Advances in Cancer Research, Volume 5

ADVANCES I N CANCER RESEARCH VOLUME V

This Page Intentionally Left Blank

ADVANCES IN CANCER RESEARCH Edited by

JESSE P. GREENSTEIN National Cancer Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland

ALEXANDER HADDOW Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London, England

Volume V

@

1958

ACADEMIC PRESS INC., PUBLISHERS, NEW YORK, N. Y.

COPYRIGHT 0 1958 BY

ACADEMICPRESSINC. 111 FIFTHAVENUE NEWYORK3, N. Y. All Rights Reserved N o part of this book may be reproduced in any form by photostat, microfilm, or any other means without written permission from the pztblishers. Library of Congress Catalog Card Number 52-13360

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME V I .c

20

0

.-

0

0 a

0

5

10

0

30

40

50

Time of incubation (min.)

FIG.8a and b. Intracellular distribution of radioactivity in Krebs I1 ascites tumor cells after incubation in vitro with glycine-C14. The washed cells were incubated in a bicarbonate medium under aerobic conditions a t 37°C. After incubation, the cells were disrupted by ultrasonic disintegration and the components fractionated in a sucrose medium (from Campbell el al., 1957).

138

P. N. CAMPBELL

Littlefield and Keller (1957) have followed the incorporation of L-Valine-C14 into Ehrlich ascites cells incubated in fortified ascitic fluid. After lysis of the cells, the microsome fraction was treated either with deoxycholate or sodium chloride. The results obtained are shown in Fig. 9. As with liver, this shows that the ribonucleoprotein particles are the initial site of incorporation. When reticulocytes are incubated with ~-leucine-C’~, the initial incorporation of radioactivity is into the microsomal protein (Rabinovitz and Olson, 1956). The microsomes had a content of 20% ribonucleic acid. After treatment with deoxycholate, the residue had a content of 30% ribonucleic acid and the protein contained more radioactivity than that of the untreated microsomes.

r

Deoxycholate- sol.

6 50E

0

5

10

15

20

25

30

Time of incubation (min.1

FIQ.9. Time curve of incorporationof ~-valine-Cl~ into proteins of ascites tumor cells incubated in aacitic fluid fortified with glucose. The curve for the “pH5 enzyme” fraction was almost. identical with that of the whole cell. The per cent RNA of the deoxycholate-insoluble particles is indicated on the chart for each sample. The per cent RNA averaged 8 for the whole cells, 4 for the “pH5 enzyme,” and 11 for the deoxycholatesoluble fraction of the microsomes (from Littlefield and Keller, 1957).

B. Slices and Brei. When rat liver slices are incubated in a bicarbonate medium under aerobic conditions in the presence of glycine-C14,the most active cell fraction is associated with the microsomes, as under in vivo conditions (Campbell et al., 1957). Typical results are shown in Fig. 10. If regenerating liver taken 40 hours after partial hepatectomy, when the rate of protein synthesis is maximal, is used, the incorporation into all the fractions is greatly enchanced compared with the normal liver as shown in Fig. 10. If, however, slices of liver tumor are incubated under the same conditions, the incorporation into the microsomes, supernatant fraction, and mitochondria is similar to normal liver; but the incorporat.ioninto the nuclear fractions of the tumor greatly exceeds that into the same fraction

PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES

139

of normal liver. Therefore as with ascites, the nuclear fraction appears to be relatively more important in the tumor than normal tissue. Since the incorporation into the nuclear fraction is not preferentially enhanced in the regenerating liver, this effect does not appear to be associated with growth. Hendler (1956) fractionated coarse minces of hen oviduct which had been incubated in a medium containing radioactive bicarbonate. He found a

In

b

0)

Normol Liver

x

.-

5

Microsomes

Liver Tumor Microsomes

160

120 Mitochondria I00 80

._ E

60

0

40

+

.-

0

Nuclei

10

30

50

40 20

0

10

30

50

U

Time of incubotion (min.)

lx

t 600u ._

Regeneroting Liver

f

2 500-

.a

c .-

>

0

10

20 Time of lncubatlon ( m i d

60

FIG.10. Intracellular distribution of radioactivity in tissue slices after incubation in a medium containing glycine-Cl*. The liver tumor (b) was obtained from rats fed 4 dimethylaminoazobenzene and the regenerating liver (c) from rats 40 hr. after partial hepatectomy. The slices were incubated in a bicarbonate medium under aerobic;conditions at 37°C. (from Campbell et al., 1957).

140

1'.

ti. CAMPBXLL

that, when the usual method of fractionation was applied, the most active fraction centrifuged down with the cell debris. This fraction, contrary t.0 that found in other tissues, contains most of the ribonucleic acid and light and electron microscopy has shown it to contain the microsomes (Hendler, 1957). Thus under these conditions, the initial site of incorporation of radioactivity is also the ribonucleoprotein rich fraction. On the other hand when rat pituitary glands are incubated in a medium containing methionineSas, the most active fraction after 1 and 2 hours incubation was the supernatant followed by the microsomes and then the mitochondria (Ziegler and Melchior, 1956). It seems possible that if shorter incubation times were used the microsomes might then be the most active fraction. I n a previous discussion, it was concluded that incorporation of radioactive amino acids into specific proteins was a good indication of de novo protein synthesis under in vitro conditions. From the experiments just described, i t will be clear that the distribution of radioactivity in the subcellular constituents under these in vitro conditions is very similar to that taking place in vivo. These experiments, therefore, support the idea that protein synthesis can be studied'under in vitro conditions. I t now remains to be seen what can be learned about. this process from incorporation studies in subcellular fractions.

4. Incorporation into Subcellular Fractions A. Hornoyenates. The first report of the incorporation of a radioactive amino acid int.0 a tissue homogenate was by Winnick et al. (1948). This showed that g 1 y ~ i n e - Cwas ~ ~ incorporated into homogenates of rat tissue under aerobic conditions and that incorporation appeared to be enzymatic in nature. Under these conditions, the incorporation of amino acids was faster with homogenates of fetal rat liver and mouse mammary carcinoma than with adult rat or mouse liver (Winnick, 1950). Our knowledge of the mechanism of the incorporation of amino acids by homogenates was advanced considerably by the work of Siekevitz (1952). He found that when rat liver homogenates were incubated aerobically with alanine-C14, the addition of a-ketoglutarate greatly increased the uptake of labeled alanine into the protein. The distribution of radioactivity into the subcellular constituents is shown in Fig. 11. If each cell fraction was incubated alone, there was no uptake of radioactivity. However, when the microsomal fraction was added to the mitochondria1 fraction, uptake occurred. It was also found that factors necessary for the optimal oxidation of a-ketoglutarate (Le., adenosine-5-phosphate1 MgC12, and phosphate) were necessary for optimal uptake of alanine. Since the presence of dinitrophenol reduced incorporation, it appeared that the uptake of alanine was coupled with phosphorylation and not oxidation per se.

PROTElN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES

141

As the result of such experiments, it became clear that the incorporation of amino acids into the protein of homogenized tissues depended on an energy source and appeared to be enzymatic. Moreover, since the requirements were similar to those found for incorporation into the protein of tissue slices, it was tempting to consider the process as being a step in protein synthesis. However, since the homogenates possessed very much less activity for incorporation than the slices, objections were raised that incorporation of radioactivity represented adsorption or coupling of the amino acids with protein by bonds other than peptide. Such objections were gradually overruled, but so far the synthesis of a specific protein has not been demonstrated in a cell-free system. 120 105

d

tEE

.-

> c 0

90

Microsomes

75

6ol /

45

Mitochondria

15

0

10

20

30

40

Time of incubation ( m i n )

FIG.11. Incorporation of radioactivity into the proteins of the various fractions of a rat liver homogenate. The homogenate was incubated under aerobic conditions in the

a-ketoglutarate and adenosine-5-phosphate (from Siekevitz, presence of ~~-alanine-l-C14, 1952)

