNA of Epstein-Barr virus. 11. Comparison of the molecular weights of restriction endonuclease fragments of the DNA of strains of EBV and identification of end fragments of the B95-8 strain. J . Virol. 23,421-429. Henderson, E., Miller, G., Robinson, J., and Heston, L. (1977). Efficiency of transformation of lymphocytes by Epstein-Barr virus. Virology 76, 152- 163. Henderson, E., Heston, L., Grogan, E., and Miller, G. (1978). Radiobiological inactivation of Epstein-Barr virus. J . Virol. 25, 51-59. Henle, G.. Henle, W., Clifford, P., Diehl, V., Kafuko, G. W., Kirya, B. G.. Klein, G., Morrow, R. H., Munube, G . M. R., Pike, P., Tukei, P. M., and Ziegler, J. L. (1969). Antibodies to Epstein-Barr virus in Burkitt’s lymphoma and control groups. J . N a i l . Cancer Inst. 43, 1147-1 157. Henle, W.. Henle, G., Ho, H.-C., Burtin, P., Cachin, Y., Clifford, P., de Schryver. A , . de-The, G., Diehl. V., and Klein, G. (1970a). Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups. J . Niitl. Cancer Inst. 44, 225-23 I . Henle, W., Henle, G . . Zajac, B. A., Pearson, G., Wdubke, R., and Scriba, M. (1970b). Differential reactivity of human serums with early antigens induced by Epstein-Barr virus. Sciencr 169, 188- 190. Henry, B. E., Glaser, R., Heuetson, J . , and O’Callaghan, D. (19781. Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein-Barr virus. I n press. Hinuma, Y.,Konn, M . , Yamaguchi, J., Wudarski, D. J . , Blakeslee, J . R., Jr., and Grace, J. T., Jr. (1967). lmmunofluorescence and Herpes-type virus particles in the P3HR-I Burkitt lymphoma cell line. J. Virol. 1 , 1045-1051. Hinuma, Y., and Katsuki, T. (1978). Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients. Int. J . C n n w r 21,426-43 I . Hinze, H. (1971a). New member of the herpesvirus group isolated from wild cottontail rabbits. Infect. Irnmnn. 3, 350-354. Hinze, H. C. (1971b). Induction of lymphoid hyperplasia and lymphoma-like disease in rabbits by Hivp,svirr/.s syhiliigrrs. Int. J . Cnncrr 8, 5 14-522. Huber, C., Sundstriim, C. C.. Nilsson, K., and Wigzell, H. (1976). Surface receptors on human haematopoietic cell lines. Clin. Exp. Immrtnol. 25, 367-378. Hunt, R. D., Melendez, L. V . , King, N. W., Gilmore, C. E., Daniel, M. D., Williamson, M. E., and Jones, T. C. (1970). Morphology of a disease with features of malignant lymphoma in marmosets and owl monkeys inoculated with Hc,rpi,.si’ir//s suiniiri. J . Natl. Cunccv Inst. 44,447-465. Jackson, C. A. W.. Biggs, P. M., Bell, R. A , , Lancaster. F. M., and Milne. B. S. (1974). A study of vaccination against Marek’s disease with an attenuated Marek‘s disease virus. Ai~icrnPutIiuI. 3, 123-144. Jondal, M., and Klein, G. (1973). Surface markers on human B and T lymphocytes. I J . Presence of Epstein-Barr virus receptors on B lymphocytes. J . Exp. Med. 138, 136S1378. Kaschka-Dierich. C . , Falk. L.. Bjursell, G., Adams, A., and Lindahl, T. (1977). Human lymphoblastoid cell lines derived from individuals without lymphoproliferative disease contain the same latent forms of Epstein-Barr virus DNA a s those found in tumor cells. I n / . J . Cirnciv 20, 173-180. Katsuki, T., and Hinuma, Y . (1975). Characteristics of cell lines derived from human leukocytes transformed by different strains of Epstein-Barr virus. 1/11. J . C u n c w 15, 203-210.


273

Katsuki, T., Hinuma, Y.,Yamamoto, N., Abo, T., and Kumagai, K. (1977). Identification of the target cells in human B lymphocytes for transformation by Epstein-Barr virus. Virology 83, 287-294. Kawamura. H., King, D. J., and Anderson, D. P. (1969). A herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13, 853-863. Kelly, R. B., Cozzarelli, N. R., Duetscher, M. P., Lehman, 1. R., and Kornberg, A. (1970). Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replciation of duplex deoxyribonucleic acid by polymerase at a single strand break. J. B i d . C h e m . 245, 39-45. Klein, G. (1973). The Epstein-Barr virus. In “The Herpesviruses” (A. S. Kaplan, ed.), pp. 521-555. Academic Press, New York. Klein, G., Giovanella, B. C., Lindahl, T., Fialkow, P. J., Singh, S., and Stehlin, J. S. (1974). Direct evidence for the presence of Epstein-Barr virus DNA and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of the nasopharynx. Proc. N a i l . Acad. Sci. U . S . A . 71, 4737-4741. Klein, G. (1975). Virus-induced antigens-a review. In “Oncogenesis and Herpesvirus 11” (G. de-The, M. A. Epstein, and H. zur Hausen, eds.), Pt. I , pp. 293-308. International Agency for Research on Cancer, Lyon. Klein, G., Svedmyr, E., Jondal, M., and Persson, P. 0.(1976). EBV-determined nuclear antigen (EBNA)-positive cells in the peripheral blood of infectious mononucleosis patients. lnt. J. Cancer 17, 21-26. Klessig, D. F. (1977). Two adenovirus mRNAs have common 5 ‘ terminal leader sequence encoded at least 10 Kb upstream from their main coding regions. Cell 12, 9-21. Kraiselbard, E., Gage, L. P.,and Weissbach, A. (1975). Presence of a herpes simplex virus DNA fragment in an L cell clone obtained after infection with irradiated herpes simplex virus 1 . J . Mol. B i d . 91, 533-542. Kumagai, K . Abo, T., Sekizawa, T., and Sasaki, M. (1975). Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37°C. J . Inirnunol. 115, 982-987. Landon, J . C., and Malan, L. B. (1971). Seroepidemiologic studies of Epstein-Barr virus antibody in monkeys. J . N a t l . Cancer Inst. 46, 881-884. Landon, J. C., Ellis, L. B. Zeve, V. H., and Fabrizio, D. P. A. (1968). Herpes-type virus in cultured leukocytes from chimpanzees. J. Natl. Cancer Inst. 40, 181-192. Lee, L. F. (1972). Induction of deoxyribonucleic acid synthesis and the oncogenicity of Marek’s disease virus. J . Virol. 10, 167-170. Lee, L. F., Kieff. E. D., Bachenheimer, S. L., Roizman, B.. Spear, P. G., Burmester, B. R., and Nazerian, K. (1971). Size and compostion of Marek’s disease virus deoxyribonucleic acid. 1. Virol. 7 , 289-294. Lee, L. F.. Nazerian, K . , Leinbach, S. S . , Reno, J. M., and Boezi, J . A. (1976). Effect of phosphonoacetate on Marek’s disease virus replication. J. Nut/. C a n w r Inst. 56, 823827. Leinbach, S. S . , Reno, J . M., Lee, L. F., Isbell, A. F., and Boezi, J. A . (1976). Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase. Biochemistry 15, 426-430. Lemon, S. M., Hutt, L. M . , Shaw, J . E., Li, J.-L. H., and Pagano, J . S. (1977). Replication of EBV in epithelial cells during infectious mononucleosis. Nurure (Londo17)268, 268270. Lenoir. G . , Berthelon, M.-C., Favre. M.-C., and de-The, G. (1976). Characterization of Epstein-Barr virus antigens. I . Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen. J . Virol. 17, 672-674. Lindahl, T., Klein. G., Reedman, B. M., Johansson, B., and Singh. S. (1974). Relationship

274

BILL SUGDEN ET AL.

between Epstein-Barr virus (EBV) DNA and the EBV-determined nuclear antigen (EBNA) in Burkitt lymphoma biopsies and other lymphoproliferative malignancies. Int. J. Cancer 13,764-772. Lindahl, T . , Adams, A, Bjursell, G., Bornkamm. G. W., Kaschka-Dierich, C.. and Jehn, U. (1976). Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J . Mol. Biol. 102, 51 1-530. Long, P. A., Kaveh-Yamini, P.. and Velicer, L. F. (197Sa). Marek’s disease herpesviruses. I . Production and preliminary characterization of Marek‘s disease herpesviruses A antigen. J. Viro/. 15, 1182-1191. Long, P. A., Clark, J. L., and Velicer, L . F. (1975b). Marek’s disease herpesviruses. 11. Purification and further characterization of Marek’s disease herpesvirus A antigen. J. viroi.15,1 1 9 2 - i m . Longenecker, B. M., Pazderka, F.. Gavora, J . S . , Spencer, J . L., and Ruth, R. F. (1976). Lymphoma induced by herpesvirus: Resistance associated with a major histocompa~ l 401-407. ~ l 7 ~ ~ ~ ~ . ~ tibility gene. / ~ l l ~ l / / l l ~ J 3, Luka. J . , Siegert, W., and Klein, G. (1977). Solubilization of the Epstein-Barr virusdetermined nuclear antigen and its characterization as a DNA-binding protein. J. Virol. 22, 1-8. Luka, J., Lindahl, T., and Klein, G. (1978). Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines. J . Virol. 27, 604-6 I I. Mao, J. C.-H., Robishaw, E. E., and Overby, L . R. (1975). Inhibition of DNA polymerase from herpes simplex virus-infected Wi-38 cells by phosphonoacetic acid. J. Virol. 15, I28 1- 1283. Matsuda, H . , Ikuta. K., and Kato, S. (1976). Detection of T-cell surface determinants in three Marek‘s disease lymphoblastoid cell lines. Biken J. 19, 29-32. Matsuda, H.. Ikuta, K., Miyamoto, H.,and Kato, S. (1976). Demonstration of a Marek’s disease tumor-associated surface antigen (MATSA) on six cell lines derived from Marek’s disease lymphomas. Bikrn J. 19, 119-123. Matsuo, T., Nishi, S., Hirai. H.,and Osato, T. (1977). Studies on Epstein-Barr virusrelated antigens. 11. Biochemical properties of soluble antigen in Raji Burkitt lymphoma cells. Int. J. Cancer 19, 364-370. Melendez, L. V.. Daniel, M. D., Hunt, R. D., and Garcia, F. G. (1968). An apparently new herpesvirus from primary kidney cultures of the squirrel monkey. Lab. Aiiiin. Care 18, 374-38 I . Melendez, L. V., Hunt, R. D., King, N. W., Barahona, H.H., Daniel, M. D.. Fraser, C. E. 0..and Garcia. F. G. (1972).Herpesvirm atrles, a new lymphoma virus of monkeys. Nature (LOnd(J11)N f w Biol. 235, 182- 184. Miller, G. (1974). The oncogenicity of Epstein-Barr virus. J . Infkct. Dis. 130, 187-205. Miller, G., and Lipman, M. (1973). Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natl. Acad. Sci. U.S.A. 70, 190-194. Miller, G., Shope. T., Coope, D., Waters, L., Pagano. J . , Bornkamm, G. W., and Henle, W. ( 1977a). Lymphoma in cotton-top marmosets after inoculation with Epstein-Barr virus: Tumor incidence, histologic spectrum antibody responses, demonstration of viral DNA, and characterization of viruses. J . Exp. Med. 145, 948-967. Miller. R. L., Glaser, R., and Rapp, F. (1977b). Studies of an Epstein-Barr virus-induced DNA polymerase. Virology 76, 494-502. Mizuno, F..Aya, T., and Osato, T . (1974). B-Lymphocytes as target cells for E.B. virus transformation. Br. Med. J. 3, 689. Mizuno, F.. Aya, T., and Osato, T . (1976). Growth in semisolid agar medium of human


275

cord leukocytes freshly transformed by Epstein-Barr virus. J . Nail. Cancer Inst. 56, I7 1- 173. Moore, G . E., Gerner, R. E., and Franklin, H. A. (1967). Culture of normal human leukocytes. J . Am. Med. Assoc. 199, 519-524. Moss, D. J . , and Pope, J . H. (1972). Assay of the infectivity of Epstein-Barr virus by transformation of human leucocytes in Vi/ro.J . Cen. Virol. 17, 233-236. Nazerian. K. (1973). Marek’s disease: A neoplastic disease of chickens caused by a herpesvirus. A h * . Cancer Res. 17, 279-3 15. Nazerian, K., and Lee, L. F. (1974). Deoxynbonucleic acid of Marek’s disease virus in a lymphoblastoid cell line from Marek‘s disease tumours. J . Cen. Virol. 25, 3 17-32 I . Nazerian, K., and Sharma, J . M. (19751. Detection of T-cell surface antigens in a Marek‘s disease lymphoblastoid cell line, J . Nail. Cancer Inst. 54, 277-279. Nazerian, K., and Witter, R. L. (1970). Cell-free transmission and in Vivo replication of Marek’s disease virus. J . Virol. 5, 388-397. Niederman, J. C., McCollum, R. W., Henle, G., and Henle, W. (1968). Infectious mononucleosis: Clinical manifestations in relation to EB virus antibodies. J . A m . Med. Assoc. 203, 205-209. Niederman, J . C . , Miller, G., Pearson, H. A., Pagano, J . S . , and Dowaliby, J. M. (1976). Infectious mononucleosis: Epstein-Barr- virus shedding in saliva and the oropharynx. N . Enyl. J . Med. 294, 1355-1359. Nilsson, K., Giovanella, B. C., Stehlin, J . S . , and Klein, G . (1977). Tumorigenicity of human hematopoietic cell lines in athymic nude mice. int. J . Cancer 19, 337-344. Nonoyama, M., and Pagano, J . S. (1972). Separation of Epstein-Barr virus DNA from large chromosomal DNA in non-virus-producing cells. Nature (Londun)New Biol. 238, 169- I7 I . Nonoyama, M., and Pagano, J . S. (1973). Homology between Epstein-Barr virus DNA and viral DNA from Burkitt’s lymphoma and nasopharyngeal carcinoma determined by DNA-DNA reassociation kinetics. Natirre (London)242, 44-47. Okazaki, W., Purchase, H. G . , and Burmester, B. R. (1970). Protection against Marek’s disease by vaccination with a herpes virus of turkeys. Avian Dis. 14, 413-429. Orellana, T., and Kieff, E. (1977). Epstein-Barr virus specific RNA. I I . Analysis of polyadenylated viral RNA in restringent, abortive, and productive infection. J . Virol. 22, 321-330. Otto, B.. and Fanning, E. (1978). DNA polymerase a is associated with replicating SV40 nucleoprotein complexes. Nucl. Acids Res. 5 , 1715-1728. Pattengale, P. K . , Smith, R. W., and Gerber, P. (1973). Selective transformation of B lymphocytes by E. B. virus. Lancet 2, 93-94. Payne, L. N., Powell, P. C . . and Rennie, M. (1974). Response of B and T lymphocytes and other blood leukocytes in chickens with Marek’s disease. Cold Spring Harbor Svmp. Qrtuni. Biol. 39, Pt. 2, 817-826. Pope, J . H., Horne, M. K., and Scott, W. (1968). Transformation offoetal human leukocyte in Vitro by filtrates of a human leukaemic cell line containing herpes-like virus. int. J . Cancer 3, 857-866. Powell, A. L. T., King, W., and Kieff, E. (1979). Epstein-Barr virus specific RNA. 111. Mapping of DNA encoding viral RNA in restringent infection. Submitted for publication. Pritchett, R. Hayward, S. D., and Kieff, E. (1975). DNA of Epstein-Barr virus. I . Comparison of DNA of virus purified from HR-I and B95-8 cells. J . Virol. 15, 556-569. Purchase, H. G . , Burmester, B. R . , and Cunningham, C. H. (1971). Pathogenicity and antigenicity of clones from strains of Marek’s disease virus and the herpesvirus of turkeys. infect. Immiin. 3, 295-303.

276

BILL SUGDEN ET AL.

Purtilo, D. T., Hutt, L., Bhawan, J., Yang, J . P. S., Cassel, C., Allegra, S., and Rosen, F. S. (1978). Immunodeficiency to the Epstein-Barr virus in the X-linked recessive lymphoproliferative syndrome. Clin. Immuttol. Jmtnunopathol. 9, 147- 156. Rabin, H., Neubauer. R. H., Hopkins, R. F., Dzhikidze, E. K., Shevtsova, Z. V., and Lapin, B. A. ( 19771. Transforming activity and antigenicity of an Epstein-Barr-like virus from lymphoblastoid cell lines of baboons with lymphoid disease. Intcwirology 8, 240-249. Rabin, H . , Neubauer, R. H., Hopkins, R. F., and Nonoyama, M. (1978). Further characterization of a herpesvirus-positive orang-utan cell line and comparative aspects of in Vitro transformation with lymphotropic old world primate herpesviruses. Int. J. Cunccr 21, 762-767. Rasheed, S., Rongey, R. W., Bruszweski, J., Nelson-Rees, W. A., Rabin, H., Neubauer, R. H., Esra, G., and Gardner, M. B. (1977). Establishment of a cell line with associated Epstein-Barr-like virus from a leukemic orangutan. Scirncr 198, 407-409. Reedman. B. M., and Klein, G. (1973). Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. I n t . J . Cancer 11, 499-520. Rickinson, A. B., Jarvis, J . E., Crawford, D. H., and Epstein, M. A. (1975). Observations on the nature of Epstein-Barr virus infection of peripheral lymphoid cells in infectious mononucleosis. I n “Oncogenesis and Herpesviruses I I ” (G. de-ThC, M. A. Epstein, and H. zur Hausen, eds.), Pt. 2, pp. 169-177. International Agency for Research on Cancer, Lyon. Rickinson, A. B., Finerty, S., and Epstein, M. A. (1977). Mechanism of the establishment of Epstein-Barr virus genome-containing lymphoid cell lines from infectious mononucleosis patients: Studies with phosphonoacetate. Int. J. Cancer 20, 861-868. Robinson, J . E., Andiman, W. A., Henderson, E., and Miller, G. (1977). Host-determined differences in expression of surface marker characteristics on human and simian lymphoblastoid cell lines transformed by Epstein-Barr virus. Proc. Natl. Accid. Sci. U . S . A . 74, 749-753. Ross, L. J . N., Biggs, P. M.. and Newton, A. A. (1973). Purification and properties of the ‘A’ antigen associated with Marek’s disease virus infections. J . Gcn. Virol. 18, 291304. Rowe, D. S., Hug, K., Forni, L., and Pernis, B. (1973). Immunoglobulin D a s a lymphocyte receptor. J. Exp. Med. 138, 965-972. Rymo, L., and Forsblom. S. (1978). Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRl, Hind111 and B a d . Nucl. Acids Res. 5 , 1387-1402. Sakamoto, K., and Hinuma, Y. (1978). Demonstration of a surface antigen on EpsteinBarr virus genome-carrying lymphoid cells: Distinction from the virus-determined membrane antigen. J . Immunol. 120, 1702-1708. Sambrook, J., Westphal, H., Srinivasin, P. R., and Dulbecco, R. (1968). The integrated state of viral DNA in SV40-transformed cells. Proc. Nut/. Acad. Sci. U.S.A. 60, 12881295. Sambrook, J., Botchan, M., Gallimore, P., Ozanne, B., Pettersson, U . , Williams, J . , and Sharp, P. A. (1974). Viral DNA sequences in cells transformed by simian virus 40, adenovirus type 2 and adenovirus type 5 . Cold Spring Harbor Symp. Quunt. Biol. 39, Pt. I , 615-632. Schulte-Holthausen, H., and zur Hausen, H. (1970). Parital purification of the EpsteinB a n virus and some properties of its DNA. Virology 40, 776779. Sheldrick, P., Laithier, M., Lando, D., and Ryhiner, M. L. (1973). Infectious DNA from herpes simplex virus: Infectivity of double-stranded and single-stranded molecules. Proc. Natl. Acad. Sci. U . S . A . 70, 3621-3625.


277

Shope, T., Dechairo, D., and Miller, G. (1973). Malignant lymphoma in cottontop marmosets after inoculation with Epstein-Barr virus. Proc. Natl. Acud. Sci. U . S . A . 70, 2487-249 I . Stockert, E., Old, L. J . , and Boyse, E. A. (1971). The GlXsystem../. Exp. M u d . 133, 13341355. Strauch, B., Siegal, N . , Andrews, L.-L., and Miller, G. (1974). Oropharyngeal excretion of Epstein-Barr virus by renal transplant recipients and other patients treated with immunosuppressive drugs. Luncet 1, 234-237. Sugden, B. (1977). Comparison of Epstein-Barr viral DNAs in Burkitt lymphoma biopsy cells and in cells clonally transformed in Vitro. Proc.. Nut/. Acud. Sci. U . S . A . 74, 465 1-4655. Sugden, B., and Mark, W. (1977). Clonal transformation of adult human leukocytes by Epstein-Barr virus. J. Virol. 23, 503-508. Sugden, B., Summers, W. C., and Klein, G. (1976). Nucleic acid renaturation and restriction endonuclease cleavage analyses show that the DNAs of a transforming and a nontransforming strain of Epstein-Barr virus share approximately 90% of their nucleotide sequence. J . Virol. 18, 765-775. Svedmyr, E., and Jondal, M. (1975). Cytotoxic effector cells specific for B cell lines transformed by Epstein-Barr virus are present in patients with infectious mononucleosis. Proc. Natl. Acud. Sci. U . S . A . 72, 1622-1626. Takada, K., and Osato, T. (1978). Analysis of the transformation of human leukocytes by Epstein-Barr virus: Sequential occurrence from the virus-determined nuclear antigen synthesis to blastogenesis to DNA synthesis. In press. Tanaka, A., and Nonoyama, M . (1974). Latent DNA of Epstein-Barr virus: Separation from high-molecular-weight cell DNA in a neutral glycerol gradient. Proc. Nut/. Acad. Sci. U . S . A . 71, 4658-4661. Tanaka, A., Silver, S., and Nonoyama, M . (1978). Biochemical evidence of the nonintegrated status of Marek’s disease virus DNA in virus-transformed lymphoblastoid cells of chicken. Virology 88, 19-24. Temin, H. (1971). The role of the DNA provirus in carcinogenesis by R N A tumor viruses. I n “The Biology of Oncogenic Viruses” (L. G. Silvestri, ed.), pp. 17h-187. NorthHolland Publ., Amsterdam. Thorley-Lawson, D. A., and Strominger, J . L. (1976). Transformation of human lymphocytes by Epstein-Barr virus is inhibited by phosphonoacetic acid. Nutrire (Londorr) 263, 332-334. Thorley-Lawson, D. A., Chess, L., and Strominger, J. L. (1977). Suppression of in Vitro Epstein-Barr virus infection. A new role for adult human T lymphocytes. J. Exp. Med. 146,495-508. Thorley-Lawson, D. A., and Strominger, J . (1978). Reversible inhibition by phosphonoacetic acid of Human B lymphoctye transformation by Epstein-Barr virus. Virology 86, 423-43 I . Tijan, R. (1977). The binding site on SV40 DNA for a T antigen related protein. Cell 13, 165- 179. Tooze, J. (ed.) (1973) “The Molecular Biology of Turnour Viruses.“ Cold Spring Harbor, New York. Traul, K. A., Stephens, R., Gerber, P., and Peterson, W. D. (1977). Productive EpsteinBarr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus. In/. J . Cancer 20, 247-255. Velicer, L. F., Yager, D. R., and Clark, J . L. (1978). Marek’s disease herpesviruses. 111. Purification and characterization of Marek’s disease herpesvirus B antigen. J. Virol. 27, 205-217.

278

BILL SUGDEN ET AL.

Veltri, R. W., Heyl, L. W., and Sprinkle, P. M. (1977). Epstein-Barr virus genome carrying lymphoctye subpopulations of human palatine tonsils. Proc. Soc. Exp. Biol. Med. 156, 282-286. Vitetta. E. S . , and Uhr. J. W. (1975). Immunoglobulin-receptors revisited: A model for the differentiation of bone marrow-derived lymphocytes is described. Science 189, 964969. von Biilow, V. (1971). Diagnosis and certain biological properties of the virus of Marek's disease. Am. J. Vet. Res. 32, 1275-1288. Wallen, W. C., Neubauer, R. H., Rabin, H., and Cicmanec, J. L. (1973). Nonimmune rosette formation by lymphoma and leukemia cells from herpesvirus saimiri-infected owl monkeys. J. Narl. Cancer I n s t . 51, 967-975. Werner, F.-J., Bornkamm, G. W., and Fleckenstein. B. (1977). Episomal viral DNA in a Irrrpes~~irus sainriri-transformed lymphoid cell line. J. Virol. 22, 794-803. Werner, F. J., Desrosiers, R. C., Mulder, C., Bornkamm, G. W., and Fleckenstein, B. ( 1978). Physical mapping of viral episomes in Herpesvirr4.v saimiri transformed lymphoid cells. UCLA Meeting on Persistemt Viruses, Keystone, Colorado (in press). Wising, P. J. (1942). Successful transmission of infectious mononucleosis to man by transfusion of heparinized blood'! Acta Med. Sccind. 109, 507-513. Witter. R . L. (1971). Marek's disease research-history and perspectives. Porrlt. Sci. 50, 333-342. Witter, R. L. (1972). Epidemiology of Marek's disease-A review. I n "Oncogenesis and Herpesviruses" (P. M . Biggs, G. de-The, and L. N . Payne, eds.), pp. I 11-122. International Agency for Research on Cancer, Lyon. Witter, R. L.. Solomon, J . J., and Burgoyne. G. H. (1969). Cell culture techniques for primary isolation of Marek's disease-associated herpesvirus. Airinn Dis. 13, 101- 118. Witter, R. L., Nazerian. K . , Purchase, H. G., and Burgoyne, G. H. (1970). Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Ain. J. Vet. R e s . 31, 525-538. Witter, R . L.. Stephens, E. A., Sharma, J. M., and Nazerian, K. (1975). Demonstration of a tumor-associated surface antigen in Marek's disease. J . Iinmunol. 115, 177-183. Yajima. Y.. Marczynska, B., and Nonoyama, M. (1978). Transforming activity of EpsteinBarr virus obtained by superinfection of Raji cells. Proc. Nut/. Acud. Sci. U.S.A. 75, 2008-20 10. Yamamoto, N., and Hinuma, Y. (1976). Clonal transformation of human leukocytes by Epstein-Barr virus in soft agar. f l i t . J. Cancer 17, 191-196. zur Hausen. H. (1975). Oncogenic herpes viruses. Biochitn. Biophvs. Actu 417, 25-53. zur Hausen, H.. and Fresen, K.-0. (1977). Heterogeneity of Epstein-Barr virus. 11. Induction of early antigens (EA) by complementation. Virology 81, 138-143. zur Hausen, H., and Schulte-Holthausen, H. (1970). Presence of EB virus nucleic acid homology in a "virus-free" line of Burkitt tumour cells. Natrrri, (London) 227, 245248. zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, W., Henle, G., Clifford, P., and Santesson, L. (1970). EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature (London) 228, 1056-1058. zur Hausen, H., Diehl, V., Wolf, H., Schulte-Holthausen, H., and Schneider, U. (1972). Occurrence of Epstein-Barr virus genomes in human lymphoblastoid cell lines. Natrrrc (London) N i w B i d . 237, 189- 190. zur Hausen, H.. O'Neill, F. J., Freese, U . K., and Hecker, E. (1978). Persisting oncogenic herpesvirus induced by the tumor promoter TPA. Nature (London) 272, 373-375.

