The Comparative Enzymology and Cell Origin of Rat Hepatomas I

The Comparative Enzymology and Cell Origin of Rat Hepatomas I. Deoxycytidylate Deaminase and Thymine Degradation*

VAN R. POTTER, HENRY C. PITOT,~ TETSUO ONO,$ AND HAROLD P. MORRIS

(McArdle Memorial Laboratories, The Medical School, University of Wisconsin, Madison, Wis.; and National Cancer Institute, National Institutes of Health, Bethesda, Md.)

SUMMARY Enzyme assays for deoxycytidylate deaminase and thymine catabolism were carried out on ten rat hepatomas, including the Novikoff, Dunning L-C18, McCoy MDAB, and the Morris 3683, 3924A, and 5128 hepatomas, plus primary hepatomas produced by feeding ethionine or 8'-methyl-4-dimethylaminoazobenzene, and hepatomas derived from the primary tumors induced with ethionine. Normal liver, Dunning L-C18 hepatoma, and Morris 5123 hepatoma were similar in having almost no deoxycytidylate deaminase, whereas the Novikoff hepatoma was shown to be a rich source of the enzyme, converting 400-600 #moles substrate/gm protein/hr. Other hepatomas had values between 50 and 500 gmoles/gm protein/hr. The Morris 5123 hepatoma contained "thymine reductase" with a capacity of 9-23 gmoles substrate/gm protein/hr, which was very near the capacity of normal liver. All other hepatomas appeared to lack the capacity to convert carbon 2 of thymine to CO2, even in the coenzyme-fortified assay system employed. On the basis of the two enzyme assays mentioned, hepatomas were classified as D + if the deaminase were present and T + if the "reductase" were present, the letter and sign indicating presence of readily measurable enzyme activity. A tentative chart of relationships was shown, with the hepatomas classified into three types: Novikoff, D+T-; Dunning, D-T-; and Morris 5123, D-T +.

The Novikoff rat hepatoma has been studied discovered deoxycytidylate deaminase was found rather extensively and has been shown to lack to be present in the Novikoff hepatoma and absent lnany of the enzymes that are found in rat liver (13, from normal adult rat liver (10). However, it was 19), whereas another rat hepatoma, the Dunning present in livers containing large numbers of pro- L-C18 (4), has been reported to resemble normal liferating bile duet epithelial cells, but absent from liver cells more closely than do the cells of the the Dunning hepatoma (17). The two hepatomas Novikoff hepatoma both microscopically and bio- resembled each other in lacking enzymes for the chemically (16), although still differing consider- reductive catabolism of thymine (21), a process ably from normal or regenerating liver (8, 16). almost exclusively limited to liver, whereas the In contrast to the above studies, the newly Novikoff hepatoma differed from the Dunning * This work was supported in part by a grant (No. C-646) by being unable to convert thymidine to thymine from the National Cancer Institute, National Institutes of (5). In terms of the deletion hypothesis (18, 191) Health, U.S. Public Health Service. it might be suggested from the biochemical studies t Lillian Israel Memorial Fellow in Cancer Research of mentioned that the Novikoff hepatoma represents the American Cancer Society, 1957-1960. multiple deletions from a bile duct epithelial cell :~ On leave from the Cancer Institute, Japanese Foundation (or a more involved process in a liver cell), while for Cancer Research, Tokyo, Japan. the Dunning hepatoma represents at the least Received for publication April 4, 1960. a deletion from a parenchymal liver cell of one 1~55