On the basis that the incorporation of radioactive amino acids represents a step in the path of the synthesis of protein, the results obtained with isolated fractions of subcellular constituents will now be reviewed. B. Nuclei. Although the incorporaOion of radioactivity into the nuclear fraction of liver homogenates is very small, this may be due to damage to the nuclei during the process of homogenization. Thus Stern and Mirsky (1953) in a comparison of the nuclei of calf thymus and liver and of rat liver found t ha t there was a much greater loss of protein from the liver than from the thymus nuclei. When such nuclei were incubated with

142

1’. N. CAMPBELL

alanine-C14 and a-ketoglutarate, there was quite a subst,antial incorporation of alanine into the nuclear protein (Allfrey, 1954). These studies on isolated nuclei have been extended by Allfrey et al. (1957). I n particular the role of deoxyribonucleic acid has been studied, and the nuclear proteins have been fractionated. The quantitatively small protein fraction which is strongly bound to deoxyribonucleic acid was the most active fraction. The role of the nucleus in protein synthesis has recently been reviewed by Brachet and Chantrenne (1956). There seems no doubt that under certain conditions isolated nuclei can actively incorporate amino acids. C. Microsomes. I n view of the rapid uptake of radioactive amino acids into the microsome fraction of liver tissue both in vivo and in vitro, the immediate objective was to find conditions under which incorporation would t’ake place in isolated suspensions of microsomes. Siekevitz (1952) had shown that microsomes plus mitochondria were effective. Zamecnik and Keller (1954) next showed that the mitochondria could be replaced by the presence of ATP plus a substrate such as phosphoenolpyruvate (PEP) which would maintain the level of ATP. Under these conditions, aerobic conditions were no longer necessary. Nevertheless, in order for incorporation into microsomes to take place, a soluble, heat labile, nondialyzable fraction from the supernatant of the liver homogenate (known as the cell sap) was required. The activity of this fraction was present in a precipitate obtained by lowering the pH to 5.2 (called the “pH5 enzyme”) provided GDP or GTP were added (Hoagland el al., 1956). A summary of the fractionations described up to this point is shown in Fig. 12. If supernatant A is incubated at 37°C. with radioactive amino acid plus ATP plus PEP, incorporation into the protein precipitated with trichloroacetic acid takes place. If, however, the microsome pellet is mixed with a small amount of cell sap, i.e., the concentration of microsomes in the medium is increased, the efficiency of the incorporation increases. I n either case if the microsomes are separated from the medium after incubation, the activity in the microsomal protein is found to be many times greater than that of the cell sap protein. In favor of the view that the incorporation of amino acids into isolated microsomes is connected with protein synthesis in vivo are the results obtained with deoxycholate. When C14-amino acid-containing microsomes were treated with deoxycholate, the activity of the insoluble fraction was up to twenty-two times that of the soluble fract.ion (Littlefield el al., 1955). Against the view are the results of Simkin (1957) who found that when the C“-amino acid-containing microsomes were fractionated by salt extraction, the pattern of incorporation was different under in vivo and in vitro conditions. If the relative rates of incorporation of amino acids into the microsomal

PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES

143

Ti iue Homogenize in wcrose Spin 12,OOO g

I

Cell debris Nuclei Mitochondria

Supernatant A

Spin 105,000 g

Rlicrosome pellet Treat with deoxycholate

1

Insoluble (ribonucleoprotein)

Soluble

pH 5.2 spin 12,000 g

1

Supernatant B

“pH5 enzyme”

FIG.12. Fractionation of subcellular particles.

fraction of tissue slices and isolated microsomes are compared, conflicting results are again obtained. Thus, from the experiments with slices, microsomes from regenerating liver would be expected to incorporate amino acids more rapidly than microsomes from normal liver. That this is so is shown in Table XVI (Campbell and Greengard, 1957). On the other hand, whereas normal liver and liver tumor microsomes had similar activities in slices, the tumor microsomes have only very low activity when isolated. Littlefield and Keller (1957) also found that the activity of microsomes from ascites was less than those from liver, whereas the reverse was true under in vivo conditions. Prom ultracentrifugal studies, they found that component C of Petermann was rapidly lost on incubating the microsomes, and this may account for the lower activity of the ascites mkrosomes. They also found that, whereas the ribonucleoprotein particles from liver obtained by treating the microsomes with deoxycholate failed to incorporate amino acid on incubation, similar particles from ascites microsomes did so.

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TABLE XVI Incorporation of Glycine-C14into Microsome Preparations from Normal Rn.t Liver, Livcr Tumor, and Regenerating Liver"** Normal liver Liver tumor Regenerating liver

Microsomes Cell sap Microsomes Cell sap Microsomes Cell sap

Radioactivity of proteinc

+- +- -- + - + - +

+

10

69

236

+ + -34

+ + -10

++ -

+ --

-

+

135 110

From Campbell and Greengard, 1957. Similar amounts of the microsome pellet suspended in cell ERP from the three types of tissue were incubated for 50 min. at 37°C. with glycine-C", ATP, and phosphoenolpyruvicacid. * Radioaotivity is expressed ae counts per min. at infinite thickness. 4

b

In their work with isolated liver microsomes, Zamecnik and Keller (1954) found that the incorporation of a radioactive amino acid was unin-

fluenced by the presence of a full complement of amino acids in the medium. This is a rather surprising result., for the amount of free amino acid in the microsomes would not be expected to be very large and if complete polypeptide chains were undergoing synthesis, a requirement for additional amino acids would be expected. Recently Zamecnik and his colleagues have made two important discoveries concerning the mechanism of the incorporation of amino acids into

Ad 0-

I HO-PII 0

0-

0

0

- IIPI - 0 - P I 0

I

9-

6

II 0

0-

R I HC-NH:

I

HO-P-0-P-0'

I

-O-i

0

0%-

C,

H~-NH:

I : I P - 0 AC 4 \ II

0

0-

0

FIG.13. Schematic representation of amino acid carboxyl activation by ATP and the "pH5 enzyme". Ad = adenosine. 0 indicates the attacking carboxyl oxygen which would remain with the nucleotide moiety upon subsequent splitting of the activated compound (dash line) (from Hoagland et al., 1956).

PROTEIN SYNTHESIS WITH REFERENCE TO GROWTH PROCESSES

145

liver microsomes. First, they found that i n tlhe presence of “pH5 enzyme” the amino acids were activated before being incorporated into the microsomes (Hoagland et al., 1956). The reaction in which ATP, amino acid, and enzyme take part is illustrated in Fig. 13. The evidence for the activation reaction is mainly based on two observations. First, if radioactive pryophosphate is added to the system, there is an exchange of P32with ATP. Secondly, in the presence of high concentrations of hydroxylamine, hydroxamic acids and pyrophosphate accumulate. Since the “pH5 enzyme” is needed both for the activation step and for the incorporation of amino acids into microsomes, it seemed likely that amino acid activation was a step in the overall incorporation reaction. That this is so is made more likely by the fact that hydroxylamine inhibits the incorporation of amino acids into the microsomes. The degree of inhibition roughly parallels the aminohydroxamic acid formation with increasing concentration of hydroxylamine. Evidence that such an amino acid activation occurs with other biological systems is accumulating. Thus Littlefield and Keller (1957) have found it in ascites, and Cole et al. (1957) have tested a large number of tissue extracts from different animals and found it in many of them. They found the best source was guinea pig pancreas. DeMoss and Novelli (1956) have also demonstrated the presence of amino acid activation systems in many microorganisms and, moreover, have shown that leucyl adenylate behaves like an intermediary in this reaction (DeMoss et al. , 1956). Scarano and Maggio (1957) have found the system in unfertilized sea urchin eggs. The question arises as to whether there is a different enzyme responsible for the activation of each individual amino acid or whether one enzyme catalyzes the activation of all amino acids. Preliminary fractionation of the “pH5 enzyme” suggested that the former was the case (Hoagland et al., 1956). The isolation of an enzyme specific for tryptophan by Davie et al. (1956) and Cole et al. (1957) also suggests that there is one enzyme for each amino acid. If the kinetics of the incorporation of radioactive amino acids into the protein of the isolated liver microsomes is studied, evidence is obtained of a two-step reaction (Hultin, 1956; Hultin and Beskow, 1956). The explanation of this finding is provided by Hoagland el al. (1957) and concerns the presence in the “pH5 enzyme” fraction of ribonucleoprotein. This is a low molecular weight substance which contains only a very small quantity of protein relative to ribonucleic acid. It therefore differs from the ribonucleoprotein of the microsomes and may be denoted S-RNA. It has now been found that when 1e~cine-C’~ is incubated with ATP and “pH5 enzyme,” the S-RNA rapidly becomes labeled. The amino acid linkage is acid stable, but alkali labile. However, when this labeled S-RNA is incubated