ADVANCES I N CANCER RESEARCH. VOL. 30

VIRAL XENOGENIZATION OF INTACT TUMOR CELLS

Hiroshi Kobayashi Laboratory of Pathology, Cancer Institute. Hokkaido University School of Medicine, Sapporo, Japan

I. Introduction ...........................................................

279

.......... . . . . . . . . . . . . . . . . 280 A. Evidence for Acquisition of a New Antigen ............................ 280

11. Acquisition of a Virus-Specific Antigen

B. Acquisition of a Virus-Specific Antigen Resulting in the Immunological Regression of Tumor Cells ........................................... C. Definition of the Xenogenization of Tumor Cells . . . . .... 111. Increase in the Antigenicity of a Tumor-Specific Antigen . . . . . . . . . . . . . . . . . . . A. Increase in the Irnmunogenicity of Tumor Cells by Infection with a Lytic Virus . . . . . . . . . . .

280 286 287 287 290

C. Increase in th

A. Rejection of Xenogenized Tumor Cells and the Immune Response . . . . . . . B. The Mechanism Responsible for the Increase in the Antigenicity of Tumor V. Summary. . . . . . . . . . . . . . . . References . . . . . . .

................

................

290 29 I 292 292 293 295 297

I. Introduction

One approach to tumor immunotherapy is to modify tumor cells so that they become more foreign to the host. This review will discuss how investigators have attempted to make tumor cells foreign to the host by increasing their antigenicity. The review has been divided into two parts: the first part reviews attempts to induce new foreign proteins on tumor cells, and the second part reviews attempts to increase the antigenicity of existing tumor-specific or tumor-associated antigens (TSA or TAA) on tumor cells. By both of these methods tumor cells may be made more highly immunogenic or more easily immunosensitive to the immune responses of the host. 279 Copyright 0 1979 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN OI-I?-OOM30-0

280

HIROSH I KOBAYASHl

II. Acquisition of a Virus-Specific Antigen

A. EVIDENCE FOR

ACQUISITION OF A

NEWANTIGEN

Several investigators have described the acquisition of new antigens by tumor cells following infection with viruses. These virus-specific new antigens have been detected in mice and hamsters by use of the complement-dependent cytotoxicity assay (Stuck et af., 1964), transplantation resistance (Hamburg and Svet-Moldavsky, 1964; Sjogren and Hellstrom, 1965; Hosokawa et al., 1967; Eiselein and Biggs, 1970), or the immunofluorescence technique (Pasternak and Pasternak, 1967). Svet-Moldavsky and Hamburg (1964) performed experiments which showed that several virus-infected tumor cell lines were rejected by a host which had been immunized against the virus. Svet-Moldavsky labeled this phenomenon “heterogenization,” while Stuck et al. called it “antigenic conversion” and Sjogren and Hellstrom referred to it as the “acquisition of a virusspecific new antigen.” Many studies have since confirmed these first studies and these are summarized in Table I (A).

B. ACQUISITION OF A VIRUS-SPECIFIC ANTIGENRESULTINGI N THE IMMUNOLOGICALREGRESSION OF TUMOR CELLS[Table I (B)] If tumor cells are infected with a virus, they generally express the viral antigen on their cell surface. If the virus used to infect the tumor cells is foreign to the host, then the virus-infected tumor cells often undergo regression. Kobayashi et al. were successful in inducing the immunological regression of rat tumors which had been artificially infected with murine leukemia viruses, such as the Friend virus or the Gross virus (Kobayashi et al., 1969a,b, 1970; Sendo et al., 1970). Virus-infected transplanted tumors showed no tumor growth when inoculated into either a syngeneic or an autochthonous host. In autochthonous hosts, the tumors were surgically removed, artificially infected with the virus either in vivo or in vitro, and then reinoculated into the animal. The tumors occasionally showed some tumor growth, but eventually regressed [Tables I(B), 11; Figs. 1 , 2). It was observed that rat tumor metastases regressed if the tumor cells had been infected with murine leukemia virus (Kodama et al., 1974). The only exception to this rule of virus-induced regression was a transplantable leukemia and very rapidly growing carcinomas. In the carcinomas, it was felt that tumor proliferation was so rapid that it did not allow time for the effects of the immune response to


28 1

,,,-------t ,./'

15

20

25

DLT

30

35

Days after transplantation

FIG. I . Growth curve of Friend virus-infected and noninfected lung tumor (DLTI cells in Donryu rats.

occur, whereas with the leukemia there was initial regression of the injected cells but a subsequent relapse. If the host was first made tolerant through the neonatal injection of Friend or Gross virus, then the virus-infected tumor grew and eventually killed the tolerant host. The virus-infected tumor also grew progressively in a host which had been immunosuppressed by chemicals or irradiation. The explanation for regression of rat tumors infected with murine leukemia virus was that the tumors had acquired a highly antigenic virusspecific antigen (VSA or VAA). Since this antigen was recognized as foreign to the host, a strong immune response occurred against it, resulting in the death of the virus-infected tumor cells. The author called this phenomenon the "xenogenization" of tumor cells (Kobayashi et al., 1969a,b). The observation that tumors regress after they have been artificially infected with a virus has not been limited to the rat [Table I(B)]. Holtermann and Majde (1971) reported that the lethal growth of an adenocarcinoma in SWWJ mice was decreased 50% if the tumor cells were first infected with the LCM virus. Barbieri et al. (1971) reported that the lethal growth of mouse tumors was decreased by 46%-77% when the tumor cells were infected with Rauscher leukemia virus. Greenberger and Aaronson (1973) reported the regression of tumors which had originally been induced by Moloney sarcoma virus (MSV) (nonproducer) and also reported the regression of tumors which had been infected in tissue culture with the C-type helper virus. Similar results have been observed

ACQUISITION OF

A

TABLE I VIRUS-SPECIFIC NEW ANTIGENI N TUMOR CELLS

Animal

N

w

Species

Strain

(A) Inhibition of tumor growth after previous immunization: Mouse Stuck et a / . (1964)

Hamburg and Svet-Moldavsky (1964)

Hamster and mouse

Sjogren and Hellstrom

Mouse

Virus

Tumor

EL4

Rauscher

Herpes, polyoma, SV,, adeno Lymphoma

Polyoma

OMT-6

Friend virus

(1965)

Hosokawa ct al. (1967) Pasternak and Pasternak (1967) Eiselein and Biggs ( 1970)

Mouse Mouse Mouse

ddiOm

Note Antigenic conversion (cytotoxicity test) Heterogenization (transplantation) Acquisition of a virus-specific new antigen Fluorescence

Ehrlich

LCM

Regression of tumors without previous immunization: Kobayashi ef a / . (1969) Rat WKA, Donryu

KMT-68, WST-5 (sarc.), Takeda (sarc.), DLT (sq. carc.), AH-I09 MCA primary tumor Adenocarcinoma (spont.) P4bic (sarc.), TBLC2 ( MCA-sarc.) SSI (sarc.) G M*

Friend

Sendo et a / . (1970) Holtermann and Majde (1971) Barbieri ef af. (1971)

Rat Mouse

WKA SWWJ

Mouse

C57BL

Basombrio (1972) Yamada and Hatano ( 1972) Greenberger and Aaronson (1973) Kuzumaki and Kobayashi ( 1976)

Mouse Hamster

BALBIc

Mouse

NIH, Swiss BALB/c

MSV tumor

Mouse

C3HlHe

Kuzumaki et a / . (1978)

Rat

BDIX

Takeichi et a / . (1978) Takeyama ef al. (1978) Ishimoto (1978) Reed (1978) Kodama (1978)

Mouse Mouse Hamster Mouse Rat

Wi starIFu BALBIc BDF, nude WKA

MH134 (hepatoma), RaLV, MSV, Human Meth A (MCA-sarc.) measles virus, LLFV MBDB (fibrosarc.), 290-T (neurogenic) Endogenous mouse PW 41 (sarc.) E4 (sarc.) MSV L1210 (lymphoma) HVJ-pi THK Friend BHK, HeLa Mumps DEAE-D + Friend KMT-17

Xenogenization (transplantation, cytotoxicity test, etc.)

LCM Rauscher MSV HVJ, rubella RNA C-type

Regression of growing tumor

284

HIROSHI KOBAYASHI

FIG. 2. (a) Friend virus-infected KMT-17 rat tumor tissue 4 days after inoculation showing an ordinary pattern of KMT-17 tumor cells ( ~ 1 5 0 ) . (b) 7 days after inoculation. Note degenerative changes of tumor cells and infiltration of fibroblasts accompanied by lymphoid cells ( X 100).(c) 10 days after inoculation. Tumor cells have completely disappeared ( x 150).


285

TABLE I1 LATERAL GROWTHOF VARIOUSRAT TUMORS ARTIFICIALLY INFECTED W I T H FRIEND VIRUS I N SYNGENEIC RATS Tumors infected with virus Spontaneous sarcoma WST-5 Takeda KST- I MCA sarcoma KMT-17 KMT-I9 KMT-50 K MT-68 AMC-60 4NQ0 lung cancer DLT N B U breast cancer KBT- I KBT-2 DAB hepatoma AH I09 KDH-8 NBU leukemia KNL- 1 KNL-2 Total

Lethal growth (96) 0126 6123 0125 01443 0180 0/17

0/24 0110

0128

0112 018

218 0158

(25.0) (0)

0115

014 8/781

Tumors recurred 2-3 months after the regression in all the cases.

by Basombrio (1972), Kuzumaki and Kobayashi (1976), and Takeichi ef al. (1978a) by infecting several mouse tumors with MSV. Ishimoto (personal communication) demonstrated that the hamster tumor, THK, regressed after infection of the tumor with Friend virus. The xenogenization phenomenon, therefore, has been observed not only in rats but also in mice and hamsters. One could generalize and say that the immunological regression of virus-infected tumor cells might be observed in many species. Two conditions must be met if the virus-infected tumor cells are to undergo regression. The first is that the virus must be a nonlytic surfacebudding virus, and the second is that the host must be capable of mounting an immune response against the virus-associated new antigen. It would be desirable to use a nononcogenic virus to produce the immunological regression of tumors. There have been no reports of neoplasms produced by the murine oncornavirus in adult xenogeneic o r allogeneic hosts, but it must still be considered theoretically possible that

286

HI ROSH I KOBAY ASH1

oncornavirus could produce neoplasms in the normal adult host. A few recent reports have described the regression of tumor cells after infection with a nononcogenic virus. Holtermann and Majde (1971) have reported that infection with the LCM virus caused regression of tumors, but that the virus occasionally caused lethal inflammatory changes in the brain of the host. Yamada and Hatano (1972) used nononcogenic viruses in the hamster model system. They showed a decrease in the lethal growth of tumors following infection with the HVJ virus and the regression of tumors if they were infected with the rubella virus. Takeyama et ul. (1978) infected L1210 leukemia cells with HVJ-pi virus and showed that tumors regressed in about one-half of inoculated BDF, mice. Kuzumaki et al. (1978) observed that rat tumor cells regressed when artificially infected with an endogenous mouse virus obtained from nude mice. M. L. Reed (personal communication) infected HeLa cells with the mumps virus and transplanted them into nude mice. The mumps virus-infected viable cells grew only if the nude mice had been X-irradiated. The possible use of xenogenization of human tumor cells has great potential clinical application for cancer treatment. Murine leukemia virus has been shown to be infectious for human neoplastic cells, This was done using the ferritin conjugate technique (Wright and Korol, 1969: Kodama rt ul., 1970). It is interesting to speculate whether it would be possible to xenogenize or virally infect an existing tumor, so that regression would occur without its surgical removal. For this to succeed, the virus would have to infect the growing tumor cells rapidly and efficiently since it is expected that the virus would be quickly eliminated by the host immune response. Kodama ( 1978) has observed that the intralesional inoculation of DEAED before direct infection with a virus produces a higher rate of regression of primary tumors. It is also interesting to speculate whether certain chemicals might in some way modify tumor antigenicity. If this were possible, then such chemicals could extend the concept of xenogenization. N o report has described the regression of inoculated tumors first modified in vitro by exposure to chemicals. Even if a foreign antigen were produced on a tumor cell following exposure to a chemical, the antigen would most likely be eliminated after a number of tumor cell divisions.

c. DEFINITION OF THE XENOGENIZATION OF TUMOR CELLS The word "xenogenization" was used in 1969 to define the immunological regression of tumors after infection of tumor cells with a nonlytic budding virus (Kobayashi et ul., 1969b, 1970, 1977: Sendo et al., 1970).


287

Xenogenization more recently has been widely used to designate an acquisition of foreign antigen regardless of the regression of tumors, as well as to designate an increase in the antigenicity of existing tumorspecific antigens as is described in Section 111. The term xenogenization, therefore, has become a general term used to describe any foreignness that tumor cells have acquired due to either a n acquisition of a new foreign antigen or to an increase in the antigenicity of the existing tumorspecific antigen. Xenogenization in its broadest sense may be very similar to SvetMoldavsky’s term heterogenization (Svet-Moldavsky and Hamburg, 1964, 1967). The main aim of the heterogenization experiments was to produce a new antigen, whereas xenogenization has attempted to increase the antigenicity of the already existing tumor-specific antigens in addition to the acquisition of a new antigen. Both processes, either the production of a new antigen or the increase in the antigenicity of weak tumor-specific antigens, should facilitate the recognition of the tumor as foreign to the host. The term heterogenization could be applied to the regression of tumors secondary to inoculation with BCG and other microorganisms. In this case, the growth of tumor cells may be interfered with by a reaction to the microorganisms which occurs in the stroma of the tumor tissues (Svet-Moldavsky, 1978). The definition of xenogenization may then be limited to a phenomenon that occurs on the surface of the tumor cells, and does not include the suppression of tumor growth caused by the inoculation and mixture of microorganisms.

Ill. Increase in the Antigenicity of a Tumor-Specific Antigen

A. INCREASE I N THE IMMUNOGENICITY OF TUMOR CELLSBY INFECTION WITH A LYTICVIRUS[Table III(A)] Lindenmann and Klein (1967), and Lindenmann (1974) showed that the immunogenicity of tumor cells increased after virus infection. They immunized mice with either a lysate obtained from influenza-infected Ehrlich tumor cells (oncolysate) or a lysate from noninfected Ehrlich tumor cells. They observed an increased level of resistance to rechallenge in the animals immunized with the oncolysate. This was confirmed by Eaton et al. (1967, 1973) using Newcastle virus-infected lymphoma cells in mice. Boone et al. (1972a,b, 1974) infected SV40 transformed BALB/3T3 cells (E4 cells) with influenza virus and demonstrated a higher immunogenicity of homogenates from virus-infected cells as compared to homogenates from noninfected cells.

INCREASE IN

THE

TABLE 111 ANTIGENICITY OF TUMOR-SPECIFIC ANTIGENBY INFECTIONWITH

A

VIRUS

Animal Species N W 00

Strain

( A ) Increase in the immunogenicity of tumor cells by infection with a lytic virus Lindenmann and Klein Mouse A2G (1%7)

Tumor

Ehrlich

Eaton et a/. ( 1967)

Mouse

C3H

Lymphoma

Boone and Blackman (1972) Beverley et a / . ( 1973) Takeichi et a / . ( 1978)

Mouse

BALB/c

SV3T3-T4

Mouse Mouse

CBA. BALBIc BALB/c

337 E4

Materials and methods

Note

Influenza A virus (oncoly- Immunization with sate) oncolysate or crude membrane Newcastle disease virus (oncolysate) Influenza A virus (homogenate) Newcastle disease virus MSV, influenza (crude membrane)

(B) Increase in the immunogenicity of tumor cells by infection with a nonlytic budding virus: Kobayashi et a / . (1970) Rat WKA Donryu

WST, KMT DLT

Friend virus

Immunization with viable xenogenized tumor cells

Takeichi rt a / . (1978) Kuzumaki ef al. (1978) Takeyama ef a/. ( 1978) Katoh et a / . ( 1978)

Mouse Rat Mouse Rat

BALBIc BDIX BDF, WKA

E4 MBDB, 290-T L1210 KMT-17

MSV Endogenous mouse virus HVJ-pi Friend virus (inactivated by MMC, glutaraldehyde, and formalin)

Increase in the immunosensitivity of tumor cells by infection with a nonlytic budding virus: Rat Donryu Shirai r f a / . (1971)

DLT

Friend virus

Gotohda ct a/. ( 1978)

Rat

WKA

KMT-17

Moriuchi r f ctl. ( 1978)

Rat

WKA

KMT-I7

Hosokawa ef a / . (1978)

Rat

WKA

KMT-17

Takeichi rt a / . (1978)

Mouse

BALBlc

E4

Friend virus (lateral fluidity) Friend virus (antigenic modulation of TSA) Friend virus (51Cr release assay) MSV

Complement-dependent cytotoxicity test

Cell-mediated immunity

290

HlROSHl KOBAYASHI

The difference in the immunogenicity of oncolysates as compared to tumor lysates has been quite small. Most likely this has been due to the fact that homogenates of tumor cells frequently result in a destruction of the membrane-bound tumor-specific antigen with a major loss in the immunogenicity of such preparations. There have been several reports on the clinical application of oncolysates in combination with chemotherapy (Sauter et ul., 1972: Murray er al., 1977: Sinkovics et al., 1977: Wallack and Steplewski, 1977), but results do not as yet seem satisfactory.

B.

INCREASE I N THE IMMUNOGENICITYOF TUMORCELLSBY INFECTION W I T H A NONLYTIC BUDDING VIRUS[Table III(B)]

Kobayashi et a / . (1969a) have studied the immunogenicity of virusinfected tumor cells. The immunogenicity of virus-infected and nonvirusinfected cells was compared after the tumor cells had been killed by exposure to various chemicals. Katoh rt al. (1978) used mitomycin C , glutaraldehyde, and formalin to treat tumor cells and showed that there was a slightly higher immunogenicity with virus-infected tumor cells. Kuzumaki et a / . (1978) treated virus-infected rat tumor cells with irradiation and showed that these cells were more antigenic than nonvirusinfected tumor cells. A characteristic of xenogenized tumor cells defined by Kobayashi et al. is that viable tumor cells can be used to immunize the host as the tumor cells grow and regress. Following the process of regression of tumor cells, a strong specific immunity is produced. LTD,, when immunized with viable xenogenized tumor cells was approximately 1000 times higher than when not immunized. LTD,, when immunized with viable xenogenized tumor cells is further enhanced from 1000 to 1800 times by the previous administration of cyclophosphamide (Kobayashi et al., unpublished data). Attempts of tumor immunotherapy with viable xenogenized tumor cells are described in Section I I 1 , D .

c . INCREASE IN THE IMMUNOSENSITIVITY OF TUMOR CELLS BY BUDDINGVIRUS[Table III(C)] INFECTION WITH A NONLYTIC

Many studies have been performed to attempt to increase the immunogenicity of tumor cells. There have been a few reports that have described the immunosensitivity of tumor cells infected with a nonlytic virus. Shirai r t a / . (1971) first reported an increase in the immunosensitivity of tumor cells infected with a nonlytic virus. They reported that


29 I

nonlytic budding virus-infected tumor cells had a higher sensitivity to antitumor immune cells than noninfected tumor cells using the complement-dependent cytotoxicity assay. Gotohda er al. (1978) and Moriuchi et al. (1978) investigated the lateral mobility of tumor-surface antigens as it is related to the immunosensitivity of tumor cells. Virus-infected tumor cells have been reported to have a higher immunosensitivity to antitumor lymphocytes. Hosokawa er al. ( 1978) demonstrated that Friend virus- or Gross virus-infected rat tumor cells had an increased level of cytotoxicity compared to noninfected tumor cells when incubated with T lymphocytes obtained from tumor-bearing rats. Takeichi er al. (1978b) made similar observations using MSV-infected E4 tumor cells. It can be suggested that virus-infected tumor cells may be a valuable aid in the detection of weaker tumor antigens by using either the cellular or humoral assay system.

D. APPLICABILITY OF VIABLE XENOGENIZED TUMORCELLSI N THE TREATMENT OF CANCER Xenogenization as defined by Kobayashi et al. includes two phenomena; the first is that tumor cells infected with a nonlytic budding virus undergo regression which is immunologically mediated, and the second is that virus-infected cells when compared to noninfected cells are capable of inducing an increased antitumor immune response. Xenogenized tumor cells, because they are good immunogens, may be useful in im-

FIG.3. Ferritin labeling on the membrane surface of a Friend virus-infected KMT-17 cell treated with an antivirus-specific antigen (VSA) or VAA serum absorbed with purified plasma viruses. Mature type-C virus particles are not labeled (x50,OoO).

292

HlROSHl KOBAYASHI

munotherapy. Xenogenized tumor cells have been used in animal immunotherapy experiments (Kaji et a / . , 1969; Kobayashi et al., 1970) but with limited effects. Immunization had to be started in the earliest stage of tumor growth when there was a minimal tumor burden present. Studies in a rat tumor model showed that surgical removal of a tumor followed by immunotherapy with viable xenogenized tumor cells resulted in a higher survival rate when compared to animals having had only surgical or imrnunotherapy treatment (Kobayashi et al., 1975). A combination of chemotherapy and immunotherapy with viable xenogenized tumor cells was effective in causing the tumor growth inhibition of a slow growing tumor (Hosokawa et ul., 1971). When a rapidly growing tumor was studied, most rats died of tumors when immunotherapy or chemotherapy alone was performed, while the combination of these treatments resulted in a definite increase in the antitumor effect. Gotohda et al. (1974) performed studies with a rapidly growing rat tumor, and showed that active immunization with xenogenized tumor cells and the passive transfer of lymphocytes obtained from rats immunized with xenogenized tumor cells used either with or without chemotherapy resulted in an increased survival rate. Xenogenized tumor cells could be used for human immunotherapy experiments. It would be desirable to use viable xenogenized tumor cells as they are a more efffective immunogen than killed xenogenized tumor cells. There is, however, the remote possibility that viable xenogenized tumor cells might grow in immunologically compromised tumor patients, and therefore, much safer irradiated xenogenized tumor cells should first be used to immunize such patients. IV. Immune Responses against Xenogenized Tumor Cells

A. REJECTION OF

XENOGENIZED

TUMOR CELLS

A N D THE IMMUNE

RESPONSE The rejection mechanism responsible for the death of viable xenogenized tumor cells has not been completely investigated. Host reaction to the foreign virus-specific antigen (VSA) present on the surface of the tumor cells may be the most likely explanation as to why xenogenized tumor cells are killed. Although the host may become immunized against the weak tumor-specific or tumor-associated antigens, it is unclear how much this response contributes to the rejection of the tumor. Kobayashi et al. have demonstrated that xenogenized tumor cells can be inhibited by the humoral immune response. Xenogenized tumor cells were placed in a three-layer diffusion chamber and then inserted into the abdominal


293

cavity of rats. As the animals became immunized, the number of xenogenized tumor cells in the chamber gradually decreased and eventually all of the tumor cells disappeared. Repeated transfer of immune serum also retarded the growth of xenogenized tumor cells grown in immunologically tolerant rats (Itaya et af.,unpublished data). These experiments indicated that the humoral immune response was responsible for the rejection of xenogenized tumor cells. However, the cellular immune response may also play a very important role in rejection of xenogenized tumor cells. Hosokawa et al. (1978) showed with in vitro studies that xenogenized tumor cells were rejected by lymphocytes from the tumor-bearing host. Experiments by Hosokawa et al. showed that the growth of xenogenized tumor cells was inhibited in irradiated rats which had received an intravenous transfer with normal spleen cells, but not in animals transferred with normal thymus o r bone marrow. However, it has not yet been determined what types o r subsets of immune-competent cells play a major role in inhibiting xenogenized tumor cells. It is of interest to note that repeated immunization with viable xenogenized tumor cells has not produced as strong a level of immunity as has the surgical excision of a tumor mass with subsequent repeated immunization with viable nonxenogenized tumor cells. From this data, one can conclude that there are many unknown factors that can influence the development of tumor immunity following immunization with viable xenogenized tumor cells. Some preliminary studies by Kobayashi et al. have shown that suppressor cells may be produced during the regression of viable xenogenized tumor cells and this may be one explanation for the above phenomenon. Further studies to elucidate what might be the most effective means of immunization with xenogenized tumor cells and what mechanisms are responsible for such immunization are necessary.

