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ADVANCES IN CANCER RESEARCH VOLUME 61

This Page Intentionally Left Blank

ADVANCES IN CANCERRESEARCH Edited by

GEORGE F. VANDE WOUDE ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 61

ACADEMIC PRESS, INC. Harcourt Brace EL Company

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS,INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-4311 United Kingdom Edition published by

Academic Press Limited 24-28 Oval Road. London NWI 7DX

International Standard Serial Number: 0065-230X International Standard Book Number: 0- 12-006661-0

PRINTED IN THE UNITED STATES OF AMERICA 9 3 9 4 9 5 9 6 9 1 9 8

QW

9 8 1 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 61 . . . . . . . . . . . .

.......

....

ix

Cancer Prevention Research Trials PETER G R E E N W A L D ,

WINFRED F. M A L O N E , HARRIET R. S T E R N

MARY

E.

CERNY,

AND

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

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

11. Basic Research Stiidies: Surveying earch ..................... 111. ’The Research Strategy: Froin Basic Research to Huinan Applications ............................... ....... IV. Progress i i i CIinirdl Trials . . . . ................................. .............. ........ V. C:onclusions . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . ..................................

I 2

7 I:!

‘Lo 21

Molecular Genetic Changes in Human Breast Cancer MARCJ.VAN I. Introduction

DE Vl.JVER

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

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

I l l . Genetic Changes in Hum;in Breast (hnc ........................ I V. Genetic Predisposition t o Breast Ca~icer. . . . . . . . . . . . . . .

V. (:onclutling Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... ............. References . . . . . . . . . . .

V

30

50

vi

CONTENTS

Molecular Approaches to Cancer Therapy

MARKA. ISRAEL 1. I l i t rod uct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Cancer as a Molecular Disorder ...........................

I l l . Molecular Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Molecular Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Regulation and Mechanism of Mammalian Gene Amplification GEORGER. STARK Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Amplitication ........ Primary Mechanisnis o f An1 Hypotheses Integrating Regulation of Amplifcation with the Chroniatidic Teloniere Fusion Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Evolution of Amplifecl DNA ................................... References ..................

1. 11. 111. IV.

87 89

10.1 108

Ill

Unraveling the Function of the Retinoblastoma Gene

ELDADZACKSENHAUS, ROD B R E M N E R , Z H E J I A N G , R. MONTGOMERY G I L L , MICHELLEMUNCASTER, MARYSOPTA, ROBERTA. PHILLIPS, A N D BRENDAL. (;AI.LIE I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Genetics of Retinoblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. I l l . The K B 1 Gene ;IIKI Protein Protluri

I V. Binding o f Viral Oncoproteins t o pl IOft”’ ............................ V. Mechanisnis of R B I Gene Regulation ......................... VI. Interaction i)f pl lO/(/J/ with Cellular P VII. Functions of‘ pl IOU/’/ . . . . . . . . . . . VIII. Tissue Specific Susceptibility to K B IX.

115 I 1 (i

I I8 I ‘LO

vii

CONTENTS

Tumor Promotion by Inhibitors of Protein Phosphatases 1 and 2A: The Okadaic Acid Class of Compounds HIROTAFUJIKIA N D MASAMISUCANUMA Introduction . . . . . . . . . . Okadaic Acid Class 1000

> 1000 > 1000 0.6 40.0 0.7

a Phosphorylated histone HI (10,000cpm/pg) was incubated with fraction containing PP-2A partially purified from mouse brain and test compounds for 10 min at 30°C. The inhibitory effects of PP-I with these compounds closely resembled those presented in this table.

TUMOR PROMOTION BY INHIBITORS OF PROTEIN PHOSPHATASES

161

199la). Three murine monoclonal antibodies against okadaic acid were prepared (Usagawa et al., 1989). These antibodies will provide similar results.

G. APPARENT“ACTIVATION” OF PROTEIN KINASES Okadaic acid enhanced incorporation of 32Pinto histone III-S in vitro by incubation with [Y-~~PIATP, protein kinases and protein phosphatases, as reported in Section 11, B. We called this the apparent “activation” of protein kinases (Sassa et al., 1989). Next we studied the consequence of inhibition of PP-1 and PP-PA by okadaic acid in the cells. A hyperphosphorylated protein with a molecular mass of 58 kDa was found in cell lysates of primary human fibroblasts treated with okadaic acid or dinophysistoxin-1 at concentrations of 100 nh4 for 2 hr (Fig. 8). The hyperphosphorylated 58-kDa protein was not induced by teleocidin, one of the TPA-type tumor promoters. Therefore, this hyperphosphorylation was thought to be the result of inhibition of PP-1 and

FIG.8. Hyperphosphorylated vimentin in primary human fibroblasts treated with the okadaic acid class compounds: control ( I ) , okadaic acid (2), dinophysistoxin-1 (3) and calyculin A (4), at a concentration of 100 nM.

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HIROTA FUJIKI A N D MASAMI SUGANUMA

PP-2A by okadaic acid or dinophysistoxin-1 in primary human fibroblasts (Yatsunami et al., 1991b). The hyperphosphorylated 58-kDa protein in the cell lysates specifically reacted with monoclonal and polyclonal anti-vimentin antibodies. In addition, in vitro phosphorylation of vimentin was enhanced dose-dependently by okadaic acid or dinophysistoxin-1 in the presence of PP-2A and protein kinases; that is, it reproduced the apparent “activation” of protein kinases in primary human fibroblasts by treatment with okadaic acid or dinophysistoxin-1 (Yatsunami et al., 1991b). Vimentin is one of the intermediate filaments, mainly found in various mesenchymal cells (Steinert and Roop, 1988). Various types of intermediate filaments can be hyperphosphorylated in various cells treated with okadaic acid. When human keratinocytes (PHK 16-1 cells), immortalized by human papilloma virus type 16 DNA (HPV 16 DNA), were treated with okadaic acid or dinophysistoxin- 1, six hyperphosphorylated proteins with molecular masses of 60, 58, 56, 52, 42, and 27 kDa were found. Immunoprecipitation and Western blot analysis revealed that the hyperphosphorylated proteins, except 27 kDa, were identified as cytokeratin peptides CK5, CK6, CK7, CK16, and CK19, respectively, and the 27-kDa protein was a heat-shock protein, HSP 27 (Yatsunami et al., 199313). Similar phosphorylation patterns in cytokeratins were not induced in human keratinocytes treated with TPA o r teleocidin. However, the phosphorylation of HSP 27 was induced by TPA or teleocidin. These results indicated that a greater number of target proteins are phosphorylated by the okadaic acid pathway than by the PKC pathway, and some proteins, such as HSP 27, are commonly phosphorylated by both pathways. Hyperphosphorylation of intermediate filaments is associated with morphological changes and is suggested to affect cell cycle regulation directly. In these experiments, the cells were treated with okadaic acid or dinophysistoxin-1 at concentrations of 10 to 100 nM, to obtain marked hyperphosphorylation and morphological changes. These concentrations are reasonable in view of the fact that the intracellular concentrations of PP-1 and PP-2A are often in the range 100 nM to 1 pA4 (Cohen et al., 1990). Therefore, we think the biochemical reaction and morphological changes caused by okadaic acid might be ongoing, to some extent, in the cells, at concentrations even lower than 100 nM. What protein kinases are involved in hyperphosphorylation of intermediate filaments is not well known. There are some reports that ~ 3 4 ‘ ~kinase ‘~ phosphorylates vimentin during mitosis (Chou et al., 1990) and that CAMP-dependent protein kinase, cyclic nucleotide-independent protein kinase, PKC, and Ca2 -calmodulin-dependent pro+


163

tein kinase I1 phosphorylate vimentin and cytokeratins (O’Connor et al., 1981; Gilmartin et ad., 1984; Huang et al., 1988; Tokui et al., 1990). We think the sites phosphorylated by these protein kinases are sustained by inhibition of PP-1 and PP-2A in the cells by treatment with okadaic acid. To summarize our understanding of the okadaic acid pathway: okadaic acid binds to PP-1 and PP-2A in the cell membrane; is incorporated into the cells; and inhibits activities in the cytosol and nuclei, resulting in an increase of phosphorylation of proteins, such as hyperphosphorylation of intermediate filaments in the cells. In the nuclei, okadaic acid causes sustained activation of gene expression, as well as hyperphosphorylation of suppressor gene products (Fujiki, 1992; Guy et al., 1992; Yatsunami et al., 1993a). H. DISTRIBUTION OF [3H]OKADAIC ACID On the basis of the evidence that okadaic acid causes diarrhetic shellfish poisoning, the effects of okadaic acid on rat intestinal epithelium were studied (Edebo et al., 1989). In this section, the preliminary results of [SH]okadaicacid distribution given by two different routes, PO and ip, are presented. Oral administration of [3H]okadaic acid (14 pCi/0.2 ml sesame oil) into the stomach of mice was achieved. Most of the radioactivity (>77%) was found in the contents of the gastrointestinal tract 3 hr after intubation. Nineteen hours later, 4% was found in the contents of the gastrointestinal tract and 30% was found in the feces. At 3 and 19 h r after intubation, 1% of the radioactivity was found in the liver. These results suggested that most of the [3H]okadaic acid remained in the gastrointestinal tract and some had interacted with cell membranes (R. Nishiwaki et al., unpublished results). The effects of ip administration of okadaic acid were first reported by Terao and associates (1986). They found that severe injury to the absorptive epithelium of the duodenum and upper part of the small intestine of suckling mice was induced within 15 min of administration of dinophysistoxin- 1. A visible increase in the permeability of the villial vessels of the small intestine, the presence of numerous vesicles in the cytoplasm of the epithelium, and marked destruction of the Golgi apparatus were observed (Terao et al., 1986). [3H]Okadaic acid (28 pCi/0.2 ml saline solution) was injected ip into mice. Most of the radioactivity (33.3%) was found in the contents of the gastrointestinal tract 3 hr after intubation and 5% was found 19 hr later. However, 27.4% of the radioactivity was found in the liver 3 hr after injection and 15.9% was found 19 hr after, suggesting that ip-administered [3H]okadaic acid was

164

HIROTA FUJIKI AND MASAMI SUGANUMA

excreted through hepatobiliary circulation (R. Nishiwaki et al., unpublished results). Intraperitoneal administration of 50 and 100 pg/kg okadaic acid into rats released glutamic pyruvic transaminase (GPT) from the liver into the blood serum, with 38 and 50 IU/liter 24 hr later, respectively, whereas that of the control saline released levels of 18 IU/Iiter. The results indicated that ip administration of okadaic acid has some effects on the liver (Ohta et al., manuscript in preparation). However, the main target tissue of okadaic acid administered by the two routes was the epithelium of the gastrointestinal tract, which contains high amounts of the okadaic acid receptors, as has been previously reported (Suganuma et al., 1989).

I. BIOCHEMICAL AND BIOLOGICAL EFFECTS Although okadaic acid has a simple mechanism of action, the okadaic acid pathway includes the discordant regulation of both protein kinases and protein phosphatases, resulting in induction of various biochemical and biological effects in various cell lines. This is called the pleiotropic effects of a tumor promoter. In this section, biochemical and biological effects related to tumor promotion, as well as unique properties induced by okadaic acid, are summarized briefly. Okadaic acid, at concentrations of 10 to 100 ng/ml (12.4 to 124 nM), stimulated prostaglandin E, production in rat peritoneal macrophages. T h e potency at a concentration of 12.4 nM was as strong as TPA at 16.2 nM, 20 hr after incubation. However, okadaic acid required a lag phase before stimulation. Since cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonicacid-labeled macrophages and prostaglandin E, production, protein synthesis was a prerequisite reaction for stimulation of arachidonic acid metabolism (Ohuchi et al., 1989). Okadaic acid induced angiogenesis in the chorioallantoic membrane of the chick embryo. T h e potency was one order of magnitude stronger than that of TPA. There was a difference between the time courses of angiogenesis induction by okadaic acid and TPA (Oikawa et al., 1992). Okadaic acid stimulated mouse macrophages to produce colony-stimulating factors (CSFs), which induced granulocyte macrophage colony-forming unit (CFU-GM) colony formation. However, okadaic acid inhibited the erythroid colony-forming unit (CFU-E) colony formation induced by erythropoietin (Oka et al., 1989). Although we now know that okadaic acid binds to the catalytic subunits of PP-1 and PP-2A, the interaction of okadaic acid with a lipid bilayer membrane was studied to obtain information on its incorporation into the target cells. Okadaic acid was not easily distributed into a


165

dipalmitoyl phosphatidylcholine membrane and did not significantly change the membrane structure. Okadaic acid freely permeated through the lipid membrane of multilayer vesicles in a liquid-crystalline state, supporting the evidence that okadaic acid gains access to receptors in the cytosol (Nam et al., 1990). Mammalian cells treated with okadaic acid at concentrations of 0.1 to 1 CLM showed the morphological characteristics accompanying apoptotic death: condensation of chromatin, shedding of cell contents through surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. Okadaic acid showed internucleosomal DNA fragmentation in rat promyelocytic IPC-8 1 cells and neuroblastoma cells. However, the extent of DNA fragmentation was much less than that by CAMPat concentrations equipotent for inducing morphological apoptosis and cell death (Bge et al., 1991). In addition, okadaic acidtreated cells did not accumulate in early S-phase, but rather decreased the rate of transition from G, to S, indicating that okadaic acid inhibited DNA replication by regulatory influence rather than by direct DNA damage (Bge et al., 1991). However, okadaic acid inhibited apoptosis induced by either heat treatment or by ionizing radiation exposure to the human B-cell lymphoma cell line, and inhibited dephosphorylation of some proteins involved in apoptosis (Baxter and Lavin, 1992). Okadaic acid increased in vitro phosphorylation of the 100- and 30kDa proteins present in a cytosolic fraction of the mucosa of the rat Adenosine glandular stomach by in vitro incubation with [Y-~~PIATP. diphosphate (ADP) ribosylation by incubation with diphtheria toxin and [3*P]NAD revealed that the 100-kDa protein, which is ADP-ribosylated, is elongation factor-:! (EF-2) (Suganuma et al., 1992b). In addition to protein phosphatase, Ca2 -calmodulin-dependent protein kinase 111, which phosphorylates EF-2 specifically, was present in homogenated Ehrlich ascites tumor cells. Phosphorylation of EF-2, at defined stages of the cell cycle, in the presence and absence of okadaic acid showed that the highest protein kinase activity was found in cells from the early Sphase, whereas protein phosphatase activity was most pronounced during the G, plus M phases (Carlberg et al., 1991). Treatment of cells with okadaic acid increased phosphorylation of various proteins: the EGF receptor, in several cell types (HernandezSotomayor et al., 1991); nuclear proteins, such as a 43-kDa protein in mink lung CC1 64 cells (Kramer et al., 1991) and in human leukemia K562 cells (Zheng et al., 1991); histone H3 and a 33-kDa protein (pp33) in C3H/lOT1/2 mouse fibroblasts (Mahadevan et al., 1991); a progesterone receptor in the chicken oviduct (Denner et d., 1990); and the +

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HIROTA FUJIKI AND MASAMI SUCANUMA

a-subunit of the inhibitory guanine nucleotide binding protein Gi2 (Bushfield et al., 1991). Some of the unique properties induced by okadaic acid are as follows: stimulation of glucose transport and 2-deoxyglucose uptake (Haystead et al., 1989; Tanti et al., 1991); insulin-mimetic action (Haystead et al., 1990); maturation and maturation promoting factor (MPF) formation in Xenopzls laevis oocytes (Goris et al., 1989); lymphocyte proliferation (Grove and Mastro, 1991); neurotoxicity against rat cerebellar neuron in primary cultures (Fernandez et al., 1991); increase of the amplitude of the synaptic response in the frog (Abdul-Ghani et al., 1991); differentiation of human breast tumor cells to the cells associated with mature phenotypes (Kiguchi et al., 1992); absence of inhibition of junctional communication in hamster embryo cells (Rivedal et al., 1990) and in BALB/3T3 cells and human and mouse keratinocytes (Katoh et al., 1990); and absence of induction of Epstein-Barr virus early antigen in Raji cells (S. Yoshizawa et al., unpublished results). J. GENEEXPRESSION AND TRANSCRIPTIONAL REGULATION

Since 1986, understanding of gene regulation by tumor promoters, such as TPA, has grown enormously (Rahmsdorf and Herrlich, 1990). Herschman and associates studied induction of mRNA of the TPAinduced-sequence (TIS) genes, which are rapidly and transiently induced by TPA, in C3H/lOT1/2 cells and Swiss 3T3 cells (Lim et al., 1987). They first reported that okadaic acid induced TIS 1 and TIS 8 mRNA expressions in the cells. Their maximum mRNA accumulations were found at 3 hr for okadaic acid and at 1 h r for TPA, indicating the presence of qualitative and quantitative differences between gene expressions by okadaic acid and TPA (Herschman et al., 1989). Kim and associates ( 1990) found that induction of collagenase gene expression by okadaic acid, like TPA, requires the AP-1 consensus sequence in the collagenase promoter. Okadaic acid induced transcription of the c-fos gene dramatically, and of the cjun gene to a lesser extent, both in human synoviocytes and in the human 'lung adenocarcinoma cell line A-549 (Kim et al., 1990). On the other hand, in Jurkat cells, it induced transcription of the c-jun gene dramatically and of the c-fos gene to a lesser extent (ThCvenin et al., 1991). Okadaic acid was a potent inducer of NF-KB in Jurkat cells. Treatment of the cells with okadaic acid resulted in the dissociation of NF-KB and a cytoplasmic inhibitor, IKB, within the cytoplasm. The NF-KB translocates to the nucleus, where it


167

recognizes an ll-bp DNA sequence that is present in the immunoglobulin K light chain enhancer (Sen and Baltimore, 1986; Atchison and Perry, 1987; Thkvenin et al., 1990). Okadaic acid inhibited myogenesis of mouse myoblast C2C12 cells by both extinguishing the expression of the myogenic determination gene MyoD 1 and inducing the expression of the inhibitor of the differentiation gene Id. MyoDl and Id expression required a prerequisite protein synthesis (Kim et al., 1992). Okadaic acid induced AP-1 binding activity in C2C12 cells. This is suggested to be one of the mechanisms by which okadaic acid regulates the expression of MyoDl negatively and inhibits myogenesis ( h r k et al., 1992). The expression of both early and secondary response genes to okadaic acid was studied in mouse skin. A topical application of okadaic acid induced expression of the c-fos gene, biphasic, 6 hr and 48 to 72 hr after treatment and of the c-jun gene, to a lesser extent, as early response genes. Okadaic acid also induced expression of secondary response genes, such as transin or stromelysin, and plasminogen activator-type urokinase (Holladay et al., 1992). In the study of mouse papilloma cell line 308, okadaic acid induced higher transcripts of c-fos and c-jun genes than an equimolar dose of TPA. It was apparent that their expression induced by okadaic acid continued over a longer period of time. Okadaic acid, like TPA, induced expression of the secondary response genes described above in cell line 308. These studies showed that okadaic acid and TPA affect gene expression in mouse keratinocytes through different pathways (Holladay et al., 1992). These results supported the previous evidence that disruption of the normal balance between members of the AP-1 complex induces malignant cell transformation (Schiitte et al., 1989). Recently, Bowden and associates identified a TPA-inactive mutated TPA response element, ,TGACTCC,, in the human collagenase promoter and named it the okadaic acid response element (ORE). The TPA response element (TRE) had already been identified as ,TGAGTCA,. The ORE is transactivated by both mouse c-jun A and cjun B. Both the consensus sequence of the ORE and possible new transcription factors that bind to the ORE are now under investigation (Levy et al., 1991, 1992). Similar studies have been pursued in various systems. Okadaic acid markedly potentiated the heat-induced expression of a human HSP 70 promoter linked to a CAT gene transfected into N-18 mouse neuroblastoma cells. Analysis using mutant constructs of the HSP 70 promoter revealed that the promoter activity by okadaic acid was not dependent on the heat shock-element, but on a new enhancer element (Huang et al., 1992).