140

P. N. CAMPBELL

with hydroxylamine, leucine hydroxamic acid is formed. Moreover, if the labeled S-RNA is incubated with microsomes, the label is transferred to the microsomes provided GTP is present. The incorporation of amino acid into the S-RNA, unlike the amino acid activation reaction, is sensitive to ribonuclease. The results on which these observations are based are shown in Table XVII. TABLE XVII Transfer of Leucine-C14from Prelabeled Activating Enzyme Fraction to Microsomal Proteina Total Counts in RNA Protein Complete system (before incubation) Complete system (after incubation) Complete system, minus GTP* Complete system, minus generating system Complete system, minus both GTPband generating system Complete system, minus generating system but with 5 X GTPb Complete system, CTPb replacing GTPb Complete system, UTPb replacing GTPb Complete system, plus 0.005 M C12-leucine a

489 180 111 72 23 145 96 101 183

30 374 40 155 30 129 44 53 314

From Hoagland el al., 1957

* ATP, GTP, CTP, UTP are the triphosphates of adenosine, guanosine, cytidine, and uridine respectively.

That the S-RNA plays an important role under i n vivo conditions is shown by results obtained with ascites tumor. I n these experiments, in which ascites cells were labeled i n vivo, the S-RNA fraction became labeled more rapidly than the ribonucleoprotein particles of the microsomes and fulfilled the requirements of a precursor. Sachs and Waelsch (1956) previously showed that if labeled microsomes were incubated with pyrophosphate, some of the microsome fraction was solubilized, and this fraction has a very much higher radioactivity than the untreated microsomes. These two findings of Zamecnik and his colleagues suggest, therefore, that the incorporation of amino acids into microsomes takes place in three steps as summarized in Fig. 14. One further aspect of these results is of interest, for it suggests a possible link between nucleic acid and protein synthesis. If ATP-C14 is incubated with “pH5 enzyme,’’ then the S-RNA becomes labeled (Zamecnik et aZ., 1957). This reaction is sensitive to ribonuclease and may be connected with the finding of Holley (1957) that a fraction obtained from “pH5 enzyme” catalyzes the conversion of adenylic acid-C14to ATP-C14 in the presence of alanine.

PROTEIN SYNTHESIS WITH I~EFERENCE TO GROWTH PROCESSES

Amino acid

I

147

+ enzyme + ATP Activation Stage

Amino acid-AMPenzyme I

+ PP

.1

Amino acid-AMP-(SRNA)

Ribonucleoprokin particle in microsomes

FIG.14. Steps in the incorporation of amino acids into liver microsomes.

VI. CONCLUSIONS Owing to the intriguing nature of the subject of the biosynthesis of proteins, there has been a tendency in the past to expect progress to be made on a rather dramatic scale, and for the lack of such progress to be counted as a failure. An attempt has been made in this review to assemble the known facts concerning protein synthesis within the limitations set out in the introduction. It is hoped that it will be apparent that steady progress has been made to elucidate the problems involved. In particular, much is now known about the struct.ure of proteins and the ways in which protein synthesis may be followed; both aspects of the subject which are essential preliminaries to an understanding of the way in which proteins are synthesized. Both in carbohydrate metabolism and fat metabolism, the most rapid advances had to await the development of methods involving cell-free systems. This knowledge has acted as an incentive to develop similar methods for the study of protein synthesis, but so far it has not been possible to demonstrate the synthesis of a specific protein in a cell-free system. It is, a t least, possible th at the integrity of the whole cell is required for such a synthesis. If this is so, then progress will indeed be difficult. It seems more likely that many subcellular components play a part in the synthesis of a complete protein. The isolation of such components and the understanding of their function will require the close collaboration of the electron microscopist and the biochemist. It is encouraging that progress in this direction has recently been reported by Palade and Siekevitz and others.

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P. N. CAMPBELL

I n the meantime Zamecnik and his group have shown that even if the synthesis of a complete protein cannot be demonstrated in cell-free systems, the incorporation of amino acids into proteins may be studied under these conditions. To what extent this incorporation is connected with protein synthesis is a debatable point, but the correlation between the cell-free systems, in vitro systems (slices, etc.), and in viz~ostudies is sufficiently close t o suggest that steps in the synthesis of complete protlein are being studied. Among other things, the work of Zamecnik has clearly shown that there are various types of ribonucleic acid in the cell with different biochemical functions. This may explain why many of the attempts to correlate the synthesis of protein and nucleic acid have been rather inconclusive. The trend a t the moment is to replace theoretical discussions by practical experiments, and the fact that these are now possible is a measure of the progress of the subject in recent years. A comparison between the synt,hesis of protein in tumor and normal tissue has had to await developments in the field as a whole. I t is clear that in certain diseases the tumor cells synthesize proteins which differ from those synthesized by normal cells and that some tumor cells have an enhanced ability to utilize plasma protein for the synthesis of their tissue proteins. It is not known, at present, whether the mechanism of protein synthesis in tumor cells differs from that in normal cells. The need now is for careful comparisons between normal and tumor tissue to be made employing the techniques which have been developed in recent years. ACKNOWLEDGMENTS

I would like to thank Professor F. Dickens, F.R.S., Dr. I. M. Roitt, Mrs. 0. Greengard, and Mrs. N. E. Stone for many helpful discussions and Miss B. Kernot for preparing the figures.

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THE NEWER CONCEPT OF CANCER TOXIN W a r 0 N a k a h a r a a n d Fumiko Fukuoka Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Toxohormone Production a Universal Property of Cancer Cells.. . . . . . . . . 111. Isolation of Toxohormone from Materials Other than Cancer Tissue. . . . . IV. Normal Liver Catalase Level. . . V. Chemical Nature of Toxohormon VI. Mode of Action of Toxohormone VII. Toxohormone in General Tumor-Host Relations . . . . . . . . . . . . . . . . . . . . . . . VIII. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Referenccs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION What are the characteristics of cancer cells as distinct from normal cells? This is a n important question, to which the present state of our knowledge can give only a partial answer. Classical pathology has long since established atypia, as to the cancer cell itself, and heterotopia, in relation to other structural elements of the body, as characterizing cellular malignancy. These terms are descriptive of the fundamental phenomena of malignancy, though subject to the limitation inherent in the nature of morphology itself. A great stride was made in the understanding of the properties of cancer cells when Otto Warburg, some thirty years ago, demonstrated a high aerobic and anaerobic glycolysis as an outstanding biochemical feature of these rells. In spite of t,he fact that the greatly increased glycolytic capacity has since been found to be not strictly specific to cancer cells but t,o be shared also with certain normal cells, such as embryonic and placental cells, Warburg’s original observations stand to this day as the most salient, single contribution to cancer cell biochemistry. During the past several years an interesting phase of this problem has been uncovered in a somewhat unexpected manner. This newer knowledge stemmed from studies on the systemic effect of cancer growth instead of from direct attacks on t.he cancer cell itself. We refer to the discovery of a characteristic toxic substance which is produced by cancer cells and which is responsible for certain systemic changes constantly occurring in cancer bearing hosts. The toxic substance in question is toxohormone (Nakahara 157

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and Fukuoka, 1948), and the systemic change it produces is represented by bhe marked decrease of liver catalase activity. It can be said that the demonstration of what may truly be regarded as cancer toxin opened up a new vista of promise, although some of the more important implications of the cancer toxin concept do not seem to be as generally recognized as they should be. Indeed, there has been an extremely cautious attitude in some quarters toward the toxohormone theory as the explanation of the marked decrease of liver catalase activity in cancer bearing animals. Accumulating evidence, however, seems to suggest that such extreme caution may serve only to hinder progress by blinding one to a patent fact which could otherwise be profitably exploited. I n the present article, we will attempt to outline the current status of the problem, to indicate the points which require further elucidation, and to emphasize the prospect of the future development of the subject. To a great extent, these grounds have already been covered by Greenstein in the second edition of his “Biochemistry of Cancer” (1954), and an effort will be made to minimize unnecessary duplications in this article. Newer experimental results will be considered more fully, especially those originating from Japanese laboratories where much work of interest has been done of late. The term “toxohormone” will be used throughout for the characteristic substance which is produced by cancer tissue and which is nssayable by its marked action in decreasing liver catalase activity in vivo. The substance is frequently referred to in the literature by various terms such as ‘(liver catalase inhibiting tumor factor,” and “tumor agent depressing liver catalase activity.” These terms, however, not only fail entirely to connote the basic significance of this unique substance, but they are also inconveniently long. Even for this latter reason alone, the term “toxohormone” may be useful, a t least until such a time as when the substance can be designated by its proper chemical name.