B. THEMECHANISMRESPONSIBLE FOR THE INCREASE IN THE ANTIGENICITY OF TUMOR CELLSAFTERINFECTIONWITH A NONLYTIC BUDDING VIRUS The ability to produce immunity against VSA is necessary to produce regression of xenogenized tumor cells and to indirectly develop immunity against the TSA in the normal host. Rats immunized with xenogenized tumor cells develop both TSA and VSA immunity, and indeed the level of tumor-specific immunity has been high in animals immunized with xenogenized tumor cells. The mechanism responsible for the increased tumor-specific immunogenicity of virus-infected tumor cells is not known

294

HlROSHl KOBAYASHI

(Boone et al., 1974, 1978: Lindenmann, 1978; Bromberg et af., 1978: Hamaoka et af., 1978; Lachmann and Sikora, 1978). There are several possible explanations for this phenomenon. The first is that there is a quantitative and qualitative change of the tumor-specific antigen. Absorption experiments performed using tumor-specific antiserum have not been able to detect a quantitative change in the amount of tumor-specific antigen present on xenogenized or nonxenogenized tumor cells. The possibility that there is a qualitative change of the tumor-specific antigen has not been studied. The second possibility is that there is an adjuvant effect of xenogenized tumor cells which results in an increase in the immune response. There are reports that have described the adjuvant activity of viruses, but none of these experiments has shown an increased transplantation resistance following viral xenogenization (Kobayashi ef al., unpublished data). The third possibility is the "hapten carrier effects" of the tumor-specific antigen conjugated with a carrier antigen (Hamaoka et al., 1978; Lachmann and Sikora, 1978). The virus-specific new antigen present on xenogenized tumor cells may stimulate the immune response against the haptenic tumor-specific antigen. There are several reports that have described the hapten carrier effect, but this hypothesis has not always been considered (Beverley ef al., 1973: Evermann and Burnstein, 1973, particularly in the viral xenogenization experiment (Kobayashi et d . , unpublished data). Associative recognition of virus-specific and tumor-specific antigens might be in some way responsible for the stronger immunogenicity produced by xenogenized tumor cells (Boone et d . , 1978: Lindenmann, 1978; Bromberg ef af., 1978). In other words, virus-specific antigen may play a role as a helper determinant. The final possible explanation as to why xenogenized tumor cells are more antigenic, particularly more immunosensitive, may be that changes in the mobility of the tumor cell surface occur in xenogenized tumor cells. Tumor-specific antigens may be able to move more easily and may actually cluster if the tumor cells have first been infected with a virus. That clustering of tumor-specific antigen occurs on xenogenized tumor cells has been shown by the complement-dependent cytotoxicity assay, using monospecific syngeneic antiserum to the tumor-specific antigen (Moriuchi et al., 1978). Immunoelectron microscopic examination of xenogenized tumor cells using ferritin-labeled tumor-specific antibody also has shown a clustering of tumor antigenic determinants on xenogenized tumor cells as compared to nonvirus-infected tumor cells (Figs. 4, 5 ) . It is provable that the clustering of the tumor antigens may be responsible for the increase in the immunosensitivity of tumor-specific antigens on xenogenized tumor cells to antitumor lymphocytes and tumor-specific


295

antiserum. Further studies are needed to define more precisely why there is an increased cell-mediated cytotoxicity of virus-infected tumor cells.

V. Summary

Tumor cells have been shown to be of low antigenicity and as such are not readily recognized as foreign by the host. This review has summarized studies that have explored how the antigenicity of tumor cells can be

FIG.4. (a) TSA on the surface of a KMT-17 cell distributed sparsely ( ~ 6 0 , 0 0 0 )(b) . TSA (arrow) on the surface of a Friend virus-infected KMT-17 cell easily clustered ( ~ 6 0 , 0 0 0 ) .

296

HIROSHI KOBAYASHI

tumor cell infected with Friend virus

tumor cell

FIG.5 . Clustering of TSA with antibody in Friend virus-infected KMT-17 tumor cells.

increased by biological modification. By the process of xenogenization of tumor cells infected with a nonlytic budding murine virus (Friend or Gross virus), the author has been able to cause the regression of tumors when inoculated into normal hosts owing to an acquisition of virus-specific antigen that is foreign in the host. Concurrently, the inoculated animals developed a level of tumor-specific immunity that was higher than that observed when immunization was done with X-irradiated nonxenogenized tumor cells. Both in vitro and in vivo studies provided additional evidence that the tumor-specific antigenicity of viral infected tumor cells was increased (Table IV). It is hoped that in the future such modified tumor cells will prove to be more effective in both immunotherapy and in the immunodiagnosis of tumors. The primary focus of this review has been to summarize studies performed on tumor cells that

TABLE IV OF XENOGENIZED TUMOR CELLS ANTIGENICITY Ordinary tumor cells Acquisition of a new antigen (VAN Regression (primary, metastasis) lmmunogenicity (TAA) with viable cells with inactivated cells Immunosensitivity (TAA) against humoral antibody against lymphocytes

Xenogenized tumor cells

None

Yes

None

Yes

Difficult to perform Low

Very strong Slightly increased

Low Low

Increased Increased


297

have been infected with a nonlytic budding virus (xenogenized tumor cells). Xenogenization was the general term that was used to describe the process of making a tumor cell antigenically foreign (Greek, x e n m foreign) to the host.

REFERENCES Barbieri, D., Belehradek, .. and Barski, G. (1971). t . J . Cancer 7 , 4-37 Basombrio, M. A. (1972). Proc. Am. Assoc. Cancer Res. 13, 74. Beverley, P. C. L., Lowenthal, R. M., and Tyrrell, D. A. J. (1973). Inr. J . Cancer 11,212223. Boone, C. W.. and Blackman, K. (1972a). Cancer Res. 32, 1018-1022. Boone, C. W., and Blackman, K. (1972b). Nail. Cancer Ins/. Monogr. 35, 301-307. Boone, C. W., Paranjpe, M., Orme, T., and Gillette, R. (1974). In?. J. Cuncer 13,543-551. Boone, C . W., Takeichi, N., Austin, F. C., Gotohda, E., Oikawa, T., and Gillitte, R. ( 1978). Gann Monogr. Cancer Res. 23 (in press). Bromberg, J . , Brenan, M., Clark, E. A., Lake, P., Mitchison, N. A., Nakashima, I., and Sainis. K. B. (1978). Gann Monogr. Cancer Res. 23 (in press). Eaton, M. D., Levinthal, J. D., and Scala, A. R. (1967). J . Natl. Cancer I n s t . 39, 10891097. Eaton, M. D., Heller, J . A., and Scala, A. R. (1973). Cancer Res. 33, 3293-3298. Eiselein, J., and Biggs, M . W. (1970). Cancer Res. 30, 1953-1957. Evermann, J. F., and Burnstein, T. (1975). Int. J. Cancer 16, 861-869. Gotohda, E., Sendo, F., Hosokawa, M., Kodarna, T., and Kobayashi, H. (1974). Cuncer Res. 34, 1947-1951. Gotohda, E., Sendo, F., Nakayarna, M., Hosokawa, M., Kawarnura, T., Kodama, T., and Kobayashi, H. (1975). J. Nut/. Cancer Inst. 55, 1079-1083. Gotohda, E., Moriuchi, T., Kawamura, T., Akiyama, J., Oikawa, T., Sendo, F., Hosokawa, M., Kodama, T., and Kobayashi, H. (1978) (accepted by J. Nut/. Cancer I n s t . ) . Greenberger, J. S., and Aaronson, S. A. (1973). J. N u / / . Cancer Inst. 51, 1935-1938. Harnaoka, T., Fujiwara, H., Tsuchida, T., Kinouchi, T., and Aoki. H . (1978). Gunti Monogr. Cancer Rvs. 23 (in press). Hamburg, V. P.. and Svet-Moldavsky, G. J . (1964). Natirre (London) 203, 772-773. Holterrnann, 0. A., and Majde, J. A. (1971). Transplantation 11, 20-29. Hosokawa, M., Kodama, T., Sendo, F., Takeichi, N., and Kobayashi, H . ( 1967).J. Cancer Immirnopathol. 3, 42-46. Hosokawa, M., Sendo, F., Gotohda, E., and Kobayashi, H. (1971). Cann 62, 57-60. Hosokawa, M., Kasai, M., Yamaguchi, H., and Kobayashi, H. (1978). Gunn Monogr. Cancer Res. 23 (in press). Ishimoto, A. Personal communication. Kaji, H., Sendo, F., Shirdi, T., Saito, H., Kodama, T., and Kobayashi, H. (1969). Mod. Med. 24, 1329-1333. Katoh, H., Ikeda, K., Minarni, A., Hosokawa, M., Kodama, T., and Kobayashi. H. (1978). ( i n preparation). Klein. G., and Klein, E. (1977). Proc. Narl. Acud. Sci. U.S.A. 74, 2121-2125. Kobayashi, H. (1970). In "Immunity and Tolerance in Oncogenesis" (L. Severi. ed.), pp. 637-659. 4th Perugia Quadr. Int. Conf. on Cancer, 1969, Perugia.

298

HIROSHI KOBAYASHI

Kobayashi, H., Sendo, F., Shirai, T..Kaji, H., Kodama, T., and Saito, H. (1969a).J . Nail. Cancer Inst. 42, 413-419. Kobayashi, H . , Kodama. T., Shirai, T., Kaji, H., Hosokawa, M., Sendo, F., Saito, H., and Takeichi, N . (1969b). Hohhaido J . Med. Sci. 44, 133-134. Kobayashi, H., Sendo, F., Kaji, H., Shirai, T., Saito, H., Takeichi, N . , Hosokawa, M., and Kodama, T. (1970). J . Nut/. Cancer 1 m t . 44, 11-19, Kobayashi, H., Gotohda, E.. Hosokawa, M., and Kodama, T . ( 1975). J . Nail. Cuncer I n s t . 54, 997-999.

Kobayashi, H., Kodama, T., and Gotohda, E. (1977). 111 "Xenogenization of Tumor Cells," Vol. 9. pp. 1-124, Hokkaido University Med. Libr. Ser. Hokkaido University School of Medicine, Sappporo. Kodama, T. (1978). Gunn Monogr. Cancer Rc,s. 23 (in press). Kodama, T., Kobayashi, H., Saito, H., Shirai, T., and Matsumiya, H. (1970). Gunn 61, 2 19-22 I . Kodama, T., Gotohda, E., Takeichi. N . , Kuzumaki, N . , and Kobayashi, H. (1974). J. Natl. Cancer I m t . 52, 931-939. Kodama, T., Katoh, H., Gotohda, E., Kobayashi, H., and Sendo, F. (1978). J . Nut/. Cancer I n s t . (brief communication; in press). Kuzumaki, N., and Kobayashi, H. (1976). Transp/antation 22, 545-550. Kuzumaki, N . , Fenyo, E. M., Giovanella. B. C., and Klein, G. (1978). 1nt. J . Cuncc,r 21, 62-66.

Lachmann, P. J., and Sikora, K. (1978). Native (London) 271, 463-464. Lindenmann, J. (1974). Biochim. Biopliys. Acra 335, 49-75. Lindenmann. J. (1978). Gann Monogr. Cancer R e s . 23 (in press). Lindenmann, J., and Klein, P. A. (1967). J. Exp. Med. 126,93-108. Moriuchi, T., Gotohda. E., Hosokawa, M., Kodama, T., and Kobayashi, H. (1978). J . Null. Cancer I n s t . 62, 579-583. Murray, D. R., Cassel, W. A., Torbin, A. H., Olkowski, Z. L., and Morre, M. E. (1977). Cancer 40, 680-686. Pasternak, G., and Pasternak, L. (1967). J. Narl. Cancer Inst. 38, 157-168. Reed, M. L. Personal communication. Sauter, C., Gerber, A., Lindenmann, J., and Martz, G. (1972). Schweiz. Med. Wochensclir. 102, 285-290.

Sendo. F., Kaji, H., Saito, H.,and Kobayashi, H. (1970). Gann 61, 223-226. Shirai, T., Kaji, H., Takeichi, N.. Sendo, F., Saito, H., Hosokawa, M.. and Kobayashi, H. (1971). J . Nut/. Cancer Inst. 46, 449-460. Sinkovics, J. G.. Plager, C.. McMurtrey, M. J., Romero, J. J., and Romsdahl, M. M. (1977). Proc. A m . Assoc. Cancer Rcs. 18, 86. Sjogren, H. O., and Hellstrom, I. (1965). Exp. Cell Res. 40, 208-212. Stuck, B., Old, L. J., and Boyse, E. A. (1964). Nature (London) 202, 1016-1018. Svet-Moldavsky, G. J. (1978). Gann Monogr. Cancer Res. 23 (in press). Svet-Moldavsky, G. J., and Hamburg, V. P. (1964). Nature (London) 202, 303-304. Svet-Moldavsky, G. J., and Hamburg, V. P. (1967). UICC Monogr. 2 (Specific Tumor Antigens), 323-327. Takeichi, N . , Boone, C. W., Holden, H. T., and Herberman, R. B. (1978a). Int. J . Cancer 21, 78-84.

Takeichi, N . , Austin, F. C . , Oikawa, T., and Boone, C. W. (1978b). Cancer Res 38,45804584.


299

Takeyama, H . , Kawashima, K . , Yamada, K . , and Ito, Y . (1978). (submitted for publication). Wallack, M. K . , and Steplewski, Z. (1977). Proc. A m . Assoc. Cancer Rrs. I%, 18. Wright, B. S., and Korol, W . (1969). Cancer Rrs. 29, 18861888. Yamada, T., and Hatano, M . (1972). Gann 63, 647-655.


ADVANCES IN CANCER RESEARCH, VOL. 30

VIRUS AUGMENTATION OF THE ANTIGENICITY OF TUMOR CELL EXTRACTS

Faye C. Austin a n d Charles W . Boone Cell Biology Section, Laboratory of Viral Carcinogenesls, National Cancer Institute. National Institutes of Health, Bethesda. Maryland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Virus Therapy of C a n c e r . . . . . . . . . . . . . A. Virus Therapy in Animals.. .......................................... B. Virus Therapy in Human Cancer Patients. 111. Augmented Immunogenicity of Virus-Infected .......... A. Animal Model Studies ............ B. Immunotherapy of Human Cancer: s ...................... C. Immunodiagnosis of Human Cancer with Virus-Augmented Skin Test Antigens: Clinical Trials ................................................ IV. Mechanisms of Virus Augmentation of TATA Activity . . . . A. Relationship of Viral Antigens to Host Cell Antigens . . B. Augmented Induction of the Primary Antitumor Immune Response C. Virus-Augmented Delayed Hypersensitivity Skin Tests . . . . . . . . . . . . . . . . . . V. Prospects for the Application of Virus-Augmented Tumor Antigens in Immunodiagnosis and Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Immunodiagnosis ...... B. Immunotherapy ................................................. VI. Summary.. . . . . . . . ..................................... References ............................................................

30 I 303 303 305 308 308 320 327 329 329 334 338 338 338 339 339 340

I. Introduction

It has been known for many decades that certain ascites tumors of mice can be made to disappear completely by inoculating them with a cytolytic virus such as influenza virus. More recently, it was noted that mice which survived such “viral oncolysis” therapy were remarkably immune to an additional challenge with viable tumor cells. Given the facts of viral oncolysis and “postoncolytic immunity,” Lindenmann and Klein (1967a) took the next logical step and showed that normal mice could be immunized against tumor graft challenge by inoculating them with influenza virus oncolysate (residual membranous debris remaining in the peritoneal cavity after destruction of ascites tumor by influenza virus) but not by inoculating them with the membranous debris of mechanically disrupted tumor cells. Lindenmann later (1974, 1977) applied 301 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved ISBN 0-12-006630-0

302

FAYE C. AUSTIN A N D CHARLES W . BOONE

the term “viral adjuvanticity” to this effect to emphasize that although immunization is performed by inoculating a combination of “tumor-associated transplantation antigen” (TATA) and virus, the improved immune response of interest is directed against the TATA alone. “Adjuvanticity” carries with it the connotation that the virus augmentation effect might occur by a mechanism akin to the way BCG or Freund’s adjuvant operates, and that a simple mixture of virus with tumor homogenate might be effective. This is not true, however. Only the membranes of tumor cells actively infected with the virus exhibit augmented immunogenicity. We therefore prefer to use the term “virus augmentation of TATA” for the phenomenon described by Lindenmann. To emphasize the fact that the virus antigen on the tumor cell membrane must be in the molecular vicinity of the TATA to enhance the immunogenicity of the latter, the term “vicinal adjuvanticity” is also useful. There are two quite different groups of investigators interested in the use of viruses to augment the immunogenicity of TATA. One group, whose interests form the subject of this review, bases its activity on the initial findings of L.indenmann (described above) and deals exclusively with tumor cell extracts or simple homogenates of virus-infected tumor cells. This group has as one of its major goals the purification of virusaugmented TATAs to high specific activity, thereby increasing their potential usefulness in immunodiagnosis (as skin test antigens) and in immunotherapy. The other group deals exclusively with viable proliferating tumor cells and seeks to improve their immunogenicity for use in immunotherapy by productively infecting them with a surface-budding noncytopathic virus, frequently a type-C retrovirus. The details of this approach are described by H. Kobayashi (1979) in another chapter in this volume. Kobayashi Pf NI. (1969) were apparently the first to demonstrate the virus augmentation of TATA on intact tumor cells. They inoculated rats with syngeneic methylcholanthrene (MCA)-induced tumor cells that had previously been infected with Friend leukemia virus. Whereas uninfected tumor cells grew to lethal size, the Friend virus-infected tumor cells produced tumors which enlarged for about 2 weeks and then regressed completely. The “postregression” rats were strongly immune to challenge with uninfected tumor cells. Here, as in immunization with membrane extracts of influenza virus-infected tumor cells, primary immunization with the combination of TATA and virus produced an augmented immune response to the TATA. Kobayashi introduced the term “artificial xenogenization” to describe this phenomenon. Unfortunately, his term is easily confused with one used earlier by Svet-Moldavsky and Hamburg (1964), ”artificial heterogenization,” which they used to describe the

VIRUS AUGMENTATION OF TUMOR CELL EXTRACTS

303

different concept of initiating a virus infection in a tumor as it is growing in the host, thereby inducing an immune response to the virus antigen in the tumor cell membrane which brings about the destruction of the tumor cell. Note that in this case the presence of a TATA is not required. A naturally occurring virus infection in a growing tumor, with its attendant alteration of the cell surface by insertion of a virus antigen, Svet-Moldavsky ( 1979) termed “natural heterogenization.” Kobayashi has described still another phenomenon using the term “natural xenogenization” (Kobayashi et al., 1977) which he defines in relation to the regression of murine leukemia virus-induced tumors in rats. This confusion of terms has been further compounded by a tendency of both SvetMoldavsky and Kobayashi, and others [including Boone (1972)] to use the words “heterogenization” and “xenogenization” in their literal sense, i.e., “to make different o r foreign.” Thus, there appear terms such as viral xenogenization (or heterogenization), chemical xenogenization (or heterogenization), xenogenization with enzymes, etc. We feel that eventually the term “xenogenization” in the general sense of “to make foreign” may take hold, but for the present and also to emphasize that this review concerns the enhanced immunogenicity of tumor cell extracts and not whole cells, we will continue to use the term “virus augmentation of TATA.” II. Virus Therapy of Cancer

Awareness of the phenomenon of virus augmentation of TATA immunogenicity evolved directly from attempts to treat tumors with cytopathic viruses. It is therefore germane to describe the development and current status of virus therapy of cancer in animal models and in humans.

A. VIRUSTHERAPYI N ANIMALS The oncolytic properties of several groups of viruses [e.g., arboviruses (togaviruses, rhabdoviruses, Bunyamwera viruses), myxoviruses, pox viruses (vaccinia)] have been demonstrated in various animal systems. Early experiments, which have been reviewed by Moore (1954, 1960), Southam (1960), and Lindenmann and Klein (1967b), demonstrated a lack of consistency in virus-tumor interactions: i.e., no tumors were uniformly susceptible to oncolysis by all viruses, and no viruses were uniformly oncolytic for all tumors. In early studies, Sharpless et al. (1950) noted that certain neurotropic

304

FAYE C . AUSTIN A N D CHARLES W . BOONE

arboviruses had an antagonistic effect on lymphoid tumors in chickens, with resulting tumor immunity. Koprowski et ( i f . (1957) studied the oncolytic effect of several arboviruses on 12 ascites tumors of mice and found that not all types of tumors supported virus growth and only certain types of viruses would thrive on malignant cells. However, neoplastic tissues showed enhanced susceptibility to virus infection. They noted that mice which had survived viral oncolysis were more immune to subsequent tumor graft challenge. Lindenmann (1963) reported that after influenza virus oncolysis of growing Ehrlich ascites (EA) tumors in mice, a strong postoncolytic immunity resulted. Oncolysis of EA tumors by vesicular stomatitis virus was also demonstrated (Lindenmann, 1970). Although the above studies used oncolytic viruses with marked neurotropism, Cassel and Garrett (1965, 1966) demonstrated that a strain of Newcastle disease virus (NDV) with minimal neurotropism could effect a cure of mouse ascites tumors which had reached up to 41% of their total possible development, with resultant tumor immunity. Studies of oncolysis with NDV, vaccinia, and influenza virus led Castle and Garrett ( 1967a,b) to postulate a relationship between viral neurotropism and oncolytic activity. Klein (1967) reported the induction of antibody-mediated immunity to transplantable tumors following oncolysis with reovirus, the mechanism of which is uncertain since reovirus is not a surface-budding virus and is not known to induce cell surface antigens in the infected cell. Interpretation of these experiments is difficult because they have not been repeated in a syngeneic system. Taylor ct uf. (1970, 1971) and Sedmak et ul. (1972) determined that bovine enterovirus caused a rapid regression of nonspecific transplanta. ble tumors in outbred mice. The mechanism of initial tumor cell destruction was due to direct viral cytolysis, while the destruction of any residual tumor was attributed to the development of postoncolytic immunity. The specificity of virus for tumor cells i n vilw was shown to correlate with cytopathic effect (CPE) observed in tumor cells infected in vitvo, which might allow for the in vitvo screening of various viruses to assess their oncolytic potential. In specificity studies, bovine enterovirus was shown to adsorb only to tumor cells of other species and to normal bovine cells. The authors postulated that this specificity of adsorption to the membranes of tumor cells might possibly be due to their increased negative charge. Rukavishnikova and Alekseyeva (1976) studied the use of a vaccine strain of influenza A virus to inhibit the growth of ascites tumor cells in outbred rats and inbred mice. They produced complete regression of 35% of tumors in outbred rats by daily inoculation of virus intraperitoneally


305

(i.p.1 and intramuscularly (im.) for 1 week. With a syngeneic mouse ascites lymphoma, however, inoculations of tumor with influenza virus resulted only in prolongation of the survival time of tumor-bearing mice. Recently, Eiselein et al. (1978) reported on the treatment of transplantable mouse mammary tumors, both in solid and ascites form, with an unidentified virus isolated from an ascites form of mammary tumor which had unexpectedly regressed without treatment. This viral agent was adapted to grow in tumor cells in v i m , and has been shown to cause oncolysis of ascites tumors when injected i.p., but not when injected intravenously (i.v.), i m . , or subcutaneously (s.c.). Injection of the virus into solid tumors did not produce the dramatic lysis observed with ascites tumors. However, the addition of cyclophosphamide (CTX) to the treatment schedule significantly improved the cure rate (14% cures with virus alone, 83% cures with virus plus CTX). The virus treatment was effective even with tumors containing several grams of tissue. The authors postulated that in order for this therapy to be effective against tumor metastases, it will be necessary to develop a way to deliver the virus to the metastatic lesions. Work is in progress to identify the virus.