168


IV. Calyculins A. STRUCTURE A N D BIOCHEMICAL ACTIVITY Calyculin A was isolated from a marine sponge, Discodermia calyx, and was a strong inhibitor of starfish development and a strong toxic conipound against L 1210 leukemia cells (Kato et al., 1986). Karaki and associates reported that calyculin A induced contraction of intact and skinned fibers (Ishihara et al., 1989b). Its structure contains an octamethyl-polyhydroxylated C28 fatty acid that is linked to two y-amino acids and esterified by phosphoric acid (Fig. 1). Seven calyculins, B to H, were additionally isolated from the same sponge and their structures were elucidated (Fig. 9) (Kato et al., 1988; Matsunaga et al., 1991). Recently, Matsunaga and Fusetani (1991) determined the absolute stereochemistry of the calyculin molecules. Our first evidence was that calyculin A, provided by Fusetani, bound to the okadaic acid receptors in particulate and cytosolic fractions of mouse skin, although its structure was unrelated to that of okadaic acid (Table V) (Fujiki et al., 1989a, 1991; Suganuma et al., 1989, 1990). As reported in Section 11, B, calyculin A is a potent inhibitor of PP-1 and PP-PA. Distinct from okadaic acid, calyculin A was found equally effective against PP-1 and PP-2A (Ishihara et al., 1989a; Suganuma el al., 1990, 1992a). T h e effective doses for 50% inhibition of specific [3H]okadaic acid binding by calyculins A through H ranged from 2.5 to 9.9 nM, and their IC,, values toward PP-1 and PP-2A in the cytosolic fraction of mouse brain ranged from 0.6 to 7.5 nM and from 2.6 to 14.0 nM, respectively (Table V). Since their potencies were all within a similar range, the seven additional calyculins, B to H, like calyculin A, might be also tumor promoters on mouse skin, indicating that the geometrics of the tetraene portion and the presence or absence of a methyl group on C-32 did not have any effects on activity (Matsunaga et al., 1991). In addition, decahydrocalyculin A isomers, which are hydrogenated derivatives of calyculin A, retained the activity. Calyculin A acetonide, an isopropylidene derivative formed of the hydroxyl groups between C-1 1 and C-13, was chemically synthesized by treatment of calyculin A with 2,2-dimethoxypropane, and its IC,, value indicating I, that these two hydroxyl groups toward PP-2A was > 1 @ were involved in the activity (S. Matsunaga, unpublished results). Calyculin A inhibited specific [%H]okadaicacid binding to particulate and cytosolic fractions of mouse skin, dose-dependently (Fig. 10). T h e binding affinities of calyculin A to the receptors in both fractions were compared with those of okadaic acid. T h e effective doses for 50% inhibition (1C5,) to the receptors in the particulate fraction were 2.5 nM for calyculin A and 45 nM for okadaic acid. However, the IC,,, to the


Calyculin Calyculin Calyculin Calyculin

A B C D

Rl CN H CN H

Calyculin Calyculin Calyculin Calyculin

E F G H

CN H CN H

R1

Rz H CN H CN

R2

H CN H CN

169

R3

H H CH3 CH3

R3

H H CH3 CH3

FIG.9. Structures of calyculins A, B, C, D,E, F, G , and H.

receptors in the cytosolic fraction were 2.8 nM for both calyculin A and okadaic acid. That is, calyculin A showed a binding affinity to the particulate fraction about 10 times stronger than okadaic acid, whereas calyculin A showed the same affinity to the cytosolic fraction as okadaic acid (Suganuma et al., 1990). T h e binding of calyculin A and of okadaic acid to the cytosolic fraction were in agreement with the results of

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HIROTA FUJIKI A N D MASAMI SUCANUMA

TABLE V BIOCHEMICAL ACTIVITIES OF CALYCULINS A TO H

Compounds Calyculin A B C D E F G H

Inhibition of specific ISHIokadaic acid binding [IC5o

Inhibition of PP-1 I1C50 (&)I

Inhibition of PP-2A [ G o (&)I

1.4 1 .0 0.6 4.0 I .4 1.4 6.4 7.5

2.6 3.6 2.8 4.8 5.2 4.8 8.5 14.0

2.5 4.6 3.7 9.9 6.0 6.0 9.0 9.6

inhibition of PP-2A present in the fraction of the cytosolic fraction of mouse brain eluted with 0.2 M NaCl on DEAE-cellulose column chromatography. That is, the dose-dependent inhibitory curve of calyculin A toward PP-2A in the cytosolic fraction was similar to that of okadaic acid (Fig. 10) (Suganuma et al., 1990).These results raised a question about which binding affinity (toeither the particulate or the cytosolic fraction) is correlated with the tumor-promoting activity of calyculin A on mouse skin. B. TUMOR PROMOTION ON MOUSESKIN The tumor-promoting activity of calyculin A was studied at two doses, 0.1 pg (0.1 nmol) and 1 pg (1 nmol) per application, according to the same experimental procedures of the two-stage carcinogenesis experiment. Table VI summarizes the results of the two groups treated with DMBA plus calyculin A compared with those of the group treated with DMBA plus okadaic acid. Calyculin A showed as strong a tumor-promoting activity as okadaic acid at an equimolar dose. Thus, the binding affinities of calyculin A and okadaic acid to the receptors in the cytosolic fraction were well correlated with their tumor-promoting activities, and the inhibition by calyculin A of protein phosphatases in the cytosolic fraction of mouse brain and skin well reflected a tumor-promoting activity. As Table I11 shows, DNA isolated from tumors of the group treated with DMBA plus calyculin A had a mutation at the second nucleotide of codon 61 in the c-Ha-ras gene, similar to that of the tumors of the group


I

17 1

Particulate

I

Cytosolic

I

PP-2A

I

Concentration ( n M 1

FIG. 10. Inhibition of specific [3HH]okadaicacid binding to particulate or cytosolic fractions of mouse skin and inhibition of PP-2A by calyculin A (0)and okadaic acid (0).

treated with DMBA plus okadaic acid (Fujiki el al., 1989d). Like okadaic acid, various concentrations of calyculin A increased phosphorylation of vimentin in primary human fibroblasts (Fig. 8) and in mouse embryo fibroblasts BALB/3T3 (Chartier et al., 1991) and BHK cells (Eriksson et al., 1992), and of cytokeratins of various molecular weights in human karatinocytes transfected with HPV 16 DNA (Section 111, G). Therefore, calyculin A and okadaic acid showed the same effects on human

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HIROTA FUJIKI AND MASAMI S U G A N U M A

TABLE VI TUMOR-PROMOTING AcrIvITY OF CALYCULIN A COMPARED WITH THAT OF OKADAlC ACID Amounts per application (nmol)

% of tumorbearing mice in Week 30

Average No. of tumors per mouse in Week 30

Calyculin A

0.1 1.o

Okadaic acid

1.2

13.3 86.7 80.0

0. I 4.3 7.2

Tumor promoters

Note. The groups treated with DMBA alone, calyculin A alone, or okadaic acid alone did not produce any tumors.

keratinocytes, as well as on mouse skin, with similar potencies. If the binding affinity to the particulate fraction reflects the binding to PP- 1 and that to the cytosolic fraction reflects that to PP-BA, based on the relative potencies of calyculin A and okadaic acid against PP-1 and PP-'LA, the inhibition of PP-PA in the cytosolic fraction rather than that of PP-1 in the particulate fraction seems to be an essential biochemical reaction for tumor promotion (Suganuma et al., 1990; Fujiki et al., 1992).

V. Microcystins and Nodularin A. STRUCTURE A N D BIOCHEMICAL ACTIVITY In 1987, three microcystins, microcystin-LR, -YR and -RR, were provided to us by two Japanese scientists so that we could study the biochemical mechanisms of action (Harada et al., 1988; Watanabe et al., 1988). Microcystins, isolated from colonial and filamentous algae, cyanobacteria, Macrocystis aeruginosa, M. viridis, Anabena jos-aquae, and Oscillatoria agardhii (Carmichael and Mahmood, 1984), attracted our attention for two main reasons: potent hepatotoxicity and unique structure. Toxic blue-green algae containing the microcyst ins pose an increasing environmental hazard in several areas of the world, because death of cattle and liver damage to humans have been caused by drinking water containing the algae. Structurally, the microcystins are cyclic heptapeptides containing two variable L amino acids and an unusual amino acid, E), 6(E)-dienoic 3-amino-9-methoxy- 1O-phenyl-2,6,8-trimethyl-deca-4( acid (Adda) (Botes el al., 1984; Rinehart et al., 1988). T h e microcystins differ primarily in the two variable amino acids and their nomenclature is based on these two L amino acids (Carmichael et al., 1988a). Microcystin-LR contains leucine and arginine in the variable positions (Fig. 1 1).


173

Microcystin-YR and -RR contain tyrosine and arginine, instead of the leucine in microcystin-LR. Although the structures of 42 microcystins have been determined to date, all these microcystins commonly contain the Adda molecule, which is necessary for activity. Geometrical isomers at C-7 in the Adda molecule of microcystin-LR and -RR,named 6(Z)-Adda microcystin-LR and -RR, have been isolated as minor components, with maternal microcystin-LR and -RR,from samples containinghficrocystlsspecies (Fig. 1 1) (Harada et al., 1990a,b). Namikoshi and associates (1992) have identified nine new microcystins. R

\

ClU

Mdha

Microcyst in- LR

G(Z)-Adda Microcystin-LR

/

,/-

Am

i

I

1

I . .

lrCY

Nodularin FIG. I 1. Structures of microcystin-LR, 6(Z)-Adda microcystin-LR and nodularin. Microcystin-LR contains two L amino acids, leucine and arginine; two D amino acids, alanine and glutamic acid; erythro-P-methylaspartic acid (Masp); methyldehydroalanine (Mdha); and 3-amino-9-methoxy-1 O-phenyl-2,6,8-trimethyl-deca-4,6-dienoic acid (Adda).

174


Nodularin was isolated from the toxic brackish-water cyanobacterium Nodularia splmigena (Carmichael et al., 198813; Rinehart et al., 1988) and has a cyclic pentapeptide, which contains Adda but lacks one of the L and D amino acids found in the microcystins (Fig. 11). When we first looked at the structure of the microcystins, it was apparent that the cyclic structure of heptapeptides was roughly similar to the flexible cavity of okadaic acid (Fig. 7). Without any further evidence, we subjected microcystin-LR, -YR, and -RR to the test of okadaic acid receptor binding. These three microcystins dose-dependently inhibited specific [3H]okadaicacid binding to the cytosolic fraction of mouse liver. As Table VII shows, the IC,, values of 50% inhibition were between 1.3 and 2.7 nM for the three microcystins and 3.2 nM for okadaic acid. Nodularin also inhibited specific [SH]okadaicacid binding to the cytosolic fraction of mouse liver with an IC,, of 2.3 nM. Three microcystins and nodularin also dose-dependently inhibited specific [SH]okadaicacid binding to the particulate fraction of mouse liver. Thus, the microcystins and nodularin bound to the okadaic acid receptors, although their structures are not related to that of okadaic acid (Yoshizawa et al., 1990). Microcystin-LR, -YR, and -RR and nodularin inhibited dose-dependently PP-2A, which had been partially purified from a cytosolic fraction of mouse liver on DEAE-cellulose column chromatography. As Table VII shows, the IC,, values were between 1.4 and 3.4 nM for the microcystins, and 0.7 nM for nodularin. The well-known hepatotoxic compounds a-amanitin and phalloidin did not show any inhibitory effects on protein phosphatases similar to those of the microcystins and nodularin. TABLE VII BIOCHEMICAL EFFECTS OF THREE MICROCYSTINS A N D NODULARIN COMPARED WITH THOSE OF OKADAIC ACIDAND THE HEPATOTOXIC COMPOUNDS

Compounds

Inhibition of specific [sH]okadaic acid binding [Ic50 ( d ) 1

Inhibition of protein phosphatase activity [IC5O (nM)l

Increase of protein phosphorylation IED50a

microcyst in-LR Microcystin-YR Microcystin-RR Nodularin Okadaic acid a-Amanitin Phalloidin

1.3 2.7 2.0 2.3 3.2 > 10,000 > 10,000

1.6 1.4 3.4 0.7 1.2 > 10,000 > 10,000

2.5 1.3 3.4 1.2 2.5 > 10.000 > 10.000

The concentration causing 50% of maximal activation


175

These results indicated that the hepatotoxicity of the microcystins and nodularin is induced by the okadaic pathway. We presented the results with the microcystins and nodularin at the 1988 Gordon Conference on Marine Natural Products (Yoshizawa et al., 1990). Similar results using purified catalytic subunits of PP-1 and PP-2A were presented later (Honkanen et al., 1990, 1991; Mackintosh et al., 1990). It is important to note that microcystin-LR is the most potent inhibitor of PP-1 as well as PP-2A, compared with okadaic acid, calyculin A, and tautomycin (Table I) (Suganuma et al., 1992a). Based on this significant property, microcystin-LR affinity chromatography was developed to purify PP-2A as a holoenzyme. This chromatography was not suitable for PP- 1, however, and okadaic acid affinity chromatography did not give us good purification of protein phosphatases (Nishiwaki et al., 1991). Since the microcystins and nodularin inhibited PP-1 and PP-2A in the cytosolic fraction of mouse liver, these compounds also increased the incorporation of 32P into histone H 1, referred to as the apparent “activation” of protein kinases by okadaic acid, during in vitro incubation with [Y-~~PIATP, protein kinases, and PP-2A (Sassa et al., 1989). T h e ED,, values for the increase of protein phosphorylation were between 1.3 and 3.4 nM for the three microcystins and 1.2 nM for nodularin (Table VII). That is, the microcystins and nodularin showed the same biochemical activities as did okadaic acid, with similar specific activities (Yoshizawa et al., 1990). (22.7 Ci/mmol), Using a newly synthesized [3H]dihydr~micr~cy~tin-LR the specific binding of the cytosolic and particulate fractions of rat liver was studied. The K , value was 0.03 nM for receptors in the cytosolic fraction and 0.4 nM for those in the particulate fraction. Specific [3H]dihydromicrocystin-LR binding to the cytosolic fraction was inhibited by microcystin-LR and -RR. As Fig. 12 shows, the IC,,, values of microcystin-LR and -RR were 0.38 and 0.42 nM, respectively. 6(Z)-Adda microcystin-LR and -RR inhibited binding 100 times more weakly than their maternal microcystins. T h e IC,, values were 32 and 52 nM for both 6(Z)-Adda microcystin-LR and -RR (R. Nishiwaki et al., manuscript in preparation). Next, inhibitory potencies of the two 6(Z)-Adda microcystins toward PP-2A in the cytosolic fraction of mouse brain were compared with those of their maternal compounds. The IC,, values of microcystin-LR and -RR were 0.28 and 0.78 nM, respectively, whereas those of 6(Z)-Adda microcystin-LR and -RR were both 80 nM. Intraperitoneal administration of the maternal microcystins into rats rapidly released GPT from the liver into blood serum. T h e amounts of 50 Fg/kg microcystin-LR and -RR released levels of GPT similar to those of 500 pg/kg 6(Z)-Adda microcystin-LR and -RR, within 24 hr after

176


a

I 1 00

50

i

E

B

0

-

v)

lo-'

10 0

1 02

104

Concentration ( nM 1

FIG. 12. Inhibition of specific [3H]dihydromicrocystin-LR binding to cytosolic fraction of rat liver by microcystin-LR (0) and -RR (A) and G(Z)-Adda microcystin-LR (0)and -RR (A).

administration (Nishiwaki-Matsushima et al., 199l), indicating that the conjugated diene with 4(E),6(E) geometry in the Adda molecule is important for the interaction with PP-1 and PP-2A.

B. TARGET TISSUE Our first evidence showed that the microcystins and nodularin did not induce hyperphosphorylation of vimentin in primary human fibroblasts, whereas other protein phosphatase inhibitors, okadaic acid and calyculin A, did. Moreover, the microcystins and nodularin, which in vitro bind to the okadaic acid receptors in the cell membrane, did not induce ODC in mouse skin. These results suggested that the microcystins and nodularin have difficulty penetrating the cells. In a test, we utilized as a parameter the evidence that okadaic acid at a concentration of 0.1 pbf induced morphological changes of primary human fibroblasts, from a spindle-like to a round form within 2 hr of incubation, whereas microcystin-YR did not, at concentrations up to 9.6 p M . On the basis of our understanding that the morphological changes were induced by the inhibition of PP-1 and PP-2A by okadaic acid in primary human fibroblasts, we microinjected a microcystin-YR solution at a concentration of 670 pbf into about 50 fibroblasts. All of the injected cells caused morphological changes from a spindle-like to a round form, 45 min after injection, whereas noninjected cells did not show any morphological changes. It was noteworthy that morphological changes in-


177

duced by injection of microcystin-YR were similar to those induced by incubation with okadaic acid. These results support our idea that the microcystins and nodularin proceed along the okadaic acid pathway in the cells after microinjection, whereas okadaic acid can penetrate readily the cells (Matsushima et al., 1990). Since the microcystins and nodularin are hepatotoxic compounds, we studied whether microcystin-YR could enter rat primary-cultured hepatocytes and also cause morphological changes of the cells. Treatment with microcystin-YR at a concentration of 9.6 pA4 induced morphological changes of primary hepatocytes from the normal round form to fused cells within 2 hr of incubation. Similar changes were also induced by okadaic acid at a concentration of 1.2 pM. These results indicated that microcystin-YR was about eight times less effective than okadaic acid in its action on rat hepatocytes. Microcystin-YR and -LR and nodularin induced hyperphosphorylation of proteins of various molecular weights as the result of inhibition of PP-1 and PP-2A, as did okadaic acid (Yoshizawa et al., 1990). Recently, the main hyperphosphorylated proteins in rat primary-cultured hepatocytes were identified to be cytokeratins 8 and 18, one type of intermediate filament (Ohta et al., 1992). Microcystin-LR, 7-desmethyl-microcystin-RR, and nodularin increased the level of protein phosphorylation in hepatocytes and induced changes in morphology and in the cytoskeleton of hepatocytes (Eriksson et al., 1990a). These results indicated that the microcystins and nodularin can penetrate the liver cells. Livestock deaths due to ingestion of algae are well documented (Francis, 1878; Beasley et al., 1983; Botes et al., 1984; Carmichael, 1988). T h e evidence shows that the microcystins contained in the algae can reach the liver from the digestive tract. Although we have not yet obtained results with po-administered [3H]dihydromicrocystin-LR, the ip administration of [3H]dihydromicrocystin-LR (1 1.4 pCi/0.2 ml saline solution) into mice has been studied. The radioactivity in the liver increased continuously from 5 min to 1 hr after injection, and was 82% 1 hr after. T h e second order of radioactivity, 4% of the radioactivity, at 5 min, was found in the lower small intestine and declined to 0.5% after 1 hr, suggesting that the toxin had adhered to the small intestine before absorption (Ohta et al., manuscript in preparation; Robinson et al., 1989). Eriksson and associates ( 1990b) reported the preferential uptake of the toxin by the multispecific bile acid transport system across the ileum and into the hepatocytes. T h e study of tissue distribution by iv-administered [3H]microcystin-LR into mice revealed long-term hepatic retention of radiolabeling associated with cytosolic components, 85 5 % at Day 1

*

178


and 42 2 11% at Day 6 after injection (Robinson et al., 1991). These results suggest that the target tissue of the microcystins is the liver, in which microcystins are accumulated. PROMOTION IN THE LIVER C. TUMOR The microcystins and nodularin have a unique liver organotrophy. Tumor promotion of microcystin-LR was shown in rat liver with diethylnitrosamine (DEN) and was followed by a partial hepatectomy at the end of third week of the experiment. The doses used for the experiment were below the acute toxicity level. Repeated ip injections of microcystin-LR induced a significant increase of both parameters: increase in numbers and percentage areas of positive foci of glutathione S-transferase placental form (GST-P) in rat liver (Table VIII). In a separate experiment, 0.05% phenobarbital in the diet induced 38.1 & 10.9 foci/cm2 and 1.09 0.5% area of foci. Thus, microcystin-LR is one of the strongest liver tumor promoters found to date (Nishiwaki-Matsushima et al., 1992b). We think that microcystin-LR has a tumor-promoting activity in rat liver, because only a few foci were observed in initiated rats. In the present experiment, no detectable mutation was found in codon 61 of the c-Ha-ras gene in DNA isolated from rat livers of the group treated with DEN plus microcystin-LR. T h e tumor-promoting activity of microcystin-LR may involve human liver cancer through drinking water supplies. Yu (1989) reported that the incidence of primary liver cancer in Qidong County, People’s Republic of China, where people drink pond and ditch water, was about eight times higher than that in populations who drink well water. T h e water of the ponds and ditches of Qidong County is contaminated by

*

TABLE VIII TUMOR-PROMOTING ACTIVITY OF MICROCYSTIN-LR Microcystin-LR Positive foci of GST-P

( P g w

Partial hepatectomy Initiator

Before

After

+ + + +

10 10 10

-

-

10

10 25 50 50

No. of focilliver (No./cm2)

Area of foci

13.4 f 4.2 17.4 2 3.8 32.7 + 11.1 44.4 2 10.3 0.4 f 0.3

2.7 f 3.1 1.9 f 0.5 6.8 f 3.8 29.6 f 12.9 0.1 f 0.2

(%)


179

blue-green algae, which produce microcystins (W. W.Carmichael, personal communication). It was reported that the microcystins are not affected by normal chlorination, flocculation, and filtration procedures used by water treatment facilities (Krishnamurthy et al., 1989). Epidemiological study of primary liver cancer in relation to exposure to the microcystins is anticipated. Similar concerns about how serious this threat may be to the human population are becoming global in scope, including countries such as Australia (Falconer et al., 1983), Norway (Skulberg etal., 1984), and the United States (Billing, 1981; Beasley et al., 1989).