11. TOXOHORMONE PRODUCTION A UNIVERSAL PROPERTY OF CANCER CELLS Blumenthal, Brahn, and Ilosenthal early reported that liver catalase is markedly reduced in tumor bearing humans and animals (Blumenthal and Brahn, 1910; Rosenthal, 1912; Brahn, 1914, 1916). The significance of this fact was not fully realized until Greenstein and co-workers, thirty years later, (Greenstein, 1942, 1943; Greenstein and Andervont, 1942, 1943; Greenstein et al., 1941, 1942), demonstrated conclusively that the observed decrease of liver catalase activity is specifically attributable t o the presence of the growing tumor itself, and not to any secondary cause to which the tumor’s presence may give rise. Von Euler and Heller (1949) showed, by means of centrifugal fractionation of liver homogenates. that catalase

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activity is normally about equally divided between mitochondrial and microsomal supernatant fractions and that it is mostly in the latter fraction that the enzyme activity becomes greatly reduced in tumor bearing animals; decreased activity in the mitochondrial fraction is also observed after a rapidly growing tumor has reached a certain size. A new milestone in the progress of this study was established when a potent chemical fraction, capable of strikingly decreasing liver catalase activity when injected into normal animals (Nakahara and Fukuoka, 1948), was isolated from cancer tissues. The isolation of the active fraction was first attained by a simple method of extracting human tumor tissues with water under heat and precipitating with alcohol; this yielded crude material active for mice in 50-100 mg. doses. This alcohol precipitate was later purified by reprecipitation with one of the conventional protein precipitants, and the most active of such fractions produced a n unequivocal effect in mice in 5-mg. amounts. In these early studies, all the samples of cancer tissue examined, without exception, yielded the active material. They included 14 gastric carcinomas, 5 rectal carcinomas, 2 carcinomas of the sigmoid region, 3 mammary carcinomas, and 1 each of carcinoma of the transverse colon, lymph node metastasis of liver carcinoma, bladder carcinoma, mammary fibrosarcoma, and lymphosarcoma, all human surgical materials CLf confirmed histological diagnosis. Necrotic portions of cancer tissue yielded no more active fraction than fresh, nonnecrotic portion, nor did decomposition in vitro of cancer tissue increase the activity or yield of the fraction. Similar fractions from 14 samples of normal human tissue were uniformly inactive. It is the active substance of these cancer tissue fractions that we tentatively designated “toxohormone” in the hope of indicating its singular biological status of being a cell-borne substance which is released into circulation and which produced a clearly definable biochemical lesion (namely, decreased catalase activity) in the target organ. It may be conceived of as a pathological counterpart of hormones in that its presence produces a pathological condition, and its withdrawal restores the normal state. We did not call it “cancer toxin” in order to avoid possible confusion with miscellaneous alleged “cancer toxins’’ of the past literature. Our original experimental results have since received full confirmation from all sides. An active alcohol-precipitable fraction was isolated by Greenfield and Meister (1951), in Greenstein’s laboratory, from mammary carcinoma 3CHb of mouse and from Walker sarcoma and fibrosarcoma AXCMCA2 of rat; by Okushimu (1952a) from several gastric, rectal, and mammary carcinomas of man; and by Kawamorita et al. (1951) from Brown-Pearce carcinoma and Kato sarcoma of rabbit, to mention the earlier reports. Kuzin et al. (1955) duplicated some of these findings. Osawa

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(1954) isolated alcohol precipitable fraction from the Chiba strain of chicken sarcoma, as well as from the original Kous No. 1, and found them to be active for mice in 50-mg. doses. Sat0 and Yunoki (1951) reported separating a similar liver catalase reducing fraction by adsorbing an extract of human gastric carcinoma with kaolin a t pH 5.4, eluting with ammonia water (pH 9.0), and finally precipitating the eluate with acetone. This fraction was found to be active in 30-mg. amounts for mice. I n a series of experiments, Adams (1950b, 1951a) did not attempt to separate the active substance, but found that injections of whole homogenate of mouse sarcoma 37 or of mouse carcinoma 63 produced a marked depression of the liver catalase in normal mice, and he inferred that some chemical components of the cancer tissue must be responsible for the enzymatic change. According to the recent study by Nakagawa and Nakagawa (1956), centrifugal fractionation of tumor homogenates, using Yoshida sarcoma and ascites hepatoma of the rat, yields a supernatant fraction consisting of soluble protein and microsome which seems to be the main carrier of toxohormone, whereas the nuclear or mitochondria1 fraction failed to show clear evidence of the liver catalase depressing action. I n later studies, not only have many of the animal tumors already mentioned been tested butothers also, and all have been found to be good sources of toxohormone. Umeda’s rhodamine sarcoma of the rat, Murphy’s lymphoma, Ehrlich’s carcinoma (both ascitic and solid forms) Nakahara and Fukuoka’s N F sarcoma and N F carcinoma of the mouse are now being used successfully for the purpose of separation and concentration of this interesting substance. It should be noted that in all these experiments no malignant tumor was encountered, carcinoma or sarcoma, which, upon experimentation, did not yield active toxohormone fraction. This is in keeping with the fact that greatly decreased liver catalase activity has been demonstrated in all the cases of malignant tumors, so far as these have been investigated, and suggests very strongly that the production of toxohormone may be a universal property of all the malignant tumors.

111. ISOLA4TION O F TOXOHORMONE FROM MATERIALS OTHER THAN CANCERTISSUES As may perhaps be expected of a substance elaborated by cancer cells and thence thrown into circulation, toxohormone has been demonstrated t o occur in several types of body fluid in cancer bearing humans and animals. The first of this group of data emerged when Nakagawa (1952) reported the isolation of the liver catalase depressing fraction from the urine of

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cancer patients. By means of the benzoic acid adsorption method followed by precipitation with alcohol, this author obtained a material which was found to reduce markedly liver catalase activity in normal mice. The amount in terms of dry weight of the material necessary to produce the positive effect was not given, but it was stated that the yield from 50-200 cc. of urine was injected per mouse. The urine used in these experiments came from 42 different cancer patients of confirmed diagnosis. Fractions similarly prepared from urine of patients with diseases other than cancer did not show the catalase depressing effect in comparable doses. These findings were confirmed by Sat0 el al. (1953) who separated a similarly active fraction from cancer patients’ urine by the kaolin adsorption method which they had previously used for the isolation of toxohormone from cancer tissues. The yield from 200 cc. of the urine was injected per mouse. Both Nakagawa and Sat0 and the latter’s co-workers failed to detect any property which distinguishes the urinary factor from toxohormone of cancer tissue. More recently, Fuchigami et al. (1956), in our laboratory, reexamined the subject and confirmed the excretion of toxohormone in the urine of cancer patients. According to their results, however, the concentration of the active substance in the urine is generally very much lower than the two previous reports had given to underttand. Cancer ascites is a source from which toxohormone may reasonably be expected t o be recovered. Hirsch and Pfutzer (1953) reported that not only the cells but also the cell-free fluid of Ehrlich ascites carcinoma and of Yoshida sarcoma are active in decreasing liver catalase activity when injected into normal mice. There seems to be no doubt that toxohormone occurs in cancer ascites in man, but the amount present may be expected to vary greatly according to the case. Miyajima (1955) isolated the alcohol precipitable fraction from 46 cases and in doses of 25-100 mg. (mostly 50 mg.) found the fraction indisputably active for mice only in 28 of these cases. Gastric juice of patients with gastric cancer is another material which may be expected to contain toxohormone. The first evidence of the fact was brought forward by Kawamorita et al. (1951) working in the laboratory of Nakagawa. They used gastric juice from 10 gastric cancer cases and prepared the toxohormone fraction by alcohol precipitation; injecting the yield from 30-40 cc. gastric juice per mouse, they discovered that the fraction from 4 of these cases produced an indisputable effect. The urine from these cases also yielded a highly active fraction. None of the similarly prepared fractions from the gastric juice of 11 patients suffering from diseases other than gastric cancer showed any activity. These results were confirmed by Iwatsuru et al. (1945) who, in addition, also obtained active