B. VIRUSTHERAPY I N HUMANCANCER PATIENTS The wave of initial interest in treating human cancer with viruses that occurred between 1950 and 1967 has been reviewed by Moore (1954, 1960), Southam (1960), and Lindenmann and KIein (1967b). Many viruses, primarily arboviruses, were used to treat patients with advanced cancers who were not responsive to any established method of treatment. In general, clinical applications of virus therapy as presented in these early reviews did not produce significant improvement in the clinical course of the disease. Smith rt a / . (1956) reported on the use of adenovirus to treat carcinoma of the cervix, which resulted in sloughing of the tumor after viral oncolysis, but with no appreciable modification in the course of the disease. Suskind rt a / . (1957) studied the oncolytic effect of Coxsackie B virus, an enterovirus, on human cancer cells (HeLa and KB) which had been heterotransplanted into irradiated, cortisone-treated rats. Evidence indicated that the virus did not proliferate in tissue other than the tumor in this system and adaptation of the virus to the tumor tissue enhanced the oncolytic effect. According to Southam (1960), Egypt 101 strain of West Nile virus was the only virus among many tested which produced any convincing evidence of oncolytic effect in clinical trials. Cassel and Garrett (1965) attempted virus therapy of a cervical carcinoma with New-

306


castle disease virus, chosen because of its low degree of neurotropism. Inoculation of the virus into a carcinoma of the cervix resulted in extensive sloughing of the tumor and shrinkage of the lymph node metastases, with no evidence of the virus attacking nerve tissue. Webb et al. (1966) reported results of a clinical trial of virus therapy with Langat and Kyasanur Forest disease viruses. The patients in these studies had advanced disease and little effect was noted. Later, Webb and Gordon Smith (1970) discussed the possible beneficial effects of virus therapy in the treatment of human cancer. Examples of tumor-regressive effects of virus infections in cancer patients continue to be reported (Csatary, 1971; Pasquinucci, 1971: BIuming and Ziegler, 1971). The replication in vitro of surface-budding viruses in human cancer cells has been studied (Gerber et d.,1973a,b; Sauter P I a / . , 1973, 1975: Illiger et d., 1975). Sauter et al. (1972) treated a patient with acute myelogenous leukemia (AML) with avian influenza virus adapted to leukemia cells and reported that although the patient had a poor prognosis, a striking hematological improvement was produced. Clinical trials with virus-assisted immunotherapy are continuing. Lindenmann (l979a) and colleagues tested avian influenza virus, adapted in iyifro to the particular cell type involved, for its ability to replicate in i ~ h ~ o when injected into a large tumor mass. In one patient, the virus was inoculated into a carcinomatous pleural effusion, causing regression of the pleural effusion and palpable lymph nodes (Sauter and Lindenmann, 1976). I n another patient (C. Mayer, in preparation, cited in Lindenmann, 1979a) virus therapy appeared to have a positive effect on a pleural exudate and tumorous infiltrates around the neck. Although objective improvement was not long lasting, viral replication was demonstrated in \ t i i v , with effects on the tumor at both local and distant sites. In the U.S.S.R., Muceniece (1972, 1978) studied the suitability of human enteroviruses for viral therapy of tumors and established that about 70% of these viruses possessed oncotropic and oncolytic properties in heterotransplants of human angiosarcoma and lymphoma. Experiments in vitr’o confirmed that various human tumors adsorbed human enteroviruses. The spectrum of viruses adsorbed depended on the individual tumor. The oncotropic and oncolytic activities of human enteroviruses were then assessed in patients with advanced cancer. After i.m. injection, viral antigens and cytopathic changes typical of enteroviruses were detected in about 50% of excised tumor cells. No infectious virus could be isolated. Susceptibility to oncolysis was found to be dependent on tumor type. “Gastrointestinal tract cancer,” some melanomas, and certain sarcomas were found to be sensitive to oncolytic enteroviruses, while breast and lung cancers were insensitive. Viral oncolytic activities ceased one


307

week after therapy due to the induction of antiviral immunity. Muceniece (1978) pointed out that virus therapy can be used in conjunction with chemotherapeutic agents if the latter are used in subtherapeutic doses that are not immunosuppressive. In fact, it has been shown in animal studies that virus-infected tumor cells are more susceptible to chemotherapy (Eiselein and Biggs, 1970). Svet-Moldavsky (1974, 1979) reported negative results in attempts to treat patients having widely disseminated cancer with adenovirus therapy. Roenigk et al. (1974) reported positive results of immunotherapy of malignant melanoma with intralesional inoculation of vaccinia virus. Major regression of the tumor was observid in 8 out of 8 patients with stage I1 disease. In studies of patients with stage I11 disease, however, little or no regression occurred. Other studies of vaccinia virus therapy have demonstrated positive results, primarily in patients without extensive tumor involvement (Belisario and Milton, 1961; Milton and Brown, 1966; Hunter-Craig et ul., 1970). In Japan, Asada (1974) described the oncolytic effect of mumps virus on rat tumors. His results appeared to warrant performing mumps virus therapy in humans with advanced cancer. Of 90 patients with terminal cancer of various types, treatment was assessed as very good in 37 (tumor disappeared or decreased in size to less than half of initial size, subjective symptoms improved) and good in 42 (some suppression of tumor growth, subjective symptoms improved). Depending on the patient’s general condition and the type, size, and location of the tumor, mumps virus was applied either externally, locally, orally, rectally, intravenously, or by inhalation. Administration of virus produced few side effects. The initial antineoplastic effect of mumps virus therapy seemed to occur rapidly and strongly in proportion to the growth rate of the tumor, an observation possibly related to increased virus replication in rapidly growing cells (DeMarchi and Kaplan, 1977). The oncolytic effect of the virus came to an end in a short time because of the induction of antiviral immunity. However, there was little additional proliferation of the tumor cells for a long period of time, even if the remaining tumor was left untreated, a phenomenon probably related to postoncolytic immunity. The recent reports described above indicate that the use of surfacebudding viruses in virus therapy may possibly involve the host’s immune system in active immunization, resulting in postoncolytic immunity to residual tumor cells. An enhanced immunological response to the tumor antigen during the course of viral oncolysis would obviously be of great advantage during virus therapy, for the host’s immune system will soon interfere with continued oncolysis by the virus. There are many hazards involved in virus therapy, especially the possibility of viral pathogenicity

308


in an immunosuppressed patient. The selection of a virus is critical, for a successful agent must be oncotropic, oncolytic, and nonpathogenic. Preimmunization of the patient with inactivated virus would diminish any possibility of viremia (Lindenmann, 1963). The success of potential virus therapy will also depend on careful attention to dose, route, and timing of inoculations: the effect of preimmunization with virus: and coordination with other types of therapy. Ill. Augmented lmmunogenicity of Virus-Infected Tumor Cell Extracts

A. ANIMALMODEL STUDIES

I . Criidc Mrmbranr Extracts The first extended experiments to evaluate postoncolytic immunity were performed by Lindenmann and Klein (1967a). When the WSAIAo strain of influenza virus was inoculated i.p. into EA tumors in A2G mice, which are genetically resistant to infection by the virus, the ascites collapsed within 48-72 hours and an agglutinated membranous mass of lysed tumor cells, the oncolysate, was found in the peritoneal cavity. Dilutions of the mechanically dispersed oncolysate up to 1/15 protected normal mice against challenge by lo3 tumor cells, whereas mechanical lysates of the EA tumor gave no protection at all. A number of significant experiments gave insight into the mechanism of the virus augmentation, or viral adjuvanticity , phenomenon..Postoncolytic immunity could be transferred to normal mice by either serum or lymphoid cells (Lindenmann, 1964). The serum was found to contain a high titer of antibodies directed against an alloantigen ( E antigen) present in the tumor cell membrane and in normal tissues of other mouse strains (Klein and Lindenmann, 1965). Later, when other investigators performed similar experiments in syngeneic tumor systems, rather than with the nonspecific allogeneic EA tumor, the immunity produced by virus-augmented TATA was transferrable with lymphoid cells, but never with serum. In spite of the fact that the EA tumor was allogeneic, Lindenmann and Klein (1967a) were able to determine a number of important facts about virus augmentation that were later confirmed in syngeneic systems. Viral oncolysates did not lose their augmented immunogenicity after fixation with 0.08% formaldehyde, which completely inactivated virus infectivity. Simple mixtures of egggrown virus with mechanical lysates of tumor cells were not immunogenic: Actual virus infection of the tumor cells was required before


3 09

augmentation of immunogenicity occurred. Two different types of experiments both appeared to show that the immune system was involved in the virus augmentation phenomenon: ( 1 ) Preimmunization with egggrown virus enhanced even more the immune response to immunization with oncolysate. (2) When viral oncolysates were mixed with rabbit antiserum against egg-grown influenza virus, the resultant mixture did not induce either antiviral or antitumor responses. Immunization with egg-grown virus alone produced no tumor protective effect. Thus, the mechanism of virus augmentation was not due to a nonspecific adjuvant effect nor to any changes produced by live virus. Lindenmann (1970) extended his studies of TATA augmentation to include vesicular stomatitis virus (VSV), a rhabdovirus which also matures by budding at the cell surface and which has only one envelope antigen, a hemagglutinin, compared to two, a hemagglutinin and a neuraminidase, possessed by influenza virus. VSV was chosen because it is easily adaptable to many different tissues, in contrast to influenza virus, and is known to contain antigens derived from the host cell. The finding that inbred A2G mice could be immunized against the EA tumor by inoculation with viral oncolysates prepared from VSV-infected EA tumor cells led to the conclusion that VSV acted as an immunological carrier for EA tumor cell antigens which had become incorporated into the coat of the mature virion. In these experiments, it is noteworthy that although the infectivity titer of VSV in viral oncolysates reached a maximum within 24 hours, the immunogenicity of the oncolysates was greatest at 48 hours. It appeared that immunogenic material had accumulated between 24 and 48 hours after infection without concomitant increase in the number of infectious virus particles. Lindenmann (1970, 1973) speculated that a suitable virus could be grown in cancer tissue obtained from a patient and then, after inactivation, be injected into the patient to increase his defenses against the specific antigens of his own tumor. The patient could be protected from any adverse viral infection by a previous vaccination with virus. How much of Lindenmann's speculation has come to pass will be presented in a later section on the application of virus-augmented tumor antigens to human cancer immunotherapy. Since these initial studies, many reports of virus-augmented TATA in animal model systems have appeared (Table I). Hakkinen and Halonen (1971) showed that oncolysates could be prepared from EA tumor cells grown in suspension culture and infected in v i m with either influenza virus or VSV. Unconcentrated cell culture supernatant, purified concentrated virus, and a 10% suspension of virus-infected EA tumor cells all were able to induce antitumor immunity.

S U M M A R Y OF PUBLISHED

Virus

TABLE I DATAON VIRUS-AUGMENTED TUMOR CELL EXTRACTS (ANIMALSTUDIES)

Preparation of infected cells

Tumor

Host

Influenza

In iYi-o

Ehrlich ascites (EA)

A2G mice

Influenza (Hong Kong strain)

117 i.iiw

EA

A2G mice

Influenza

In

EA

BALB/c mice

In ritro

SV4013T3

BALB/c mice

IIT isitro In i'itro

MCA-induced fibrosarcoma MCA-induced fibrosarcoma Lewis lung carcinoma WEHI-I 1 fibrosarcoma

BALB/c mice C57BL/6 mice C57BL/6 mice BALB/c mice

vitro

111 i i t r o Iri

isitru

Reference Lindenmann and Klein (1967a): Lindenmann (1971, 1973, 1974, 1977) Haller and Lindenmann (1975) Hakkinen and Halonen (1971) Boone et 01. (1971, 1973, 1974, 1979a): Boone (1972, 1974); Boone and Blackman (1972); Boone and Gillette (1977): Takeichi et al. 1978 Boone ( 1972) Klein (1974) Griffith e / a / . (1973 Griffith et a / . (1975)

Newcastle disease viius (NDV)

SV40-transformed human cells Sarcoma-37 (S 37) Gross ascites lymphoma

Hamster

Axler and Girardi (1970)

CBA mice C3H/Bi mice

Beverley et ai. (1973) Eaton et a / . (1973); Eaton and Almquist (1975)

EA

A2G mice

Lindenmann (1970)

EA

BALB/c mice

SV4W3T3

BALB/c mice

SV40-transformed TSV-5 CI-2

Inbred Syrian hamsters

L1210 leukemia

C57BL16 mice

Hakkinen and Halonen (1971) Boone et al. (1974): Gillette and Boone ( 1976) Ansel(l974); Ansel er ai. (1977); Huet and Ansel ( 1977) Wise (1977)

Sendai virus

Gross ascites lymphoma

C3H/Bi mice

Eaton et al. (1973)

Semliki Forest virus

WEHI-11 fibrosarcoma

BALB/c mice

Grifith el ai. (1975)

Vaccinia

SV40-transformed peritoneal macrophage tumor

BALB/c mice

Wallack et a/.(1977)

Vesicular stomatitis virus (VSV)

(I

lmmunogen consisted of purified VSV grown in the tumor cells.

3 12


In the first studies of virus augmentation in a syngeneic tumor system, Boone et al. (1971) demonstrated that BALB/c mice could be made immune to tumor challenge with syngeneic SV4O-transformed fibrosarcoma cells (E4) by vaccinating them with homogenates of these cells that had been infected in vitro with influenza virus. No immunity was induced by vaccinating mice with homogenates of either uninfected cells or of influenza virus-infected normal (3T3) cells. The syngeneic cultured tumor cell line (E4, formerly called SV3T3-T4) was obtained as an explant from a solid fibrosarcoma in a BALB/c mouse inoculated with SV40-transformed Balb/3T3 cells. The use of the E4 cell line allowed for a direct comparison with its normal counterpart, Balb/3T3 cells. Boone et al. (1971) used the same WSA strain of influenza A virus that had been used by Lindenmann but with further adaption to growth in E4 cells. The SV40 TATA is a strong antigen, and immunization with X-irradiated E4 cells induced complete immunity to a tumorigenic challenge dose of viable E4 tumor cells. Inoculation of normal 3T3 cells in the same manner did not. The immunogenicity of SV40 TATA is destroyed if the tumor cells are homogenized, but retained in homogenates of tumor cells previously infected with influenza virus. A typical tumor protection experiment is illustrated in Table 11. Tumor cell challenge after immunization with virus-augmented E4 cell extracts resulted in a tumor incidence of 24% as compared to 85% for control mice, and 82% for mice immunized with a homogenate of uninfected E4 cells. The immunogenicity of virusinfected crude membranes (CM) was still less than that of X-irradiated intact tumor cells. The immunizing dose of CM was equivalent to apTABLE I1 AUGMENTED TATA ACTIVITY OF CRUDE MEMBRANES OF E4 TUMOR CELLSINFECTED W I T H INFLUENZAVIRUS Immunogen"

lofi X-irradiated (4000 rad) E4 cells Uninfected E4 CMb, 500 pg FIu-E~CM,b*'500 pg Saline

Tumor incidence after challenge"

'

1/20 (5%) 14/17 (82%) 4/17 (24%) 17/20 (85%)

Immunization (s.c.) on days 0 and 3. Crude membranes prepared by centrifugation (100.000 g , I hour) of a nucleus-free Dounce homogenate. Protein concentration determined by Lowry method. Cells harvested 24 hours after infection with influenza virus, multiplicity of infection: 20. HA titer of infected CM: 4096. Ratio of mice with tumor to mice challenged with lofi E4 cells 14 days after immunization. Tumor incidence recorded 28 days after challenge. a


313

proximately lo7 cells. Virus-augmented tumor cell homogenates retained their immunogenicity after inactivation with 3% formalin (Boone and Blackman, 1972). To evaluate the relative immunogenicity of various subcellular fractions, influenza virus-infected E4 tumor cells were homogenized and fractionated by sucrose density gradient centrifugation. Maximum immunogenic activity was found in the 33%-40% (plasma membrane fragments, intact influenza virus) and 40%-45% (plasma membrane fragments, rough endoplasmic reticulum, ribosomes, mitochondria, lysosomes) sucrose fractions. It was postulated that most of the augmented TATA in the 33%-40% fraction may exist as virus antigen-containing plasma membrane microvesicles, rather than as virus with hostcell TATA incorporated into its envelope (Boone, 1972; Boone ef al., 1973). The relative roles of viral antigens incorporated into the tumor cell membrane versus tumor antigens incorporated into the virus envelope in the virus augmentation phenomenon is still unresolved. (See Section 111,A,3 .) In further studies, Boone et al. (1974, 1979a) demonstrated the augmented immunogenicity of homogenates of tumor cells infected with the Hong Kong strain of influenza virus and with VSV. UV inactivation of the virus-infected tumor cell homogenates did not destroy TATA activity. Evidence was presented in support of the “helper antigen” hypothesis: that the viral antigen works through the host’s immune system to induce an augmented response to the TATA of the tumor cell. It was also noted that although homogenates of uninfected tumor cells elicited a delayed hypersensitivity (DH) response when injected into the footpads of tumorimmune mice, homogenates of virus-infected tumor cells produced a more intense footpad DH reaction. This observation was later confirmed (Austin el al., 1977) and formed the basis for the development of virusaugmented human skin test antigens (Boone rf al., 1978, 1979b; see Section 111,C). Influenza virus was also shown to augment the immunogenicity of homogenates of MCA-induced tumor cells, but the effects seen with this chemically induced tumor were not as striking as those seen for the SV40-transformed E4 tumor cell line (Boone, 1972). This may have been because the influenza virus stock had not been fully adapted to grow in this cell line. In another study of chemically induced tumors, Klein (1974) found that the same WSA strain of influenza virus which had been used in earlier studies with EA tumors failed to grow consistently in MCA-induced tumors of C57BL/6 mice. Following a period of adaptation, however, this virus could be grown to high titers in most but not all lines, resulting

3 14


in lysis of the cultured tumor cells. VSV and encephalomyocarditis virus grew in all tumor lines tested without adaptation. Klein (1974) reported the augmented immunogenicity of one line of cultured MCA-induced tumor cells following influenza virus infection. This immunoprophylaxis experiment was performed in virus-immunized mice because earlier observations had indicated that virus-primed mice are more sensitive indicators of virus-augmented tumor cell immunogenicity (Lindenmann and Klein, 1967a). Although Lindenmann had originally stressed the necessity for working with viruses that were fully adapted to grow in tumor cells, Haller and Lindenmann (1975) reported the augmentation of antigens of EA tumor cells produced by infection with the Hong Kong strain of influenza A virus that had not been previously adapted to this tumor. Infection of tumor cells in i i i r v with this strain of influenza virus resulted in oncolysis, and the resultant oncolysates had high levels of hemagglutinin, but low titers of infectious virus, indicating incomplete virus growth. Nevertheless, these oncolysates induced solid antitumor immunity against EA tumors in various mouse strains. This virus strain had also been shown to augment TATA immunogenicity in a syngeneic tumor system, although the yield of infectious virus in this system was not determined (Boone et id., 1974, see above). Of course, augmentation of TATA immunogenicity by an incomplete virus growth cycle would eliminate the complications arising from the presence of large quantities of infectious virus. This approach to the preparation of virus-augmented tumor antigens has obvious potential in human cancer immunotherapy. In other basic studies, Beverley et ul. (1973) immunized mice with NDV-infected tumor cells or cell membrane fractions of a transplantable ascites sarcoma and found that the virus-infected cell membranes were more immunogenic than similar fractions prepared from uninfected cells. The immunity conferred was not very strong. Complete virus replication did not occur in the tumor cells and the authors considered the possibility that the infection by NDV was aborted at a stage prior to the development of a hapten carrier association. In this system preimmunization with virus abrogated the virus-augmented immunogenicity of tumor cell extracts. Eaton et ul. (1973) also studied the effect of NDV, as well as parainfluenza 1 (Sendai strain), to augment the immunogenicity of a Gross virusinduced ascites lymphoma in syngeneic C3H/Bi mice. Multiple injections of NDV-infected cell extracts were required for successful immunization. If the first injection was mixed with complete Freund’s adjuvant (CFA), it was found that fewer injections of antigen were required for immunization. The result that one or two injections of extract with CFA failed to immunize was consistent with the weak immunogenicity of NDV-


3 15

augmented extracts reported by Beverley et al. (1973). Experiments with cells treated with nonreplicating virus again confirmed that the growth of virus was essential to obtain virus-augmented TATA immunogenicity. Tests of various cell fractions indicated that the major portion of TATA activity was in the membrane fraction, with none in the cytoplasmic supernatant. Virus and other small particles concentrated from the tissue culture supernatant by ultracentrifugation were less effective than membranes in inducing tumor immunity. Further studies (Eaton and Almquist, 1975) of NDV-infected tumor cells demonstrated that tumor immunity was not produced by the high-speed supernatant of infected cell homogenates, by the virus fraction of tissue culture supernatants, or by detergent extracts of the membrane fractions of infected tumor cells. Griffith et al. (1975) used influenza virus or Semliki Forest virus (SFV) infection to augment the immunogenicity of extracts of cultured WEHI11 cells, a fibrosarcoma of BALB/c mice. In addition, the life span of C57/B1 mice inoculated with Lewis lung carcinoma cells was prolonged if the mice were preimmunized with membranes of the cells which had been infected in vitro with influenza virus. The influenza virus-infected membrane preparations were inactivated by exposure to UV light. Membrane preparations containing infectious SFV were inactivated by heating at 56°C for 1 hour or by treatment with 0.1% sodium deoxycholate. In the U.S.S.R., Rukavishnikova and Alekseyeva (1976) demonstrated that vaccine strains of influenza A virus inhibited the growth of a syngeneic virus-induced ascites tumor in BALB/c mice. Also, immunization with the viral oncolysates protected recipient mice against subsequent tumor challenge. Wise (1977) studied the potentiation of immunogenicity of L1210 mouse leukemia cells after in vitro infection with VSV and also demonstrated the virus-augmented immunogenicity of extracts of VSV-infected tumor cells. All homogenates were UV-inactivated in these experiments. Various aspects of the interaction of the virus with cellular antigens in this study will be discussed in subsequent sections (Sections 111,A,4 and IV,A). Wallack et al. (1977) demonstrated the use of vaccinia virus to augment the immunogenicity of an SV40-transformed mouse peritoneal macrophage tumor. In searching for a model system for the development of immunogenic viral oncolysates with application to the treatment of human cancer, they tested six live virus vaccines (measles, mumps, smallpox, rubella, yellow fever, rabies) against four human tumor cell lines (ovary, lung, melanoma, colon) and noted that only vaccinia (smallpox vaccine) duplicated the oncolytic action of influenza virus on EA tumor cells as previously described by Lindenmann (1963). Vaccinia has

3 I6


also been used in attempts to treat melanoma patients, with some positive results (Burdick, 1960; Burdick and Hawk, 1964; Roenigk et a / . , 1974: see Section 11,B).There was no need to inactivate infectious virus in the vaccinia virus oncolysate since the safety of the vaccine strain had been established. The choice of vaccinia virus to augment tumor cell extract immunogenicity is at first surprising, since vaccinia is not known to be a surface-budding virus. However, vaccinia appears to enter the host cell by fusion of the virus envelope with the plasma cell membrane, followed by distribution of vaccinia antigens in the entire cell surface membrane (Chang and Metz, 1976). In addition, during the course of virus replication, virus-induced cell surface antigens do appear (Burns and Allison, 1977) and can function as helper antigens for the TATA of the tumor cell. Wallack demonstrated that mice which were immunized with the vaccinia oncolysate were rendered solidly immune to challenge with viable tumor cells. Preimmunization with vaccinia virus alone prior to treatment with vaccinia oncolysate did not interfere with the induction of tumor immunity by viral oncolysates. In addition to immunoprophylaxis, Wallack demonstrated therapeutic effects of virus-augmented tumor cell extracts on growing tumors in mice. These successes led him to attempt active specific immunotherapy of human cancer with vaccinia oncolysates. The results of his clinical studies, and other ongoing clinical trials with virusaugmented tumor cell extracts, will be described in Section III,B.

2. Solirble Extructs c,f Virus-Infected Tumor Cells The preparation of soluble TATAs has been the object of a vast amount of research in recent years, impelled by the hope that they might provide standardized reagents for immunotherapy and also facilitate understanding of their biological role in carcinogenesis. Progress has been generally frustrating and only marginally successful because of the heterogeneity and striking lability of most TATAs (for review, see Law and Appella, 1975). Methods that were successful for purification of histocompatibility antigens have also been applied to the isolation of tumor antigens (e.g., 3 M KCl extraction, nonionic detergent solubilization, etc.). In comparing various methods of antigen solubilization, we have found that soluble extracts of influenza virus-infected tumor cells produced by high-speed centrifugation of Dounce homogenates, by sonication of membranes, or by 3 M KCI extraction of cells still exhibit augmented TATA activity (Boone et a / . , 1979a; Austin et ul., in preparation). The augmented immunogenicity of a representative soluble antigen preparation obtained after high-speed centrifugation (lO0,OOO g , I hour) of a nucleus-free Dounce hornogenate is shown in Table 111. The homogenate was sepa-

3 17


TABLE 111 VIRUS-AUGMENTED TUMOR ANTIGENS BY HIGH-SPEED ( 100,000n ) OF NUCLEUS-FREE DOUNCE HOMOGENATES

PREPARATION OF SOLUBLE CENTRIFUGATION

Results of tumor challengeb Immunogena

Tumor incidencec

Mean tumor weight(gm) 2 S E

loBX-irradiated E4 cells E4 homogenate Flu-E4 homogenate E4 crude membranes Flu-E4 crude membranes E4 soluble Flu-E4 soluble Saline

0120 (0%) 8/13 (62%) 3/13 (23%) 17/20 (85%) 4120 (20%) 13/20 (65%) 4114 (2%) 17/20 (85%)

0.39 ? 0.18 0.45 2 0 . I I 0.67 0.13 0.09 0.06 0.67 2 0.14 0.36 ? 0.25 1.03 ? 0.13

* *

pd

< .01 < ,005 < .05 < ,0005

< .05 < ,025

Mice were immunized with lo6 X-irradiated (4000 rad) E4 cells or 500 pg of antigen S . C . on days 0 and 3. Mice challenged with 106 viable E4 cells, s.c., on day 14. Ratio of mice with tumors to mice challenged, 28 days after tumor challenge. " Comparison of mean tumor weight (of animals with tumors only) with that of control group (saline) by Student r test.

rated into a cytoplasmic soluble supernatant fraction and a crude membrane pellet fraction, both of which were tested for immunogenicity according to the protocol in Table 111. Soluble extracts from virus-infected tumor cells were more immunogenic than those from uninfected cells. Augmented TATA activity of the soluble supernatant had not been found in early studies (Boone et al., 1973). However, the present studies were performed with improved virus stocks that grew more efficiently in the tumor cells and may have produced a stronger augmenting effect. Our criteria for solubility have been lack of precipitation after centrifugation at 100,000g for 1 hour, or inclusion in the bed volume of 200,000 molecular weight cutoff gel filtration media. We have also solubilized tumor antigens by physical (sonication of cell membranes) and chemical (3 M KCI extraction) methods and found that after further fractionation by gel filtration, some fractions retained virus-augmented TATA immunogenicity (Austin et al., in preparation). Further study of the chemical nature of the virus-augmented soluble antigens should shed additional light on the mechanism of virus augmentation of TATA.