D. MOLECULAR MODELING T h e microcystins and nodularin showed specific binding to the okadaic acid receptors, inhibition of PP- 1 and PP-2A and hepatotoxicity, with similar specific activities. Therefore, we think that the microcystins and nodularin have similar specific molecular interaction with PP-1 and PP-2A due to a similar conformation in some parts of their molecules. The recent results with computer models of the three-dimensional structures of microcystin-LR and nodularin showed that their peptide rings formed an angle of 90" to each other, when Adda in both microcystin-LR and nodularin were fitted together (Lanaras et al., 1991). It was difficult to explain from these results, however, the similar potencies of microcystin-LR and nodularin. Computer graphic analyses by Quinn and associates indicated a different conclusion and provided a rationalization for receptor binding properties for the microcystins and nodularin. The lower energy values and the three-dimensional similarity of these compounds suggest that they are more reasonable computer models than those previously reported (Taylor et al., 1992). Figure 13 depicts the optimized structures of microcystin-LR and nodularin showing the planarity of both peptide rings and the relative spatial alignments of the Adda and arginine side chains. Rigid superimposition of the two molecules defined the microcystin-LR and nodularin model (Fig. 13) (Taylor et al., 1992). T h e structures of microcystin-LR and nodularin have arginine and Adda in common. To determine the relative importance of the arginine residue to biochemical activity, microcystin-LA, which contains alanine, rather than arginine, as in microcystin-LR, was tested. T h e IC,, values toward PP-1 in the cytosolic fraction of mouse brain were 0.90 and 0.44 nM for microcystin-LA and -LR, and those toward PP-2A in the cytosolic fraction of mouse brain were 0.38 and 0.32 nM, respectively. They also

180


Microcystin-LR

Nodularin

Superimposition FIG. 13. Three-dimensional structures of rnicrocystin-LR and nodularin and their superimposition.

inhibited specific ["Iokadaic acid binding to both PP-1 and PP-PA in the cytosolic fraction of mouse brain with similar IC,, values. These results showed that the arginine residue does not significantly interact with the enzymes and can be substituted by other amino acids without loss of activity (Nishiwaki-Matsushima et al., 1992a). It had already been reported that Adda is essential for activity (Rinehart et al., 1988). As w e previously reported, 6(Z)-Adda microcystins showed 100 times weaker activity than the maternal 6(E)-Adda microcystins (Nishiwaki-Matsushima et al., 1991). Computer models of the three-dimensional structures of microcystinLR and nodularin should be further extended to the three other types of the okadaic acid class compounds. Quinn and associates (1993) are now investigating molecular modeling that allows common regions of okadaic acid, calyculin A, and microcystin-LR to be recognized. Preliminary results meet well with our findings that the F and G rings of the okadaic acid molecule are located outside of the functional group. Computer-assisted molecular modeling will provide us with important information about functional parts of the okadaic acid class compounds.


181

VI. Tautomycin A. STRUCTURE AND BIOCHEMICAL ACTIVITY Tautomycin was isolated from Streptomyces spiroverticillutus as an antibiotic (Fig. 1) and is toxic to various cells, including fungi, yeast, and animal cells (Cheng et al., 1987). Tautomycin induced bleb formation on the surface of human chronic leukemia K562 cells and slightly enhanced protein phosphorylation through activation of PKC in vitro, although it did not bind to the phorbol ester receptor in K562 cells (Magae et al., 1988). In 1987, Isono provided us with tautomycin, one part containing a six-membered spiroketal moiety that is similar to some parts of the okadaic acid molecule, and the other part being similar to aplysiatoxin, one of the TPA-type tumor promoters. Following up on his unique observation, we subjected tautomycin to the two tests, activation of PKC in vitro and specific binding to the okadaic acid receptors. We soon obtained conclusive results; that is, tautomycin did not directly activate PKC in vitro, but inhibited specific [3H]okadaic acid binding to the particulate fraction of mouse skin. Thus, tautomycin was further subjected to inhibition of PP-1 and PP-PA in the cytosolic fraction of mouse brain. T h e results revealed that tautomycin is an additional okadaic acid class compound. T h e activity of tautomycin was compared with that of okadaic acid: the IC,, values of specific [3H]okadaic acid binding to a particulate fraction of mouse skin were 180 nM for tautomycin and 59 nh4 for okadaic acid. T h e IC,, values for tautomycin and okadaic acid toward PP-1 in the cytosolic fraction of mouse brain were 2.2 and 45 nM, and toward PP-2A were 1.8 and 0.5 nM, respectively (S. Nishiwaki et al., unpublished results). As described in Table I, tautomycin inhibited activity of the catalytic subunits of PP-1 and PP-2A with IC,, values of 0.7 and 0.65 nM, respectively (Suganuma et al., 1992a). Similar results with tautomycin were reported by various research groups (Mackintosh and Klumpp, 1990; Hori et ul., 1991). One of the reasons tautomycin is weaker than okadaic acid might be its 2,3-dialkylmaleic anhydride structure, which is in equilibrium with an open ring dicarboxylic acid in neutral solution (K. Isono, personal communication). Tautomycin induced hyperphosphorylation of cytokeratins in human keratinocytes, with one-tenth the potency of okadaic acid (unpublished results). Tautomycin also enhanced protein phosphorylation of 15 and 25-kDa in K562 cells. Since the phosphorylation of these two proteins was also enhanced by okadaic acid and dinophysistoxin- 1, but not by okadaic acid tetramethyl ether, we thought it might be the so-called apparent “activation” of protein kinases due to inhibition of PP-1 and

182


PP-2A (Magae et al., 1990). Furthermore, tautomycin and okadaic acid were shown to share the same biochemical and biological activities in vitro and in vivo, suggesting that tautomycin might also be a tumor promoter of the okadaic acid class in mouse skin or other organs. B. ABSENCE OF TUMOR PROMOTION ON MOUSESKIN Tumor promotion of tautomycin was tested in a two-stage carcinogenesis experiment on mouse skin following our standard experimental procedure. Initiation was achieved by a single application of 100 pg DMBA. T h e dose of tautomycin to be used per application was carefully considered before tumor promotion was started. We had learned from the study of calyculin A that its potency to inhibit PP-2A in the cytosolic fraction correlates more significantly with tumor-promoting activity than that to inhibit PP-1. Thus, because the relative potency of tautomycin toward PP-2A was about 10 times weaker than that of okadaic acid, 30 pg (36 nmol) of tautomycin per application was the chosen dose, compared with 1 pg (1.2 nmol) of okadaic acid. The group treated with DMBA plus tautomycin did not produce any tumors on mouse skin up to Week 30, the same as in the two control groups, treated with DMBA alone or tautomycin alone. (H. Fujiki, et al., unpublished results). If tautomycin, like okadaic acid, acts on PP-2A in the cells of mouse skin, tautomycin, based on its specific activity, would have shown tumor-promoting activity. We attributed the absence of tumor-promoting activity to the chemical nature of tautomycin; it is probable that the difficulty in penetrating the basal cell layer of mouse skin is due to the polarity of the tautomycin molecule. In contrast to okadaic acid and calyculin A, tautomycin was not an irritant and not able to induce ODC in mouse skin. The absence of irritancy also indicates that tautomycin is unable to reach the basal cell layer through the corneum and does not penetrate the cells of mouse skin. This was further supported by the absence of ODC induction and tumor promotion in mouse skin. C. EFFECTS ON DIGESTIVE TRACT

As microcystin-LR showed tumor promotion in the liver, tautomycin was expected to be a tumor promoter in tissues other than mouse skin. To find these other target tissues, a solution of tautomycin was passed to the stomach through intubation. Surprisingly, 4 hr after intubation, tautomycin induced ODC induction in the glandular stomach to an extent similar that of okadaic acid, but at higher doses (data not shown).


183

Recently, Tatematsu and associates demonstrated that a single administration of tautomycin into the stomach of rat induced enhanced incorporation of 5-bromodeoxyuridine (BrdU) in DNA of the cells in the stomach, small intestine, and colon, suggesting induction of cell proliferation (M. Tatematsu et al., manuscript in preparation). A two-stage carcinogenesis experiment on tautomycin is ongoing in the rat glandular stomach, and a tumor-promoting activity is anticipated. Our recent understanding of ODC induction should be briefly discussed. Ornithine decarboxylase induction is partly regulated through TRE in the ODC gene, when PP-1 and PP-2A in the cytosolic fraction are inhibited in the cells. It was previously reported that ODC induction was strongly induced by TPA-type tumor promoters (OBrien et al., 1975), and its induction was strongly inhibited by pretreatment with retinoic acid, and this was well correlated with inhibition of tumor promotion (Verma and Boutwell, 1977). In this regard, ODC induction was thought to be a significant biochemical activity in tumor promotion as a rate-limiting enzyme for polyamine biosynthesis, followed by cell proliferation. However, study of the okadaic acid class compounds provided ODC induction with an additionally significant indication; that is, ODC induction is an important parameter of tumor promotion that signal transduction has occurred at the gene level. To summarize our results, signal transduction from tautomycin takes place in the glandular stomach, but not in mouse skin, whereas, with okadaic acid, signal transduction is found in both the glandular stomach and mouse skin. As discussed previously, okadaic acid induced ODC about 10 times less strongly than TPA, with a similar time course, whereas their tumorpromoting activities were almost the same. Although potency has not been directly correlated to tumor-promoting activity, ODC induction has become a significant biochemical parameter in estimating stimulation of gene expression, indicating the presence of signal transduction for tumor promotion in the responsive gene (Suganuma et al., 1992b). VII. Hypotheses in Relation to Human Cancer By using tumor promoters of the okadaic acid class as chemical probes, we identified a general biochemical mechanism of tumor promotion applicable to various organs. How the okadaic acid pathway is related to human cancer should be considered from at least three aspects (Fig. 14). The first possibility is from exposure to the okadaic acid class compounds. The second is that the okadaic acid pathway might possibly be pursued by endogeneous protein inhibitors. The third is that the effects of the okadaic acid pathway can be mimicked, in part, by t w o

184


87gpPhosphorylation

Function

Inactive

8

8

Transformation

+

Protein

Gene

Mutatio:

I ,

Inactive

+

FIG. 14. Schematic illustration of inactivation of suppressor function by the okadaic acid pathway compared with genetic changes of a suppressor gene.

major monocyte/macrophage-derived lymphokines, interleukin 1 (IL1) and tumor necrosis factor (TNF). 1. Okadaic acid and dinophysistoxin- 1 are tumor promoters in the rat glandular stomach and are also causative agents of diarrhetic shellfish poisoning in humans. These compounds accumulate in the hepatopancreas of mussels and shellfish (Yasumoto, 1990). Consumption of these organisms causes diarrhetic shellfish poisoning, which has been reported in several countries, such as Japan, Chile, Norway, T h e Netherlands, and Spain. In Japan, government regulation sets the maximum allowable levels of toxins in shellfish meat at 0.05 mouse unit/g meat, which corresponds to 0.16 pg okadaic acid/g meat. Thus, it is possible for humans to avoid acute toxicity of okadaic acid and dinophysistoxin- 1. T h e evidence of chronic exposure to okadaic acid and dinophysistoxin- 1 has not been well documented. Chronic exposure to the microcystins or nodularin through drinking water supplies seems to be linked to high incidences of human primary liver cancer. Yu (1989) described that the incidence of human primary liver cancer decreased after the source of water was changed from ditch water to well water in Qidong County, People’s Republic of China; so intake of microcystins might be correlated with development of human liver cancer. Epidemiological investigation of the relationship between the ingested amounts of toxin and incidences of human liver cancer becomes increasingly important. Continuous exposure to the potent liver tumor


185

promoters present in drinking water supplies possibly promotes human liver cancer, initiated by aflatoxin, in certain areas. Protectionary measures based on the scientific studies are needed. 2. As for the second possibility, it should be noted that there are endogenous protein inhibitors of PP-1, called inhibitors-l and -2, in the cells (Cohen, 1989). Inhibitor-1, an 18-kDa protein, is activated by CAMP-dependent protein kinase and inhibitor-2, a 22-kDa protein, is an active form without any phosphorylation necessary. Based on molar ratios, the inhibitory activity of inhibitor-2 toward PP-1 was as strong as that of okadaic acid, but inhibitor-2 was not effective on PP-2A (S. Nishiwaki et al., unpublished results). The level of inhibitor-:, is reported to oscillate during the cell cycle, peaking at the S phase and during mitosis in rat fibroblasts (Brautigan et al., 1990). Whether the levels of inhibitors-l and -2 are deregulated in the developmental process of cancer cells remains to be investigated. One particularly striking finding was that a regulatory subunit and the catalytic subunit of PP-2A form stable complexes with the simian virus 40 small T antigen and the small and middle T antigens of polyoma virus (Pallas et al., 1990; Walter et al., 1990). In the stable complexes, the small T antigen acted as an inhibitor of PP-PA, resulting in hyperphosphorylation of p53 and large T antigen, which are both inactive forms (Scheidtmann et al., 1991). The small T antigen also inhibited dephosphorylation of the Rb protein. It is accepted that inactivation of the suppressor function can be caused by hyperphosphorylation of suppressor gene products; both p53 and the Rb protein are phosphorylated by p34'dC2, the cell division cycle protein kinase. This protein kinase is also involved in phosphorylation of vimentin (Chou et al., 1990), the dephosphorylation of which was inhibited by okadaic acid and calyculin A, as reported previously (Yatsunami et al., 1991b). We recently demonstrated that treatment of primary human fibroblasts with okadaic acid induced hyperphosphorylation of p53 and the Rb protein dose-dependently (Yatsunami et al., 1993a). How a protein similar to the small T antigen is constitutively expressed in the cancer cells remains to be clarified. 3. Okadaic acid and I L 1 stimulated the steady-state levels of collagenase transcripts in human synoviocytes through different mechanisms (Postlethwaite et al., 1988; Kim et al., 1990). Okadaic acid and TNF-a induced NF-KBin Jurkat cells (Meichle et al., 1990; Thevenin et al., 1990). In addition to these molecular biological results, I L 1 , TNF-a, and okadaic acid all induced a similar phosphorylation pattern within 15 min in human fibroblasts (Guy et al., 1991, 1992). I L 1 , a 17-kDa protein, binds to I L 1 receptors of an 80-kDa protein present in T lymphocytes,

186

HIROTA FVJIKI AND MASAMI SUGANUMA

fibroblasts, and other connective tissue cells (O’Neillet al., 1990). Tumor necrosis factor a, another 17-kDa protein, binds to two distinct TNF receptors of 56- and 75-kDa proteins in a variety of cell types (Loetscher et al., 1990; Schall et al., 1990; Blank et al., 1992). These two lymphokines are recognized polypeptide mediators of inflammation and cellular immune responses (Old, 1985; Beutler and Cerami, 1988). Therefore, their effects, such as protein phosphorylation, expression of transcription factors (Guy et al., 1992; Viltek and Lee, 1991), and inflammatory response, were in partial mimicry of those of okadaic acid. However, there are distinct differences in the time course of effects between the lymphokines and okadaic acid. That is, I L 1 and TNF directly activate multiple protein kinases through their receptor bindings and their protein phosphorylations were maximum at 15 min after treatment and later declined, whereas hyperphosphorylation of vimentin and cytokeratins by okadaic acid increased gradually and reached a maximum at 2 hr after treatment. It is assumed that the lymphokines function like tumor promoters, although their signaling effects are temporary, compared with those of the okadaic acid class tumor promoters. We recently found that mouse TNF-a stimulated transformation of MCAinitiated BALB/3T3 cells (A. Komori et al., manuscript in preparation).

VIII. Future Perspectives The discovery of the okadaic acid class tumor promoters led us to recognize the significance of protein phosphatase activity and inhibition in cancer research. Although the okadaic acid pathway is biochemically distinct from the PKC pathway, both pathways ultimately involve upregulating the expression of members of the fos and jun gene families, resulting in disruption of the normal balance between members of the AP-1 complex. If a pattern o r series of gene expressions for tumor promotion can be demonstrated, chemical tumor promoters will still be useful tools for the investigation. T h e interaction between Jun and Fos in the initiated cells in relation to protein phosphorylation needs to be clarified. As the stable complex of PP-2A and viral T antigens has indicated, the nature and function of the regulatory subunits of protein phosphatases remain to be further characterized. As depicted in Fig. 14, it should be investigated in initiated cells how the sustained hyperphosphorylation of a tumor suppressor gene product is induced. Although the okadaic acid pathway only deals with PP-1 and PP-2A, the calcium/calmodulin-dependent protein phosphatase-2B, known as calcineurin, was recently found to be inhibited by an immunosuppressantreceptor complex, consisting of cyclosporin A and cyclophilin A (Liu


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et al., 1991; McKeon, 1991). This evidence opens u p a significant role for

PP-PB. How activity of a protein phosphatase is related to those of the other protein phosphatases is not well known. T h e evidence that four structurally different types of compounds of the okadaic acid class bind to a common binding site in the catalytic subunits of PP-1 and PP-2A provides a new research objective for the study of computer modeling. How to inhibit the okadaic acid pathway in the initiated cells and how to recover the deregulated expression of the proto-oncogenes are also important future projects in cancer research. IX. Conclusion Since the discovery that okadaic acid is a tumor promoter as potent as TPA through different mechanisms of action, we have extended studies on okadaic acid derivatives, the calyculins, the microcystins, and tautomycin. T h e evidence that these tumor promoters are all potent inhibitors of PP-1 and PP-2A provided a new area in tumor promotion research and increased interest due to its distinction from the activation of PKC by TPA-type tumor promoters. Present understanding of gene expression of the proto-oncogenes has assisted in interpreting their mechanisms of action at the molecular level and to put these studies in the limelight. Important new pieces of evidence related to tumor promotion have been summarized in this review article. Just after our second publication, in which okadaic acid was shown to have potent tumor-promoting activity, we received numerous requests for okadaic acid, since it was not commercially available at that time. It was very exciting for us to find so many scientists in various research fields who were interested in protein phosphatase activity and its inhibition. Although our research on tumor promoters is a small field in cancer research, we are convinced that it is vitally interrelated with various other research fields, due to the pleiotropic effects of tumor promoters. It is anticipated that this review will provide information to those scientists as well.

ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Cancer Research, Overseas Scientific Research Program (Cancer Program) from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare of Japan, and a Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan, and by grants from the Foundation for Promotion of Cancer Research, the Princess Takamatsu Cancer Research Fund, the Uehara Memorial Life Science Foundation, and the Smoking Research Foundation of Japan. We thank Dr. T. Sugimura for encouragement during the course of this work and Japanese and American scientists cited in the refer-

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ences for collaborations. To the Japanese scientists, including Drs. Y. Hirdta, K. Yamada, T. Yasumoto, D. Uemura, N. Fusetani, K-I. Harada, M. F. Watanabe, and K. Isono, and the many scientists from abroad, including Drs. R. K. Boutwell, I. B. Weinstein, L. Levine, S. H . Yuspa, M. B. Sporn, T. J. Slaga, W. Troll, G. T. Bowden, H. R. Henchman, E. Huberman, M. R. Rosner, S. -J. Kim, W. W. Carmichael, R. E. Moore, Y. Hokama, L. Edebo, E. Hecker, H. zur Hausen, and R. J. Quinn, we express gratitude for fruitful discussions.