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material by precipitation from concentrated cancerous gastric juice with the same volume of 10% trichloroacetic acid solution. It would seem quite certain that toxohormone is present in gastric juice of gastric cancer patients. Unfortunately, however, the actual amount of the active fraction injected was not given by any of these authors. The presence of toxohormone in the blood of tumor bearing hosts is to be expected, even without the experiments of Lucke et al. (1952) on parabiotic rats, but we are somewhat doubtful that it may occur in sufficient concentration to be demonstrable by the usual in v i m test. Okushima (1952b) prepared the alcohol precipitable fraction from serum of cancer patients and found it to be without effect. However, Plaza de 10s Reyes et al. (1953) reported that fresh plasma of cancer patients injected into normal rats reduced liver catalase activity, and Kuzin et al. (1955) also found that the alcohol precipitable toxohormone fraction separated from the blood of tumor bearing animals exerted a similar effect. With refinement of technique, the toxohormone in blood, even very small in amount, must eventually become demonstrable. It may be recalled that Greenstein el al. (1942) early noted that kidney catalase activity is lowered relatively little and blood catalase activity not at all in tumor bearing animals. Possibly the apparent relative insusceptibility of kidney catalase may be due to a rapid excretion of toxohormone, since we now know that toxohormone occurs in cancer urine in demonstrable amounts. The failure of the blood catalase to be affected may be explained by the insufficient concentration of toxohormone in the blood. However, according to Theorell et al. (1951) and Schmid et al. (1955), erythrocyte catalase has a far longer life-span than liver catalase, and it is possible that the apparently different behavior towards toxohormone of the two types of catalase may be accounted for by this fact. Also, if we follow the view that the liver constitutes the major site of catalase synthesis in an organism, blood catalase may be expected to be affected differently from liver catalase by the inhibition of the synthetic mechanism which is localized in the liver. There are obvious reasons for assuming that the attempts to demonstrate toxohormone in blood or urine were made with an eye to the possible utilization of the findings for the clinical diagnosis of cancer. Theoretically, the anticipated toxohormone test as a means of detecting the presence of cancer has the rationale not usually found in the numerous so-called cancer reactions that have been proposed in the past. Practically, however, since the production of toxohormone is a function of cancer cells, small cancer nodulen, such as are the objects of early diagnosis, cannot be expected to yield a ltirge amount of the substance. The most urgent need for diagnostic purposes, therefore, may be the discovery of some method whereby toxohormone in a very low concentration can be detected.

I\’. NORMAL LIVEHC r r r \ L . \ m LEVEL Thc possibility may be pointed out licrc that toxohormone, simply as a chemical substance, may not be strictly specific to cancer cells, but may occur in normal tissues in negligible amounts-negligible because, even if it occurs, it can serve a t most merely to maintain the normal level of catalase where it is. Although, as abundantly demonstrated, normal tissue fractions prepared by methods similar to those yielding potent toxohormone from cancer tissue fail to depress the liver catalase in comparable dosage, it is not impossible that they may do so in larger amounts. Greenfield and Meister (1951) stated in the summary of their paper that fractions obtained from normal tissues “possessed considerably less ability to lower liver catalase,” apparently implying that a small amount of active material may be present in such fractions. Obviously granting that small amounts of toxohormone may occur in normal tissues, Greenstein (1955) stated in his recent review that “that which is a toxin in a tumor due to abnormal production may be only a normal regulator of enzyme levels in a normal tissue.” It must be admitted, however, that there is nothing in our present knowledge to show that toxohormone in normal tissues actually plays a role in the regulation of the normal catalase level. The idea is merely conceivable. I n this connection, observations by Day et al. (1954) showing depression of liver catalase activity in mice injected with homogenate of normal mouse spleen may be referred to. This observation is a t variance with the previous results of Adams (1950b, 1951s) who injected homogenates of a variety of normal tissues, including embryo tissue, producing no significant change. It is also contradicated by the more recent results of Nakagawa and Nakagawa (1956) who found homogenates of neither spleen, liver, nor kidney from normal rats to reduce liver catalase. Of special interest is the work of Sawasaki (1953) who prepared the toxohormone fraction from human placenta according to the original method of Nakahara and Fukuoka and found it to be entirely without effect in 100-mg. doses. It may be recalled that embryonic and placental tissues are among the nonmalignant tissues whose metabolism resembles that of cancer tissue. It is possible that the discrepancy may have arisen from the difference in the source of normal tissue in relation to the test animal (mice) or that what little effect the normal tissue homogenates exerted was not regarded by these latter authors as significant. In any event, it must be admitted that the catalase depression reported by Day and associates is only of a small degree, and it is hardly to be compared with the marked effect of cancer tissue homogenate or of toxohormone concentrates. When Day and collaborators said that “the nature of the depression . . . involve[s] n rather complex physiological process, n dynamic process apparently

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involving a delicate compensatory mechanism under stress,” they probably had the normal mechanism of regulating liver catalase level in mind. Adams (1952, 1953, 1955) carried out extensive experiments concerning the maintenance of liver catalase activity in normal mice, and he arrived a t the conclusion that testicular and adrenal hormones, as well as traces of copper and manganese, are of importance in this mechanism. He also showed that testosterone, which is responsible for the sex difference in catalase activity in normal mice, requires the presence of riboflavin for its action on the enzyme level. Although it seems probable that there exists some sort of hormonal control to regulate the normal level of liver catalase, it does not necessarily follow that toxohormone operates through the mediation of such a mechanism. Nor does Adams’ idea that the function of toxohormone may be “to protect this liver enzyme (catalase) from hormonal influences” seem quite lucid. Whether or not these hormonal influences are capable of exerting any protection against the catalase depressing action of toxohormone is a more pertinent question. Begg (1951b) reported that injections of testosterone do not restore the catalase level in rats bearing large tumors. It would seem probable that in tumor bearing animals the supposed normal regulatory mechanism may no longer be operative, being overwhelmed by the active production of toxohormone.

V. CHEMICAL NATURE OF TOXOHORMONE The principal steps in the purification of toxohormone as originally carried out by us consisted of (1) extraction with water under heat and removing the heat coagulable matter, (2) precipitation with two volumes of absolute alcohol, and (3) removal of ether-soluble substances. The active substance of this fraction is precipitable by half saturation with ammonium sulfate and also by other usual protein precipitants such as trichloroacetic acid, picric acid, cupric sulfate. From these facts, we assumed a t the outset that toxohormone may be protein-like in nature and probably a polypeptide. From the dialysis and ultracentrifugal experiments, Greenfield and Meister (1951) suggested that the active agent might itself be of, or be associated with particles with, a molecular weight of a t least 40,000. The active fraction, when digested with 6 N HC1, was found to yield by paper chromatography the following amino acids : alanine, glycine, serine, proline, aspartic acid, arginine, valine, threonine, phenylalanine, hydroxyproline, a-aminobutyric acid, tryptophan, lysine, leucine, isoleucine, and glutamic acid. Okushima (1952a) hydrolyzed with 10% HC1 the original alcohol precipitate from human cancer tissue and established, also by paper chromatography, similar amino acid composition. He did not find isoleucine, hydroxyproline, a-aminobutyric acid and tryptophan, but noted