3 . Antigenicity of Tumor-Grown Viruses

To assess the role of host cell TATA incorporated into the envelope of surface-budding viruses, Ansel (1974) studied the immunogenicity of

318


VSV which was grown in SV40-transformed hamster cells. When this virus was highly purified and inactivated, it possessed SV40 TATA activity as demonstrated by its ability to induce specific transplantation immunity to tumor cells which possessed the SV40 TATA. The presence of host cell antigens in this purified virus was also demonstrated by the ability of rabbit antiserum against purified tumor-grown virus to react specifically with SV4O-transformed cells in a complement-dependent 51Cr cytolysis assay. Ansel concluded that the presence of SV40 TATA activity in tumor-grown viruses was due to the incorporation of TATA into the virus envelope. In further studies (Ansel et al., 1977; Huet and Ansel, 1977), treatment of tumor-grown VSV with proteolytic enzymes did not destroy the virus-augmented TATA activity associated with the virus, although the VSV lost its ability to induce virus-neutralizing antibody, thus demonstrating that the augmented TATA activity was not dependent on the presence of the viral glycoprotein. Phospholipase C, however, did suppress the TATA activity of tumor-grown VSV, but not the antigenicity of the glycoprotein of the virus. Since the protein components of virus membranes are thought to be entirely virus specific, while lipids, glycolipids, and the carbohydrate moiety of the glycoprotein originate from the host, these results led to the hypothesis that SV40 TATA activity may be conveyed by the lipid core of the virus envelope. In contrast to Ansel’s experiments, Griffith et a / . (1975) found that partially purified SFV which was grown in WEHI-11 fibrosarcoma cells also protected mice from tumor cell challenge. However, neither highly purified SFV nor the glycoprotein from the envelope of this virus conferred protective immunity to mice. From these results, it was concluded that SFV-augmented immunogenicity was not due to covalent linkage of TATA to the viral envelope protein, but was most probably due to the apposition of viral glycoprotein and cellular TATA in the plasma membrane of the tumor cells. These results, which are in conflict with those of Ansel, may be due to a difference in the molecular structure of the TATA in these different tumors or to a difference in the composition of viral envelopes of the different viruses studied. Other studies have shown partially purified virus to be less effective than crude infected cell homogenates for the induction of antitumor immunity (Hakkinen and Halonen, 1971: Beverley et ul., 1973; Eaton et d..1973). 4. The Ncitirre of Virrrses Thut Augment TATA Activity Table I lists the several viruses that have been shown to augment TATA activity of tumor cell extracts by their growth in the cells prior to their disruption. The viruses listed are known to induce virus-specific


319

cell-surface antigens during their replicative cycle. In a study using the SV40-transformed cell line E4 described above, comparison was made of the ability of influenza virus and VSV to augment the immunogenicity of SV40-induced TATA (Gillette and Boone, 1976). Membranes from influenza virus-infected cells were immunogenic at ooth the dose required for membranes from VSV-infected cells. Subcutaneous inoculation was better than the intraperitoneal route of administration. Maximum protection against tumor cell challenge was afforded by two inoculations of VSV-infected cell membranes spaced 3 days apart or by a single inoculation with influenza virus-infected cell membranes. The use of Freund’s adjuvant produced either no effect or a slight enhancement of tumor growth. The viruses listed in Table I differ from those that have been used in studies of viral xenogenization of intact tumor cells (see H. Kobayashi, this volume) in that they were selected for their oncolytic effect on tumor cells, whereas the viruses chosen to augment the TATA activity of intact tumor cells produce a persistent infection with little or no cytopathic effect on the host cell. Augmenting viruses in the latter category include Sendai virus (Eaton et al., 1973), measles virus (Evermann and Burnstein, 1975), lymphocytic choriomeningitis virus (Eiselein and Biggs, 1970), and type-C retroviruses (Kobayashi et a / ., 1969, 1970; Kobayashi, 1970; Sendo et al., 1970; Kuzumaki and Kobayashi, 1976; Al-Ghazzouli et al., 1976; Kuzumaki et al., 1978; Kobayashi and Sendo, 1979). Most of this work is reviewed by Kob’ayashi et al. (1977) and in this volume (H. Kobayashi, 1979). To evaluate the effectiveness of type-C retroviruses in augmenting the immunogenicity of TATA in tumor cell extracts as contrasted to virusaugmented TATA on intact tumor cells, Takeichi et af. (1978) compared the augmented immunogenicity of E4 tumor cell membranes prepared after infection of the cells with influenza virus as opposed to infection with Moloney sarcoma virus (MSV). As expected, crude membrane extracts from influenza virus-infected E4 cells were markedly more immunogenic than extracts from uninfected cells, as measured either by the ability to induce protective immunity against tumor graft challenge or by heightened lymphocyte-mediated cytotoxicity against tumor cells in vitro. Next it was shown that E4 cells productively infected with MSV were rendered so immunogenic that they would grow only in X-irradiated syngeneic mice. In spite of this, the crude membrane extracts from these MSV-infected cells showed no augmented TATA activity such as was seen after infection with influenza virus. Thus, it appears that MSV, and possibly other type-C viruses, may augment the TATA activity of tumor cell extracts in a different way than lytic surface-budding viruses do.

320

FAYE C. AUSTIN A N D CHARLES W. BOONE

Other reports also point to this possibility. Eaton et al. (1967) had shown that syngeneic mice could survive repeated injections of NDVinfected Gross virus-induced lymphoma cells, for they were no longer capable of producing tumors. However, repeated injection of the virusmodified intact tumor cells did not induce immunity against challenge with uninfected Gross virus-induced lymphoma cells. Al-Ghazzouli et al. (1976) reported that the enhanced immunogenicity of type-C virus-infected intact tumor cells was destroyed after X-irradiation, but Kuzumaki et af. (1978) showed that some but not all tumors infected with endogenous type-C mouse virus retained augmented antigenicity after X-irradiation. Of interest is the report by Wise (1977) which demonstrated that although a single injection of X-irradiated L1210 leukemia cells protected mice against tumor cell challenge, the same dose of VSV-infected cells was not immunogenic after X-irradiation. On the other hand, homogenates of VSV-infected L1210 cells were immunogenic, but those of uninfected cells were not.

B. IMMUNOTHERAPY OF HUMAN CANCER: CLINICAL TRIALS The positive results obtained in immunoprophylaxis (tumor protection) experiments with virus-augmented tumor extracts, described in preceding sections, have led several investigators to attempt immunotherapy in human cancer patients with virus-augmented tumor cell extracts (viral oncolysates). A survey of the published reports of clinical trials to date (Table IV) reveals that initial results have not been particularly striking. However, the studies were performed in patients with advanced disseminated disease, where regressive effects on tumor growth are harder to detect. Sinkovics and his colleagues (Sinkovics, 1977; Sinkovics et al., 1974a,b, 1977: McMurtrey ef ul., 1976) implemented an immunotherapy protocol, in conjunction with chemotherapy, in attempts to stimulate the patient’s immune responses to his own tumor. They have established in culture many cell lines from human sarcomas and have prepared viral oncolysates from most of them by infecting them with the PR8 strain of influenza A virus. Initial studies showed an increase in antitumor cell cytotoxic lymphocytes and conversion from blocking to potentiating serum factors in the peripheral blood of patients who were receiving immunotherapy with either X-irradiated allogeneic sarcoma cells or with viral oncolysates of allogeneic sarcoma cells. In further studies, the effect of tumor-specific active immunization with viral oncolysates in combination with chemotherapy and nonspecific immunization with BCG was investigated (Sin-

TABLE IV SUMMARY OF PUBLISHED REPORTSOF IMMUNOTHERAPY WITH VIRUS-AUGMENTED H U M A NTUMOR EXTRACTS ~

Virus

Tumor

Viral oncolysate

Influenza (PR8)

Sarcomas, melanoma

Allogeneic, UVinactivated

Influenza (fowl plague virus) Influenza (A2 or B)

AML"

Newcastle disease virus

Melanoma

Allogeneic, formaldehydeinactivated* Autologous, and allogeneic, formalininactivatedc Allogeneic, noninactivated'

Vaccinia

Carcinomas, melanoma

Osteosarcoma

Acute rnyelogenous leukemia. Given along with chemotherapy. Given along with chemotherapy in some patients.

Autologous, noninactivated

Effect of treatment Increased lymphocytemediated cytotoxicity: slowed progress of disease No apparent effect Increase in cellular and humoral antitumor responses Increased lymphocyte cytotoxicity: decreased size of skin nodules and/or diseases lymph nodes (6 of 13 patients): no effect on visceral disease Evidence of control of tumor growth (9 of 29 patients)

Reference Sinkovics (1977): Sinkovics et al. (1974a,b, 1977): McMurtrey et al. (1976) Sauter et al. (1978); Lindenmann (1979a) Green et al. (1976)

Cassel et al. (1977): Murray et al. (1977)

Wallack (1979a,b): Wallack et al. (1977)

322


kovics, 1977; Sinkovics et ul., 1977). Patients with melanoma or metastatic soft tissue sarcomas received intermittent chemotherapy with or without BCG (6 x IOR viable units scarified twice monthly) or identical chemotherapy, BCG, and viral oncolysate [equivalent of 10' tumor cells injected intracutaneously (i.c.) in the region of BCG scarification twice monthly]. Chemotherapy was given on days 1 through 5; immunotherapy was given on days 17 and 24. Courses were repeated at 28-day intervals. Viral oncolysates were prepared from allogeneic cultured sarcoma cells infected in \iituo with influenza virus (PR8). At the end of the first year of treatment, the results were as follows: of 48 patients with metastatic sarcomas receiving chemotherapy only, 10 had complete remission (21%), and 6 had partial remission or stable disease status (13%). In 32, the disease progressed, often resulting in death (66%). Of 10 patients with metastatic sarcomas receiving chemotherapy and BCG, one had complete remission (lo%), 5 had partial remission or stable disease status (50%), and 4 had disease progression, some resulting in death (4%). Of 14 patients with metastatic sarcomas receiving chemotherapy, BCG, and viral oncolysates, 6 had complete remission (43%), 6 had partial remission or were in stable disease status (43%), and 2 had disease progression (14%). Similar results were reported following treatment of melanoma patients. Of 12 patients receiving chemotherapy and BCG, 6 had disease progression (50%), whereas only 7 out of 27 patients (26%) receiving comparable chemotherapy with BCG and viral oncolysates had disease progression. These results clearly indicated a trend favoring active tumorspecific immunization with influenza virus-augmented allogeneic tumor cell extracts and should be confirmed by observation of these patients for a longer period and by prospective randomized trials. Sauter et ul. (1978) and Lindenmann (1979a) performed a randomized clinical trial to assess the effect of immunization with viral oncolysates in AML patients in remission. They used avian influenza A virus (fowl plague virus) which had been adapted to several types of human tumor cells including human leukemic myeloblasts (Sauter Pt ul., 1973, 1975: Illiger ef u l . , 1975). Patients with AML were chosen for this study because, as soon as they had achieved complete remission induced by chemotherapy, they retained only a minimum residual tumor mass. Also, since the median remission time in this disease is less than 1 year, results of additional therapy would be seen within a short period of time. Patients achieving complete remission were randomized into two groups: one receiving chemotherapy alone, and the other receiving identical chemotherapy plus immunization with viral oncolysates. Chemotherapy was given for a 5-day course every 4 weeks throughout duration of this study. In addition, patients receiving immunotherapy received, on day 15 of


323

each 4-week cycle, 1.0 ml of viral oncolysate (0.2 ml i.c. and 0.8 ml s.c.). One milliliter of viral oncolysate was equivalent to log leukemic myeloblasts. The virus was inactivated with formaldehyde (0.2%). No side effects attributable to the injection of viral oncolysate were observed; however, DH skin reactions were observed in most patients at the first injection of viral oncolysate. After an average follow-up period of 13.5 months, there was no significant difference between the two groups with respect to the probability of staying in remission or staying alive. In a critique of this work, Lindenmann (1979a) pointed out that although formalin treatment did not impair the immunogenicity of viral oncolysates in animal model studies, it is not known whether this finding can be extrapolated to human tumor antigens, which may be destroyed by formalin. Inactivation was deemed necessary to avoid the possibility of producing recombinant strains of influenza virus in patients who might accidentally acquire an infection with a common human influenza virus strain. Although it is theoretically possible that such a recombinant could give rise to a new pandemic variant, Lindenmann now feels that this is not a great danger. To investigate the possibility that the allogeneic immunizing material may not have had the same TATA as the patient’s own tumor, a pilot study is planned in which only autologous viral oncolysates will be used. It should be noted that Sinkovics and colleagues (described above) used a more intensive immunotherapy program and a different method of virus inactivation than were used in these studies. Green et af. (1975, 1976) designed a Phase 1 study to determine the toxicity and general effectiveness of a vaccine prepared from influenza virus-infected osteosarcoma tumor cells in osteosarcoma patients. The study included patients with measurable tumors who were resistant to several chemotherapeutic drugs. Twelve patients were chosen: 6 did not receive antitumor agents during or after the administration of the vaccine: the remaining 6 received intravenous methotrexate every 3 weeks while receiving the vaccine. The tumor lines used were established in culture from osteosarcoma tumor tissue taken from three of the patients in the study. The viral oncolysates used were produced with the AYPort Chalmers and B/Michigan strains of human influenza virus. No attempt was made to adapt virus strains to replicate in the tumor cells. The viral oncolysates were inactivated by treatment with 0.01% formalin. Two milliliters of the oncolysate vaccine were injected i.m. at 2-week intervals. All patients, including those receiving autologous tumor cell vaccine, developed antibodies to both tumor and virus antigens. There was a direct correlation between the responses to viral and tumor cell antigens, but no correlation between the titer of antibody and the clinical course of disease either before or after immunization. In assessing the

3 24

FAYE C. AUSTIN A N D CHARLES W. BOONE

cellular immune response of 6 patients with extensive disease it was found that an immune response was generated only in patients who received autologous tumor cell vaccine, leading to the initial conclusion that autologous vaccine was necessary to stimulate cell-mediated immunity. However, further studies on 6 patients with less advanced disease indicated that 3 out of 4 patients who received allogeneic vaccine also developed cell-mediated immune responses and that the responses to autologous and allogenic vaccines were comparable in these patients with minimal disease. The 6 patients with minimal disease were receiving high-dose methotrexate chemotherapy. However, there was no evidence of suppression of the immune response in these patients receiving concurrent chemotherapy and viral oncolysate vaccine. The response to immunization correlated with disease status during the immunization. It was postulated that although the drug may have suppressed what would have been an even better response in the absence of chemotherapy, the drug may also have controlled the tumor to some extent and permitted patients to respond better to the vaccine. On the basis of these results, the authors are planning prospective imrnunotherapy trials. The use of recently isolated influenza virus strains in the preparation of viral oncolysate raises the question of the effect of viral antibodies in the patients' serum on the stimulation of immunity by viral oncolysates. Several patients did not have preexisting antiinfluenza antibody and responded anamnestically to the viral antigen present in the vaccine. The overall effect of this response on the effectiveness of the viral oncolysate in stimulating tumor immunity was not assessed. Antibody to the virus is known to inhibit the release of virus from infected cells (Dowdle et ul., 1974) and also, by neutralizing virus infectivity, would protect the patient from any spread of infectious virus. The effect of preexisting virus antibody on the ability of viral oncolysates to stimulate protective tumor immunity in animal model systems varied with the system studied (Lindenmann and Klein, 1967a; Boone rt al., 1974). Cassel et al. (1977) and Murray et ul. (1977) have studied the use of NDV-augmented melanoma cell extracts in the treatment of malignant melanoma. Because of the known oncolytic capability and minimal pathogenicity of this strain of NDV (Cassel and Garrett, 1965, 1966), viral oncolysates were administered without inactivation of the virus. The viral infectivity titers of the oncolysates ranged from lo3.' to 105.55070 egg-infectious doses per 0.05 ml. Viral oncolysate was prepared from primary explants of autochthonous melanoma and administered as a 10fold concentrate prepared in an ultrafiltration cell. The average protein concentration was 39 mg/ml. One milliliter of the 10-fold concentrate was the product of approximately 8 x lo6 cells. In a typical schedule, the


325

patients received 2 ml of the concentrated oncolysate S.C. every week. Distinct changes were observed in cell-mediated immunity. Lymphocyte cytotoxicity against cultured allogeneic melanoma cells increased in all patients within 1 to 2 weeks. Most patients showed a tendency for sustained increase both in relative and absolute levels of circulating T lymphocytes. T-lymphocyte levels in melanoma patients not receiving immunotherapy were low and decreased progressively until the patient died. Of the 13 patients with metastatic melanoma who were treated with viral oncolysate, 6 patients showed a decrease in the size of skin nodules or diseased lymph nodes. However, visceral lesions were not favorably influenced to any marked degree. This reduction in size in skin nodules and a noticeable lack of increase in size of existing superficial lesions in other cases were considered to be uncommon occurrences by the investigators. They noted that although the course of disseminated melanoma is often unpredictable, there is an inexorable fatal termination. The one surviving patient had the least advanced disease of all those studied, being the only one with no evidence of visceral disease. The patient had been unsuccessfully treated by surgery and chemotherapy with recurrent disease for over 4 years. At the time of the published report, the patient had no evidence of disease. The primary consideration in this pilot study was the assessment of the safety of viral oncolysates prepared from NDV-infected melanoma cells, without subsequent virus inactivation. No adverse responses were encountered, including evidence of any adverse effect on the central nervous system or any shedding of virus into the environment. The overall findings of this study suggested that the use of viral oncolysates may augment the immune response to malignant melanoma and produce benefit to the patient. As with all other studies, it appears that the patients could have been better helped if oncolysate treatment had been instituted earlier in the disease, particularly after removal of major tumor burden. The investigators in this project plan to extend these studies to high-risk cases with limited regional lymph node involvement. Wallack et a / . (1977), after demonstrating the effectiveness of vaccinia oncolysates in the immunoprophylaxis and immunotherapy of SV40-induced mouse peritoneal macrophage tumors, extended these findings to the study of vaccinia virus-augmented human tumor cell vaccines for active specific immunotherapy of human cancer (Wallack, 1979a). Since the safety of the vaccine strain of vaccinia virus has been established, it was considered unnecessary to inactivate any live virus in the oncolysates. Vaccine was prepared from autochthonous tumor cells after surgical removal, followed by preparation of the cells as a single-cell suspension culture. The cells were infected with vaccinia virus in vitro and

326


incubated for 96 hours while both tumor cell lysis and virus titer were monitored. The cells were harvested, homogenized, and suspended to effect a concentration equivalent to los cells per milliliter. Doses of 1 ml of the vaccinia oncolysate were injected intradermally (i.d.) every 2 weeks for 3 months. Thereafter, 1-ml booster doses were given every month until recurrence of the tumor or exhaustion of the vaccine supply. A total of 29 tumor patients were reported to receive vaccinia oncolysate immunotherapy, representing 10 cases of melanoma, 9 cases of colon carcinoma, 2 cases of gastric carcinoma, 1 case of fibrosarcoma, and 1 case each of cancer of the cervix, lung, liver, ovary, kidney, breast, and thyroid. Although these patients probably had preexisting antivaccinia antibodies (from smallpox vaccination), experiments with mice had shown that preimmunization with virus alone prior to administration of vaccinia oncolysate did not abrogate the immunogenicity of the oncolysate. Side effects of the therapy were minimal, except for pain and inflammation at the injection sites. The patients' responses to vaccinia oncolysate immunotherapy were summarized as follows: (1) 0 out of 29 patients had generalized vaccinia, allergy, anaphylaxis, or tumor growth at injection sites: (2) 20 out of 29 patients died; (3) 15 out of 29 patients had delayed hypersensitivity reactions at vaccine injection sites, and of those, 9 of the 15 remained alive with controlled tumor growth at the time of this report: (4) 9 out of 9 patients with controlled tumor growth had elevated vaccinia antibody titers-a measure of nonspecific immunostimulation. Although all patients involved in this study had advanced metastatic disease, they did show DH reactions to one or more recall antigens and had tumors that could be safely excised in the operating room. The initial Phase 1 trials demonstrated that the vaccinia oncolysate was safe, nontoxic, and may even have been effective in stimulating the patients' tumor-immune response (9 out of 29 patients with evidence of controlled tumor growth). Wallack (1979b) has begun randomized prospective trials to determine the true efficacy of this approach in the treatment of carcinoma of the colon and rectum, Studies are currently underway at M. D. Anderson Hospital to assess the efficacy of influenza virus-augmented tumor cell extracts in the treatment of carcinoma of the vulva and cervix (R. s. Freedman and J . Bowen, in preparation). Allogeneic cultured tumor cells derived from both a primary squamous carcinoma of the vulva and a primary squamous carcinoma of the uterine cervix were infected with influenza A (PR8) virus. Although the virus was not adapted to grow in these cells, both human tumor lines readily supported replication of the virus. As monitored by membrane immunofluorescence, maximal detectable surface antigenicity preceded maximal release of progeny virus by several hours.


327

The cells remained intact during this period, but plating studies showed that infected cells were unable to proliferate. Influenza virus-augmented tumor cell extract (1.5 mg/0.3 ml) was administered every 2 weeks. Seven patients received extracts of virus-augmented vulva1 carcinoma cells. All patients had been treated with a radical vulvectomy and lymphadenectomy, and had two or more positive regional lymph nodes. In 3 out of 7 cases, deep nodes were also involved. Five patients received X-ray therapy at 3 to 4 months after surgery. Historically, at the M. D. Anderson Hospital, patients with two or more positive lymph nodes have an 80% risk of recurrence, and 60% of recurrences will occur within 12 months of primary therapy. Preliminary evaluation of the patients who received virus-augmented immunotherapy indicates that 5 of the 7 have had no recurrence after more than 34 weeks. No patients have developed recurrence while on therapy, and local or systemic toxicity has not been observed. Tumor-enhancing antibodies were not detected in a lymphocyte-mediated cytotoxicity assay. A randomized prospective study is now in progress in which the effectiveness of radiation therapy plus virusaugmented extracts will be compared with that of radiation therapy alone in the treatment of squamous cervical carcinoma.

C. IMMUNODIAGNOSIS OF HUMAN CANCER WITH VIRUS-AUGMENTED SKINTESTANTIGENS: CLINICAL TRIALS The radioisotopic footpad assay in tumor-immune mice has proved to be a useful animal model for DH reactions to tumor cells and tumor cell extracts (Paranjpe and Boone, 1972; Boone er al., 1974, 1979b; Austin et al., 1977, and submitted for publication). In this assay, tumor-immune mice inoculated in a rear footpad with tumor extracts developed a DH response which was quantitated by determining the increase in leakage of radioiodine-labeled albumin from the blood vessels in the test foot as compared to the contralateral control foot. It appears that a significant proportion of cancer patients give positive DH skin test reactions to CM or soluble extracts from their own tumors. Some of these reports show that CM from allogeneic tumor cells also give positive cancer-specific skin test reactions (see review by Burdick et al., 1975). In studies of virus augmentation of tumor rejection antigens (TATA) in mice, we found that the DH response of tumor-immune mice to tumor cell CM was augmented if the CM were prepared from cells that had been infected with influenza virus (Boone et al., 1974, 1979b; Austin et al., 1977). Although CM extracts of uninfected E4 tumor cells did not induce protective tumor immunity, they did elicit a DH response in tumor-immune

328


mice. However, as stated above, extracts of influenza virus-infected E4 tumor cells elicited a greater footpad DH response. This result was not obtained with egg-grown influenza virus alone or with CM from uninfected cells mixed with egg-grown virus (Austin et al., unpublished observation). We therefore turned to the evaluation of virus-augmented allogeneic tumor cell CM as cancer-specific skin test antigens in humans. We chose melanoma patients because the existence of a tumor tissue-specific antigen has been documented in this disease (Fass et al., 1970; Morton et a / . , 1970; Shiku et a / . , 1976; Liao et a / . , 1978). VSV was chosen as the augmenting virus because of its relatively low pathogenicity in humans (mild flulike syndrome; Fields and Hawkins, 1967). To evaluate crossreactivity of melanoma antigens, we used three different established cell culture lines of human melanoma. Since UV-inactivation of the virus did not abrogate virus-augmented antigenicity in the E4 tumor system in mice (Austin et a / . , in preparation), we used UV-irradiation to inactivate the VSV in these preparations. Unadapted virus sometimes took 4 to 5 days to produce a recognizable CPE. After one passage through the respective melanoma line, the virus always produced marked CPE within 24 hours. Such facile adaption may be due to the acquired membrane compatibility between virus and host cell membranes after tumor cell passage (Young and Ash, 1974). We found that the presence of bovine serum proteins in tumor cell extracts frequently caused a nonspecific reaction when present in skin test materials. The fetal bovine serum was therefore removed from the cell cultures and replaced with 5% human serum several days prior to virus infection. Irie rt a / . (1974) have shown that bovine antigens can be incorporated into the plasma cell membrane during growth of the cells in the presence of bovine serum. Virus-augmented human melanoma cell extracts produced positive skin tests in 41 out of 58 (71%) trials in 20 melanoma patients. Identical CM extracts from the same melanoma lines that had not been infected with VSV gave positive skin tests in 8 out of 58 (14%) trials in the same 20 melanoma patients. In 18 control patients with other cancers or normal volunteers, the virus-augmented extracts were positive in only 3 out of 58 (6%) trials. The virus-augmented CM extracts thus exhibited markedly greater sensitivity without significant loss of specificity, as compared to the unaugmented extracts, when used as tumor-specific melanoma skin test antigens (Boone et nl., 1978, 1979b). Several cultured cell lines of human lung and breast cancer are now being evaluated for their VSV-augmented antigenicity in DH responses of patients with the respective tumor type. The testing of various established cell lines of the same histological type will indicate any cross-reactivity between individual tumors of the same


329

type. Methods that have been developed for the isolation of SV40 TATA of mouse tumor cells (Austin et af., submitted for publication; see Section 111,A,2) are now being applied t o the preparation of soluble virus-augmented human tumor skin test antigens.