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ONCOGENIC BASIS OF RADIATION R ESISTANCE Usha Kasid,' Kathleen Pirollo,t Anatoly Dritschilo,* and Esther Changt-* 'Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007; and Departments of *Pathology and *Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

I. Introduction 11. Radiation Response Phenotype A. Relative Radioresistance and Radiosensitivity: Radiation Survival Parameters B. Clonal Nature of the Radiation Response 111. Human and Rodent Cell Model Systems A. Radioresistant Human Squamous Cell Carcinomas B. Skin Fibroblasts from a Cancer-Prone Family C. NIH13T3 Transfectants D. Other Cell Lines IV. Mitogenic Signals and Radiation Response V. Transformation and Radiation Resistance VI. Radiation-Resistant Phenotype: Cause or Effect A. Multifactorial Nature of Radiation Response: Differential Gene Expression B. Molecular Targets of Ionizing Radiation VII. DNA Damage and Repair Cascade: Biochemical and Cellular Factors VIII. Modulation of Radiation Resistance: Therapeutic Implications of Oncogene Strategy IX. Conclusion References

1. Introduction

Evidence continues to accumulate implicating oncogenes in the development of neoplasia. T h e normal counterparts of these genes (protooncogenes) are involved in numerous vital cellular functions. The products of many of these protooncogenes have been shown to interact with one another as components of a signal transduction pathway that involves transmission of molecular/biochemical signals from the membrane to the nucleus directing the cells to divide or to differentiate (Weinstein, 1988a; Nigg, 1990; Bishop, 1991; Cantley et al., 1991). Ionizing radiation induces multiple cellular and biological effects either by direct interaction with DNA or through the formation of free 195 ADVANCES IN CANCER RESEARCH. VOL. 61

Copyright 0 199.3 by Academic Press. Inc. All rights of reproduction in any form reserved.

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radical species leading to DNA damage (Painter, 1980). These effects include cell cycle-specific growth arrest, repair of DNA damage, radicalscavenging proteins, gene mutations, malignant transformation, and cell killing. The mechanisms underlying sensitivity or resistance of certain mammalian cells to the toxic effects of y-radiation have been a topic of a number of studies (Taylor et al., 1975; Paterson et al., 1976; Weichselbaum et al., 1977; Arlett and Harcourt, 1980; Cox and Masson, 1980; Little et al., 1989; Little and Nove, 1990). T h e individual molecular events and specific genes involved in the resistance to y-radiation will affect both normal cellular protection from radiation damage and the failure of tumors to respond to radiation therapy. In recent years, several lines of investigation have coalesced to demonstrate a link between certain oncogenes and the phenomenon of cellular resistance to ionizing radiation. T h e raf-l l protooncogene has been associated with radiation-resistant human laryngeal squamous carcinoma-derived cells (Kasid el al., 1987a, 1989a),as well as radiation-resistant noncancerous skin fibroblasts from a specific cancer-prone family with Li-Fraumeni syndrome (Chang et al., 1987; Pirollo et al., 1989). Transfections not only of the ruf- 1 oncogene, but also of other protein-serine kinase oncogenes, mos and cot, have been shown to confer the radiationresistant phenotype on the recipient cells (Pirollo et al., 1989; Suzuki et al., 1992). An increase in level of radiation resistance was also demonstrated by transfection of Ha-, Ki-, or N-ras oncogene into murine hematopoietic cells or NIH/3T3 cells (FitzCerald et al., 1985; Sklar, 1988), and a synergistic increase in the level of radiation resistance of primary rat embryo cells was seen by cotransfection of ras and m y oncogenes (Ling and Endlich, 1989; McKenna et al., 1990b). Therefore, there is a growing body of evidence indicating that oncogenes play a major role in cellular resistance to ionizing radiation. Here we will review some of that evidence and attempt to formulate the themes underlying the oncogenic basis of radiation resistance. II. Radiation Response Phenotype

A. RELATIVE RADIORESISTANCE AND RADIOSENSITIVITY: RADIATION SURVIVAL PARAMETERS T h e central hypothesis concerning the outcome of the y-irradiation of eukaryotic cells suggests that loss of clonogenic capacity and cell death I Italicized, three-letter code refers to the gene (e.g., ruf); three-letter code with first letter in uppercase refers to the protein (e.g., Raf).

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result from damage to the structure and function of genomic DNA (Painter, 1980). T h e clonogenic assay for studying responses of cells to radiation is based on the method described by Puck and Marcus (1956). T h e effect of increasing doses of radiation on the clonogenic capacity is described by a radiation dose-survival curve (Alper, 1979), which generally consists of an initial curved component in the low dose range (the shoulder) and of an exponential component (the terminal slope). The single-hit, multitarget (target model) and the linear quadratic model are most commonly used to analyze cellular radiation survival. A graphic representation of the single-hit, multitarget model and the linear quadratic model of radiation survival are shown in Figs. 1A and 1B. T h e target model is based on the parameters Do and 6,where Do is the inverse of the terminal slope of the survival curve and fi, reflects the extrapolation of this slope to the ordinate (Fertil et al., 1980, 1988; Steele et al., 1983). Another parameter, D, is the measure of the shoulder of the survival curve as the terminal slope line intersects the abscissa (Withers, 1987). T h e linear quadratic model has two major parameters: a, the linear component characterizing the radiation response at low doses; and p, the quadratic component predominating at higher doses. T h e higher the value of a,the more linear is the response of cells to low doses of radiation and the more sensitive are the cells to the cytotoxic effects of X-rays (Hall, 1988). In addition, using a model-free parameter, the mean inactivation dose (b,the area under the survival curve plotted on linear coordinates; Fig. 1 C) has been employed as a measure of the intrinsic radiosensitivity of human cell lines (Fertil et al., 1984). Finally, a distinction between the relative radioresistant and radiosensitive phenotypes can be made by comparison of the values of the survival fraction following exposure to 2 Gy (SF,), the dose most usually delivered per session of radiotherapy (Fertil and Malaise, 1985). Parameters that describe the exponential component are used to define the radiosensitivity of cells, whereas those parameters that describe the shoulder reflect the capacity to repair radiation lesions and to restore clonogenicity. Quantitative evaluations of the cellular capacity to repair radiation-induced sublethal damage, potentially lethal damage, DNA single-strand breaks and DNA double-strand breaks are routinely performed employing the previously described and/or modified procedures (Elkind and Sutton, 1959; Philips and Tolmach, 1966; Belli and Shelton, 1969; Little, 1969; Kemp et al., 1984; Wlodek and Hittleman, 1987; Iliakis and Seaner, 1988; Iliakis et al., 1991). In this review, the definition of relative radioresistance (or radiosensitivity) of different cell types, o r transfectants derived thereof, applies to the slope and/or the shoulder of the radiation survival curve. Changes

A

C

B

0

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5.0

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Radiation dose (Gy) 1.o

1.0 C

0

0.5

-

c

0

E

5

.-

2

.c

2.0

4.0

6.0

8.0

10.0

12.0

Radiation dose (Gy) 2.0

4.0

6.0

8.0

10.0

12.0 t

0.5

0

E

LL

-

*' ,

Radiation dose (Gy)

LL

0.1 -

5 0.05 -

0.005

-

0.001

-

0.01

m

.? 0.1

E

$ 0.05

FIG. 1. AnalysiLof a standard radiation survival curve using the multitarget model (A), the linear quadratic model (B), and the concept of mean inactivation dose (D)(C) (adapted from Fenil and Malaise, 1985).

ONCOCENIC BASIS OF RADIATION RESISTANCE

199

in these regions of the curve may result in greater (or lesser) survival after radiation exposure.

B. CLONAL NATUREOF THE RADIATION RESPONSE Clonal heterogeneity has been reported in both normal and neoplastic cells. This phenomenon includes such diverse features as tumor histology, metastatic phenotype, growth characteristics, antigens, and transplantability (Poste et al., 1981; Brattain et al., 1981; Rubin et al., 1983; Heppner, 1984). Heterogeneity of the radiobiological response(s) among clonally derived cell lines has been the focus of several investigations (Hill et al., 1979; Leith et al., 1982; Weichselbaum et al., 1988; Kasid et al., 1989~).These studies have reported significant variations in the radiation survival parameters of clonally derived cell lines representing such diverse cell types as colon and lung carcinomas, human squamous cell carcinomas, and NIH/3T3 cells. Earlier reports have revealed that the cotransfection of human tumor DNA and pSV2Neo plasmid DNA into NIH/3T3 cells results in G418resistant NIH/3T3 clones that demonstrate a spectrum of radiation responses ranging from a relatively radioresistant phenotype (Do = 2.28 Gy) to a relatively radiosensitive phenotype (Do = 1.36 Gy) compared to the untransfected parental NIH/3T3 strain (Do = 2.02 Gy) (Kasid et al., 1989~).However, heterogeneity in radiation response was also observed when the untransfected single cell-derived NIH/3T3 clones were studied (range, Do = 1.06 Gy to 2.38 Gy) (Kasid et al., 1989~).In a recent report, the y-radiation survival of Syrian hamster (SHOK) cells was shown not to be affected by transfection of the neomycin gene (Suzuki et al., 1992). The above studies are in contrast to an earlier report suggesting that transfection of a neomycin resistance marker and clonal selection could impart radioresistance to both normal and tumor cells (F’ardo et al., 1991). In the latter report, these investigators also did not find significant heterogeneity in the radiation response of clonally derived untransfected primary rat embryo cells or glioblastoma cells. Because clonal cell lines derived from both NIH/3T3 and human tumor cell populations exhibit sufficient heterogeneity in radiation survival responses to make interpretation of oncogene effects exceedingly difficult (Leith et al., 1982; Weichselbaum et al., 1988; Kasid et al., 1989c), it seems reasonable to circumvent the heterogenous component of the radiation response phenotype by using pooled transfectant cell populations for studies of the effects of oncogenes on radiation survival response (Kasid et al., 1989a,b; Pirollo et al., 1989). However, in several reports discussed in this review, oncogene-transfected clonally derived

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cell lines have been used to investigate changes in radiation sensitivity as a function of oncogene expression. Therefore, for the sake of simplicity, this review will be based solely on the interpretations of the radiation survival data as they have been reported in the literature.

111. Human and Rodent Cell Model Systems The initial approach used to identify the genetic factors associated with relatively radiation-resistant cells both from a human squamous cell carcinoma and from nontumorigenic skin fibroblasts (NSFs) of certain cancer-prone individuals was based on cotransfection of the representative human DNA and pSV2Neo plasmid DNA into NIH/3T3 cells followed by G418 selection and screening of the G418-resistant NIH/3T3 transfectant clones for the presence of human counterparts of the known oncogene sequences (Kasid et al., 1987a; Chang et al., 1987; Pir0110 et al., 1989). Subsequently, cloned cDNA of the candidate human protooncogene (raf-1) was expressed in human squamous carcinoma cells in an attempt to determine the phenotypic changes in the recipient human cells (Kasid et al., 1989a). A similar approach was used in studies using other human cell lines and rodent cell lines in order to determine the radiobiological consequences of the expression of a variety of protooncogenes (Pirollo et al., 1989, Table I). This section will (a)summarize the various cell systems that have been studied to date and (6) identify the candidate oncogenes that appear to have a potential role in the regulation of radiation resistance. A. RADIORESISTANT HUMAN SQUAMOUS CELLCARCINOMAS Relatively radioresistant tumor cell lines (SQ-2OB, SCC-35, JSQ-3) were established in culture from squamous cell carcinomas of head and neck origin following the full course of radiotherapy (Weichselbaurn et al., 1989). DNA-mediated gene transfer was used to investigate the genetic factors associated with these tumor cells. The human raf-1 sequences were found in the NIH/3T3 clones transfected with these DNAs. A majority of the NIH/3T3 transfectants were highly tumorigenic in athymic mice (Kasid et al., 1987a, 1993). Significantly, the NIH/3T3 transfectant clone lacking the kinase region (cl 21) was nontumorigenic, as were the control untransfected NIH/3T3 cells (Kasid et al., 1987a). The identification of the loss of the regulatory domain and retention of the kinase domain in the highly tumorigenic clones supports the hypothesis that deletion of the regulatory region results in the

20 1


TABLE 1

ONCOGENE EFFECTS ON RADIATION RESPONSE Oncogeneu v-abl

Cell type used for oncogene transfection

N 1H/3T3

Changes in responseb NC

Sklar el al. (1986); Pirollo et al. (1989) FitzCerald et al. (1991)

t

Suzuki et al. (1992)

NlH/3T3 and murine hematopoietic cells, 32D c13 c-cot

Syrian hamster OsakaKanazawa (SHOK) cells

FitzGerald et al. ( 1990) Suzuki et al. (1992) Pirollo et al. ( I 989)

32D c13 SHOK cells v-fes V Y P

c-fm

v-fos v-mas c-myc

References

NIH/3T3 SHOK cells


N I H13T3 32D c13

Sklar et al. (1986) FitzGerald et al. (1991)

NI H/3T3

Sklar (1988) FitzGerald et al. (1991)

N IH/3T3 NlH/3T3 SHOK cells N 1H /3T3 Rat embryo cells (REC)

FitzGerald et al. (1990) Pirollo et al. (1989) Suzuki et al. (1992) Pirollo et al. (1989) Ling and Endlich (1989) McKenna et al. (1990a) Kasid el al. (1989b)

lmmortalized human bronchial epithelial cells (Beas-2B) SHOK cells


v-myc

32D c13

FitzGerald et al. (1991)

c-raf- 1

Human SCC, (SQ-2OB) NIH/3T3 Beas-PB Beas-2B

Kasid et al. (1989a) Pirollo et al. ( I 989) Kasid el al. (1989b)

c-raf-I and c-myc c-raf-I (AS) c-H-rm

SQ-20B NlH/3T3

Kasid et al. (1989b)

.1

Kasid et al. (1989a)

NC

Sklar (1988) Pirollo at al. (1 989); Samid et al. (1991) Grant et al. (1990)

t Transformed human embryo retinal cells (HER)

NC

(continued)

202

USHA KASID ET AL.

TABLE I (Continued )

Oncogenea EJ-Tu

v-H-T~

Cell type used for oncogene transfection

Changes in responseb

References

N I H13T3

t

REC

f (m)

Human mammary epithelial cells, (HBL 100) Immortalized human keratinocytes (HaCaT)

NC

NC

Mendonca et al. (1991)

NIH/3T3

t t (Id)

Sklar (1988) FitzGerald et al. (1990) Suzuki et al. (1992)

t

Sklar (1988) Harris et al. (1990) Szuki et al. (1992)

Sklar (1988); Pirollo et al. ( 1989) Ling and Endlich (1989); McKenna et al. (1990a) Alapetite el al. (1991)

SHOK cells

NC

NIH/3T3 Rat kidney epithelial cells SHOK cells

J. NC

N I H13T3

t

HER SHOK cells REC

NC

REC

t

McKenna et al. (1990a)

V-Sic

32D c13

NC

FitzGerald et al. (1990)

v-STC

N I H13T3 32D c13 Rat fibroblast cells (LA-24) Multidrug-resistant LA-24 cells

J.

NC

FitzGerald et al. ( 1990) FitzGerald et al. (1990) Shimm el al. (1992)

t

Shimm et al. (1992)

v-K-T~s

N-rm

EJ-rm and c-my EJ-rm and

t t

FitzGerald et al. (1985); Sklar (1988) Grant et al. (1990) Suzuki et al. (1992) Ling and Endlich (1989)

v-my

t (Id)

Oncogenes are alphabetically arranged on the basis of their three-letter code. AS, antisense cDNA. t , evidence suggesting increase in relative radiation resistance (Do or D, value); 1, evidence suggesting decrease in relative radiation resistance (Do value); NC, no change reported compared to the experimental control; f (m). moderate increase in relative radioresistance; t (Id). increase in radioresistance reported at low dose range y-irradiation.


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catalytic activation of the kinase domain (Kasid et al., 1987a; Pfeifer et al., 1989a; Stanton et al., 1989; Heidecker et al., 1990). Although the identification of human m f - 1 sequences in the NIH/3T3 transfection assay has been reported using a variety of tumor DNA samples (Shimizu et al., 1985; Fukui et al., 1985; Ishikawa et al., 1986; Stanton and Cooper, 1987), the presence of human ruf-1 sequences in all of these human a h containing NIH/3T3 transfectant clones derived from transfection of these radioresistant tumor cell-derived DNAs is remarkable. Because the clonally derived NIH/3T3 cell lines (untransfected) present a heterogenous population in terms of their radiation sensitivities (Do values), a direct correlation between activated raf-1 and radiation response was not feasible in the above-described NIH/3T3 transfectants (Kasid et al., 1989~). Given the complex nature of cellular radiation sensitivity, one way to demonstrate a correlation between raf- 1 activation and resistance to ionizing radiation in tumor cells is to perform radiation survival analysis on a pooled cell population in which rafi 1 expression has been inhibited by transfection of antisense human rafil cDNA. Indeed, this antisense RNA approach has been successfully used to demonstrate that the down-regulation of endogenous rafi 1 expression leads to decreased tumorigenicity and enhanced radiation sensitivity of human squamous carcinoma-derived cells, SQ-20B (Kasid et al., 1989a) (Fig. 2). These data provide evidence for the role of raf-1 function in radiation resistance. Furthermore, these studies demonstrate that the antisense vector-based strategy to inhibit the biological outcome of a specific gene function is also applicable to the radiation response phenotype. T h e modulatory effect on radioresistance due to antisense c-raf- 1 expression was evident only during the early passages in culture, as has been observed by other investigators using antisense RNA constructs (Bolen et al., 1987). Nevertheless, modifications of the antisense RNA strategy using inducible vectors or inhibition of Raf- 1 function by antisense deoxyoligonucleotides (Kasid et al., 1991a) are promising approaches for further investigations into the oncogenic basis of radiation resistance. B. SKINFIBROBLASTS FROM

A

CANCER-PRONE FAMILY

T h e cancer family syndrome originally described by Li and Fraumeni (1969)is characterized by a constellation of tumor types including breast carcinoma, soft tissue sarcoma, brain tumors, osteosarcoma, and leukemias. These diverse neoplasms, which occur in a dominantly inherited

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0.005

0.001

I -

0 100

300

500

700

900

Radiation Dose (cGy)

FIG. 2 . Clonogenic radiation survival curves for c-raf-1 (S) and c-raf-1 (AS) cDNAtransfected SQ-2OB cell populations (adapted from Kasid el al., 1989a). The experimental points are plotted &SEM. The Do values were 310 and 191 cCy for c-raf-I (S) cDNA and for c-raf-1 (AS) cDNA-transfected SQ-POB cell populations, respectively.

pattern, develop at an early age with multiple primaries appearing in the same individual. In some instances they appear to be related to carcinogenic exposures, including ionizing radiation. Recently, inherited germline mutations in the tumor suppressor gene p53 were simultaneously identified by two different groups in a total of six different LiFraumeni families (Malkin et d.,1990; Srivastava et al., 1990). These mutations, which are located in a highly conserved region of the gene, are believed to represent the primary inherited defect predisposing these individuals to develop cancer. One particular Li-Fraumeni family has been studied more exten-


205

sively than others. This specific family, originally described by Blattner et al. (1979), involves 18 affected descendants of a single individual through six generations. Neoplasms in 3 members of the family may have been induced by occupational exposure or therapeutic radiation. One member developed polycythemia Vera after working for 5 years in a factory producing heavy water, a second family member with lung adenocarcinoma worked in a foundry, and an osteosarcoma was diagnosed in a third individual within the field of radiotherapy for an earlier neurilemmoma. In addition to the inherited germline mutation in codon 245 of p53 identified in this family (Srivastava et al., 1990), examination of nontumorigenic skin fibroblast (NSF) cell lines from individuals in the cancer-prone lineage revealed a three- to eightfold elevation in the level of c-myc expression relative to that found in unrelated control fibroblast cells (Chang et al., 1987). Moreover, by means of the NIH/3T3 transfection assay, the presence of an activated raf-1 oncogene has also been detected in the NSFs from at least one family member (Changet al., 1987). It is also noteworthy that the NSF cell lines from most members of this family have been found to display the unusual property of resistance to the killing effects of ionizing radiation (Bech-Hansen et al., 1981) (Fig. 3). Five of the family members examined demonstrated an increased level of radiation resistance relative to the normal controls. In one instance multiple cell lines representing biopsies taken from the same individual but at different times were included in the study. T h e differences in the D,,values were statistically significant (P< 0.001) with the fibroblast line from individual IV-19 having one of the highest levels. Furthermore, no correlation was found between the presence of the inherited p53 mutation and the radiation-resistance phenotype observed in individuals in this family. These results support the notion that the radiation resistance may be one of the inherited defects in this specific cancer-prone family, although this clearly is not the case for all LiFraumeni families (Little et d., 1987). Moreover, heterogeneity does exist among these kindred inasmuch as not all of the families previously identified as having Li-Fraumeni syndrome were found to contain a germline mutation in the tumor suppressor gene p53 (Santibanez-Koref et al., 1991). An association between the radiation-resistant phenotype present in these family members and the activated r a . 1 gene was demonstrated when a tertiary NIH/3T3 transformant derived from DNA of the NSF cell line with one of the highest levels of radiation resistance was assessed for its resistance to killing by y-radiation. This human raf- 1-containing mouse cell line demonstrated an increased level of radiation resistance

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USHA KASID E T A L .