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the presence of methionine and a large spot of what appeared to be “underglutamic acid.” There is some indirect evidence which seems to support the view that toxohormone may be of polypeptide nature. Our early experiments showed that the toxohormone content of tumor tissue, as roughly estimated by the relative amounts of tissue homogenate necessary to produce liver catalase depression in normal mice, can be perceptibly increased by injecting a whole protein hydrolyzate or a mixture of pure amino acids into the tumor bearing animals (Fukuoka and Nakahara, 1953). The amino acids necessary for insuring this increased toxohormone content of the tumor were found to be alanine, proline, aspartic acid, arginine, phenylalanine, lysine, leucine, and glutamic acid. We interpreted the findings as indicating that these eight amino acids may be the major components of toxohormone polypeptide and that the increased toxohormone content may be due to the supply of an extra quantity of the building material. It is possible that in order to reveal the effect of the extra supply of amino acids, the basal diet, on which the tumor bearing animals are maintained, should be of protein subdeficient type, as was used in our experiments. If the dietary protein furnishes the optimum amounts of all the necessary amino acids, the superposition of additional amino acids may find no expression in the amount of toxohormone synthesized. The role of amino acids in the synthesis of toxohormone was demonstrated more clearly in our subsequent in vitro experiments (Nakahara and Fukuoka, 195413). Under the influence of the recent discovery of the ATPdependent biosynthesis of the tripeptide glutathione by an enzyme system from liver tissue, we speculated that biosynthesis of toxohormone may be possible in a somewhat similar manner. Upon experimentation, it was readily found that a substance highly active a s toxohormone can be producecl in vitro by incubating a t 37°C. for 24 hours fresh tumor tissue slices in a solution of amino acid mixtures in the presence of ATP. The active material thus obtained was heat stable, water soluble, and alcohol precipitable, exactly as was the toxohormone fraction directly separated from tumor tissue. The alcohol precipitate was active for mice in about one-fifth the amount of the material similarly prepared from the same tumor tissue ( N F strain of mouse sarcoma) the slices of which were used in the biosynthesis experiments. An interesting fact emerging from these experiments was that only three specific amino acids need be included in the reaction mixture, and these three turned out to be arginine, phenylalanine, and leucine. It is important, however, to take into account the fact that small amounts of many amino acids are carried into the reaction mixture b y the tumor slices and hence the special role of these three amino acids cannot be strictly upheld. It is also to be admitted that it is merely an assumption that the,

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active substance produced is a condensation product of the amino acids, and, therefore, is actually a polypeptide. The real value of these biosynthesis experiments may consist, after all, in the control experiments which demonstrated the failure of normal tissue slices to produce toxohormone under the conditions in which tumor tissue slices so readily elaborate the same substances. As may be inferred from the method used, the toxohormone concentrate originally obtained b y us contained much nucleic acid, often amounting to 30% of the weight, Nakagawa et al. (1955), in their attempt to purify toxohormone from Brown-Pearce rabbit carcinoma, followed and confirmed Nakahara and Fukuoka up to the copper sulfate precipitate; they then separated this precipitate into protein and nucleic acid fractions by means of hot tricholoroacetic acid solution and found the latter to be more active than the former. Ent!irely analogous results were also obtained using human lung and stomach cancer tissues. Nakagawa and associates claimed that their nucleic acid fraction was active for mice in doses as low as 0.1-0.2 mg. The fraction gave a negative biuret reaction and a strongly positive Molisch reaction, but an examination by means of the chloroform gel method revealed the presence of a small quantity of protein or peptide. It may be mentioned here in passing that, according to Fuchigami el al. (1956) who investigated in our laboratory the chemical properties of the active material isolated from the urine of cancer patients b y the original method of Nakagawa et al. (the benzoic acid adsorption and alcohol precipitation), neither RNA nor DNA fraction of this material shows the ultraviolet absorption characteristic of nucleic acid. They identified the following amino acids as components of the polypeptides of the material: aspartic acid, glutamic acid, serine, threonine, proline, alanine, valine, leucine, phenylalanine, lysine, cystine, and glycine. Nucleic acid is not the active constituent of toxohormone. As a matter of fact, tumor nucleic acid isolated b y means of Clark-Schryver’s method was found to be devoid of toxohormone activity (Endo, 1954). Moreover, it was possible to obtain a n active fraction free from nucleic acid contamination. From the fact that nucleic acid is attached to many toxohormone preparations, Ono et al. (1955), in our laboratory, assumed that the toxohormone may be a basic polypeptide. They have isolated a basic polypeptide from tumor tissues in the form of a white amorphous powder which was active as toxohormone in doses as low as 10 mg. and which showed no definite absorption peak a t around 280 mp. I n separating this nucleic acid-free toxohormone fraction, they followed the method for the preparation of corticotropin, involving the processes of defatting with acetone, extracting with methanolacetic acid mixture, and precipitating with ether. It is interesting to observe that the nucleic acid-free toxohormone is not precipitable from its solution by the addition of two volumes of alcohol

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a t pH 4.5. The fact that Nakahara and Fukuoka originally achieved the isolation of toxohormone by means of alcohol and acid precipitations may be attributable t o the nucleic acid moiety of their fractions. Greenfield and Meister (1951), incidental to confirming the isolation of toxohormone, reported an unexpected fact that digestion of the active alcohol precipitate with 6 N HCl at 100°C. for 1 2 4 8 hours did not affect the activity and that the active substance in the digest was dialyzable. A complete hydrolysis should have reduced the protein to amino acids, but, as Greenfield and Meister themselves showed, a mixture of all the amino acids detectable in the hydrolyzate of the toxohormone concentrate failed to depress liver catalase activity. Two alternative possibilities, therefore, come t o mind in the interpretation of this experimental result, namely, either that the hydrolysis was incomplete and enough active peptide remained intact, or that toxohormone is a substance not attacked by the protein hydrolyzing process. It is important to point out here that, according to our observations, freshly prepared extract of tumor tissue contains a small amount of dialyzable substance which depresses liver catalase activity when injected into normal mice (Nakahara and Fukuoka, 1954a). By separating the dialyzate into alcohol precipitate and filtrate, it was found that the filtrate, instead of the precipitate, was active. Yields varied, but on an average the total dialyzate amounted to about 10 mg. per gram of fresh tumor tissue, and the ratio of alcohol precipitate to filtrate was something like 1:30. The filtrate was brownish yellow, sticky, hygroscopic matter, and active for mice in 50-mg. doses. Toxohormone as originally isolated by us, or by Greenfield and Meister, is nondialyzable, but it is interesting to note that this nondialyzable toxohormone fraction, when digested with papain or pepsin, yields a dialyzable substance which is potent i n vivo, exactly as the active substance in the dialyzate of tumor tissues (Nakahara and Fukuoka, 1954a). Digests were heated a t lOO"C., coagulum formed was centrifuged off, and the clear solution was dialyzed against 200 cc, of distilled water for 5 days a t the temperature of 2-3°C. Dialyzates were then precipitated by alcohol, yielding about 40 mg. of precipitate and 200 mg. of filtrate per 1 gm. of the starting material before digestion. These facts definitely suggest that toxohormone occurs in two forms, one nondialyzable and the other dialyzable, and that the latter may be derived from the former b y the enzymatic splitting process. Taking advantage of the knowledge that toxohormone in the form of alcohol precipitate can be largely converted into dialyzable form without damage to its activity, Ono et al. (1956a) subjected their nucleic acid-free toxohormone fraction to enzymatic hydrolysis for 4 days. They used 1% pepsin and, adjusting the reaction of the digest to the slightly alkaline side,