IV. Mechanisms of Virus Augmentation of TATA Activity

A. RELATIONSHIP O F VIRAL ANTIGENS TO HOST CELL ANTIGENS In contrast to earlier impressions, it is now generally assumed that the envelope of surface-budding viruses contains little or no host protein (Holland and Kiehn, 1970), but that the carbohydrates and lipids of the virus envelope are host-derived (Laver and Webster, 1966; Klenk and Choppin, 1969). Many virus infections result in the appearance of new virus-induced proteins in the cell surface membrane which can serve as foreign antigens in the host without further modification or after they have been glycosylated by host cell enzymes. Much work has been done to determine the effect of virus replication on cell surface membranes. Extensive reports characterizing influenza virus-induced proteins within the cell surface membrane have appeared (Lazarowitz et al., 1971; Hay, 1974; Meier-Ewert and Compans, 1974), and a vast literature describing influenza virus biosynthesis has been dealt with in several reviews (White, 1974: Compans and Caliguiri, 1975; Klenk et a/., 1975; Ehrnst and Sundqvist, 1976). An excellent summary of the surface antigens of virus-infected cells and methods for their detection are presented in a recent review by Burns and Allison (1977). During influenza virus infection, two nonstructural and seven structural proteins are synthesized. Of the latter, three are glycoproteins that occur on or in the plasma membranes of the infected cell and the envelope of intact virions: the hemagglutinin (HA), neuraminidase (NA), and membrane (M) proteins. The other four are internal proteins (White, 1974). HA appears to be synthesized on the rough endoplasmic reticulum. Glycosylation with glucosamine ensues, followed by migration of the molecule to the smooth endoplasmic membranes where glucose and other sugars are added (Lazarowitz ef nl., 1971: Compans, 1973). Posttranslational cleavage of the HA differs for various virus strains, is host dependent, and occurs in the plasma membrane (Compans, 1973: Lazarowitz et al., 1973). Influenza virus-induced antigens have been demonstrated to move in the plane of the plasma membrane and are present within the entire plasma membrane of the infected cells (Rutter and Mannweiler, 19731, a finding which is consistent with the currently accepted fluid

330


mosaic model of cell membranes (Singer and Nicholson, 1972). It has been suggested that the nucleoprotein (NP) antigen, formerly known as S antigen, may also interact with the plasma membrane, since it is known that large quantities of NP antigen accumulate in the extracellular medium or allantoic fluid and must, therefore, in some way pass through the plasma membrane. N P can be detected by immunofluorescence on the plasma membrane of cultured cells several hours after infection (Virelizier et a / . , 1977). NDV and other paramyxoviruses have many similarities to the orthomyxoviruses (influenza viruses). These viruses have been shown to alter the host cell plasma membranes in several ways, including exposing receptor sites for lectins and inducing rearrangement of receptor sites (Rott et af., 1975: Reeve et a / . , 1975) and producing changes in the phospholipid content of the plasma membrane (Semmel et al., 1975). Semliki Forest virus is a member of the alphavirus group of togaviruses (formerly group A arboviruses), which contain only three or four structural proteins and are the simplest of the enveloped viruses. Viral protein is synthesized on membrane-associated ribosomes, and two proteins are found in the plasma membrane as glyoproteins. Budding of alphaviruses occurs primarily, although not exclusively, at the plasma membrane (reviewed in Fenner e f al., 1974: and in Burns and Allison, 1977). VSV, a well-studied rhabdovirus, codes for two viral proteins which are associated with the plasma membrane shortly after their synthesis. One is not glycosylated and probably makes little contribution to surface antigenicity of infected cells, while the other is a glycoprotein with HA activity (Wagner et a / . , 1971; David, 1973; Morrison and Lidish, 1975). All of the above-described viruses are known to mature by budding through the plasma membrane of the infected cell. By contrast, pox viruses, which are the largest animal viruses, acquire a lipid membrane coat not by budding through cell membranes but by a poorly understood process of membrane biogenesis in cytoplasmic virus factories (Dales and Mosbach, 1968; Ichibashi et al., 1971). Vaccinia virus enters cells by a process of direct fusion between the virus envelope and the plasma membrane, followed by rapid dispersion of viral antigens in the plasma membrane (Chang and Metz, 1976). During maturation, the virus migrates to the Golgi area and is internalized by Golgi cisternae. It then migrates to the cell surface where the plasma membrane and cisternal membrane fuse and eject the virus particle. The virus envelope and cell membranes are probably separated at all times (Dales and Mosbach, 1968). HA is produced on the surface of cells infected with pox virus, but unlike the situation for all other viruses that produce HA, it is found in areas away from where virus is being released (Ichibashi and Dales, 1971).


33 1

According to White (1974), there are at least four distinct mechanisms whereby virus-coded protein could be inserted into the plasma membrane of the infected cell. (1) Viral proteins could be inserted only into newly synthesized membranes. (2) They could be substituted in place of the membrane proteins which are being turned over. (3) Viral proteins could be initially incorporated into areas of cytoplasmic membranes which are noted for their vigorous synthetic activity, namely Golgi vesicles and adjacent areas of smooth membranes. HA and M proteins of influenza virus are found not only in plasma membranes but in other cytoplasmic smooth membranes as well (Holland and Kiehn, 1970; Compans, 1973; Klenk et al., 1975). (4) Viral proteins could be inserted into the plasma membrane by displacing existing protein. Much work has been done to study this latter mechanism, with varying results. Holland and Kiehn (1970) reported that influenza virus envelope proteins completely replaced host membrane proteins only in those spots where the virus buds out, while most of the original host protein was retained. Hay (1974) studied the incorporation of influenza (fowl plague) virus-specific polypeptides into the plasma membrane of infected chick cells and found no apparent displacement of any host cell membrane protein as the result of virus infection. Hecht and Summers (1976) have studied the interaction of VSV with histocompatibility antigens of mouse cells and found a loss of 70% of H-2 activity after VSV infection. A large part (75%) of this activity was recovered in the purified VSV. In experiments described above demonstrating augmented TATA activity after infection with VSV, Wise (1977) found a 50% decrease in H-2 activity of tumor cells after virus infection. By attempting to block the absorption of cytotoxic anti-H-2 antibodies to VSV-infected tumor cells by adding excess antiserum to VSV, no steric blocking of absorption of anti-H-2 antibodies was observed, indicating no spatial association between H-2 and VSV surface antigens. On the other hand, Schrader et al. (1975) studied the functional interaction of viral and H-2 antigens on the surface of infected tumor cells and found that these antigens copatch and cocap as hybrid antigens when exposed to specific antiserum. Since the insertion of virus proteins has been reported to cause a decrease in host cell histocompatibility antigens, it is reasonable to consider that virus infection may also decrease the concentration of TATA on the surface of tumor cells. The relationship between histocompatibility antigens and tumor antigens has been studied and an inverse relationship has been demonstrated between the two antigen concentrations (Haywood and McKhann, 1971; Ting and Herberman, 1971; Cikes et al., 1973; Fujimoto ef al., 1973; Tsakraklides et al., 1974). It has been suggested that tumor-specific antigens may be modified histocompatibility antigens

332


(Germain et al., 1975; Invernizzi and Parmiani, 1975: Thomson et al., 1976). However, these interpretations are uncertain since several investigators have reported the separation of tumor antigens from histocompatibility antigens by affinity chromatography (Appella et ul., 1976; Henriksen et a f . , 1977) or by gel filtration (Siegert et al., 1977). Kon et ul. (1976) were able to distinguish between DH responses to tumor antigens and to H-2 antigens by a footpad swelling assay and found that only tumor antigens were detected in extracts of cultured tumor cells. They could not specify, however, if the tumor antigen inducing DH responses was the TATA since the tumor-rejection activity of the antigen was not assayed. In studies of melanoma patients, Pellegrino et ul. (1977) found no inverse relationship between HLA antigens and melanoma antigens. Therefore, it remains to be determined whether the appearance of viral antigens in the tumor cell surface membrane causes a decrease in the actual number of TATA molecules. In fact, Wise and Acton (1978) recently reported that infection of T-lymphoblastoid cells of AKR mice with VSV had no effect on the expression of Thy-1, H-2K, or gp70 surface antigens, but produced an increase in Gross cell surface antigens (GCSA) and p30 antigens. This selective increase in GCSA followed VSV maturation at the cell surface but preceded the cytopathogenic effect of this virus. It is interesting that enteroviruses, which have been studied for their oncolytic and oncotropic capabilities (described above), have not been shown to induce cell surface antigens in infected cells, even though cytoplastic membranes play an important role in their replication and assembly. Also no reports demonstrating virus-augmented TATA activity of enterovirus oncolysates have appeared. If virus-induced surface antigens are not formed in any detectable amount, then immune responses against the tumor cells in which the virus has replicated are not likely to be important. Neutralizing serum antibody appears to play the major role in defense against enterovirus infections (Allison, 1974). With the above background in mind, several general questions regarding the mechanism of virus augmentation of TATA may now be addressed. 1. Is fully adapted virus necessary to augment TATA activity? If the insertion of viral antigens in the tumor cell surface membrane, juxtaposed to the TATA of the tumor cell, were a sufficient condition to augment TATA activity, then the answer to this question would be “no.” In fact, a virus that could replicate just to the point of inducing a full complement of virus-induced surface antigens, but with a defect in late gene functions related to virus maturation, would be preferable to a fully adapted virus because it would eliminate the problem of infectious virus in the resultant


333

oncolysate. Several studies have demonstrated augmented TATA activity after incomplete virus replication (Hailer and Lindenmann, 1975: Green et a l . , 1976). However, it is not certain that the mere presence of virusinduced antigens is the only factor required for maximum augmentation of tumor antigens. For example, early work by Lindenmann (1970) demonstrated that although VSV-infection of tumor cells was complete at 24 hours after infection, maximum augmentation of TATA immunogenicity did not occur until 48 hours after infection. To ascertain the need for virus adaptation, it would be necessary to perform carefully controlled studies to compare augmentation of TATA immunogenicity at various time points after virus infection. That point in the virus replicative cycle which contributes most to augmenting TATA activity may vary with the type of virus studied. 2. Which is the augmented immrrnogen: The tumor-grown virus or thc virus-infected host-cell membrane;? Which virus-infected cell fraction is most immunogenic? Most studies have shown that the major portion of immunogenic activity resides in the plasma membrane fraction of virusinfected cells, with little activity in soluble cytoplasmic fractions or in highly purified virus grown in tumor cells. Original studies which demonstrated TATA activity in tumor-grown viruses did not claim to have purified the virus to any great extent (Lindenmann, 1971, 1973). However, in studies of an SV40-transformed tumor cell line, Ansel(l974) and colleagues (Ansel et al., 1977: Huet and Ansel, 1977) demonstrated immunogenicity of purified tumor-grown VSV and attributed this immunogenicity to the incorporation of host cell SV40 TATA into the lipid core of the virus. The role of lipid may prove important since it has been recently reported that lipophilic agents can increase the immunogenicity of chemically modified tumor cells and soluble antigens (Prager and Gordon, 1978). The role of intracellular membranes has not been well studied, but it would not be surprising if these membranes also possessed augmented TATA activity. The recent finding of TATA activity in nuclear fractions of SV40-transformed tumor cells (Anderson et al., 1977: Rogers et a l . , 1977) raises the question of whether this phenomenon is characteristic of SV40-transformed tumors only, of virus-induced tumors in general, or whether it may also have relevance to chemically induced or spontaneous tumors. The role of the host cell nucleus in the replicative cycle of influenza virus is still unclear. 3 . What is the nature of the association between iiiriis antigen and tumor antigen? Is there a special spatial relationship.?Are they coi~ulently linked? Can they be copurified and retain augmented TATA activity:) There have been conflicting results on the nature of the association of these antigens. Work in progress indicates that antigens solubilized from

334


virus-infected tumor cells can be further fractionated while still retaining augmented TATA activity (Austin and Boone, unpublished results). Methods successful for purification and characterization of histocompatibility antigens have not been as useful for the isolation of tumor antigens. Most attempts to purify tumor antigens have been frustrated by low yields or loss of antigenic activity (see review by Law and Appella, 1975). It is important to keep in mind that soluble antigens may induce an altered immune response solely because of differences in the form in which they are presented to the host’s immune system. 4. What is the effect qf inrrctivntioii of infectious virus in viral oncolvsates? Most published studies claim little decrease of virus-augmented immunogenicity after inactivation of virus infectivity either by UV-irradiation or inactivation with formalin. However, further controlled studies would be helpful to establish more exactly what, if any, contribution to augmented activity is due to the infectious nature of the viru.s. In summary, many of the conclusions concerning the interaction of virus-induced antigens with host cell antigens have been reached by inductive reasoning from results produced in similar systems. These conclusions will remain conjectural until more definitive experiments have been performed that elucidate the interaction between the cell surface antigen induced by the augmenting virus and the TATA of the host tumor cell.

B. AUGMENTED INDUCTION RESPONSE

OF THE

PRIMARY ANTITUMOR IMMUNE

1 . Biological Mechanisms Several possible mechanisms have been considered to explain the augmented TATA activity of extracts of virus-infected tumor cells, including ( 1) simple adjuvant action, (2) chemical stabilization, (3) neuraminidase action, and (4) helper antigen activity. The role of the virus acting as a simple adjuvant was ruled out in early experiments which demonstrated that merely mixing egg-grown virus with homogenates of uninfected tumor cells did not augment the immunogenicity of the tumor cell extracts (Lindenmann and Klein, 1967a; Boone et a l . , 1974). The requirement for virus replication in the tumor cells to augment TATA immunogenicity has been repeatedly demonstrated. Boone ez al. (1974) considered the possibility that a structural alteration of the tumor cell plasma membrane might occur during viral replication that could result in stabilization of the TATA against chemical degradation following cell disruption. How-


335

ever, this mechanism was ruled out by the demonstration that mice made immunologically tolerant to influenza virus (by pretreatment with Cytoxan plus virus) could no longer be immunized against the tumor by injection with influenza virus-infected tumor homogenates. The role of the enzymic activity of the viral neuraminidase was also considered, since neuraminidase has been shown to unmask antigenic components of tumor cells (Simmons and Rios, 1972; Rios and Simmons, 1976). However, treatment of tumor cells with purified neuraminidase preparations did not result in augmented TATA activity (Boone et al., 1974). Many subsequent studies have involved viruses which do not possess neuraminidase activity, e.g., VSV (Lindenmann, 1970; Hakkinen and Halonen, 1971; Boone et al., 1974; Ansel, 1974; Wise, 1977). This is not to say, however, that the neuraminidase glycoprotein spike of the influenza viruses may not interact in some other antigenic fashion to augment TATA activity. It is interesting to note that Gillette and Boone (1976) found that influenza virus-infected tumor cell homogenates were more immunogenic than those prepared from VSV-infected tumor cells. Most of the evidence to date points to a helper antigen role for virus antigens in stimulating an increased response to TATA in extracts of infected tumor cells, as discussed by Mitchison (1970). In addition to the above-mentioned experiment demonstrating that mice made tolerant to influenza virus could no longer be immunized with virus-infected tumor cell homogenates, Boone et al. (1974) showed that priming mice with egg-grown virus reduced the degree of tumor immunity that could be induced with virus-infected tumor cell homogenates. Similar results were reported in a study of the immune response to vaccinia virus-augmented xenoantigens (Bandlow and Koszinowski, 1974). Although these findings are opposite to those of Lindenmann and Klein (1967a) who demonstrated increased induction of immunity in virus-primed mice, both sets of results demonstrate involvement of the host’s immune system in the virus augmentation phenomenon. The variable effects of priming with virus may be due to differences in dose, route, and timing of the priming and immunizing inoculations. The early finding that the addition of antivirus antibody abrogated the augmented immunogenicity of viral oncolysates (Lindenmann and Klein, 1967a) has been interpreted as evidence for juxtaposed virus antigens and tumor antigens functioning through the host’s immune system. Interestingly, Crum and McGregor (1977) recently demonstrated that soluble tumor-associated antigens were rendered immunogenic when incubated briefly with living BCG. Imrnunization with either BCG or tumor protein alone or with mixtures made under circumstances designed to impede association of tumor protein with BCG failed to stimulate an immune response. Their results imply

336


that the immunopotentiating power of BCG was related in some way to the proximity of the BCG antigens and the tumor-associated antigens. The helper antigen effect of virus-induced cell surface antigens has been shown to augment the immune response to alloantigens and xenoantigens, as well as to tumor antigens. Infection with Sendai virus, vaccinia virus, and herpesvirus simplex type I enhanced the primary humoral immune response to Thy-1 surface antigens in virus-primed congenic mice. Both antiviral priming of the host and virus infection of the immunizing cell were necessary to obtain the full response (Lake and Bromberg, 1979; Bromberg et al., 1979). In addition, guinea pigs demonstrated an increased cell-mediated immunity against xenoantigens after active immunization with vaccinia virus-infected xenogeneic cells (Bandlow and Koszinowski, 1974). The reproduction of the virus in the cell against which the increased immune response was directed was a prerequisite for its augmenting effect. 2 . Intertiction of Vir-ris and Tiitnor- Antigens with the Host's Immirne

Systcm The details of the mechanism by which virus infection of tumor cells augments the immune response to their TATA remains to be worked out. We know for certain that during virus replication, virus-specific antigens are inserted into the host cell plasma membrane in close proximity to, or possibly chemically linked with, the TATA, and that the afferent limb of the host immune system, during the process of responding to the virus antigen, also responds more strongly to the adjacent TATA than it would ordinarily have done in the absence of the vicinal virus antigen. Enough facts are known to construct the general hypothesis that the TATA and virus antigens share one or more soluble or cellular mediators, both specific and nonspecific, of the afferent immune response to the virus antigens. For example, lymphocyte chemotactic factors, macrophage migration inhibition factor, and other lymphokines released by T lymphocytes responding specifically to the virus antigen could serve to locally concentrate lymphoid cells which would also be able to participate in the afferent response to the nearby TATA. Factors similar to "allogeneic effect factor" could also be involved in enhancing the response. As another example, macrophages activated by the virus antigen could phagocytose, process, and present to helper T cells both the virus antigen and TATA. The idea is tenable that virus antigens on the cell surface membrane, especially the hemagglutinins, are "sticky" glycoproteins which could


337

facilitate the binding of adjacent TATA to lymphocytes and/or macrophages. As pointed out previously (Section IV,B,l), in virus augmentation of TATA, the virus antigens appear to operate through the host’s immune system. Lindenmann (1979b) has proposed that virus infection could alter the “antigen presentation” of the TATA either by increasing their number, conformation, or topological association on the tumor cell or on the macrophage membrane after processing. However, if this were the case, the following facts would not have been determined. ( 1 ) Priming mice with virus alone enhances (Lindenmann, 1974; Rukavishnikova and Alekseyeva, 1976) or diminishes (Beverley et al., 1973; Boone et ul., 1974) the augmented immunogenicity of virus-infected tumor cell homogenates. (2) Homogenates of virus-infected cells to which antiviral antibody has been added do not show augmented TATA immunogenicity (Lindenmann, 1974: Rukavishnikova and Alekseyeva, 1976). (3) Mice made tolerant to influenza virus could no longer be immunized with virus-infected tumor cell homogenates (Boone rt ul., 1974). The mechanisms of interaction of the various helper and effector cells in an immune response are popular subjects in current immunology research, and new theories are constantly being presented and evaluated. (For a concise and clear review of today’s knowledge of the cellular basis of the immune response, see Golub, 1977.) We know from studies of humoral immunity that helper T cells can operate via close proximity to B cells. For example, T-cell interactions with the carrier portion of an antigen facilitate B-cell responses to the hapten portion. Close association of helper T cells with effector T cells on the same stimulator cell surface has also been shown to occur in the cell-mediated immune response. It seems reasonable to postulate that the presence of a virus-induced neoantigen on the cell surface could stimulate helper T cells to assist effector T-cell precursors in recognizing adjacent antigenic determinants such as the TATA. This attractive hypothesis is supported by reports that viruses have been shown to generate helper T-cell activity for antibody production in primary immunization against certain alloantigens which would otherwise fail to induce an immune response in congenic hosts (Lake and Bromberg, 1978; Bromberg et al., 1979). To induce this response, it was shown that the helper determinant must be carried on the same cell to be effective, but not necessarily on the same molecule. Also, priming with virus increased this effect. We would expect that the net result of immunizing a tumor-bearing host with virus-augmented TATA would be the generation of a larger, or

338

F A Y E C. AUSTIN A N D CHARLES W . BOONE

a more intensely reactive, population of effector lymphocytes and humoral factors that would be active against the TATA as it appears on tumor cells within the main tumor mass away from the site of immunization.

c. VIRUS-AUGMENTED DELAYEDHYPERSENSITIVITY S K I N TESTS The proposition that the presence of a strong helper antigen within the plasma membrane, closely associated with the tumor antigen, could stimulate the generation of cellular or soluble mediators that would synergistically intensify the DH response to the tumor antigen, as previously described for virus augmentation of a primary immune response, suffers from the fact that the 48-hour time period in eliciting a DH response may be too short a time to see any helpful effect from an intense primary response to the virus antigen. In the absence of further data, we favor the working hypothesis that the virus antigen may produce a helper effect by generating a mild acute inflammatory response, producing soluble and cellular mediators that intensify the DH response to the tumor antigen. This effect apparently still requires a close interaction of virus and tumor antigens, since only a slight augmentation effect is obtained if the virus is simply mixed with tumor cell extracts before injection (Austin et NI., unpublished observation).

V. Prospects for the Application of Virus-Augmented Tumor Antigens in lmmunodiagnosis and lmmunotherapy

A. IMMUNODIAGNOSIS

It is clear that without the existence of a TATA in a given cancer patient, both nonspecific immunotherapy ,such as with BCG, and specific immunotherapy with a TATA vaccine would be impossible. Yet the presence, strength, and cross-reactivity of TATA in the different tissue types and grades of human cancer is far from sufficiently documented. In our view, skin testing with virus-augmented tumor membrane extracts is a clinically simple way to carry out this documentation. We therefore feel that the initial goal of skin test antigen development should be to screen patients with different types of cancer for the presence, strength, and cross-reactivity of their TATA, with secondary emphasis on a concurrent exploration of the use of skin test antigens to diagnose cancer and monitor its clinical course. Obviously, virus augmentation of TATA


339

can be effective only when a TATA exists. High priority should be given to the development of stable, soluble, or homogeneously dispersed, purified cancer skin test antigens.

B. IMMUNOTHERAPY A small number of clinical immunotherapy trials using virus-augmented tumor cell extracts are currently in progress (see Table IV), and there are indications that more may follow. At present, results are too preliminary to draw any definite conclusions. Therapeutic effects are often assessed by various in vitro assays of the cell-mediated immune response. However, tumor-specific DH skin test reactions have been demonstrated to correlate better with the patients’ clinical status (Leventhal et al., 1972). We feel that skin testing with autologous or, somewhat less desirably, allogeneic non-virus-augmented tumor cell extracts should be performed prior to and concurrently with virus-augmented TATA immunotherapy. Conversion of a negative tumor-specific skin test to a positive one during the course of a therapeutic regimen would be highly valuable proof of the efficacy of virus-augmented TATA therapy. In the meantime, we await further clinical results with cautious enthusiasm.

VI. Summary

Although many animal tumor cells possess TATA on their surface membrane, their immunogenicity is lost when the tumor cells are disrupted. Stemming from studies of the virus therapy of cancer, it was found that infection of tumor cells with a lytic virus that induces viral cell-surface antigens will augment the TATA activity of the tumor cell homogenate, or oncolysate. Substantial data have indicated that the mechanism of virus augmentation operates through the host’s immune system. Positive results in immunoprophylaxis and immunotherapy with virus-augmented TATA in animal model systems have led to the initiation of several attempts at immunotherapy with virus-augmented TATA in human cancer patients. In addition, virus-augmented crude membrane extracts of cultured human tumor cells have been shown to elicit enhanced delayed hypersensitivity skin tests without any loss of skin test specificity. The results of immunotherapy clinical trials are still too preliminary to draw any definite conclusions, but initial results are encouraging.

340

FAYE C. AUSTIN AND CHARLES W . BOONE

ACKNOWLEDGMENT The authors wish to especially acknowledge and thank their friend and colleague, Dr. Noritoshi Takeichi, Cancer Institute, University of Hokkaido School of Medicine, Sapporo, Japan, for his critical experimentation and high level of productivity which did much to further the progress of research on virus augmentation of TATA.