I

I

I

I

I

IIL

PV

Br

I

I

P

BT BT

BT

Br

ss

# :Single primary cancer

9I: BT

0s

in deceased female : Double primary cancer in proband

FIG.3. Partial pedigree of a specific cancer-prone family with Li-Fraumeni syndrome (modified from Bech-Hansen et al., 1981). Shown here is a branch of a much larger pedigree that traces cancers over five generations in three separate lineages from a woman who died of breast cancer in 1865 (Blattner el al., 1979). Individuals whose normal skin fibroblast cell lines were found to demonstrate an increased level of radioresistance are designated “R.”N, normal level of radiation response; OS, osteogenic sarcoma; S S , soft tissue sarcoma; BT, brain tumor; Br, breast cancer; PV,polycythemia Vera; Le, leukemia; Co, colon cancer; NL, neurilemrnoma.

relative to that of the untransfected NIH/3T3 cells, a level which approximated that observed in the radiation resistant parental NSF cell line (Pirollo et al., 1989)(Fig. 4).This report of the relationship between activated rafand radiation resistance is among the first to link this phenotype to the function of a specific oncogene. Two additional features of the NSF cell lines from members of this cancer-prone family that may be related to its radiation-resistant phenotype have been examined. The first is the level of activity of the topoisomerases in these cells. The activities of both topoisomerase I and I1 in the NSFs of the family were examined (Cunningham et al., 1991). The activity of topoisomerase I1 was found to be elevated in the radiation-resistant cell lines of this family but not in that of the non-radiationresistant cell line derived from a normal spouse (V-7).Topoisomerase I activities were comparable in all of the lines tested. The second feature is the presence of an altered DNA de novo synthesis in the NSFs of family members. The radioresistant NSF cell lines from the proband (VI-2)and his great uncle (IV-19),which both sustain and repair radiogenic DNA damage at rates comparable to those of non-


207

600-.

cA

ul

550 --

3

% 450-

T V 1 OT

400-

350-

t

rn

0-NSF 2800 A-3T3lraf 0 3T3lmos Q A-3T3IEJ-ras 0-3T3lLTR- ras

-

U

500-

0'

A

T 111

0 T

0- NIH3T3 A 3T3lmyc V-3T3Ifes 3T3labl

-

-

FIG.4. Scattergram of the D I Ovalues of various NlH/JTS-transformed cell lines demonstrating an increased level of radiation resistance for cell lines transformed by the raf, mos. or ras oncogene, relative to the parental NIH/3T3 cell line (modified from Pirollo et al., 1989). Each point represents the mean of two to five experiments 2 standard error. T h e error bars for NIH/3T3,3T3/abl, 3T3/mos, and 3T3IEJ-ras are absent due to the fact that the standard errors for these four cell lines (k3.6, 26, 2 7 , and 2 5 , respectively) are too small to be visualized within the parameters of the graph. NSF 2800 is the radioresistant normal skin fibroblast cell line derived from individual IV-19 in the family pedigree (Fig. 3) and is the cell line from which NIH13T3 transformant 3T3lrafwas derived (Chang et al., 1987).

radiation-resistant fibroblast cell lines, possess what has been described as an error-prone semiconservative DNA synthesis mechanism (Paterson et al., 1985). After treatment with y-rays (60Co), these cells displayed a longer lag time prior to initiation of synthesis and sustained a higher level of synthesis for a protracted period of time when compared to nonradiation-resistant fibroblast cells. It is of interest to note that the abnormality in DNA synthesis evident in these radiation-resistant fibroblast cell lines is the exact opposite of that seen in radiation-sensitive AT cells where DNA synthesis is appreciably reduced relative to normal cells (Houldsworth and Lavin, 1980). Therefore, this Li-Fraumeni family is a naturally occurring system that implicates the mf oncogene in the genesis of radioresistance. Since the radiation-resistant NSF cell lines are not tumorigenic, this model also provides evidence that radiation resistance and transformation are not necessarily coincidental phenomena (see also Section V). Furthermore, it suggests that events in the nucleus, such as those involved in DNA conformation, synthesis, and repair, may be important factors in cellular radiation resistance.

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U S H A KASID E T A L .

C. NIH/3T3 TRANSFECTANTS The earliest report of a possible link between an activated oncogene and a radiation-resistant phenotype in NIH/3T3 cells showed that transfection of the human N-ras oncogene was able to increase the level of relative resistance of the recipient cells at a relatively high radiation dose rate (200 cGy/min) (FitzGerald et al., 1985). Subsequent studies demonstrated that the effect of activated oncogenes on the radiation-resistance level of NIH/3T3 cells is not a generalized phenomenon but is particular to specific oncogenes (Sklar et al., 1986; Sklar, 1988; Pirollo et al., 1989; Suzuki et al., 1992); this will be discussed in greater detail below. The presence of v-H-ras, v-K-ras, EJ-ras, and N-ras oncogenes were found to significantly increase the radioresistance levels of NIH/3T3 cells, whereas NIH/3T3 cells transformed by either vfm or v-a61 did not exhibit changes in radiation sensitivity relative to the untransfected cells (Sklar et al., 1986; Sklar, 1988). A similar effect on the radiation response of NIH/3T3 cells was also demonstrated by overexpression of the normal H-ras protooncogene (Pirollo et al., 1989). Moreover, a significant degree of radiation resistance was conferred on NIH/3T3 cells expressing members of the protein-serinelthreonine kinase family (rufand mos) (Fig. 4). The effects of oncogenes appear to vary depending on the dose rate of y-radiation employed. Although the experiments using a high dose rate of the y-radiation (60Co; = 120 cGy/min) demonstrated no change in the radiation response of NI H/3T3 cells transfected with v-abl, vfm, o r v-fos (Pirollo et al., 1989), other reports using low-dose-rate y-radiation ( 13’Cs; 5 cGy/min) have indicated a significant increase in the radiation resistance of NIH/3T3 cells expressing these oncogenes (FitzGerald et al., 1990, 199 1). T h e precise mechanism(s) and significance underlying these differential effects on radiation response due to the differences in the dose rate of y-radiation employed are unclear at present. D. OTHER CELLLINES

T h e synergistic effect of EJ-ras and c-myclv-myc on the radiation-resistant phenotype has been reported in studies using primary rat embryo cells, REC (Ling and Endlich, 1989; McKenna et al., 1990b). By itself, EJ-ras had only a limited effect on the radiation response of RECs, whereas the myc gene had no effect (McKenna et al., 1990a,b). Together these two oncogenes exhibited a significant increase in radioresistance over the parental cells. These observations suggest the possibility of a putative interaction among the products of the ras and m y oncogenes in signaling mechanisms underlying the radioresistant phenotype.


209

I n a recent report, resistance to y-rays was conferred by the introduction of v-mos, c-cot (both genes encoding cytoplasmic protein serine kinases), o r N-ras gene into Syrian hamster (SHOK) cells (Suzuki et al., 1992). Interestingly, the radiation sensitivity of these cells did not change upon the transfection of the v-fgr, c-myc, v-erb-B, Ha-ras, or K-ras gene. T h e morphological transformation associated with the induction of v-src did not correlate with the radioresponsiveness of rat fibroblast cells (LA-24). However, in the multidrug-resistant clones of these rodent cells, a significant increase in radioresistance has been reported to correlate with the induction of v-src (Shimm et al., 1992). Murine hematopoietic progenitor cells, 32D c13, have also been analyzed for the oncogenic effects on radiation response using low-dose-rate y-radiation. These experiments suggest that with the exception of v-szi, the other oncogenes tested, namely v-erb-B, v-abl, v-src, c$m, and v-myc, are all able to induce radiation resistance in 32D c13 transfectant cells (FitzGerald el al., 1990, 1991). T h e increase in radiation resistance of immortalized human bronchial epithelial cells (Beas-2B) by the expression of raf-1 has also been demonstrated (Kasid et al., 1989b). These reports suggest that the increased expression of raf- 1 is sufficient to increase radioresistance in the nontumorigenic Beas-2B cells, whereas c-myc expression does not change the radiation dose response. In addition, Beas-2B cells transfected with a combination of raf-1 and c-myc genes demonstrated no further increase in the level of radiation resistance (Do value). However, this does not rule out possible synergism between rafand myc in other cell systems. Taken together, the studies to date suggest that Raf-1 plays a dominant role in the radiation-resistant response of both human and rodent cells. Finally, the role of the ras oncogene in the radiation response of human cells is somewhat intriguing. To date, three different human cell model systems have been examined in an effort to elucidate the role of the ras gene product in the radiation response of human cells. T h e N-ras and c-H-ras genes did not seem to alter the radiation sensitivity of transformed human embryo retinal cells (Grant et al., 1990) and EJ-ras transfection and expression had no effect on the radiation response of human mammary epithelial cells (HBL 100) and immortalized human keratinocytes (Alapetite et al., 1991; Mendonca et al., 1991). T h e complex basis of the ras gene-induced effects on radioresistance is also evident from another study in which the radiosensitization of rat kidney epithelial cells containing K-ras has been reported (Harris et al., 1990). Among other possibilities, the differentiation state of the cells and/or a cooperation between another gene and ras may be required to modulate

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the radiosensitivity of these cell types. Alternatively, a high level of inherent radioresistance may be a limiting factor in certain cell types. Moreover, such reports serve to remind us of the fact that these types of studies can be complicated by both the methodology and the biology of the system.

IV. Mitogenic Signals and Radiation Response The normal counterparts of many of the known oncogenic products have been shown to interact with one another as components of a proposed signal transduction pathway that serves to transmit messages from the cell membrane to the nucleus (Weinstein, 1988a). On the basis of antibody-blocking experiments, Raf- 1 has been placed downstream of Ras in this pathway (Smith et al., 1986; Rapp et al., 1988b; Morrison et al., 1989).As discussed earlier, the r a . 1 oncogene has been implicated in the expression of the radiation-resistant phenotype (Chang et al., 1987; Kasid et al., 1987a, 1989a,b; Pirollo et al., 1989). Additionally, since oncogene products upstream of Ras and Raf in the proposed signaling pathway are able to induce the resistant phenotype and reports from several other laboratories have indicated that activated ras oncogenes, or a combination of ras and myc oncogenes, can influence the radiation resistance level of cells into which they are transferred (Table I), it appears that the expression of radiation resistance may be under the control of a similar type of signal transduction mechanism. Protooncogenes have been shown to code for growth factors and growth factor receptors such as platelet-derived growth factor (PDGF) B-chain, truncated epidermal growth factor (EGF) receptor, fibroblast growth factor (FGF)-related growth factor, colony-stimulating factor (CSF-1) receptor, and nerve growth factor (NGF) receptor (Hunter, 1991).The effect of the oncogenefms, which codes for the mutant CSF-1 receptor protein-tyrosine kinase on radiation response has been examined. This gene has been found to be incapable of modulating the radiation sensitivity of NIH/3T3 cells (Sklar et al., 1986; Sklar, 1988) except at low doses of radiation (FitzGerald et al., 1990). Members of the family of nonreceptor protein-tyrosine kinases seem to be incapable of effecting the level of radiation resistance. The oncogenesfes, abl, and fgr did not alter the radiosensitivity of the recipient NIH/3T3 or Syrian hamster (SHOK) cells (Pirollo et al., 1989; Suzuki et ad., 1992).The exception appears to be the Src protein kinase. The v-src oncogene confers radioresistance on murine hematopoietic cells 32D c13 (at a low-dose-rate y-radiation) (FitzGerald et al., 1990). However, Src protein-tyrosine kinase induction did not increase the radioresistance in


21 1

rat fibroblast cells (LA-24) and appeared to increase the sensitivity of NIH/3T3 cells (FitzGerald et al., 1990). Interestingly, a significant radioresistance was noted in the multidrug-resistant variants of LA-24 cells upon induction of the Src kinase (Shimm et al., 1992). G proteins, which are involved in the reversible exchange of GTP for GDP with concomitant activation of an effector protein, also have a role in the signal transduction pathway. T h e ras p2 1 protein functions as a G protein (Trahey and McCormick, 1987; Vogel et al., 1988) and has been implicated in the activation of phospholipase C (Berridge and Irvine, 1984; Chabre, 1987; Cockroft, 1987; Marshall, 1987; Katain and Parker, 1988). T h e role of the ras gene in radiation resistance has been studied by several investigators, using not only the NIH/3T3 transfection assay (FitzGerald et al., 1985, 1990; Sklar, 1988; Pirollo et al., 1989; Samid et al., 1991), but also the primary rat embryo fibroblast cells (REC) (Ling and Endlich, 1989; McKenna et al., 1990a), Syrian hamster cells (Suzuki et al., 1992), rat kidney epithelial cells (Harris et al., 1990), and human cells (Grant et al., 1990; Alapetite et al., 1991; Mendonca et al., 1991). T h e presence of any member of the ras family, activated through either mutation o r overexpression, was sufficient to significantly increase the level of the radiation resistance in the recipient NIH/3T3 cells. N-rtls expression but not Ha-ras or Ki-ras expression was able to increase the radioresistance in Syrian hamster (SHOK) cells Suzuki et al., 1992), and only a moderate increase in radioresistance of REC was noted following the transfection of EJ-ras (Ling and Endlich, 1989; McKenna et al., 1990a,b). However, the cotransfection of EJ-ras and c-myclv-myc led to a significant increase in the relative radioresistance of REC (Ling and Endlich, 1989; McKenna et al., 1990b). In contrast, rat kidney epithelial cells containing K-ras had increased radiation sensitivity with ras activation. Human cells, however, did not show changes in the radiosensitivity in response to the ras expression. Although a cooperation between oncogenes may be required for the development of in vitro radioresistance in human cells, the evidence is not yet available. T h e cytoplasmic protein-serine/threonine kinases play a central role in signal transduction and are also implicated as one focal point in the pathway to the radiation-resistant phenotype. The prototype for such serine/threonine kinases, protein kinase C (PKC), is a principal effector for signaling mediated by phosphotidylinositol-4,5-biphosphatehydrolysis and is the vehicle by which a number of tumor promoters act (Weinstein, 1988b). Based on Raf-1 inhibition experiments, it is suggested that PKC-dependent mitogenic signals are transduced by PKCmediated activation of Raf-1 kinase in NIH/3T3 cells (Kolch et al., 1991). However, the PDGF effects on Raf-1 are seen in fibroblasts that have

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USHA KASID ET AL.

been chronically treated with a tumor promoter to down-regulate PKC (Morrison et al., 1988). Therefore, it appears that PKC is not always an effector for the activation of Raf- 1 kinase. However, the possibility exists that PKC may also be affecting the cellular response to y-radiation (Weichselbaum et al., 1991). Most important, cot, mos, and the raf family of oncogenes all encode protein-serine kinases (Aoki et al., 1991; Seth and Vande Woude, 1988; Rapp et al., 1988b) and all have been associated with the acquisition of radiation-resistant phenotype (Kasid et al., 1989a,b; Pirollo et al., 1989; Suzuki et al., 1992). Raf-1 protein kinase appears to be at a central location in the signaling pathway to radiation resistance. An association between the raf- 1 gene and the radiation-resistant phenotype in the NSFs from a specific cancer-prone family with Li-Fraumeni syndrome has been established (Pirollo et al., 1989). Transfection of antisense human raf-1 cDNA into radioresistant human squamous carcinoma cells leads to the down-regulation of endogenous raf 1 expression, delayed tumor growth, and enhanced radiation sensitivity (Kasid et al., 1989a). In addition, the transfection of human raf- 1 cDNA into immortalized human bronchial epithelial cells (Beas-2B) is sufficient to increase the radioresistance of Beas-PB-raf transfectants (Kasid et al., 1989b). These studies suggest a close link between the radiation-resistant phenotype and the function of raf-1 in human cells. Increased phosphorylation and elevated enzymatic activity of Raf- 1 protein kinase have been demonstrated in numerous cell types tested in response to a variety of ligands (Rapp, 1991). In fact, Raf-1 has been found to be complexed with at least two growth factor receptors, EGF-R (App et al., 1991) and PDGF-R (Morrison et al., 1989). Oncogenes shown to have homology to one of the EGF receptors and to PDGF (HER-2 and sis, respectively) have been shown to increase radiation resistance of NIH/3T3 cells (Pirollo et al., 1991). Further evidence for the importance of Raf-1 protein kinase in the signaling pathway leading to the radiation resistance is suggested by the report on the radioprotective effects of granulocyte macrophage-colony-stimulating factor (GM-CSF) (Waddick et al., 1991), which has been shown to increase the activity of Raf-1 protein kinase (Carroll et d., 1991). Therefore, it appears that the functional activation of Raf-1 protein kinase either via direct alteration in the product itself or via modulation of the upstream signals may be sufficient or even necessary for the induction of the molecular/biochemical events leading to radioresistance. The ultimate targets of signal transduction in the nucleus are probably the least understood part of the pathway. However, there are some


213

indications of what may be occurring there. The mitogenic signal(s) received by Raf-1 protein kinase may be forwarded to the nucleus by phosphorylation of transcription factors such as AP-1, which is an association of the products of protooncogenes fos andjun (Chiu et al., 1988; Rauscher et al., 1988; Sassone-Corsi et al., 1988; Bruder et al., 1992). Inactivation of Raf- 1 protein kinase reportedly blocks the transcription of a reporter gene from the promoter containing the AP- 1 DNA binding motif (Bruder and Rapp, 1991; Bruder et al., 1992). These studies indicate that AP-1 requires Raf-1 protein kinase for the transactivation of transcription. T h e effect ofjun on intrinsic radioresistance is not known but fos has no effect on the radiation response of NIH/3T3 cells at low doses (FitzCerald et al., 1990). Experiments using purified components suggest that the Jun but not the Fos component of AP-1 is phosphorylated by Raf-1 (Heidecker et al., 1992). The nuclear oncogene, myc, coding for Myc protein which is downstream of Raf- 1 in the proposed mitogenic signal transduction pathway, is unable by itself to increase the level of radiation resistance in NIH/3T3 cells (Pirollo et al., 1989). Similar results were obtained using primary rat embryo cells (REC) and human bronchial epithelial cells (Beas-2B) (McKenna et al., 1990a; Kasid et al., 1989b). However, when both ras and myc genes were introduced into REC, a synergistic increase in radiation resistance was noted (Ling and Endlich, 1989; McKenna et al., 1990a). In transfection experiments utilizing Beas-2B cells, cotransfection of the raf and myc genes apparently did not induce a synergistic level of radiation resistance. However, this does not necessarily rule out the possibility of cooperation between these two oncogenes. It is conceivable that there may be an upper limitation to the level of detectable in vitro radioresistance (Do value), such that, in this instance, any contribution by Myc may not have been discernible. The support for the combined role of c-myc and c-raf-1 in radioresistance is derived from several lines of investigations. Amplification of c-myc has been associated with an in vitro radiation resistance of a variant of small cell lung carcinoma (oat cell) (Carney et al., 1983; Little et al., 1983), as well as with certain human lung cancer cell lines (Carmichael et al., 1989). Interestingly, a high level of expression of the raf-1 gene, along with a concomitant activation of Raf-1 protein kinase activity, has been found in approximately 60% of all lung cancers (Rapp et al., 1988a). Moreover, an activated raf-1 oncogene was identified via the NIH/3T3 transfection assay, as the transforming gene in a human lung carcinoid (CA1-154) (Stanton and Cooper, 1987). In this same vein, the radiation-resistant NSF cell lines from a specific cancer-prone family with Li-Fraumeni syndrome exhibit both an elevated level of c-myc

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expression and the presence of an activated raf-1gene, as suggested by the NIH/3T3 transfection procedure. Furthermore, c-my expression is known to be regulated at the level of the cell cycle. Therefore, these data indicate two possibilities: (a)a cooperativity toward radioresistance may exist between the raf and the m y genes and (6) genes such as m y whose expression is also regulated in a cell cycle-dependent manner may modulate in vitro radiosensitivity. The reports available so far suggest the possibility of a signal transduction pathway for radiation resistance analogous to that suggested for cell growth and proliferation or differentiation. Although many of the intermediate steps in this pathway have not yet been identified, there is strong evidence that the cytoplasmic serinelthreonine kinases, particularly Raf-1, play a central role. Several possibilities arise by which multiple signals may interact to influence the radiation-resistant phenotype. In one instance, proteins that already exist in the activated form interact with one another to generate a cell population that is selected for after exposure to radiation. Alternatively, exposure of cells to radiation precipitates events in the nucleus through changes in chromatin. These changes may trigger a cascade of events similar to those described above, leading to cell survival. I n addition, the ability of cells to delay progression through the cell cycle following DNA damage (G, and/or G, arrest) may be an important determinant of cell survival. It is also possible that the key component of the signal directly involved in radioresistance is initiated after exposure to y-rays. For example, radiation is known to induce programmed cell death (apoptosis) (Kerr and Searle, 1980).More recently, the extent of radiation-induced apoptosis has been shown to differ markedly between radiosensitive and radioresistant tumors and correlated with their respective response to local tumor irradiation (Stephens et al., 1991).It seems very likely that the products of certain oncogenes may counteract radiation-induced apoptosis. It is tempting to speculate that this is accomplished by an oncogene-activated effector protein(s), which may direct the normal replication machinery to read through the damage, thereby allowing the cells to proliferate. In support of the role of oncogenes in apoptosis, evidence is beginning to emerge that suggests a role for v-Raf in the suppression of this physiological control mechanism (Troppmair et al.,

1992). Since much is still unknown about the oncogenic interaction(s) in signal transduction, and about the biochemical and physiological bases of radioresistance, the working hypothesis put forth here may likely be modified with the advancement of our knowledge.