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added one-third the volume of saturated solution of sodium picrate under stirring. About 30 minutes later, they noticed a gradual formation of precipitate which, upon microscopic examination, was found to consist of fine needlelike crystals. After recrystallization, the yield of the picrate was about 5 mg. from 1g. of the original material before digestion. The identical crystals were obtained upon repeated trials. I n all cases the crystalline picrate proved to be active in 0.2-mg. doses for mice. I n a spot test, this active picrate gave a blue color with ninhydrin after one week, contrary to the rapid reaction of free amino acids. It also gave one spot in paper chromatograms, with Rf of 0.2 in an acetic-butanol system and of 0.016 in a lutidine system. As determined b y paper chromatography, the amino acid composition was found to be: aspartic acid, glutamic acid, serine, threonine, proline, hydroxyproline, alanine, valine, leucine, isoleucine, phenylalanine, lysine, glysine, and arginine. Cystine, methionine, tryptophan, and tryosine were not detected. Assuming one molecule each of threonine and valine to be present and other amino acids in integral ratio, the minimum unit of constituent amino acids was calculat,ed t o be 30 moles, the molecular weight being close to 4000. The picrate crystal decomposed a t 300°C; its melting point was undeterminable. Whether or not this crystalline picrate represents that of pure toxohormone is a moot point. Peptides of many sorts must be assumed to be present in the pepsin digested material, and the probability of some of them forming a mixed picrate crystal with toxohormone cannot be regarded as small. Further studies alone can settle this point. Our present concept of the nature of toxohormone is that this unique toxin in its elementary form may be a relatively small molecule and that it may occur in cancer tissue for the most part either as aggregates of such elementary forms or in close association with some other substance, constituting the toxohormone of the usual or nondialyzable form. In the latter case, the associated substance might be nucleic acid, and the enzymatic action, which yields the dialyzable form, may simply be to dissociate the two. The dialyzable form may thus be thought to correspond to the nucleic acid-free toxohormone, neither of which is precipitated by alcohol. However, the toxohormone in the urine of cancer patients is precipitated by alcohol in spite of the absence of nucleic acid in the same fraction. Does this mean that the nondialyzable toxohormone consists of aggregates of smaller molecule forms? There is much to be done in elucidating the precise chemical nature of toxohormone.

VI. MODEOF ACTIONOF TOXOHORMONE Many substances are known to inhibit catalase activity in vitro, and animals’ tissues, normal as well as cancerous, are now known to contain such inhibitors. Hargreaves and Deutsch’s (1952) “kochsaft factor” is an

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inhibitor of this sort, representing nonspecific tissue constituents capable of reducing Fe"' of catalase to Fe" (Endo et al., 1955; Ceriotti and Spendrio, 1955) without catalase depressing action in vivo. Toxohormone differs from these substances. It does not inhibit catalase :tctivity in vitro as early demonstrated by Greenstein (1942, 1943) and by Nakahara and Fukuoka (1949). Price and Greenfield (1954) more recently reiterated that the tumor fractions which show a marked effect on liver catalase of normal animals in vivo do not lower either the catalase activity or Soret band absorption in vitro. Conclusive evidence that the decreased catalase activity of liver of tumor bearing animals is attributable to the actually smaller amount of catalase contained in the liver was obtained by Price and Greenfield (1954). By subjecting normal rat liver and the liver of tumor bearing rat to the identical treatments of absorption and elution (using calcium phosphatecellulose columns) and studying spectrophotometrically the chromatographic peaks so obtained, these authors showed that there may actually be five times as much catalase in normal liver as in the liver of the tumor bearing animal. They also isolated crystalline catalase from these two kinds of liver and noted that there is no qualitative difference whatever between the two. Prima jacie, the presence of an actually smaller amount of catalase in the liver of tumor bearing animals as compared to that of normal animals can be accounted for either by the slower rate of the synthesis of catalase or by the shorter life-span of the enzyme in these animals. Here, previous kinetic studies of Greenstein (1943) may be regarded as adequate in ruling out the second possibility. He demonstrated, among other things, that the rate of spontaneous inactivation in vitro of liver catalase is identical between tumor bearing and normal animals. There seems to be, then, no alternative but to conclude that the action of toxohormone may be to suppress the process of catalase synthesis. Hirsch and Pfutzer (1955), seeing that the dialyzate of the cell-free fluid of ascites tumor (though activc in vitro) is entirely devoid of action in vivo, made an interesting suggestion that a high molecular substance of protein nature may be acting as carrier for the catalase-inhibiting substance. They tried to explain the lack of in V ~ V Oaction of the dialyeate by assuming that the inhibitor, without this protein carrier, may pass through the liver too rapidly to attain the concentration necessary to effect the lowering of liver catalase activity. This, however, is based on the gratuitous hypothesis that the in vivo and 1'11 vitro active substances are identical. We now know that nonspecific tissue constituents of many sorts are involved in the in vitro inhibition of wttiliise, while toxohormone is active only rn uivo. How does toxohormone suppresh the synthesih o f liver catnlasc'? Having

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found that administration, either per 0s or parenterally, of ferric chloride or iron-rich materials (dried liver or blood) appreciably prevented the decrease of liver catalase activity of tumor bearing animals, we tried to visualize the mechanism of the toxohormone effect on liver catalase synthesis in terms of decrease of available iron (Fukuoka and Nakahara, 1951). The effect of injecting an isolated toxohormone fraction was also found to be almost completely nullified when test mice have been fed for one week on a diet containing 5 mg. of ferric chloride per 300 g. This latter finding was duplicated by Kosuge et al. (1951) using the toxohormone fraction from a cancer patient’s urine. Von Euler (1952), however, reported his failure to confirm fully these same results; he observed only slightly higher liver catalase activity in iron-treated, rather than untreated, tumor bearing animals. The discrepancy here may have stemmed from a difference in the type of tumors used, since the countereffect of additional iron may vary according to the amount of available toxohormone. Our experiments were carried out with the N F mouse sarcoma which is of relatively low toxohormone activity. However this may be, it must be pointed out that so far it has been impossible to demonstrate any special capacity of toxohormone to bind iron i n vitro. Resegotti and von Euler (1954) later reported that the decrease of liver catalase activity in ascites tumor bearing rats can be prevented and the activity held near normal by the injection of tartrate of trimethylcolchicine acid methylester; injections of pure colchicine showed no such effect. Since both substances blocked the growth of tumor to about the same extent, the different effects on the liver catalase cannot be ascribed to the different growth rates of tumors. Moreover, cases were found in which liver catalase activity was normal in the tartrate treated rats with profuse growth of ascites tumor. The i n evitro effects on catalase of incubating the liver homogenate from tumor bearing and normal rats with the solution of the tartrate were identical. Resegotti and von Euler suggested, purely hypothetically, that tartrate of colchicine acid methylester acts as “anti-toxohormone.” The elucidation of the mechanism of this “anti-toxohormone” effect will be of great importance. On the basis of the hypothesis of Fukuoka and Nakahara (1951) that toxohormone may suppress the synthesis of the liver catalase by impairing the utilization of iron, Ono et al. (1956b) carried out an investigation on the porphyrin level of the liver, Harderian glands, and urine of tumor bearing rats and on the effect of toxohormone on the porphyrin metabolism in normal rats. They demonstrated that t,here is a considerable increase of protoporphyrin contents in the liver and of coproporphyrin excretion in urine of tumor bearing animals. Injections of toxohormone fraction also showed nn unmistakable tendency for these prophyrins to increase in

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normal rats. According to their view, these results indicate an underutilizntion of porphyrin caused by thc impairment of iron metabolism or of the synthesis of protcin moiety of hemoproteins. KO :Idtlitional or nonphysiological porphyrin, not found in normal mts, was detected in the liver or urine of tumor bearing or toxohormone injected rats. I n order to understand the mode of action of toxohormone, it may be of help to look for some known chemical substance which can reduce liver catalase activity in viuo. One such substance seems to be allylisopropylacetylcarbamide (“sedormid”). Schmid et al. (1955) found that oral administration to rats of this substance in 80-mg. daily doses brings about a marked fall of liver catalase activity with a corresponding marked rise in hepatic porphyrin concentration. They concluded that sedormid blocks the formation of catalase in the liver and that this metabolic block may explain the markedly increased concentration of porphyrin in the liver. Heim et al. (1955) called attention to 3-amino-l,2,4-triasole with which they have been able to produce in normal animals a marked decrease of liver catalase activity. Sugimura (1956) found, however, that 3-amino-1,2,4triasole inhibits catalase activity also in vitro when in the presence of cofactors contained in liver extract. He found, moreover, that the i n vivo depression of the liver catalase following the injection of this substance is not accompanied by notable increase of porphyrin in the liver. VII. TOXOHORMONE IN GENERALTUMOR-HOST HELATIONS There is evidence for assuming that the marked decrease of liver catalase activity in cancer bearing hosts may be only a salient expression of a deep-seated and more fundamental disturbance which toxohormone brings about. It was suggested earlier that toxohormone may adversely affect, not only the synthesis of catalase, but also of other iron-containing proteins. In neoplastic disease there is a group of findings (such as anemia, lowered hemoglobin, and decrease of plasma iron), in man as well as in animals, all pointing to the disturbance of iron metabolism. This interference with iron utilization may have a t its root ttn injury to the synthetic mechanism of specific ferro-protein ferritin, and the decrease of plasma iron which precedes that of hemoglobin may also be attributable to a similar disturbance in the metabolism of t,issue iron. The relative decrease of liver ferritin seems to be demonstrable in cancer patients. The porphyrin content of the blood of tumor bearing animals is appreciably increased even before the decrease of hemoglobin becomes apparent (Sugimura el al., 1956). It may be reasonable to assume that the marked lowering of the synthesis of liver catalase cannot occur without concurrently involving changes in the general iron metabolism and particularly in the hepatic iron reserve.