REFERENCES Al-Ghazzouli, I. K., Donohoe, R. M., Huang, K. Y., Sass, B., Peters, R. L., and Kelloff, G. J . (1976). J. Inimrtnol. 117, 2239-2248. Allison, A. C. (1974). Transplmt. Rev. 19, 3-55. Anderson, J . L.. Martin, R. G., Chang, C., Mora, P. T., and Livingston, D. M. (1977). Virology 76, 420-425. Ansel, S. (1974). In/. J. Cancer 13, 773-784. Ansel, S., Huet C., and Tournier, P. (1977). I n / . J. Cancer 20, 51-60. Appella, E., Law, L . W., and Henriksen, 0. (1976). Cancer Res. 36, 3539-3544. Asada, T. (1974). Cancer 34, 1907-1928. Am. Austin, F. C., Takeichi, N., Robbins, M. L., and Boone, C. W. (1977). Annrr. MPC/. Soc. Microhiol. (Abstr.), 83. Axler, 1).A., and Girardi, A. J. (1970). Proc. Am. Assoc. Ccrncer Res. 11, 4. Bandlow, G., and Koszinowski, U. (1974). Arch. Cescrmre Virrr.~for.scIi.45, 122-127. Belisario, J. C., and Milton, G. W. (1961). Airst. J. Dawza/ol. 6, 113-118. Beverley, P. C. L., Lowenthal, R. M., and Tyrrel, P. A. J. (1973). I n / . J. Cancer 2, 212216. Bluming, A. Z., and Ziegler, J. L. (1971). Loncer 2, 105-106. Boone, C. W. (1972). Nail. Cancer Ins/. Monogr. 35, 301-307. Boone, C. W. (1974). Recent Re.s/tl/s Cancer Res. 47, 394-400. Boone, C. W., and Blackman, K. (1972). Cancer Res. 32, 1018-1022. Boone, C. W., and Gillette. R. W. (1977). In “Modulation of Host Immune Resistance in the Prevention or Treatment of Induced Neoplasias” ( M . A. Chirigos, ed.), GPO Fogarty lnternational Center Proceedings No. 28, pp. 191-194, Washington, D.C. Boone, C., Blackman, K., and Brandchaft, P. (1971). Natrrre (London) 231, 265-266. Boone, C. W., Orme, T . W., Blackman, K., and Gillette, R. (1973). J. Natl. Cancer Inst. 51, 1141-1144. Boone, C. W., Paranjpe, M., Orme, T., and Gillette, R. (1974). I n / . J. Cancer 13,543-551. Boone, C. W., Austin, F. C., Gail, M., Case, R., and Klein, E. (1978). Cancer 41, 17811787. Boone, C. W., Takeichi, N., Austin, F. C., Gotohda, E., Oikawa, T., and Gillette, R. (1979a). Cann Monogr. Cancer Res. (in press). Boone, C. W., Austin, F. C., Gail, M., Paranjpe, M., and Klein, E. (1979b). Gann Monogr. Cancer Res. (in press). Bromberg, J., Brenan, M., Clark, E. A., Lake, P.,Mitchison, N. A., Nakashima, I.. and Sainis, K. B. (1979). Gann Monogr. Cancer Res. (in press). Burdick, J . F., Wells, S. A., Jr., and Herberman, R. B. (1975). Surg. Cynecol. Ohsrtt. 141, 779-794. Burdick, K. H. (1960). Arch. Dermutol. 82, 438-439. Burdick, K. H., and Hawk, W. A. (1964). Cancer 17, 708-712.

VIRUS AUGMENTATION OF TUMOR C E L L EXTRACTS

34 1

Burns, W. H., and Allison, A. C. (1977). I n ”Virus Infection and the Cell Surface” (G. Poste and G. L. Nicholson, eds.), pp. 213-247. Elsevier North-Holland, Amsterdam. Cassel, W. A , , and Garrett, R. E. (1965). Cancer 18, 863-868. Cassel, W. A., and Garrett, R. E. ( 1966). J . Brtcteriol. 92, 792. Cassel, W. A,, and Garrett, R. E. (1967a). Crtncer 20, 433-439. Cassel, W. A., and Garrett, R. E. (1967b). Cancer 20,440-444. Cassel, W. A.. Murray, D. R., l’orbin, A. H., Olkowski, Z. L., and Moore, M. E. (1977). Cancer 40, 672-679. Chang, A., and Metz, D. H. (1976). J . G m . Virol. 32, 275-282. Cikes, M., Friberg, S., Jr., and Klein, G. (1973). J. Nut/. Crtncer I n s t . 50, 347-362. Compans, R. W. (1973). Virology 51, 56-70. Compans, R. W., and Caliguiri, L. A. (1975). In “Negative Strand Viruses” (B. W. J . Mahy and R. D. Barry, eds.), Vol. 2, pp. 573-594. Academic Press, New York. Crum, E. D., and McGregor, D. D. (1977). Int. J. Cancer 20, 805-812. Csatary, L. K. (1971). Lance1 2, 825. Dales, S., and Mosbach, E. H. (1968). Virology 35, 564-583. David, A . E. (1973). J. M o l Biol. 76, 135-148. DeMarchi, J. M., and Kaplan, A. S. (1977). 1. Virol. 23, 126-132. Dowdle, W. R., Downie, J . C., and Laver, W. G. (1974). J. Virol. 13, 269-275. Eaton, M. D., and Almquist, S. J. P. (1975). Proc. Soc. Exp. Biol. Med. 148, 1090-1094. . 1089Eaton, M. D., Levinthal, J. D., and Scala, A . R. (1967). J . Nntl. CNI?(’eI.I I I J I39, 1097. Eaton, M. D., Heller, J . A., and Scala, A. R. (1973). Cancer Res. 33, 3293-3298. Ehrnst, A., and Sundqvist, K.-G. (1976). Exp. Cell Biol. 44, 198-225. Eiselein, J., and Biggs, M. W. (1970). Cancer Res. 30, 1953-1957. Eiselein, J . E., Biggs, M. W.. and Walton, J . R. (1978). Cuncer Rcs. 38, 3817-3822. Evermann, J . F., and Burnstein, T. (1975). I n / . J . Cancer 16, 861-869. Fass, L., Herberman, R. B., Ziegler, J. L., and Kiryabuire, J . W. M. (1970). Lnncet I , 116-1 18. Fenner, F., McAuslan, B. R., Mims, C. A., Sambrook, J., and White, D. 0. (1974). “The Biology of Animal Viruses” (2nd ed.). Academic Press, New York. Fields, B. N., and Hawkins, K. (1967). N. Engl. J . M i d . 277, 989-994. Fujimoto, S., Chen, C. H., Sabbadini, E., and Sehon, A. H. (1973). J. In~rwoml.111, 10931100.

Gerber, A,, Sauter, C., and Lindenmann, J. (1973a). Arch. Geximte Viriisfor~ch.40, 137151.

Gerber, A., Sauter, C., and Lindenmann, J. (1973b). Arch. Giwtrntc, Vir/tsf0rsch. 40, 255264. Germain, R. N., Dorf, M. E., and Benacerraf, B. (1975). J. Exp. Med. 142, 1023-1028. Gillette, R. W., and Boone, C. W. (1976). Int. J. Cancer IS, 216-222. Golub, E. S. (1977). “The Cellular Basis of the Immune Response.” Sinauer, Sunderland, Massachusetts. Green, A. A., Webster, A. G., and Smith, K. (1975). Proc. Am. Assoc. Cancer Res. 16, 271. Green, A. A., Pratt, C., Webster, R. G., and Smith, K. (1976). Ann. N. Y. Acad. Sci. 277, 396-4 I I. Griffith, I. P., Crook, N . E., and White, D. 0. (1975). Br. J. Cancer 31, 603-613. Hakkinen, I., and Halonen, P. (1971). J . Natl. Cancer Inst. 46, 1161-1167. Haller, O., and Lindenmann, J. (1975). J . N u t / . Cancer Inst. 54, 459-464. Hay, A. J. (1974). Virology 60, 398-418.

342


Haywood, G. R., and McKhann, C. F. (1971). J. Exp. M e d . 133, 1171-1187. Hecht. T. T., and Summers, D. F. (1976). J . Virol. 19, 833-845. Henriksen, 0.. Law, L. W., and Appella, E. (1977). J. N u t / . Crrncer I n s t . 58, 1785-1788. Holland, J . J.. and Kiehn, E. D. (1970). Scicvici. 167, 202-205. Huet, C., and Ansel, S. (1977). I n / . J . Cuncer 20, 61-66. Hunter-Craig, I., Newton, K. A., Westbury, G., and Lacey, B. W. (1970). Br. M i d . J . 2, 5 12-5 15. Ichibashi, Y., and Dales, S . (1971). Virology 46, 533-543. Ichibashi. Y . , Matsumoto, S., and Dales, S . (1971). Virology 46, 507-532. Illiger, H . J . , Sauter, C., and Lindenmann, J. (1975). Cancer Rc~s.35, 3623-3627. Invernizzi, G., and Parmiani, G. ( 1975). Nutrrrc, (London) 254, 713-714. Irie, R. F., Irie, K., and Morton, D. L. (1974).J. N o r / . L'otri.i>rI n s t . 53, 1545-1551. Klein, P. A. (1967). Pnthol. Microhiol. 30, 222-245. Klein, P. A. (19741. Arch. Gesumte Virrrsforsch. 45, 199-208. Klein, P. A., and Lindenmann, J . (1965). Pu//iol. Microhiol. 28, 698-703. Klenk, H.-D., and Choppin, P. W. (1969). Virology 38, 255-268. Klenk, H.-D., Wollert, W., Rott, R., and Scholtissek, C. (1975). In "Negative Strand Viruses" (B. W. J . Mahy and R. D. Barry, eds.). Vol. 2 , pp. 621-634. Academic Press, New York. Kobayashi, H. (1970). Actu Ptithol. J o p u n . 20, 441-450. Kobayashi, H. (1979). Ad,'. Cancer Res. 30, 279-299. Kobayashi, H., and Sendo, F. (1979). Gun/i Monogr. Cuncer Res. (in press). Kobayashi, H., Sendo, F. Shirai, T., Kaji, H., Kodama, T., and Saito, H. (1969). J . N u t / . Crrnc~~r I n s / . 42, 413-419. Kobayashi, H . , Sendo, F., Kaji, H., Shirai, T . , Saito, H., Takeichi, N., Hosokawa, M., and Kodama, T. (1970). J . Nltrl. Cuncer Inst. 44, I I- 19. Kobayashi. H., Kodama, T., and Gotohda, E. (1977). "Xenogenization of Tumor Cells" (Hokkaido Univ. Med. Libr. Ser.), Vol. 9. Hokkaido Univ. School of Medicine, Sapporo, Japan. Kon. N. D., Forbes, J . T., and Klein, P. A. (1976). I n / . J. Cancer 17, 613-618. Koprowski, H.. Love. R . , and Koprowska, I . (1957). Ti.n-. Rep, Biol. M d . 15, 559-576. Kuzumaki, N., and Kobayashi, H. (1976). 7rrrrisplontrrtion 22, 545-550. Kuzumaki, N., Fenyo, E. M., Giovanella, B. C., and Klein, G. (1978). I n / . J. Cuncw 21, 62-66. Lake, P., and Bromberg, J . (1979). Proc. I2th Lerrkocyto C r h r c , Conf. (in press). Laver, W. G., and Webster. R. G. (1966). Virology 30, 104-115. Law, L. W., and Appella, E. (1975). In "Cancer: A Comprehensive Treatise" (F. F. Becker, ed.), Vol. 4, pp. 135-154. Plenum Press, New York. Lazarowitz, S. G . , Cornpans, R. W., and Choppin, P. W. (1971). Virology 46, 830-843. Lazarowitz, S . G . , Cornpans, R. W., and Choppin, P. W. (1973). Virology 52, 199-212. Leventhal, B. G . , Halterman, R. H., Rosenberg, E. B., and Herberman, R. B. (1972). Currcvr Rcs. 32, 1820-1825. Liao, S.-K., Leong, S. P. L., Sutherland, C. M., Dent, P. B., Kwong, P. C., and Krementz, E. T. (19781. Cancer Rcs. 38, 4395-4400. Lindenmann, J . (1963). Pro(,. Soc. E.rp. Biol. M i d . 113, 85-91. Lindenmann, J . (1964). J . I n i n n r n ~ l 92, . 912-919. Lindenmann, J . (1970). Arch. G e s u n m Virrr.s:forsch.31, 61-70. Lindenmann, J . (1971). Proc. R. Soc. London, Ser. B 176, 419-423. Lindenmann, J . (19731. C'iho Swnp. 18, 197-215. Lindenmann, J . (1974). Biochini. Biophys. Acfo 355, 49-75.


343

Lindenmann, J. (1977). I n “Modulation of Host Immune Resistance in the Prevention or Treatment of Induced Neoplasias” (M. A. Chirigos, ed.), No. 28, pp. 187-190, GPO Fogarty International Center Proceedings, Washington, D.C. Lindenmann, J. (1979a). Gann Monogr. Cancer Res. (in press). Lindenrnann, J . (1979b). Garin Monogr. Cancer Res. (in press). Lindenrnann, J . , and Klein, P. A. (1967a). J . Exp. Med. 126, 93-108. Lindenmann, J . , and Klein, P. A. (1967b). Recent Resrtlts Cancer Res. 9. McMurtrey, M. J . , Campos, L. T., Sinkovics, J . G., Romero, J . J., Loh, K . K., and Romsdahl, M. M. (1976). I n “Neoplasms of t h e Skin and Malignant Melanoma,” pp. 471-484. 20th Clin. Conf. on Cancer, M. D. Anderson Hospital and Tumor Institute, Houston, Year Book Medical, Chicago. Meier-Ewert, H., and Compans, R. W. (1974).J. Virol. 14, 1083-1091. Milton, G. W., and Brown, M. M. L. (1966). Airst. N . Z . J . S ~ i r g .35, 286-290. Mitchison, N. A . (1970). Transplant. Proc. 2, 92-103. Moore, A. E. (1954).Annu. Re\*. Microbiol. 8, 393-410. Moore, A . E. (1960). Prog. ESP. Trimor Res. 1, 411-439. Morrison, T. G . , and Lodish, H. F. (1975). J . B i d . Chem. 250, 6955-6962. Morton, D. L., Eilber, F. R., Joseph, W. L., Wood, W. C., Trahan, E . , and Ketcham, A. S . (1970). Ann. Sirrg. 172, 740-749. Muceniece, A. J . (1972). “Oncotropism of Viruses and the Problems of Virotherapy of Malignant Tumors.” Riga Publishing House, Zinatne, Riga, Latvian SSR (In Russian, summary in English). Muceniece. A. J. (1978). Workshop on I~nmiinologiculX e n o g e ~ i i a t i o nof Trimor Cells. Sapporo, Japan, June, 1978, p. 33 (Abstr.). Murray, D. R., Cassel, W. A., Torbin, A. H., Olkowski, Z. L., and Moore, M. E. (1977). Cuiicer 40, 680-686. Paranjpe, M., and Boone, C. W. (1972). J. Nail Cutrccv Inst. 48, 563-566. Pasquinucci, G. (1971). Lancet 1, 136. Pellegrino, M. A,, Ferrone, S . , Reisfeld, R. A., Irie, R. F., and Golub, S. H . (1977). C u ~ i c u 40, 36-4 1 . Prager, M. D., and Gordon, W. C . (1978). Cirncer Res. 38, 2052-2057. Reeve, P., Poste, G., and Alexander, D. (1975). [ ti “Negative Strand Viruses” (B. W. J . Mahy and R. D. Barry, eds.), Vol. 2, pp. 859-873. Academic Press, New York. Rios, A., and Simmons, R. L. (1976). Ann. N. Y . A c a d . Sci. 276, 45-60. Roenigk, H. H., Deodhar, S., St. Jacques, R., and Burdick, K. (1974). Arch. Dermutol. 109, 668-673. Rogers, M. J., Law, L. W., and Appella, E. (1977).J . Natl. Cancer Inst. 59, 1291-1295. Rott, R., Becht, H., Hammer, G., Klenk, H.-D., and Scholtissek, C. (1975). I n “Negative Strand Viruses” (B. W. J. Mahy and R. D. Barry, eds.), Vol. 2, pp. 843-857. Academic Press, New York. Rukavishnikova, G. E., and Alekseyeva, A. K. (1976). Acta Virol. 20, 387-394. Rutter, G., and Mannweiler, K. (1973). Arch. Gesamte Virri.sforsch. 43, 169-172. Sauter, C., and Lindenmann, J. (1976). I n “Erkrankungen der Myelopoese” (A. Stacher and P. Hocker, eds.), pp. 280-281. Urban & Schwarzenberg, Munich, Berlin and Vienna (cited in Lindenmann, 1979a). Sauter, C., Gerber, A., Lindenmann. J., and Martz, G. (1972). Schweiz. Med. Wochenschr. 102, 285-290 (In German, summary in English). Sauter, C., Baumberger, U., Ekenbark, S., and Lindenmann, J . (1973). Cancer Res. 33, 3002-3007. Sauter, C., Bachi, T., and Lindenmann, J . (1975). G t r . J . Cancer 11, 59-63.

344


Sauter, C., Cavalli, F., Lindenmann, J . , Gmur, J. P., Berchtold, W., Alberto, P., Obrecht, P., and Senn, H . J . (1978). In "Immunotherapy of Cancer: Present Status of Trials in Man" (W. D. Terry and D. Windhorst, eds.), pp. 355-363. Raven Press, New York. Schrader, J . W., Cunningham, B. A., and Edelman, G. M. (1975). Proc. N o t / . Acud. Sci. U.S.A. 72, 5066-5070. Sedmak, G. V . , Taylor, M. W., Mealey, J . , Jr., and Chen, T . T. (1972). Nutrrri, (London) New B i d . 238, 7-9. Semmel, M., Israel, A., and Audubert, F. (1975). I n "Negative Strand Viruses" (B. W. J. Mahy and R. D. Barry, eds.), Vol. 2, pp. 875-883. Academic Press, New York. Sendo, F., Kaji, H., Saito, H., and Kobayashi, H. (1970). Gunn 61, 223-226. Sharpless, G . R., Davies, M. C., and Cox, H. R. (1950). Proc. Soc. Exp. B i d . M d . 73, 270-275. Shiku, H., Takahashi, T., Oettgen, H . F., and Old, L. J. (1976). J. Exp. Med. 144, 873881. Siegert, W., Fenyo, E. M., and Klein, G. (1977). I n t . J . Cancer 20, 75-82. Simmons, R. L., and Rios, A. (1972). Srrrgery 71, 556-564. Singer, S. J., and Nicholson, G. L. (1972). ScicJnce 175, 720-731. Sinkovics, J . G. (1977). J . Am. Met/. Assoc. 237, 869. Sinkovics, J . G., Thota, H., Loh, K. K., Gonzales, F., Campos, L. T . , Romero, J . J . , Kay, H. D.. and King, D. K. (1974a). I n "Cancer Chemotherapy-Fundamental Concepts and Recent Advances," pp. 417-443. 19th Clin. Conf. on Cancer. Year Book Medical, Chicago. Sinkovics, J. G., Williams, D. E., Campos, L . T., Kay, H. D., and Romero, J. J . (1974b). Semin. O n d . I , 351-365. Sinkovics, J . G., Plager, C., McMurtrey, M. J . , Romero, J. J . , and Romsdahl, M. M. (1977). Proc. An?.Assoc. Crrnccv R P S .18, 86. Smith, R. R., Heubner. R. J . , R o w , W. P., Schatten, W. E.. and Thomas, L. B. (1956). Cuncxv 9, 1211-1218. Southam, C. M . (1960). Truns. N . Y . Acud. Sci. Ser. 2 22, 657-673. Suskind, R. G., Heubner, R. J . , Rowe, W. P., and Lowe, R. (1957). P r ~ cSoc. . Exp. H i d . M1.d. 94, 309-318. Svet-Moldavsky, G. (1979). Gunn Monogr. Cancer Res. (in press). Svet-Moldavsky, G . J., and Hamburg, V. P. (1964). Natrtw (London), 202, 303-304. Svet-Moldavsky, G. J., Gibadullin, R. A., Frotzova, A. E., Hamburg, V. P., Perevodchikova, N., and Bychkov, M. B. (1974). Vosprosi Virrrsol. 6, 713-719 (In Russian, Summary in English). Takeichi, N., Austin, F. C., Oikawa, T., and Boone, C. W. (1978). Ccinctr Res. 38, 45804584. Taylor, M. W., Davidson, J . N., Land, C., and Wall, R. (1970). J . N u t / . C ' u n w I n s t . 44, 515-519. Taylor, M. W., Cordell. B.. Souhrada, M., and Prather, S. (1971). Proc. Nut/. Acnd. Sci. U.S.A. 68, 836-840. Thomson, D. M . P., Gold, P., Freedman, S. 0.. and Shuster, J. (1976). Canccv Rev. 36, 3518-3525. Ting, C.-C., and Herberman, R. B. (1971). Nutiire N t w B i d . 232, 118-120. Tsakraklides, E., Smith, C., Hersey, J. H., and Good, R. A. (1974). J . N u t / . Cancw Inst. 52, 1499-1504. Virelizier, J.-L., Allison, A. C., Oxford, J.. and Schild, G. (1977). Nntrrrc (London) 266, 52-54.


345

Wagner, R. R., Heine, J. W., Goldstein, G., and Schnaitrnan, C. A. (1971). J . Virol. 7 , 274-277. Wallack, M. K. (1979a). Gann Mongr. Cancer Res. (in press). Wallack, M. K. (1979b). J . Surg. Oncol. (in press). Wallack, M. K., Steplewski, Z., Koprowski, H., Rosato, E . , George, J., Hulihan B., and Johnson, J . (1977). Cancer 39, 560-564. Webb. H. E., and Gordon Smith, C. E. (1970). Lancet 1, 1206-1209. Webb, H. E., Wetherley-Mein, G., Gordon Smith, C. E., and McMahon, D. (1966). Rr. Mecl. J . 1, 258-266. White, D. 0. (1974). Cirrr. Top. Microhiol. Irnrnrtnol. 63, 1-48. Wise, K . S. (1977). J. N u / / . Cuncer I n s t . 58, 83-90. Wise, K. S., and Acton, R. T. (1978). Proc. A m . Assoc. Cancer Rcs. 19, 137. Young, N . P., and Ash, R. J . (1974). Appl. Microhiol. 28, 26-30.