215

A majority of the relevant data in the literature is consistent with the above hypothesis. However, some discrepancies do exist. The c-myc oncogene, which was shown not to increase the radiation resistance level of NIH/3T3 cells, rat embryo cells (REC) and human bronchial epithelial cells (Pirollo et al., 1989; Kasid et al., 198913; McKenna et ad., 1990a) did so in one study using RECs (Ling and Endlich, 1989) and also in one study using the hematopoietic progenitor cell line, 32D c13 (albeit at low dose rate only) (FitzGerald et al., 1991). NIH/3T3 cells transformed by abl or fms were found not to be radiation resistant (Sklar et al., 1986; Sklar, 1988; Pirollo et al., 1989). However, in the 32D c13 cells both oncogenes were able to induce radiation resistance, again at low dose rate of y-radiation (FitzGerald et al., 1991). Evidence from clinical studies indicates that different cell types have differing responses to radiation. In fact, studies, primarily with mice, demonstrated that in whole-body irradiation, the cells of the hematopoietic and the gastrointestinal systems are the most sensitive to the killing effects of ionizing radiation (Bond et al., 1965; Bond, 1969; Broerse and MacVittie, 1984). T h e observed effect of oncogenes on radiation resistance may also vary depending on the cell types used in the study (human vs mouse cells; fibroblasts vs epithelial cells or keratinocytes), further complicating the issue. Moreover, some cell types that display a significant level of relative radiation resistance (as measured by the relatively high Do value) may be inappropriate for use in such studies. In support of the latter argument, no change in the Do value of primary human keratinocytes (Do = 2.24 Gy) was observed following immortalization by SV-40/AD-12 virus (Do = 2.43 Gy) or subsequent transformation of the immortalized human keratinocytes by KiMSV infection (Do = 2.51 Gy) (Kasid et d., 1987b). T h e presence of discrepancies, such as those discussed above, underscore the complexity of the factors contributing to radiation resistance and the importance of future studies to achieve a better understanding of the molecular mechanisms involved.

V. Transformation and Radiation Resistance Based upon the above data, it is evident that the signal transduction pathways for mitogenesis and radiation resistance may have some elements in common. Although these signals may intersect with one another at some points, they obviously represent independent pathways that are neither identical nor even concurrent. Support for this is derived from the following reports. It has been well established that ras alone is incapable of transforming primary cells (REC); a cooperative

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oncogene which can immortalize the cells such as myc o r E1A is required for transformation. However, nontumorigenic ras-containing RECs were found to be more radiation resistant than the recipient RECs (Ling and Endlich, 1989). Further evidence of the separation of the two phenotypes is seen in a study of ras-transformed and phenotypically revertant NIH/3T3 cells. In these studies both the tumorigenic and the revertant, nontumorigenic NIH/3T3 cells expressed high levels of ras and exhibited an elevated level of resistance to radiation indicating that, although ras is clearly responsible for the radiation-resistant phenotype of these cells, perhaps another gene is necessary for the transformed phenotype (Contente et al., 1990; Samid et al., 1991). In addition, a distinction between transformation and radioresistance is provided by a report on rat fibroblast cells infected with a temperature-sensitive mutant of v-src (LA-24). In these studies, induction of v-src resulted in the morphological transformation of LA-24 cells but did not change their radiosensitivity, whereas in multidrug-resistant variants, the v-src induction caused a significant increase in radioresistance (Shimm et al., 1992). In the case of the ruf oncogene also, a division between oncogenic transformation and radiation resistance is evident. As mentioned earlier, the radiation-resistant NSF cell lines from a cancer-prone family with Li-Fraumeni syndrome are clearly nontumorigenic. Moreover, immortalized, nontumorigenic human bronchial epithelial cells transfected with the human r.f-1 cDNA are significantly more radioresistant compared to the untransfected cells or the cells transfected with the Zip-neo vector alone (Kasid et al., 1989b; Pfeifer et ul., 1989b). Therefore, it appears that the Raf-1 protein kinase has a dual role, one in mediating the malignant phenotype and a second in the DNA damage and repair cascade. The dissociation between the two pathways is also apparent from the data presented in Table I. Even though capable of a significant degree of transformation, the v-fm, v-fes, v-abl, o r v - f p oncogene does not seem to contribute to the radiation-resistant phenotype. T h e final destination for the signal in the nucleus may be the most critical determinant in differentiating between the two phenotypes. T h e challenge for future studies is to elucidate the mechanism(s) of this new role for certain protooncogene products, i.e., involvement in the cellular capacity to respond to damage and repair induced by y-radiation.

VI. Radiation-Resistant Phenotype: Cause or Effect Cellular radiation sensitivity is a complex function of diverse molecular, biochemical, genetic, and/or environmental factors (Russo et al.,


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1985; Thacker, 1986; Debenham et al., 1987; Cleaver, 1989). T h e functional involvement of oncogenic factors discussed above may represent only one important level of regulation of radiation response. It is also possible that certain oncogenic factors control the radiation response phenotype by regulating enzymes or substrates (effector proteins) involved directly o r indirectly in the DNA damage and repair system. Indeed, radioresistance/radiosensitivity of human tumor cells has been linked with a differential pattern of gene expression (Kasid et al., 1989d; Ramsamooj et al., 1992). A y-radiation-induced point mutation in c-K-ras has been shown to activate its oncogenic potential (Guerrero et al., 1984). It is not clear at this time whether radiation resistance is a consequence of radiationinduced structural changes in the oncogene(s). Recent reports indicate that ionizing radiation transiently induces certain genes at the transcriptional, translational, or post-translational level (Lambert and Borek, 1988; Boothman et al., 1989, 1991; Singh and Lavin, 1990; Sherman et al., 1990; Hallahan et al., 1991; Brach et al., 1991; Papathanasiou et al., 1991). These observations are highly significant in the context of this review, since some of the genes responsive to y-irradiation code for transcription factors (AP-1, Egr- 1) with a requirement for Raf-1 protein kinase (Bruder et al., 1992; Qureshi et al., 1991).This section reviews the data suggesting (a) that an association may exist between differential expression of the various gene products and radiation response, and (b) that ionizing radiation induces intracellular signaling events involving important growth-related biological molecules. A. MULTIFACTORIAL NATUREOF RADIATION RESPONSE: DIFFERENTIAL GENEEXPRESSION The possibility that multiple genetic factors are involved in the resistance o r sensitivity of human squamous carcinoma-derived cell lines was investigated using two-dimensional polyacrylamide gel electrophoresis (Kasid et al., 1989d; Ramsamooj et al., 1992). Based on the response to radiation therapy and in vitro radiation survival analysis, the tumor cell lines were classified as relatively radioresistant o r radiosensitive (Weichselbaum et al., 1989). A set of at least 14 different proteins was found to be preferentially expressed in each of the three radioresistant tumor cell lines (SQ-20B, SCC-35, JSQ-3; Do range, 2.3 to 2.5 Gy) compared to their expression in the radiosensitive tumor cells, and a set of at least 15 different proteins was specifically expressed in each of the three radiosensitive tumor cell lines (SQ-38, SCC-9, SQ-9G; Do range, 1.3 to 1.7 Gy), compared to their expression in the radioresistant tumor cells

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TABLE 11 IDENTIFICATIONOF MARKERPROTEINS IN RELATIVELY RADIORESISTANT A N D RADIOSENSIT~IVE HUMAN SQUAMOUS CARCINOMA-DERIVED CELLLINES^

Marker protein Radiation response phenotype

Molecular mass (kDa)

PI

Fold enhancement6

Radioresistant (RR) (Dorange, 2.3 to 2.5 Gy)

92 64 24 47 17

5.5 5.2 6.6 7.4 6.2

NA 17 16 10 10

Radiosensitive (RS) (Dorange, 1.3 to 1.7 Gy)

40 36 34 32 39

7.1 6.4 6.1 6.2 6.2

NA 45 10 9 6

Ramsamooj ct af. (1992). Fold enhancement value of the RR or RS protein represents quantitative difference observed in the signal compared to the value of corresponding protein spot in RS or RR cell type, respectively. NA, not applicable due to the undetectable levels in the other radiation response category. a

(Ramsamooj et al., 1992). A representative computer-assisted quantitative analysis of the 5 most significant marker proteins identified in each response category is shown in Table 11. Some of these proteins may represent candidates belonging to the effector-protein category. The role of these proteins in radiation resistance or sensitivity awaits their structural and functional characterization. Nevertheless, these findings suggest that the complexity of the radiation response phenotype may be due to the functional interaction of multiple proteins.

B. MOLECULARTARGETS OF IONIZING RADIATION A number of X-ray-inducible factors have been reported in recent years (Lambert and Borek, 1988; Boothman et al., 1989, 1991; Singh and Lavin, 1990; Sherman et al., 1990; Hallahan et al., 1991). Some of these genetic elements are immediately early genes (cjun,fos,junB, and egr-I) that are also induced rapidly in response to growth factors. The latter genes code for transcription factors AP-I and Egr-1. The induction of egr-1 and c-jun transcription by X-rays was attenuated upon inhibition of PKC by TPA treatment or by the protein kinase inhibitor H7 (Hallahan et al., 1991), suggesting that ionizing radiation induces a signal transduction pathway involving activation of PKC (Weichselbaum et al., 1991).


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Additional evidence in support of the y-radiation induction of PKCmediated signaling events is derived from studies based on the radiation-stimulated transcription of a reporter gene coding for chloramphenicol acetyl transferase (CAT) driven by the Moloney murine sarcoma virus long terminal repeat. Preincubation of X-ray-treated cells with TPA to down-regulate PKC abolished this activation process (Lin et al., 1990). Moreover, activation of transcription and the DNA binding activity of another transcription factor, NF-KB,was noted in response to y-irradiation of human myeloid leukemia cells (Brach et al., 1991). Since activation of NF-KB also occurs in response to UV-irradiation, another DNA-damaging agent, a reverse signaling pathway induced by DNAdamaging agents, that transduces signals from the nucleus to the cytoplasm has been proposed (Brach et al., 1991; Weichselbaum et al., 1991). T h e role for transcription factors in the regulation of transcriptional events is well known (Mitchell and Tjian, 1989). Given the functional significance of protein-serine/threonine kinases in radioresistance (discussed in the previous section), it may be of specific interest to note here that the Raf-1 protein kinase is required for the transcriptional transactivation function, via specific DNA binding sites of AP-1 and Egr-1 (Bruder et al., 1992; Qureshi et al., 1991). Cells that are deficient in Raf-1 protein kinase demonstrate impaired signaling in response to growth factors, deficiency in the induction of immediate-early genes (fos, junB and egr-1), and a block of transcription by the transcription factors AP-1, Ets-1, or Egr-1 (Rapp, 1991; Heidecker et al., 1992). Therefore, the obvious question is: What is the effect of the inhibition or activation of the Raf- 1 protein kinase in the y-radiation-induced transcription of genes coding for these transcription factors? The information gained from such studies may provide a significant advance in our understanding of the role of cytoplasmic kinases in the molecular and biochemical effects of y-irradiation, a biological consequence of which may be resistance to such toxic insults. The notion that ionizing radiation induces specific molecular signals also has support from studies of growth factors, cytokines, and cell cycle control genes. Radiation treatment releases growth factors similar to PDGF-a and FGF from vascular endothelial cells (Witte et al., 1989). Basic FGF (bFGF) has been shown to induce the repair of radiationinduced potentially lethal damage (Haimovitz-Friedman et al., 1991). Precoating of culture dishes with bFGF for the postirradiation colonyforming assay caused the cells to exhibit increased repair of radiation damage. These studies have proposed that radiation induces a complete cycle of an autoregulated damage/repair pathway in bovine aortic

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endothelial cells (BAEC), initiated by radiation-induced damage to cellular DNA and followed by stimulation of bFGF synthesis and its secretion into the medium. The newly synthesized bFGF stimulates the potentially lethal damage/repair (PLDR) pathway, acting via an autocrine loop leading to the recovery of cells from radiation lesions and restoration of clonogenic capacity. Furthermore, these authors have reported that in addition to bFGF, irradiation of BAEC results in an increase in PDGF mRNA. T h e fact that Raf-1 has been observed to be complexed with the PDGF receptor again emphasizes the possibility of an autocrine mechanism of signal transduction leading to increased survival after radiation exposure with Raf-1 playing a central role. The transcriptional regulation of cytokines TNF-a or IL-1 in response to radiation has also been reported (Hallahan et al., 1989; Woloschak et al., 1990). However, the radiobiological consequences of induction of these two cytokines may be different. Whereas, TNF-a is cytotoxic via such mechanisms as free radical formation (Zimmerman et al., 1989), I L 1 is reported to protect mice from lethal doses of wholebody irradiation (Oppenheim et al., 1989). More recently, a delayed synthesis of cyclin B mRNA, which encodes a cell cycle-related protein, and absence of accumulation of the cyclin B protein were observed in HeLa cells exposed to ionizing radiation during the S and G, phases, respectively (Muschel et al., 1991). Evidence that the gene(s) associated with cell cycle checkpoints may contribute to an increase in cell survival following exposure to y-radiation is also derived from other reports (Kastan et al., 1991; Kuerbitz et al., 1992). These studies demonstrate that the levels of wild-type p53 protein in hematopoietic and nonhematopoietic mammalian cells increase and decrease in temporal association with G , arrest following irradiation. More recently, the induction of gadd45 gene following ionizing irradiation has been shown to depend on a wild-type p53 phenotype (Kastan et al., 1992). Therefore, it appears that ionizing radiation affects specific molecules with important growth-related biological functions in a variety of cell types. Further investigations are necessary to provide insight into the radiobiological significance of these radiation-inducible molecular events.

VII. DNA Damage and Repair Cascade: Biochemical and Cellular Factors Ionizing radiation is known to produce a variety of free radical species, the detoxification of which may have potential implications in the phenotypic outcome of the damage caused by irradiation. A number of intracellular radioprotective molecules with a detoxification function


22 1

have been characterized and include superoxide dismutase, catalase, glutathione (GSH) and GSH-related enzymes, protein thiols, and a number of other low-molecular-weight thiol-containing molecules (Meister and Anderson, 1983). Superoxide dismutase converts superoxide to hydrogen peroxide, whereas catalase detoxifies hydrogen peroxide to water. These two enzymes may be important in the detoxification of toxic oxygen-related species that can be produced by radiation. However, it is not clear whether high concentrations of these enzymes can protect cells from radiation damage and whether the modulations of oncogene function(s) can result in the alteration of their biochemical activities. Glutathione, a ubiquitous tripeptide, plays a critical role in several bioreductive reactions, transport, enzyme activity, protection from harmful oxidative species, and detoxification of xenobiotics (Meister and Anderson, 1983); GSH may provide radiation protection by several mechanisms including radical scavenging, restoration of damaged molecules by hydrogen donation, reduction of peroxides, and maintenance of protein thiols in the reduced state (Biaglow et al., 1983a; Clark, 1986; Mitchell and Russo, 1987; Bump and Brown, 1990).A direct correlation between intracellular GSH levels and inherent radiosensitivity has not been established (Louie et al., 1985; Mitchell et al., 1988). The depletion of cellular thiols by several reagents including diamide, N-ethylmaleimide (NEM), and DL-buthionine S,R,-sulfoximine (BSO) may render oxygenated cells more sensitive to radiation (Sinclair, 1973; Vos et al., 1976; Harris, 1979; Mitchell et al., 1983; Biaglow et al., 1983a). In this regard, the effects of BSO are of particular interest since, unlike other agents that must form a covalent bond (i.e., NEM) or oxidize GSH (i.e., diamide), BSO depletes cellular stores of GSH via a fairly specific mechanism, i.e., competitive inhibition of cysteine synthetase, a key enzyme in the biosynthesis of GSH (Griffith and Meister, 1979). Extensive cellular GSH depletion by BSO has been found to be a requirement for aerobic radiosensitization of cells (Mitchell et al., 1983; Biaglow et al., 198313; Leung et al., 1993). The mechanism of diamide-induced radiosensitization may involve oxidation of protein thiols, which are important for DNA repair (Harris, 1979).N-Ethylmaleimide possibly removes thiols from the cells or inhibits enzymes thought to be involved in the repair of lethal damage due to irradiation (Sinclair, 1973). Therefore, the possibility remains that the manipulation of the GSH and related redox systems can be effective in enhancing the cytotoxic effect of radiation and some chemotherapeutic agents in radio- and chemoresistant cells (Clark, 1986). Limited information (reviewed below) is available regarding the re-

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dox regulation of certain oncogene products via sulfhydryl modifiers (diamide, NEM), and regarding possible alterations in the intracellular levels of GSH or the GSH-related enzyme GSH S-transferase, as a result of oncogene activation o r transfection. An understanding of the role, if any, a specific oncogene(s) plays in the redox-related biochemical control mechanisms apparently elicited by y-radiation is presently lacking. A gap also exists in our knowledge of the effects of free radicals generated by y-irradiation on oncogene function(s). Several transcriptional regulatory proteins require free sulfhydryl residues for DNA binding o r transcriptional activation (Silva and Cidlowski, 1989; Levy et al., 1989). Recent studies have shown that the binding of the Jun homodimer, and the Fos plus Jun heterodimer to the AP-1 DNA binding site is inhibited by NEM. Treatment of these proteins with diamide results in their conversion to slower migrating forms most likely representing disulfide crosslinked dimers. Diamide treatment also causes inhibition of their DNA binding activities. Furthermore, a single cysteine residue in Fos and Jun was found to be important for DNA binding and that reduction was required for association with DNA (Abate et al., 1990). Sulfhydryl groups are also important in the kinase activity of p60v-src (Uehara et al., 1989). Interestingly, the last cysteine of the cysteine-finger region in the N-terminal domain of Raf-1 is critical to its dominant negative regulatory effect (Bruder et al., 1992). However, a potential role for oxidation-reduction in the control of the Raf-1 protein-serine/threonine kinase has not yet been established. It appears that a correlation between the intracellular level of GSH and activation of Raf-1 may exist. A differential modulation of intracellular GSH levels was noted as a direct response to PDGF treatment (5- 10 min) of NIH/3T3 transfectants containing different regions of transfected human raf-1 gene (Kasid et al., 1991b). Moreover, the transformation of rat liver cells with v-H-ras or v-raf is associated with expression of the multidrug resistance gene (mdr-1) and glutathione Stransferase-P, and increased resistance to cytotoxic chemicals (Burt et al., 1988). More recently, sulfhydryl modifiers, BSO and dimethylfumarate (DMF), were found to cause a graded depletion of GSH and modulation of the Raf-l-associated protein-serine/threonine kinase activity in human renal cell carcinoma-derived cells (Leung et al.,1993; U. Kasid et al.,unpublished observations). Clearly, a number of pertinent questions remain unanswered with the thiol-related biochemical regulatory mechanism(s) of Raf-1 protein kinase being one of the main issues. Another factor that may be involved in this response is the activities of DNA strand-break repair-associated enzymes topoisomerase I and 11.