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Of interest in connection with this problem is the recent findings of Iijima et nl. (1956) that toxohormone injections produce in the liver of normal mice changes in non-hemin iron frzictions similar to those occurring in the liver of gastric cancer patients. These authors prepared toxohormone concentrate from human gastric cancer tissue according to the original method of Nakahara and Fukuoka, and observed that 60 mg. was sufficient to induce, 24 hours later, (a) a decrease in the ferritin fraction to 30% from the normal level of 50%, (b) an increase in the nucleic acid iron fraction to 27% from the normal ratio of about lo%, and (c) a n increase in the hemosiderin fract,ion to about 55% from the norm of 38%. The changes they found in gastric cancer patients were quite comparable to those in toxohormone-injected mice. It would appear that toxohormone, which suppresses the hepatic catalase synthesis, brings about a t the same time an imbalance of various nonhemin iron fractions of the liver. These findings, if confirmed, may effectively connect the decrease of the liver catalase to anemia in neoplastic disease, bringing both of these important phenomena under the influence of toxohormone. It is freely admitted that obscurity prevails as to tlhe exact mechanism involved, yet there seems to be sufficient evidence already to lead us to believe that toxohormone may affect the metabolism of iron-containing proteins in general. Since these proteins include many substances of much biological importance, it seems not unreasonable to suspect that the essential cause of the so-called cancer cachexia may ultimately be found to be related to the toxohormone activity. No hasty conclusion should be drawn in dealing with such a complex phenomenon as cachexia in cancer. The subtraction of body substances by growing tumor, the effect of secondary hemorrhage or of incidental infection, the possibility of hormonal disturbances, all these may have parts to play in producing the cachectic condition as one sees it in the terminal stage of cancer. With due consideration for all these complicating factors, however, the fact remains that there is a demonstrable toxohormone-associated disturbance of iron metabolism in cancerous organisms constituting sufficient ground for suspicion that toxohormone may be fundamentally responsible for the final shaping of the cachectic condition in cancer. It may not be out of place to discuss here the marked thymus involution which, as is well known, constantly occurs in tumor bearing animals. Because thymus involution is sometimes accompanied by adrenal enlargement, attempts were made in the past to consider these phenomena in cancer in the light of the adaptation syndrome of the so-called stress reaction (Savard, 1948; Begg, 1951a). However, while involution of the thymus is a marked and constant phenomenon in tumorous animals, aclrend enlargement is often so slight as to be practically negligible. The

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enlargement of the adrenals is said to be accompanied by a marked reduction in their ascorbic acid and lipid contents and is interpreted as indicating a reduced cortical activity. The decrease of the lipid content, however, does not seem to be of great magnitude since we found (unpublished observations) that no difference in the organ’s specific gravity can be detected between normal and tumor bearing mice. An important fact here is that thymus involution can be induced in normal animals by toxohormone. By injecting into normal mice a toxohormone fraction (a copper sulfate precipitate prepared from N F mouse sarcoma tissue) in doses of 10-20-mg., Fukuoka and Nakahara (1952) found that, a t 24 hours to 3 clays after the injection, the average thymus weight was reduced to about 29 mg. per 100 g. of body weight, from the average normal of 113 mg. in males and 147 mg. in females. This reduction in thymus weight generally corresponded to that found in mice bearing tumors of 3-6 g. Injections of similarly prepared fraction from normal tissues showed no such effect. It is interesting to note that in these experiments, while marked thymus involution was constantly induced, there was no evidence of concomitant adrenal enlargement. We advocated as likely that thymus involution may be connec+ecl with the disturbance of nitrogen metabolism and that it may be looked upon as an early sign of the wasting of body protein in cancerous animals. It is well known that various conditions such as malnutrition, infection, intoxication are closely associated with thymus atrophy, and it would appear that toxohormone, through its adverse effect on protein synthesis, may bring about a similar thymus change. This point of view leads us again to the idea that toxohormone may play a significant part in producing the so-called cancer cachexia. Although both the liver-catalase-decreasing and the thymus weight reducing actions are carried by many tumor fractions so far tested, there is a possibility that the active substance concerned may not be identical (Ono et al., 1955). Further studies may make it necessary to accept a secondary cancer toxohormone of thymotropic type.

VIII. CONCLUDING REMARKS The biological significance of toxohormone has been considered largely from the point of view of its systemic effects on tumor bearing animals. This is natural, since the whole toxohormone problem originally arose as an attempt to discover the chemical basis of the tumor-host relations. There is, however, another aspect to the study of toxohormone which may well prove to be of fundamental importance but which so far has not received the attention it deserves. The ability of toxohormone to inhibit the synthesis of liver catalase

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should immediately raise the question of the consequence to the physiology of the cancer cells themselves which produce this inhibitor. We have known for years that cancer cells are notably deficient in their catalase activity. May it not be that the catalase deficiency and toxohormone production in cancer cells are causally related? Catalase, as is well known, is closely associated with aerobic cell respiration, and the almost complete absence of catalase in cancer cells may be explained on the basis of its being useless in the metabolism of these cells, Toxohormone action may provide the mechanism which brings about the deletion of catalase in cancer cells. What seems most intriguing here is the perfect harmony and adaptive interplay of the production of toxohormone, on one hand, and the development of a metabolic pattern requiring no catalase, on the other, and it. takes no great flight of imagination to conceive of these two processes taking place as necessary parts of a more complex whole, which, in its totality, characterizes the cancer cell physiology. It is from this point of view that toxohormone may assume possibly a profound significance. I n the words of Greenstein (1954), (I . . . the neoplastic transformation ends in a cell with only the two characteristics [growth and the production of a circulating toxin] to identify it. Growth is a name given to an over-all biological phenomenon, but cancer toxin is a chemical approachable by conventional methods of analysis. At the least, this latter characteristic offers an experimental handle whereby further knowledge of an unusual metabolic feature of the cancer cell may be grasped.” It may be pertinent to point out again that toxohormone is characteristic of all malignant tumors in that these tumors produce the substance in sufficient amount t o bring about certain definite systemic changes in animals bearing them. None of the normal cells does this which means that in normal cells the synthesis of toxohormone, if any indeed occurs, is of negligible proportions. There is, then, a striking difference between malignant and normal cells in respect to the synthetic mechanism. How does a cancer cell synthesize a substance with such peculiar activity? Nakahara and Fukuoka’s (195413) work on biosynthesis in vitro, already referred to, showed that toxohormone synthesis may depend upon the enzyme system, absent or not operative in normal cells. This study, however, has not even scratched the surface of the problem, since the nature of the enzyme system concerned remains entirely unknown. When the difference between the enzyme systems of cancer cells and normal cells (capable and incapable, respectively, of synthesizing toxohormone) is disclosed we will have gone far in the biochemical understanding of the cellular process of malignancy.

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Nakahara, W., and Fukuoka, I?. 1954a. Dialyzable form of toxohormone. A sixth study of tnxnhornione, etc. G m n 46, 67. Nakahara, W., and Fukuoka, 1'. l954b. I