SUBJECT INDEX A

Acute phase reactant proteins (ACRP), 143 i n animal tumors systems, 26-27 biological effects of, 28-30 in bladder cancers, 15-17 in breast cancer, 8- 10 extrahepatic synthesis and concentration of. 25-26 in GI tract cancers, 13-15 haptoglobin phenotypic variation of, 2728 in leukemia, 20-23 in liver cancer, 19 in lung cancer, 17- 19 in lymphomas, 20-23 as markers. 3 mathematical analysis of levels of, 30-37 cluster analysis. 36-37 discriminant analysis, 33-35 multivariate methods. 33-37 principal component analysis, 35-36 in peritoneal and pleural effusions, 23-24 production and half-lives of, 4-8 in prostate cancer, 8- 10 Adenomatous polyps, intestinal carcinogenesis and, 183 Animals. tumor systems phase reactant proteins of, 26-27 Antigens of intestinal tumors, 21 1-216 from virally infected tumor cells, 280287 increase of antigenicity of, 287 A-Rad leukemia virus. characteristics of, 67-68 B

Bladder cancer, acute phase reactant proteins in. 15-17 347

Bone marrow. role in leukemia induction, 49-52 Breast cancer acute phase reactant proteins in, 8- 10 hormone dysfunction in, 132- 133

C Cachexia, in cancer patient, 93 Cell-mediated immunity, in intestinal carcinogenesis. 214-2 I6 Cancer ( w e trlso Tumors) acute phase reactant proteins in, 1-43 hormonal tumor syndrome in, 92 immunodiagnosis of, by virus-augmented tumor antigen, 327-329, 338-339 virus therapy of. 303-307 Cancer embryonal antigen (CEA), in colon tumors, 21 I Carbohydrate metabolism, in tumor-host relationship, 93- 102 Carcinogenesis, hydrazine role in, 151- I64 Colon, antigens in lesions of, 213 D

Delayed hypersensitivity skin test, virusaugmented, 338 Diet, in intestinal carcinogenesis, 190- 193 Dimethylh ydrazine interaction, with cell components, 922226 as intestinal carcinogen, 168-169, 216226 modification, 221-222 tumor types, 171 metabolic pathways of, 219 DNA of herpesviruses, 259-262 in transformed cells, 264-266 DNA synthesis, in tumor hosts, 109- 112

348

SUBJECT I N D E X

D-Rad leukemia virus. characteristics of, 66-67 E

Enzymes in intestinal carcinogenesis, 207-208 in tumor hosts, 1 15- 125 Enteroviruses, in cancer therapy, 306-307 Environment, hydrazine in, I53 Epithelial tumors, classification of, 170 Epstein-Barr virus antigens from, 258-266 in human tumors, 247 properties of, 243 tissue-culture studies of, 249-258 Estrogens, intestinal carcinoma and, 213

G Gastrointestinal tract cancers, 165-237 acute phase reactant proteins in. 13- IS Genetics in intestinal carcinogenesis, 184- 185 of leukemia induction, 75-78 Glucose, tumors as traps for, 93-102, 139I40 Glycogen reserves. tumor effects on, 96 Glycoproteins, ACRPs as, 1-43 Gynecological cancers, acute phase reactant proteins in, 15- 17 H

Herpesvirus(es) (lymphotropic), 239-278 antigens from, 258-266 DNA of, 259-262 in transformed cells, 264-266 experimental tumor studies on, 248-249 lytic infections of. 251 in natural hosts, 244-249 persistant and latent infections from, 246-248 proteins of, 258-259 survey of, 240-244 tissue culture studies of, 249-258 transformation by, 25 1-258 viral diseases from, 244-246 H c q w s v i r u s ofi4i>.s.properties of, 24 I , 243

H(vpc,sviru.s of chimpanzee. properties of, 243 HrrpcJsvirus p ~ i p i properties ~. of, 243 Hcrpesvirus pongo, properties of, 243 Hrrpe.sviru.s suiiniri (HVS). 239 antigens from, 258-266 in natural host, 244-248 properties of, 242 tissue-culture studies of, 249-258 H r r p t w i r u s of turkeys (HVT), properties of. 240-24 I , 242 Hcrprsvirrts .s.dvilngu.s. properties of, 240, 242 Hodgkin's disease, hydrazine therapy of, 161 Hormonal tumor syndrome, 92 Hormones disorders in tumor hosts, 127-133 ectopic, from cancer patients, 92 Hyperthermia, as tumor therapy, 138- 139 Hydrazine( s) i n carcinogenesis. 151- 164 experiments on, 155-157 in humans, 159-160 mechanism, 158- 159 degradation of, 156 effects on liver. 152-153 in environment, 153 in Hodgkin disease therapy, 161 intestinal polyps and tumors from, 158 toxic effect of, 152

I

Immunodepression, in tumors, 133- 137 Immunology, of intestinal carcinogenesis, 189-190, 211-216 Influenza virus tumor cell antigenicity from. 3 10, 3 12315 mechanisms, 329 therapeutic use. 321 Intestinal cancer, 165-237 age factors in, 187 biochemistry of, 206-21 I cells of, kinetics of. 201-204 dietary factors in, 190- 193 electron microscopy of, 174- 177 enzyme role in, 208-21 I

349

SUBJECT INDEX

experimental models for, 166- I69 transplants, 169 tumor induction, 166- 169 factors affecting, 184- 196 genetic factors in. 184- 185 histochemistry of. 177- 179 hydrazine-induced, 158 immunological aspects of, 189- 191, 21 1. 216 antigens, 212-213 cell-mediated immunity, 2 14-216 intestinal function factors, 193- 197 kinetics of cell division in 196-201 microbiological aspects of, 187- 189 morphogenesis of, 179-184 morphology of, 169- 184 mucosal injury and, 196 nuclear proteins function in, 207-208 nucleic acid function in, 206-207 polyp role in, 183 sex hormone role in, 185-187 stem epithelial cell role in. 204-206 Infections, with cancer, 89, 92 Isonicotinylhydrazide as carcinogen, 153- 155 in humans. 159-160

L

Lactic dehydrogenase (LDH), in tumor hosts, 124 Leukemia acute phase reactive proteins in, 20-23 genetic control of, 75-78 induction of, in mice, 45-87 age-related susceptibility. 52-54 by leukemia virus, 62-81 thymus and bone marrow role, 49-52, 54-57 virus radiation induction of, 57-59 Leukemia virus radiation induction of, 57-59 variants of, 64-69 A-Rad LV, 67-68 D-Rad LV, 66-67 Rad LV, 64-66 RS-Rad LV, 68-69 target cells for, 69-72

Lipid metabolism, in tumor hosts, 112- I 15 Liver. hydrazine effects on, 152-153 Liver cancer, acute phase reactive proteins in, 19 Lung cancer, acute phase reactant proteins in, 17-19 Lymphoid tumors, radiation-induced, 5961 Lymphomas. acute phase reactive proteins in, 20-23 Lymphotropic herpesviruses, 239-278 M

Marek's disease virus (MDV), 239 antigens from, 258-266 in natural host, 244-248 properties of, 242 tissue-culture studies of, 249-258 Mathematic analysis, of acute phase reactant protein levels, 30-37 Methylhydrazine(s) as cytotoxins, 160 natural occurrence of, 153 Mice. leukemia induction in. 45-87 Microbiology, of intestinal carcinogenesis, 187- I89 Mucosa, intestinal, injury to, in carcinogenesis, 196 Mumps virus, i n cancer therapy. 307 Mushrooms, hydrazine in. 153 N

Newcastle disease virus tumor cell antigenicity from, 3 I I mechanism, 330 therapeutic use, 321 Nitrogen metabolism, in tumor patients, 102- I12 Nuclear proteins, in intestinal carcinogenesis, 207-208 Nucleoside kinases, in tumor hosts. 121

P Peritoneal effusions, acute phase reactive proteins in, 23-24

350

SUBJECT INDEX

Pleural effusions. acute phase reactive proteins in, 23-24 Polyps, adenomatous, intestinal carcinogenesis and, 183, 193- 194 Prostate cancer, acute phase reactant proteins in, 8- 10 R

Rad leukemia virus characteristics of, 64-66 immunization by, 72-75 phases of, 78-81 Radiation, leukemia induction by, 45-87 RNAs, in tumor host tissue, 125- 127 RNP complexes, in tumor host tissue, 12s- 127 RS-Rad leukemia virus, target cells for, 69-72

S Semliki Forest virus tumor cell antigenicity from, 3 I I mechanisms, 330 Sendai virus, tumor cell antigenicity from, 311 Sex hormones, in intestinal carcinogenesis. 185- I87 Small intestine tumors, antigens, 212 Stem epithelial cells, in intestinal carcinoge ne si s, 204- 206 T Tumor-associated transplantation antigen (TATA) viral augmentation of. 301-345 mechanisms, 329-338 Tumor cells antigenicity of virus augmentation, 301-341 animal studies, 308-320 human studies, 305-309, 320-327 immune response, 334-338 viral xenogenization of, 279-299 antigens of. 280-287 definition of, 286-287 immunogenicity increase in, 290 therapeutic aspects, 291-292

Thymus role i n leukemia induction. 49-52 phenotypic characteristics, 54-57 Thyroid function, in cancer patients, 132 Tissue culture. in herpesvirus studies. 249258 Tobacco. hydrazine in, 153 Transformation. by herpesviruses, 251-258 Tumors and tumor hosts ( w e trlso Cancer) cells of. sec’ Tumor cells competitive relationships with host, 93I so clinical aspects. 137- 139 effect on host tissue biology, 1 IS- 137 enzymes. 1 15- I25 RNAs, 125-127 as glucose traps. 93- 102 hormone disorders in, 127- 133 host relationships of, 89- 150 hyperthermia therapy of, 138-139 immunodeppression in, 133- 137 lipid metabolism in, 112- I IS markers for, list, 5 nitrogen metabolism i n , 102- I I2 therapy of, using viral xenogenization. 29 1-292

V

Vaccinia virus, in cancer therapy, 307. 311. 321 Vesicular stomatitis virus tumor cell antigenicity from. 3 I I mechanisms, 330 Viruses cancer therapy by. 303-307 in animals, 303-309 in humans, 305-309, 320-327 nature of, 318-320 leukemia, radiation induction of, 57-59 xenogenization of tumor cells by, 279299 X

Xenogenization of tumor cells, by viruses. 279-299 immune response against. 292-295

CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis C . A . Coirlsoii

Epidermal Carcinogenesis E . V . Coitdry The Milk Agent in the Origin of Mammary Tumors in Mice L. D l l l O C ~ l l O l t ~ S ! i i Hormonal Aspects of Experimental Tumorigenesis T . U . Grirrinu Properties of the Agent of Rous No. I Sarcoma R. .IC .. Hrrrris Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism Cho rlr s H c,itlolhcrgcr

The Carcinogenic Aminoazo Dyes J f r r i w s A . Millrr r i i i i l Eli~rihrrhC'. M i l l ~ r The Chemistry of Cytotoxic Alkylating Agents M. C . .I. Ross Nutrition in Relation to Cancer Alhcrr Toiirrc~rihairriiairrl Herhut Silwrstorlr

Plasma Proteins in Cancer Ric.liarcl .I. Wiiizler A U T H O R INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules

Carcinogenesis and Tumor Pathogenesis I . Br~rerrblur?~ Ionizing Radiations and Cancer Austin M . Brrws Survival and Preservation of Tumors in the Frozen State Jrinir.s Craigic,

Energy and Nitrogen Metabolism in Cancer Lroirarcl D. FcirilingPr aiid G. Burroirghs Mitier

Some Aspects of the Clinical Use of Nitrogen Mustards C'rrlviii T . K l o p p cirri1 Jcurlir(, C . Bcrf(,ilraii Genetic Studies in Experimental Cancer L . w. La113 The Role of Viruses in the Production of Cancer C . Ohorlirig uiid M . Giwriii Experimental Cancer Chemotherapy C . Ch(,stor Stock A U T H O R INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Richirril Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P . Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pulliiiuii o r i d B . P ~ I / / I ~ I N I I Some Aspects of Carcinogenesis

P. Rollclorli

P r t w Al~xrrriilcr

Pulmonary Tumors in Experimental Animals Mic~haolB. Shimkiii

Chemical Constitution and Carcinogenic Activity G . M . Brrclgrv

35 I

352


Oxidative Metabolism of Neoplastic Tissues Sidm,y W~,irlhousc, AUTHOR INDEX-SUBJECT INDEX

Volume 4 Advances in Chemotherapy of Cancer in Man Siclnc,y Fnrhrr. RiiiIolf Toch. Edicwd Muririirig Siwrs. urrd Doririld PiriAc4

The Use of Myleran and Similar Agents in Chronic Leukemias D. A . G . ~ n / t f J l l The Employment of Methods of Inhibition Analysis in the Normal and TumorBearing Mammalian Organism Ahrahnrii Goldiri Some Recent Work on Tumor Immunity P. A. Gowr

Inductive Tissue Interaction in Development Clij/orcl Grohs/cirr

Lipids in Cancer Frciricc,.~L . Hui’cii nrril W . R . Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . L U C U S S UN~. IP.~ ~ BUN . H o i . R . Dciiit l c ~ l .nnd F. Zujclc,lii The Hormonal Genesis of Mammary Cancer 0.Mithlhoc~X AUTHOR INDEX- SU BJ ECT INDEX

Volume 5

The Newer Concept of Cancer Toxin Wnro Ntiknhurn a r i d FiiriiiXo Fuhiiokri

Chemically Induced Tumors of Fowls P. R . Pcci COCA Anemia in Cancer V i t i ~ t ~Ei .r P r i c , arid ~ Rohrrt E . Gri~c~rrjicltl Specific Tumor Antigens L . A . Zilher Chemistry, Carcinogenicity. and Metabolism of 2-Fluorenamine and Related Compounds Eliz(rhrth K . Weishurgi,r arid Johii H . Wc,i.shrrrgc,r AUTHOR INDEX-SUBJECT INDEX

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscur Bodansky The Plant Tumor Problem Arriiiri C. Bruurr u i i c l Hidrrry N . Wood Cancer Chemotherapy by Perfusion Osc.iir C’rc,cc,h. Jr. tirrtl Et1liw-d T . KrcIill’lltZ

Viral Etiology of Mouse Leukemia Liit1nYi.k

Cross

Radiation Chimeras P . C. KolliJr. A . J . S. Dniics. urrtl S h c 4 r r M . A . Docik

Etiology and Pathogenesis of Mouse Leukemia J . F . A . P. Millcr Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid C . M . Tii?irrris Behavior of Liver Enzymes in Hepatocarcinogenesis Grorgi) W r h r r

Tumor-Host Relations R . W . Bcgg

AUTHOR INDEX-SUBJECT INDEX

Primary Carcinoma of the Liver Chur1e.s B ~ r ~ t ~ i i i i

Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P . N . C‘cirirphcll

Volume 7 Avian Virus Growths and Their Etiologic Agents .I. W . Beard

CONTENTS O F PREVIOUS VOLUMES

353

Mechanisms of Resistance to Anticancer Agents R . W . Brocknian Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M. Court Brotiln a d Ishbel M . T o u g h Ethionine Carcinogenesis Etiiiiiuiiud Furber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and H a n s L. Falh Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. NPgroIii

The Relation of the lmmune Reaction to Cancer Louis v. coso Amino Acid Transport in Tumor Cells R . M . J o h n s t o n c and P . G . S c h o l i $ d d Studies on the Development, Biochemistry. and Biology of Experimental Hepatomas Harold P . Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes. and Bone Marrow Cells 1. F . Seitz


Carcinogens. Enzyme Induction. and Gene Action H . V . Crlhoiti I i i Vitro Studies on Protein Synthesis by Malignant Cells A . Clark Gr(fli’ti The Enzymatic Pattern of Neoplastic Tissue w. Eigc,no Knox Carcinogenic Nitroso Compounds P. N. Mngc’c, crtrtl .I. M. Brrrnos The Sulfhydryl Group and Carcinogenesis J . S . Harriiigtoii The Treatment of Plasma Cell Myeloma Daniel E . Bcrgsccgel, K . M . Gri’jji’th, A . Haul. arid W . .I. StricLlcy. J r .

Volume 8 The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses i n General A . F . Hoii~atsoti Nuclear Proteins of Neoplastic Cells Harris Birsch and Willirrtii J . Stc’i40 Nucleolar Chromosomes: Structures. Interactions, and Perspectives M. J . K o ~ c Jatid ~ , Glaclys M . Mw,cyXo Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H . F . Kraybill crntl M . B . SIiin~Xin Experimental Tobacco Carci noge ne si s Enic,st L. Wyntlrr r i n d Dic,tric,h H o j $ i i ~ i i AUTHOR INDEX-SUBJECT INDEX

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Ric~k~irrl Stn/llba/rgh ( / , I d SidIiCy WOiIi/1ou.sr

AUTHOR INDEX- SU BJ ECT INDEX

Volume 10


Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethan Sitltic~yS . M i n i s / / Runting Syndromes. Autoimmunity, and Neoplasia

D. K m s t Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit

354


The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the intercellular Environment and Their Role in Cell Physiology Williuttl Rcgrlsoil Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Josc~plrC'. Arc.os otrtl Mury F.Argus AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE INDEX

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G. P a s t r r i d Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G . 1. Dc~ii~hititrtt Replication of Oncogenic Viruses in VirusInduced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hutiajirsa Cellular Immunity against Tumor Antigens Karl Erik Hc,llstrtiin atrrl Iiigiyyril Hcllstram

Perspectives in the Epidemiology of Leukemia Irving L . Kessler uritl Ahraliarn M . Lilienfeld AUTHOR INDEX-SUBJECT INDEX

Volume 13 The Role of lmmunoblasts in Host Resistance and lmmunotherapy of Primary Sarcomata P . Alexander and J . G . Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Osu~alilJarrett

The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Huii~iGiiishurg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gqjutlaii V . Sherbet The Characteristics of Animal Cells Transformed i t ~Vitro ~ ( 1 1 7 Moc.plii,r.soii Role of Cell Association i n Virus Infection and Virus Rescue J . Sl~ohoilaN I I 1. ~ Hlolrinc~k Cancer of the Urinary Tract D . B . C'luysori arid E . H . Coop1.r Aspects of the EB Virus M. A . Epstritc AUTHOR INDEX-SUBJECT INDEX

Volume 14 Active lmmunotherapy GiwrgcJs Math4 The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization E n i t s t Winocorrr Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events Gcnrgc Meycr Passive lmmunotherapy of Leukemia and Other Cancer Rolund Motta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalf' Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . 1. Abidev Low Dose Radiation Cancers in Man Alice Stritwrr AUTHOR INDEX-SUBJECT INDEX


Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J . S . Bittc,l. S . S . T(,rvthia. anil J . L . Mc.1nick

Nasopharyngeal Carcinoma (NPC) J . H. C. H o Transcriptional Regulation in Eukaryotic Cells A . J . MucGillit,ra.v, J . Pairl. arid G. Thrclfill1

Atypical Transfer RNA's and Their Origin in Neoplastic Cells Erric,.st Borpk anil Sylvia J . Kerr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip .I. Fialkoit, Electron Spin Resonance Studies of Carcinogenesis Harold M . Sttwrtz Some Biochemical Aspects of the Relationship between the Tumor and the Host V . S . Shapot Nuclear Proteins and the Cell Cycle Gary Stein and Renaro Baserga AUTHOR INDEX-SUBJECT INDEX

355

1,3-Bis(2-Chloroethyl)- I-Nitrosourea (BCNU) and Other Nitrosoureas i n Cancer Treatment: A Review St(,phcn K. Cartcv. Frank M . Schahcl. Jr.. Laii,rc,nc,c, E. Brodcr, and Thorvus P. Johnston AUTHOR INDEX-SUBJECT INDEX

Volume 17 Polysaccharides in Cancer: Glycoproteins and Glycolipids Vijai N . Nigani and Antonio Cantc,ro Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei, South Africa Gi,rald P. Warli-ick and John S . Harington

Genetic Control of Murine Viral Leukemogenesis Frank Lilly and Th~wclorc~ Pincus Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K. Nazmriun Mutation and Human Cancer Alfred G . Knudson. Jr. Mammary Neoplasia in Mice S . Nandi and Charles M. McGruth AUTHOR INDEX-SUBJECT INDEX

Volume 16 Polysaccharides in Cancer Vijai N . Nigam arid Antonio Cantero Antitumor Effects of Interferon Ion Gresscu Transformation by Polyoma Virus and Simian Virus 40 Joc, Sanibronk Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overheding? Sir Alexander Haddobiz The Expression of Normal Histocompatibility Antigens in Tumor Cells Alena LengiJrovd

Volume 18 Immunological Aspects of Chemical Carcinogenesis R . W . Baldtisin lsozymes and Cancer Fanny Schapira Physiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the Liver Yec Chu Toh Immunodeficiency and Cancer John H . Kersey, Beatrice D. Spector. and Robert A . Good

356


Recent Observations Related to the Chemotherapy and Immunology of Gestational Chonocarcinoma K . D . Bogsliaw Glycolipids of Tumor Cell Membrane Stti-itiroh Hokomori Chemical Oncogenesis in Culture C‘harlcs Hi,idelhivger AUTHOR INDEX-SUBJECT INDEX

Principles of Immunological Tolerance and lmmunocyte Receptor Blockade G . .I. V . N o s s a l The Role of Macrophages in Defense against Neoplastic Disease Mic hac4 H. Lei:\’ untl E . Fretlorich WhiJeloch

Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis P . Sims and P. L . Grovc,r Virion and Tumor Cell Antigens of C-Type RNA Tumor Viruses Hviiiz Bairn

Volume 19 Comparative Aspects of Mammary Tumors J . M. Hamilrori The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howwrcl M . Ttviiin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J . H . Coggin. Jr. and N . G . Aiitlrrsoii Simian Herpesviruses and Neoplasia Frcdrich W . Deinharilt. Laitwiice A . Falk. and L a u r ~ nG . Wo@ Cell-Mediated Immunity to Tumor Cells Ronald B . Herbermmi Herpesviruses and Cancer F r d Rapp Cyclic AMP and the Transformation of Fibroblasts Ira Pastan arid Gtorge S . Johtison Tumor Angiogenesis Jutlah Folkman SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M. C . Rapin and Max M . Burgcv

Addendum to “Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?” Sir Ali,xatrdcr Hadcloi~~ SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Micliad 8 . Shimhin arid Gary D. Storriv Cell Deazh in Normal and Malignant Tissues E. H . CoopcJr. A . J . Bi~lfi,rd,and T . E . Keiiiiy

The Histocompatibility-Linked Immune Response Genes Baruj Bc~naccrraf’mil David H . Karz Horizontally and Vertically Transmitted Oncornaviruses of Cats M . Essc,x Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms K t e f A . Rafferty. Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G . B. Clemmis The Role of DNA Repair and Somatic Mutation in Carcinogenesis James E . Trosko and Ernest H . Y . Chu SUBJECT INDEX


357

Volume 22

Volume 24

Renal Carcinogenesis J . M . Hatnilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects. Congenital Malformations, and Carcinogenic Potential Susan M . Sii+iv ant1 Richard H . Atlutnson Interrelationships among RNA Tumor Viruses and Host Cells Ragrnond V . GiIdi>n Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Rohlin. lih-Nan Chorr. atiil Ptritl H . Black lmmunodepression and Malignancy

The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology'? J . P . Lc,iy und .I. C . Lrclarc, Organization of the Genomes of Polyoma Virus and SV40 Mike Fried arid Bcivrl! E . G r i j j / n @,-Microglobulin and the Major Histocompatibility Complex

Osins Stutrllnn SUBJECT INDEX

Volume 23 The Genetic Aspects of Human Cancer W . E . Heston The Structure and Function of Intercellular Junctions in Cancer Ronald S . Weinstein. Frcdv-ick B . Mcrk. and Joscph Alroy Genetics of Adenoviruses Harold S . Gitistwrg and C . S . H . Young Molecular Biology of the Carcinogen, 4-Nitroquinoline I-Oxide Minuko Nagao and Takashi Sugimuru Epstein-Barr Virus and Nonhuman Pnmates: Natural and Experimental Infection A . Frank, W . A . Anditnun. arid G. Miller Tumor Progression and Homeostasis Richmond T . Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav Hill and Jana Hillova SUBJECT INDEX

P u A . Pctorson. Lars Rusk. untl Lurs osth(,rg

Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachirn Mark Temperature-Sensitive Mutations in Animal Cells Cluuilio Basilicw Current Concepts of the Biology of Human Cutaneous Malignant Melanoma WullacP H . Clark. J r . . Mic~hnrlJ . Mosrrangdo. Ann M. Ainsii.orth. Dai*icl B u d , Rohivt E . B~ll1,t.und Ei~rliriu A . Bcrtiurtlirio SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA F. L . Graham Malignancy and Transformation: Expression i n Somatic Cell Hybrids and Variants H a r v q ~L . O z t r and Krishna K . Jha Tumor-Bound Immunoglobulins: 111 Sitrr Expressions of Humoral Immunity Isaac P . Witz TheAh Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S . Thorgc.irssori and Danirl W . Nebert

358


Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds a n d Alkylating Agents Aritlioriy E . Pt'gg Immunosuppression and the Role of Suppressive Factors in Cancer Isno k'niri(~~ i i t Hcrrtiii/i l Fricilrtrcrri Passive lmmunotherapy of Cancer in Animals and Man Sti,wrr A . Ro.sijirhi.rg n r i t l Willicrrii I). Tiwy

The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Hirroltl B . H r n , i t t Mass Spectrometry in Cancer Research Johrr Robor Marrow Transplantation in the Treatment of Acute Leukemia E . Doriiitill Tlioirius. C . D~arr Buc,!,tii,r. Alc.wriclc,r Fqfi.r. Purr1 E . Nc,iriirrrl. crriil Rciiiior Storh Susceptibility of Human Population Groups to Colon Cancer Mrirtiri Lipkiii

SUBJECT INDEX

Natural Cell-Mediated Immunity Roiicrltl B . Hrrh(,rrirciii o r i d Hoiiuril T . H(11tlc~ri

Volume 26

SUBJECT INDEX

The Epidemiology of Large-Bowel Cancer P e I ~ y oCorroo trritl Willicriir Htierr.sz,cl Interaction between Viral and Genetic Factors in Murine Mammary Cancer . I . HilRors urid P. B i , i i t w I w i i Inhibitors of Chemical Carcinogenesis L w W . W(rito/ihi,rg Latent Characteristics of Selected Herpesviruses Jl71~AG. s/(,lY,rr.s Antitumor Activity of Coryrirhric,/c,riirr?r puri,rr/ir mil(/.^ t / / i t l Mortiir

Li/l\(i

T. S ~ o t t

SUBJECT INDEX

Volume 28 Cancer: Somatic-Genetic Considerations F . M . Brrrrict Tumors Arising in Organ Transplant Recipients Isrriol Peirii Structure and Morphogenesis of Type-C Retroviruses Roriciltl C . Moiitclnro rrrril Doni P . Bologr10.si

BCG in Tumor lmmunotherapy Rohrrt W . B d d i ~ , i ~uriil i Mokolin

Volume 27 Translational Products of Type-C RNA Tumor Viruses Johir R . S t r p h e n s o i i , Srr.sliilkuiiior G. Devnrc. nritl Fri4 H . R ~ y i i ( ~ l J~r1. . ~ . Quantitative Theories of Oncogenesis Alii,i, S. Whittomore Gestational Trophoblastic Disease: Origin of Choriocarcinoma. lnvasive Mole and Choriocarcinoma Associated with Hydatidiform Mole. and Some Immunologic Aspects J . 1. Brcwrr, E . E . Toro!,. B . D. Kaliciii, C . R . Sititiliopo. irriil B . HolpiJrri

V.

Pillllll

The Biology of Cancer Invasion and Metastasis lsaitrh .I. Fitllc,r. DoriR1u.s M . G r r s t ( ~ t i r. ~ r t l luii R . Hnrt Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis A . Burtry. F . Bcx. H . Chcriitreirtie, Y . Clcrrfrr. D. D r h c ~ g c ~.I.l . Ghysdiic.1. R . K C ~ / I I I ~ IM I I.I ILivli~rcq, . J . Leiirr(,ri. M . Mnrnriiorickx. criril D . Portotc,llc Molecular Mechanisms of Steroid Hormone Action Stopheii .I. Higgiris atid Ulriclr Gc,lrririg SUBJECT INDEX


Volume 29

Influence of the Major Histocompatibility Complex on T-cell Activation J . F . A . P . Millrr Suppressor Cells: Permitters and Promoters of Malignancy'! Dmid Nuor Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer Josc' Urii4 The Role of Glutathione and Glutathione STransferases in the Metabolism of

359

Chemical Carcinogens and Other Electrophilic Agents L . F . Chus.sc~uut1 a-Fetoprotein in Cancer and Fetal Development Erl,ki Roo,sluhti uric1 Morkkrr Srppiilii Mammary Tumor Viruses Dutr H . Moore. Curolr A . Long. AXhil B . Viiiclw. Joc4 B . Shejjii4tl. Ar/iold S . Diou. n r i t l E/ioitic, Y . L u s f u r g u ~ s Role of Selenium i n the Chemoprevention of Cancer A . C ' l d Grqjirr SUBJECT INDEX


Advances in Cancer Research, Volume 30

Advances in Botanical Research, Volume 30

Advances in Botanical Research, Volume 30

Advances in Food Research Volume 30

Advances in Drug Research, Volume 30