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These enzymes regulate the conformation of,DNA. Besides the recently described role for these enzymes as targets for anticancer drug therapy, increased levels and activities of topoisomerases have been associated with transformed cells (Heck and Earnshaw, 1986; Crespi et al., 1988; Heck et al., 1988; Schneider et al., 1990). Moreover, it has been suggested that they may be directly involved in oncogenesis (Francis, 1987; Francis et al., 1987; Crespi et al., 1988). Evidence indicating an association between the activity of these enzymes and radiation resistance is fourfold: (a) topoisomerases are known to be activated in vitro by serinelthreonine kinases (Rottman et al., 1987; Durban et al., 1983); (6) deficiency of topoisomerase I1 has been reported to be associated with the radiosensitive phenotype of cells derived from patients with the inherited disorder ataxia telangiectasia (AT) (Mohamed et al., 1987); (c) inhibitors of topoisomerases (I and 11) potentiate ionizing radiation-induced cell killing (Mattern el ad., 1991); and (d) an elevated level of activity of topoisomerase I1 was found not only in the radiation-resistant NSF cell lines from the Li-Fraumeni family, but also in radiation-resistant NIH/3T3-transformed cell lines NIH/3T3 raf and NIH/3T3 EJ-rm (Cunningham et al., 1991). An important aspect of the radiation survival response is its regulation at the level of the cell cycle. The radiation survival experiments using synchronously dividing cell cultures have established that, in general, the cells are most sensitive at or close to mitosis, most resistant in the latter part of the S phase, sensitive in G, phase, and less sensitive in G I . In cells with a long G , phase, they are resistant in early G I , and sensitive toward the end of G I (Terasima and Tolmach, 1961; Sinclair and Morton, 1963; Whitmore et al., 1965; Sinclair, 1968). These variations in radiation sensitivity during the cell cycle have been observed despite differences in intrinsic radiosensitivity (Do value) of squamous carcinoma-derived cell lines (Quiet et al., 1991). It is noteworthy that the Chinese hamster ovary cells enriched in G, phase also reveal most sensitivity to radiation-induced mutagenesis. However, the greatest level of chemical protection from radiation-induced mutagenesis was also observed for G,-enriched populations (Grdina and Sigdestad, 1992). A number of oncogenic proteins either are known to be regulated in a cell cycle-dependent manner or have been implicated in the control of cell cycle (Hunter, 1991). The oncogenes coding for some of these proteins ( m y , r a , mos, and src) have also been implicated in intrinsic radioresistance, and some others (ras plus myc; p53) in the postirradiation changes in cellcycleorDNAsynthesis(TableI; McKenna etal., 1991;Kastan etal., 1991). It is suggested that the GI arrest and/or G , arrest induced by y-radiation allows the cells to recover from the lethal effects of radiation

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prior to cell division. Thus, alterations in this response may correlate with increased or decreased resistance to radiation. T h e RAD9 control system (or checkpoint) in yeast ensures G, arrest until the damage induced by X-irradiation is repaired, whereas mutations in RAD9 gene allow cells with DNA damage to proceed through cell division (Hartwell and Weinert, 1989). Like RAD9 defects in yeast, caffeine treatment of irradiated mammalian cells permits their entry into mitosis and decreases cell viability (Schlegel and Pardee, 1986). Using the primary rat embryo system, a significant increase in the duration of the G2 block in the radiation-resistant (rm and myc cotransfected) cells was noted compared to the relatively radiation-sensitive cells transfected with myc alone (McKenna et al., 1991). By analogy to the radiobiological characteristics of cells representing the radiosensitive genetic disorder ataxia telangiectasia, the above studies have led to the proposal that alterations in the cell division delay is one mechanism by which radioresistance is conferred on oncogene (ras and my)-transformed cells. A correlation between the expression of wild-type (wt) p53 and G, arrest, as discussed in the previous section, suggests that wt p53 may participate in the control of cell cycle progression following DNA damage (Kastan et al., 1991; Kuerbitz et al., 1992). However, these findings may be more relevant to the aspects of cellular transformation than cellular radiation resistance. As an inherited germline defect, the NSF cell lines from members of families with Li-Fraumeni syndrome have been shown to possess both a point-mutated (mt) and a wt p53 allele (Srivastava et al., 1990; Malkin el al., 1990). Nontumorigenic cell lines from one family have also been shown to express equal amounts of both the mt and the wt forms of the p53 protein (Srivastava et al., 1992). T h e NSFs from this particular Li-Fraumeni family have also been found to display a radiation-resistant phenotype (Bech-Hansen et al., 1981) (Fig. 3). This relatively radioresistant phenotype was seen not only in the cell line from an individual (V-10) homozygous for wt p53 (wt/wt), but also in the cell lines established from two individuals (VI-2 and VI-4) heterozygous with respect to the p53 point mutation (mt/wt). Furthermore, two of the radioresistant NSF cell lines that carry the mt-wt genotype actually displayed a longer lag time between exposure to X-rays and the resumption of DNA synthesis compared to an unrelated, nonradioresistant (control) skin fibroblast cell line (Paterson et al., 1985). Therefore, although the presence of one mutant p53 allele may contribute to the propensity for tumor formation by increasing genetic instability (Kuerbitz et al., 1992), it may not necessarily affect the acquisition of radiation resistance. Given the fact that some other proteins (i.e., cyclins, cdc2 ser-


225

ine/threonine kinase) play a major role in the control of cell cycle (Hunt, 1989; Draetta, 1990; Hunter and Pines, 1991) in a large variety of cell types, it is conceivable that these proteins may also govern, either directly or indirectly, the radiobiological component of the oncogene function. Earlier reports have demonstrated a critical role for the mos gene product in cell cycle control by directly or indirectly stabilizing the cyclin/cdc2 complex (Roy et al., 1990). More recently, it has been suggested that the Raf-1 signaling cascade may converge in the activation of the cdc2 complex (Heidecker et al., 1992). Therefore, an alternative mechanism underlying the differential regulation of the radiation response during the cell cycle may be linked to the modulation of the cell cycle-related proteins via cytoplasmic protein-serinelthreonine protein kinases. Future studies will most likely focus on these topics for a better understanding of the connection between radiation response, the cell cycle, and oncogenes.

VIII. Modulation of Radiation Resistance: Therapeutic Implications of Oncogene Strategy As our understanding of the involvement of specific genetic factors involved in radioresistance increases, so too should our resolve to design potential therapies that can be aimed at the specific genetic o r metabolic process leading to the specific cellular manifestation. One of the most obvious long-term practical uses of the present area of research is the possibility of the radiosensitization of tumor cells by expression of a reduced level of the oncogenic protein playing a potential role in radioresistance. Since the antisense oncogene (raf 1) strategy has proven effective for the down-regulation of radiation resistance in human tumor cells (Kasid et al., 1989a), it seems logical to direct future efforts at improving upon the antisense RNA approach in order to achieve a sustained radiosensitization effect. Antisense oligonucleotides targeted at either viral o r cellular genes have been shown to be highly effective in inhibiting the expression of the targeted gene (Wickstrom, 1991; Murray, 1990; Mol and Van Derkrol, 1990). In some cases the inhibition is highly selective and the specificity reaches to that of a point mutation (Chang et al., 1991). A number of investigators have used antisense oligonucleotides as an innovative treatment strategy to block the specific genes involved in a variety of malignancies as well as in viral, inflammatory, and cardiovascular diseases. T h e development of antisense DNA technology in the last two decades has led to the present provisional approval for the first clinical trial on chronic myelogenous leukemia using an antisense strategy. In pilot

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studies, using human tumor cells, the radiosensitizing effect of the raf- 1 antisense oligonucleotides has been observed (Kasid et al., 1991a). The potential usefulness of the antisense-based experimental design may be further explored by taking advantage of the cell cycle-related variations in the radiation response and the postirradiation changes in the cell cycle. Recently, a line of transgenic mice that contains the human ruf-1 oncogene was identified as being significantly more radiation resistant than its normal, nontransgenic counterparts (K. F. Pirollo and E. H. Chang, unpublished observations). The development of such animals provides us with an in vivo model system with which to test the various potential therapies, including the antisense therapy designed to increase the therapeutic advantage of radiation-induced cytotoxicity.Thus, it is our hope that the oncogene studies may ultimately lead to the development of a specific gene-directed approach for effective radiotherapy. IX. Conclusion

There is a growing body of evidence suggesting an important role for certain oncogenes (rm,.ah cot, mos,and m y )in the regulation of cellular resistance to ionizing radiation. The observation that some of these genes demonstrate a cooperative effect toward radiation resistance is suggestive of the possibility of a selective interaction among these proteins, analogous to that involved in signal transduction leading to cell growth and proliferation or differentiation. Antisense ruf 1 cDNA transfection has been shown to cause negative regulation of radioresistance in human tumor cells, further implying that the Raf-1 protein kinase may be an important transducer of signals leading to the radiation-resistant phenotype. In addition, perturbations in the cell cycle (C, and/or G , arrest) and cell cycle-related proteins appear to be important factors contributing to cell survival. Therefore, whereas the correlation between an interaction(s) among the specific proteins and radioresistance needs to be more clearly defined, it seems likely that modifications such as serinelthreonine phosphorylation and associated transcriptional activation are critical to the radioresistant phenotype.

ACKNOWLEDGMENTS The authors acknowledge their colleagues and collaborators, especially Drs. G . Mark, A. Pfeifer, P. Ramsamooj, R. Weichselbaum, J. Mitchell, U. Rapp, D. Kaplan, and W. Anderson, for participation in the portions of studies discussed in this review; Dr. Roberta Black for helpful comments; and Elaine Miranda and Jennifer LaMontagne for excellent


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assistance in typing the manuscript. Work in the authors’ laboratories was supported by NIH Grants CA46641 and CA58984 (U.K.), CA52066 and CA45408 (A.D.),and CA45158 and CA42762 (E.C.). Additional funds were provided by National Foundation for Cancer Research Grant NFCRHUOOl (E.C.) and Uniformed Services University of the Health Sciences Grant USUHSR074DK (K.P.)

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INDEX

A

‘

Ataxia telangiectasia, and breast cancer, 47 ATF2, binding and transcription activation by p l loRBI, 128

Acanthifolicin biochemical and immunological effects, 160 isolation, 151 N-Acetyl-I-cysteine, in vitro testing, 9 Adaptive cellular therapy, 73-74 Adenovirus EIA, binding to pl l W ’ , 120-121 Amplification, gene, see Gene amplification Amplified DNA, evolution, 108-1 10 Angiogenesis, in embryo chorioallantoic membrane, okadaic acid-induced, I64 Antibodies anti-nuclear Ki-67, 4 human antimurine, development, 7172 production, network theory, 64-65 Antigens differentiation, 64 oncofetal, 64 tumor-specific, 64 Anti-idiotypic antibody therapy, 64 Antineoplastic agents, in molecular therapy, 61-63 Antioxidants, and lung cancer, 19 Antisense oligonucleotides, inhibition of gene expression by, 75-77 Antisense RNA, for radiosensitization, 225 AP-1 transcription factor, 213, 218 Aquercetin, 62 Asbestos-exposed workers, 19 Aspirin, protective role in colorectal cancer, 6

B Bacterial extracts, vaccinations from, 63 Beas-2B cells, see Immortalized human bronchial epithelial cells bek gene, amplification in breast cancer, 38-39 Biological markers of intermediate endpoints, 13 as surrogate endpoints, 20 Biologic therapy active immunotherapy, 63-66 passive immunotherapy, 66-74 Blue-green algae, microcyst in-containing, 172-1 77 Breast cancer adjuvant therapy with tamoxifen, 17 and ataxia telangiectasia, 47 Breast Cancer Prevention Trial, 17 chromosomal alterations in, 39-40 clinical management, 48-49 diagnosis, 29-30 double minutes, 40 frequency of carcinoma types, 28 gene amplifications bek and flg genes, 38-39 c-myc gene, 36-37 EGF receptor gene, 37 IGF-1 receptor gene, 37-38 list of, 31 llq13 region, 34-36 neu gene, 32-34 235

236

INDEX

genetic predisposition to, 46-47 hereditary, 46-47 histological types, 27-29 4-HPR trials, 16 inactivation of tumor suppressor genes, 40-4 1 DCC gene, 45 p53 gene, 41-43 prohibitin gene, 44-45 Rb gene, 43-44 lesions, 28-29 and Li-Fraumeni cancer syndrome, 47 lobular carcinoma in situ, 27-28 loss of heterozygosity in, 30-3 1, 4 I model of genetic changes in, 48 normal breast histology, 26-27 point mutations in, 39 sporadic, 46 tamoxifen trials, 16 treatment against altered gene products, 50 Ki-67 epitope information, 4 types of, 29-30 Bridge-breakage-fusion cycles, in CAD gene amplification, 98-99 Bystander effect, 75, 79

C CAD gene, amplification bridge-breakage-fusion cycles, 98-99 centromere recombinations, 99 telomeric fusions, 98-99 unequal sister chromatid exchange, 9697 Calcium dietary, and colorectal cancer, 6 effects on familial colon cancer, clinical trials, 13-15 Calmette-Guerin bacillus, 63 Calyculin A, biochemical and immunological effects, 160 Calyculins biochemical activity, 168-170 structure, 168-1 70 tumor promotion comparison with okadaic acid, 172 on mouse skin, 170-172

Cancer prevention carcinogenesis research, 5-6 cell biology research, 3-5 Chemoprevention Program, 2-3 Diet and Cancer Program, 2-3 drug combination studies, 1 1- 12 epidemiologic research, 6-7 molecular genetic research, 3-5 research strategies in vitro testing, 8 in vivo testing, 8-1 1 Cancer therapy molecular diagnosis, 60-61 molecular disorders, 58-59 repair of oncogenic genetic alterations, 74-75 antisense oligonucleotides, 75-77 gene therapy, 77-80 Carcinogenesis multistage model, 2 research, 5-6 Carcinomas breast, 27-28, see also Breast cancer human squamous cell marker proteins in radioresistant and radiosensitive cells, 2 18 radioresistance. 200-203 p-Carotene, clinical studies, 19 Carotene and Retinol Efficacy Trial, 19 Catalase, radiation protection by, 22 1 C2C12 cells, myogenesis inhibition by okadaic acid, 167 Cell biology, cancer prevention research, 3-5 Cell cycle machinery of cancer cells, 59 progression inhibition by p l lWB', 129 radiation survival response and, 223224 regulation, hyperphosphorylation effects, 162 Cell differentiation, modulation in cancer therapy, 59 Cell transformation in vitro, by okadaic acid class compounds, 157- 158 Cellular radiation survival, see Radiation survival Centromeres, recombinations in CAD gene amplification, 99

237

INDEX

Chemoprevention Program NSAID clinical trials, 7 research strategies, 2-3 Chromatidic telomere fusion model, integration with amplification regulation, 104-107 Chromosomal alterations, in retinoblastoma tumor cells, 117 Chromosomal analyses, of breast cancer cells, 39-40 Chromosomal rearrangements, tumorspecific, 60-6 1 Chromosome breakage-acentric element model, of gene amplification, 100-

102

Chromosome Iq, allele loss on, 45 Chromosome 7q,allele loss on, 45 Chromosome I Ip, allele loss on, 45 Chromosome 1 Iq, 1 lq13 region amplification in breast cancer, 34-36 Chromosome 13q,allele loss on, 43-44 Chromosome 13q 14,retinoblastonia susceptibility gene mapping, 117 Chromosome 17p, allele loss on, 41-43 Chromosome 17q allele loss on, 44-45 breast cancer susceptibility gene, 46 Chromosome 18q,allele loss on, 45 c-jun gene, okadaic acid-induced expression and transcription, 166 Clinical management, of breast cancer,

Clonogenic assay, for cellular response to radiation, 197 Colon cancer dietary fadfiber association, clinical trial, 18-19 pathogenesis, cell genetic changes, 3-5 Colon tumors fat effects, 5-6 fiber effects, 5-6 Colony-stimulating factors inimunotherapy with, 69 macrophage production, stimulation by okadaic acid, 164 Colorectal cancer cell turnover rates, 4 and dietary calcium and vitamin D, epidemiologic study, 6 noninvasive genetic screening, 4 protective role for aspirin and NSAIDs, epidemiologic study, 6-7 cot gene and acquisition of radiation-resistant phenotype, 212 c a t , resistance to y-rays conferred by,

209 Cyclophosphamide, enhancement of T I L effectiveness, 73-74 Cytokines for immunotherapy, 66-69 tumor cell production of, 79

48-49 Clinical trials Breast Cancer Prevention Trial, 17 Carotene and Retinol Efficacy Trial, 19 dietary fatlfiber association with colon cancer, 18- 19 Linixian esophageal cancer prevention trial, 19-20 list of chemoprevention trials, 14-15 medical setting, 13- I7 NSAIDs, 7 public health setting, 17-20 recombinant TNF-a, 68 thymidine labeling index, 4 toxicologic and safety evaluations for, 7 Clonally derived cell lines, heterogeneity in radiation survival responses, 199200

D DCC gene, 45 Dehydroepiandrosterone, in nitro testing, 9 Diet, effects on MNU-induced mammary tumors, 5-6 Diet and Cancer Program, 2-3 Differentiation antigens, 64 2-Difluoromethylornithine, in uitro testing,

9 Digestive tract, tautomycin effects, 182-

183 Dinophysk fortii, dinophysistoxin- 1 isolation, 151 Dinophysistoxin- 1 apparent activation of protein kinases,

162

INDEX

F

biochemical and immunological effects,

I60 isolation, 15 1 structure-activity relationships, 158-

161 tumor promotion on mouse skin, 151-

154 Disco&rmiu calyx, calyculin A isolation,

168 DNA amplified, evolution, 108-1 10 internucleosomal fragmentation, okadaic acid-related, 165 loss on chromosome I lq13 in breast cancer, 34-35 replication, pl10R"' effects, 131-134 synthesis & novo, alteration after y radiation, 206-207 Double minutes, 40 DRTFl transcription factor, interaction with plloRB', 125-126 Drug combinations, research, 11- 12 Ductal carcinoma in situ, 27-28 mammographic characterization, 30

E E2F transcription factor, interaction with plloRB', 125 Egr-1 transcription factor, 218 Epidemiological research, in cancer prevention, 6-7 Epidermal growth factor receptor gene amplification in breast cancer,

37 okadaic acid-stimulated phosphorylation, 165- 166 Epithelium, intestinal, okadaic acid effects, 163-164 Erbstatin, 62 Erythropoietin, immunotherapy with,

69 Esophageal cancer, vitamin/mineral supplement effects trials, 19-20 Estrogen, serum levels, fiber effects, 19 Expression libraries, for generation of new monoclonal antibodies, 72 Ex vivo genome modifications, 79-80

Familial colon cancer, calcium supplementation trials, 13-15 Familial polyposis, vitamins/fiber effects,

17-18 Fat clinical trials, 5-6, 17- 19 saturated, effects on colon tumors, 5-6 Fiber dietary, effects on MNU-induced mammary tumors, 5-6 effects on colon tumors, 5-6 Fibroblasts, nonturmorigenic skin, in LiFraumeni families altered DNA & novo synthesis, 206-

207 radioresistance, 20, 205 topoisomerase activity levels, 206 flg gene, amplification in breast cancer,

38-39 fm gene, effects on radiation response, 210 fos gene, c-fos expression and transcription, okadaic acid-induced, 166-167

G Gamma radiation, see also Ionizing radiation dose rate, and oncogene effects, 208 effects on DNA & n m synthesis in LiFraumeni syndrome, 206-207 G, or G, arrest role in radiation recovery, 223-224 induction of PKC-mediated signaling,

218-219 radioresistance after exposure to, 2 14 resistance conferred by v-mos, c-col, and N-rar genes, 209 role of redox-related biochemical control mechanisms, 222 Gene amplification in breast cancer bek gene, 38-39 c-myc gene, 36-37 flg gene, 38-39

INDEX

IGF-I receptor gene, 37-38 neu gene, 32-34 evolution of amplified DNA, 108-1 10 mechanisms bridge-breakage-fusion cycles, 98-99 centromere recombination, 99 chromosome breakage-acentric element model, 100-102 telomeric fusions, 98-99 unequal sister chromatid exchange, 96-97 regulation integration with chromatidic telomer fusion model, 104- 107 permissivity, 89-91 probability stable stimulation, 92-93 transient stimulation, 91 Gene expression differential, and radiation response, 217-2 18 inhibition, by antisense oligonucleotides, 75-77 RBI gene, regulation, 122- 123 Gene mapping, retinoblastoma susceptibility to 13q14.2, 117 Gene therapy, 77-80 Genetic counseling, for breast cancer, 49 Genistein, 62 Genomes ex uzuo modifications, 79-80 tumor-cell, manipulation, 65 Glutathione intracellular levels, and Raf-1 activation, 22 radiation protection by, 221 Glycookadaic acid, isolation, 151 Granulocyte macrophage-colonystimulating factor, radioprotective effects, 212 GSH, see Glutathione GTP antagonists, 62 GTPases, 62

H Halichundria okadai. okadaic acid isolation, 150-151

Head cancer, 13-cis-retinoicacid trial, 15 Herbimyucin, 62 Heterogeneity, clonal, in radiation response, 199-200 Heterozygosity loss in breast cancer, 30-31, 41 loss in retinoblastoma, 116 Histone H3, okadaic acid-stimulated phosphorylation, 165-166 Host tolerance, for therapy, 80 4-HPR clinical trials, for breast cancer, 16 in uitro testing, 9 tamoxifen-4HPR combination studies, 11-12 Human antimurine antibody, 71-72 Human papilloma virus, E6 and E7 genes, 80 H yperphosphorylation of cytokeratins, tautornycin-induced, 181-182 of intermediate filaments. 162- 163

I Ibuprofen, in vitro testing, 9 IGF-I, see Insulin-like growth factor-] IgG, anti-idiotypic, 64 Immortality, correlation with permissivity, 105- 106 Immortalized human bronchial epithelial cells, rafl expression-related increase i n radiation resistance, 209 Immunotherapy, passive adaptive cellular therapy, 73-74 cytokines, 66-69 monoclonal antibodies, 69-73 Insulin-like growth factor- I receptor gene, amplification in breast cancer, 37-38 Interferon-a, immunotherapy with, 66-67 Interferon-y, imrnunotherapy with, 67 Interleukin- 1, radiation-related transcriptional regulation, 68 Interleukin-2. iminunotherapy with, 67 Intermediate filaments, hyperphosphorylation, 162-163 Intestinal epithelium, okadaic acid effects, 163-164

240

INDEX

In viho testing, for chemopreventive agents, 8 In viva testing of chemopreventive agents, 8-10 for efficacy and toxicity, 9- 10 Iodine-131-mAb conjugates. 70-71 Ionizing radiation, see a h Gamma radiation DNA damage caused by, 220-225 DNA repair cascade, 220-225 induction of growth factors, cytokines, and cell cycle control genes, 219220 molecular targets of, 2 18-220 resistance, correlation with raf- 1 gene activation, 203

K Ki-67 anti-nuclear antibody, 4

L Leukoplakia, oral, 13-ci-retinoic acid trial, 15 Li-Fraumeni syndrome and breast cancer, 47 germline mutations in p53 gene, 204205 radioresistance phenotype, 205 and raf-I gene activation, 47 Linixian esophageal cancer prevention trial, 19-20 Lipid bilayer membrane, okadaic acid interaction, 164- 165 Liver cells, penetration by microcystins and nodularin, 176- 177 Liver toxicity, of microcystin and nodularin, 172-174 Lobular carcinoma in situ, 27-28 Lung cancer, antioxidant trials, 19 Lymphokine-activated killer cells, therapy with, 73 Lymphotoxin, irnmunotherapy with, 68

M Macrophages colony-stimulating factor production, okadaic acid-stimulated, 164 okadaic acid-stimulated prostaglandin E, production, 164 Mammary tumors, MNU-induced, diet effects, 5-6 Mammography, DCIS characterization, 30 Marker proteins, identification in radioresistant and radiosensitive cell lines, 218 mdr gene, 80 Mean inactivation dose, 197 Messenger RNA, RBI gene, 119-120 tissue distribution, 121-122 35-Methylokadaic acid, see Dinophysistoxin- I Methylphosphonate oligonucleotide analog, 76-77 Microcystin-LR, biochemical and immunological effects, 160 Microcystins biochemical activity, 172-176 hepatotoxicity, 174- I79 isolation, 172 molecular modeling, 179- 180 structure, 172- 176 target tissues, 176- 178 tumor promotion, in liver, 178-179 Mitogenic signals, radiation response and, 2 10-2 15 Molecular genetics, cancer prevention research, 3-5 Molecular modeling, microcystins and nodularin, 179-180 Molecular targets, of ionizing radiation, 218-220 Molecular therapies antineoplastic agents, 61-63 immunotherapy active, 63-66 passive, 66-74 tumor-specific, 60-6 1 Monoclonal antibodies combinatorial immunoglobulin gene libraries for, 72 against growth-stirnulatory receptors, 69 immunotherapy with, 69-73

24 1

INDEX

iodine-131 conjugates, 70-71 radionuclide conjugates, 70-7 1 single-chain antigen proteins, 72-73 therapies, problems confronting, 7 1-72 toxin conjugates, 70 mos gene and acquisition of radiation-resistant phenotype, 2 12 v-mos, resistance to y-rays conferred by, 209 myc gene c-myc amplification in breast cancer, 36-37 interaction with p l lORR’, 127 synergistic effect with EJ-ras on radiation-resistant phenotype, 208-209, 2 1 1 combined role with c-rafl gene for radiation resistance, 2 13 with raf gene, synergistic effects, 213 v-myc, synergistic effect with EJ-rcls on radiation-resistant phenotype, 208209, 21 1 Myogenesis, inhibition by okadaic acid (C2C12 cells), 167

N National Surgical Adjuvant Breast and Bowel Project, 17 Neck cancer, 13-cis-retinoic acid trial, 15 Network theory, of antibody production, 64-65 neu gene, amplification in breast cancer, 32-34 NF-KB induction by y-irradiation in human myeloid leukemia cells, 219 induction by okadaic acid in Jurkat cells, 166 N 1H/3T3 cells, radioresistance phenotype, rac gene activation and, 208 nm23 gene, 45 Nodularia spumigeno, nodularin isolation, 174 Nodularin biochemical activity, 172- 176 hepatotoxicity, 174- 179 isolation, 174

molecular modeling, 179-180 structure, 172-176 target tissues, 176-178 tumor promotion, in liver, 178- 179 Noninvasive genetic screening, for colorectal cancer, 4 Nonsteroidal anti-inflammatory drugs, protective role in colorectal cancer, 6 Nonturmorigenic skin fibroblasts, in LiFraumeni families altered DNA de novo synthesis, 206-207 resistance to ionizing radiation, 205 topoisomerase activity levels, 206 NSAIDs, protective role in colorectal cancer, 6 Nuclear localization signals, for pl IORH’, 123 Nuclear proteins, okadaic acid-stimulated phosphorylation, 165-166

0 Okadaic acid biochemical effects, 150-151, 160, 164166 biological effects, 164- 166 compounds synthesized from, 151 derivatives, 150-151 effects on cell morphology, 165 intestinal epithelium, 163- 164 protein phosphorylation, 165- 166 gene expression regulation, 166- 167 3H-labeled, distribution, 163-164 immunological effects, 160 interaction with lipid bilayer membrane, 164-165 simultaneous treatment with teleocidin or TPA, 155-157 structure, 150- 15 1 transcriptional regulation, 166- 167 tumor promotion comparison with calyculin A, 172 on mouse skin, 151-154 in rat glandular stomach, 154-155 unique properties induced by, 166 Okadaic acid class compounds apparent activation of protein kinases, 161- 163

242

INDEX

biochemical activities, 152 biochemical and immunological effects, 160 inhibition of PP-1 and PP-2A, 146-150 in udro cell transformation, 157-158 okadaic acid receptors, 146- 147 structure-activity relationships, 158-161 tumor-promoting activities, 152 Okadaic acid pathway relation to human cancer, 183-186 schematic, 155 summary of, 163 Okadaic acid response element, 167 Okadaic acid spiroketal I biochemical and immunological effects, 160 conformation, 159 Okadaic acid spiroketal I1 biochemical and immunological effects, 160 conformation, 159 Okadaic acid tetrdtmethyi ester, biochemical and immunological effects, 160 Oligonucleotides analogs resistant to nucleolytic cleavage, 76-77 antisense inhibition of gene expression, 75-77 radiosensitizing effects, 226 Oltipraz in uitro testing, 9- 10 preclinical testing, 10-1 1 Oncofetal antigens, 64, 69 Oncogenes activated effects variation with y radiation dose rate, 208 linkage to radiation resistant phenotype, 208 effects on radiation response, 201-202 products, redox regulation, 22 1-222 Oral leukoplakia, 13-cis-retinoicacid trial, 15

P Pandaros acanthifolium, acanthifolicin isolation, 151

Papillomavirus E7, binding to p l loRBI, 120-12 1 Passive immunotherapy adaptive cellular therapy, 73-74 cytokines, 66-69 monoclonal antibodies, 69-73 Permissivity correlation with immortality, 105- 106 in gene amplification, 89-91 p53 gene, 41-43 germline mutations in Li-Fraumeni syndrome, 204-205 wild-type, and cell cycle progression after DNA damage, 224 Phorbol- 12,I 3,didecanoate, comparison with okadaic acid in cell transformation assay, 157- 158 Phosphatidylinositol 3'-kinase, 62 Phospholipase C, 62 Phosphorothioate oligonucleotide analog, 76-77 Phosphorylation okadaic acid effects, 165-166 p l lORBf, modulation, 123-124 tautomycin effects in K562 cells, 181 Piroxicam clinical trial, 7 in vitro testing, 9 Point mutations, in breast cancer, 39 Polyoma large T antigen, binding to pl ]OR"', 120-121 p l loRBI cellular proteins associated with, 124- 125 characterization, 120 effect on DNA replication, 131-134 inhibition of cell cycle progression, 129 of cell proliferation by TGF-P,, 129131 interact ions with cellular transcription factors, 125- 128 with c-myc, 127 localization in nucleus, I23 phosphorylation modulation, 123- 124 regulation of RCBPlSpl, 128 as transcription factor, 116, 127 viral oncoprotein binding, 120- 121 Predisposition, genetic, to breast cancer, 46-47

243

INDEX

Probability, in gene amplification, 91 -93 Progesterone receptor, okadaic acidstimulated phosphorylation, 165- 166 Prohibitin gene, 44-45 Promoters, tissue- and cell type-specific, 78 Protein kinase C activation, ionizing radiation-related, 218-219 inhibitors, 62 role in radiation-resistant phenotype, 211-212 tautomycin effects on activation, 181I82 Protein kinase C pathway, schematic, 155 Protein kinases, apparent activation by okadaic acid class compounds, 161163, 181-182 Protein phosphatase 1, inhibition by okadaic acid class compounds, 146150 Protein phosphatase 2A, inhibition by okadaic acid class compounds, 146150 Pseudomom toxin-mAb conjugates, 70

R RAD9 control system, 224 Radiation resistance after exposure to y-rays, 2 14 antisense RNA approach to, 225-226 combined roles for raf-1 and m y genes, 213 defined, 197 down-regulation with antisense oncogene (raf-I), 225 of human squamous cell carcinomas, 200-203 modulation, 225-226 mutant p53 alleles and, 224 raf-1 gene function, 203 raf gene function, in Li-Fraumeni syndrome, 203-207 Raf-1 protein kinase role, 212 rac gene role, 21 1 SHOK cells, N-rac-related, 2 I 1 synergistic effects of myc and raf genes, 213

topoisomerases I and 11 role, 222223 transformation and, 215-216 v-src gene effects, 209 v-src-related, in murine hematopoietic cells, 2 10-2 1 1 Radiation-resistant phenotype EJ-rac and c-myclv-myc synergistic effects, 208-209 in Li-Fraumeni families, 205 multiple genetic factors involved in, 217-218 multiple signals for, 2 14 transfection of human N-rm oncogene into NIH/3T3 cells, 208 Radiation response clonal nature of, 199-200 fmoncogene effects, 210 of human cells, raf gene role, 209 mitogenic signals and, 2 10-2 15 multifactorial nature of, 21 7-2 I8 oncogene effects, 201-202 variation with cell type, 2 I5 Radiation survival linear quadratic model, 197- 198 multitarget model, 197- 198 Radiation survival curves, relative radioresistance or radiosensitivity, 197-198 Radionuclide-mAb conjugates, 70-7 1 Radioresistance, see Radiation resistance Radiosensitivity defined, 197 intrinsic, measurement by mean activation dose, 197 rat kidney epithelial cells, K-ras-related, 209-2 10 Radiosensitization antisense RNA approach, 225 diamide-induced, 22 1 GSH depletion by BSO and, 22 1 by taf-1 antisense oligonucleotides, 225-226 raf gene and acquisition of radiation-resistant phenotype, 212 EJ-rac, effects on NIH/3T3 radioresistance levels, 208 K-ras, radiosensitization of rat kidney epithelial cells, 209-2 10

244 with myc gene, synergistic effects, 213 N-T~ effects on NIH/3T3 radioresistance levels, 208 effects on SHOK cell radioresistance, 21 I resistance to y-rays conferred by, 209 and oncogenic transformation, 2 16 role in radiation resistance, 216 role in radiation response of human cells, 209 v-H-ru, effects on NIH/3T3 radioresistance levels, 208 v-K-rm, effects on NIH/3T3 radioresistance levels, 208 ruf-I gene activation and GSH intracellular levels, 22 and Li-Fraumeni radioresistance phenotype, 205-206 and radioresistance in Li-Fraumeni syndrome, 203-207 and resistance to ionizing radiation, 203 antisense oligonucleotides, radiosensitizing effects, 225-226 c-ruf-I, combined role with myc gene for radiation resistance, 2 13 expression in Beas-2B cells, radiation resistance conferred by, 209 function, link to radiation-resistant phenotype, 2 12 human sequences in NIHl3T3 transfectants after radiotherapy, 200-203 Raf-1 protein kinase dual role of, 216 role in radiation resistance, 2 12 rcrr gene c-Ha-rcrr activation by okadaic acid class compounds, 152-154 EJ-rcrr, synergistic effect with c-myclv-myc on radiation-resistant phenotype, 208-209, 2 1 1 mutations in breast cancer, 39 in colorectal cancer, 4 role in radiation resistance, 2 I 1, 2 16

INDEX

Rat embryo cells, radioresistance by contransfection of EJ-rcrr and c-myclv-myc, 2 I 1 synergistic effects of myc and rufgenes, 213 Rat kidney epithelial cells, K-rm-induced radiosensitization, 209-2 10 Rb gene, 43-44 RB1 gene characterization, I 18- 120 expression, 116, 122-123 germline mutations, 116-1 17 homologous sequences among vertebrates, 1 I8 inactivation, tissue-specific susceptibility to, 135-136 mRNA transcripts tissue distribution, 121- 122 plIoH*' characterization, I20 viral oncoprotein binding, 120- I2 I reconstituted cell lines, summary, 133 suppression of tuniorigenicity, 133- 135 RCBP, regulation by pl IOP', 128 Restriction enzyme fragment length polymorphisms, breast cancer carcinomas, 4 1 Retinoblastoma familial, 116 gene regulation mechanisms, 12 1 - 124 genetics, 116-1 17 heritable, I16 tumor cells, chromosomal changes in, 117 Retinoblasts. susceptibility to RBI gene inactivation, 135- 136 13-cic-Retinoic acid, clinical trials head and neck cancers, 15 oral leukoplakia, 15 Retinoids, inhibition of neoplastic conditions, 5 Retroviruses, for gene therapy, 77-78 Ricin toxin-mAb conjugates, 70

S Saturated fat, clinical trials. 5-6, 17-19 Single-chain antigen proteins, 72-73

INDEX

.Sister chromatids, unequal exchange in CAD gene amplification, 96-97 Somatomedin C, see Insulin-like growth factor- 1 Spl, regulation by p l loRBI, 128 Squamous cell carcinomas, human marker proteins in radioresistant and radiosensitive cells, 218 radioresistance, 200-203 src gene, v-scr increase in radioresistance induced by, 209 induction of radioresistance in murine hematopoietic cells, 210-21 I Staurosporine, 62 Sulindac, clinical trial, 7 Superoxide dismutase, radiation protection by, 22 I SV40, binding to plloRB’, 120-121

T Tamoxifen, 62 adjuvant therapy for breast cancer, 17 clinical trials for breast cancer, 16 4-HPR-tamoxifen combination studies, 11-12 Tautom ycin biochemical activity, 181-182 biochemical and immunological effects, 160 effects on digestive tract, 182-183 structure, 181-182 tumor promotion on mouse skin, 182 Teleocidin simultaneous treatment with okadaic acid, 155-157 tumor promotion in rat glandular stomach, 154-155 Telomeric fusions, in CAD gene amplification, 98-99 12-0-Tetradecanoylphorbol13-acetate simultaneous treatment with okadaic acid, 155- 157 tumor promotion in rat glandular stomach, 154-155 12-0-Tetradecanoylphorbol13-acetateinduced-sequence genes, okadaic

acid-induced mRNAexpression, 166 12-0-Tetradecanoylphorbol13-acetate response element, 167 Thiols, cellular depletion by BSO, and radiosensitization, 22 1 Three-dimensional structures, microcystin-LR and nodularin, 180 Thymidine labeling index, 4 TIL, see Tumor-infiltrating lymphocytes Tissue-specific susceptibility, to RBI gene inactivation, 135- 136 TNF, see Tumor necrosis factor Topoisomerases I and 11, association with radiation resistance, 222-223 Transcription factors, interactions with plloRB‘, 125-128 Transforming growth factor+, genes, activation by p l IWB’, 128 Transforming growth factor+, , inhibition of cell proliferation, pl1oRB’ role, 129-131 Tumor cells cytokine production, 79 genome manipulation, 65 Tumorigenicity, suppression by RBI, 133135 Tumor-infiltrating lymphocytes, therapy with, 73-74 Tumor necrosis factor-a immunotherapy with, 68 radiation-related transcriptional regulation, 68 Tumor necrosis factor+, immunotherapy with, 68 Tumor promotion by calyculins, on mouse skin, 170- 172 general biochemical mechanism of, 183- 186 by microcystins, in liver, 178-1 79 by nodularin, in liver, 178-179 by okadaic acid biochemical and biological effects, 164-166 on mouse skin, 151-154 by tautomycin on mouse skin, 182 TPA-type, tumor-promoting activities, 152 Tumors classification, 60 common features, 59

246 Tumor-specific chromosomal rearrangements, 60-6 1 Tumor suppressor genes, inactivation in breast cancer DCC gene, 45 p53 gene, 41-43 prohibitin gene, 44-45 Rb gene, 43-44 Tumor vaccines, 63-66 Tyrosine kinase inhibitors, development, 61-62 Tyrphostins, 62

INDEX

Vimentin calyculin A effects on phosphorylation, 171 hyperphosphorylation, 162-163 Vitamin D, dietary, and colorectal cancer, 6 Vitamin E, clinical studies, 19

W Wnt-3 gene, 45

X Vaccines, anti-tumor, 63-66, 80

I S B N O-L2-00666L-O

X-rays, egr-1 and c-jun transcription induction, 218

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