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. 1256 Cancer Research Vol. g0, September 1960 or more steps in the thymine-catabolizing path- H. E. Swim in this laboratory. 1 While the tumor way. These deductions are based in part on the was carried in culture it was inoculated back very limited distribution of deoxycytidylate de- into Fisher strain rats periodically to insure the aminase and of thymine catabolism in the tissues retention of its malignant potential. The remaining of the body, but they obviously cannot be regarded three hepatomas were supplied by Dr. H. P. as final conclusions at this time. Even with the Morris of the National Cancer Institute. The help of histological and cytological technics an tumor-bearing animals were shipped by railway unequivocal decision could not be reached, and it express from Bethesda to Madison, Wisconsin, was decided to make a survey of a number of while the tumors were quite small. The enzyme hepatomas, with primary emphasis on the alter- assays were carried out from several days to a native catabolic and anabolic pathways of py- week or more after arrival of the rats. The Morris rimidine nucleotide metabolism before making hepatomas and the carcinogens originally used further attempts to resolve the origin of the Dun- to induce them are as follows: No. 3683, induced ning or Novikoff hepatomas. The purpose was to by N-3-fluorenyldiacetamide in 1951, transplanted "seek for a cancer cell that differs from a normal for more than 150 generations, induced in and prototype as little as possible and try to narrow carried in AXC strain rats; No. 3934-A, same the spectrum of enzymatic differences that exist carcinogen, but arose in a castrate female AXC- in order to decide which differences are defini- strain rat that received testosterone during the tive" (3). induction; No. 5133, induced by N-(3-fluorenyl)- The studies were made possible by cooperation phthalamic acid (3-FPA) in a female Buffalo-strain between investigators at the McArdle Memorial rat in 1956 as described in the accompanying Laboratory and Dr. H. P. Morris of the National report (13). Cancer Institute, who provided the most extensive Homogenates and tissue extracts.--Freshly ex- source of animals bearing transplantable hepa- cised tumors were dissected free of necrotic and tomas. In the course of the study it was found supporting connective tissue and transferred to that most of the hepatomas had characteristics beakers of iced isotonic KC1. The hepatoma 51~3 intermediate between the Dunning hepatoma and contained sinusoids filled with blood, but this the Novikoff hepatoma, but one hepatoma in was rinsed away after the tumor mass was coarsely particular emerged as remarkably close to paren- minced with a scissors. Twenty per cent homoge- chymal liver cells even from the limited scope of nares were prepared and used as such, or centri- the enzyme survey. This tumor, referred to herein fuged at 105,000 X g for 40 minutes in a Spinco as the Morris hepatoma 5133, was also seen to Model L centrifuge, in the No. 40 rotor to obtain be very close to parenchymal liver cells from light a clear supernatant fluid containing the soluble (1~) and electron (14) microscope studies; it grows enzymes in the fraction usually referred to as the very slowly (13) and is the only rat hepatoma we high-speed supernatant fraction or soluble fraction. have seen that carries out the reductive catabolism Enzyme assays.--Deoxycytidylate deaminase was of thymine, as will be shown below. determined essentially according to 5~[aley and Maley (10). The reaction mixture contained 0.5 MATERIALS AND METHODS ml. soluble fraction, 0.3 ml. 0.03 M dCMP ~ dis- Hepatomas.--Primary rat hepatomas were ob- solved in 0.3M Tris:HC1 buffer, pH 8.0, and tained from Drs. James and E. Miller of the 0.3 ml. 0.1 M KF. The reaction time was 30 McArdle Memorial Laboratory. They had been minutes. The reaction was stopped by the addition induced by feeding 3'-methyl-4-dimethylamino- of 4.0 ml. of 1 • HC104. After centrifugation to azobenzene. The McCoy MDAB hepatoma was remove the precipitated protein, the supernatant was neutralized with KOH, with phenol red used originally obtained by feeding the same compound as an indicator. The tubes were left at 0 ~ C. and has been carried for over 50 transplant genera- overnight, and the resulting crystals of KCI04 tions in Holtzman rats (11). Animals bearing the were centrifuged down. The supernatant was de- tumor were kindly supplied by Dr. Alan Sartorelli of the Samuel Roberts Noble Foundation, Ard- 1H. E. Swim and H. C. Pitot, unpublished. more, Oklahoma. The ethionine-induced hepa- :Abbreviations as follows: dCMP, deoxycytidylic acid; dUMP, deoxyuridylic acid; Cdr, deoxycytidine; Udr, deoxyuri- tomas were produced by feeding a diet (5) con- dine; AMP, adenosine 5'-phosphate; ATP-K, adenosine 5'- taining 0.35 per cent DL-ethionine for 589months triphosphate, potassium salt; TPN, triphosphopyridine nucelo- and then placing the animals on a Purina chow tide; TPNH, reduced TPN; TTP, thymidine 5t-triphosphate, TDP, thymidine 5'-diphosphate; TMP, thymidine 5'-phos- diet for 5 more months. One of these tumors phate; DNA, deoxyribonucleic acid; EFS, enzyme-forming (H-l) was explanted to tissue culture by Dr. system; G-6-P, glucose-6-phosphate.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. POTTER et al.--Enzymology and Origin of Rat Hepatomas. I 1~57 canted onto 6 X 1 era. columns of Dowex 1 (X10) the Dunning and Morris tumors are of question- in the formate form, and the nucleotides and able significance, since the optical density readings nucIeosides were separated and analyzed by the of these assays were so low one could not be sure method of Brumm and Potter (unpublished) which of the validity of the spectral identification of consisted of the following elution schedule: H20, the products. X 10 ml. combined with sample effluent to give The four transplantable neoplasms with the deoxyeytidine; 0.01 ~r formic acid, 4 X 10 ml. highest deaminase activity, i.e., the Novikoff, to give deoxyuridine; 0.05 N formic acid, 4 X 10 Morris 3683, H-I, and McCoy hepatomas, all ml. to give deoxycytidylic acid; 1 N formic acid, were rapidly growing tumors having similar his- X 10 ml. to give AMP if present; 2.5 ~ formic tologic pictures. ~The Novikoff and McCoy tumors acid, 6 X 10 ml. to give deoxyuridylic acid. With were transplanted intraperitoneally, whereas 3683 fluoride in the reaction mixture almost no Cdr and H-1 were subcutaneous transplants. The H-1 or Udr was formed, and the sum of dCMP and hepatoma, which had been explanted to tissue dUMP was constant during the incubation. Opti- culture and reinoculated into animals, possessed cal densities of all eluates were measured at both a much higher speeifie activity of deoxyeytidylate s and ~80 m#. deaminase than did its parent primary ethionine- Pyrimidine breakdown was studied by a modi- induced tumor. Simultaneous with this gain in fication of the system used by Canellakis (~) for deaminase activity, there was a loss in the activity the study of uracil and thymine breakdown. The of other enzymes found in hepatic parenchymal reaction mixture contained 0.8 ml. soluble fraction; 0.3 ml. 0.045M ATP-K; 0.1 ml. 0.01 M TPN; The histopathologic characteristics of these neoplasms will be presented elsewhere. 0.~ ml. 0.03 M G-6-P; 0.~ ml. 0.154 M MgC12; 0.45/zmole of thymine-~-C t4, uracil-r t4, or thymidine-r ~4 containing 0.5 #c., 0.3 #c., 0.7 ~c. 700 of radioactivity, respectively, in 0.3 ml.; and 1.1 a:/ 600 ASSAY: ml. of buffered sucrose medium, which was com- -r- posed of ~50 ml. of 0.~ M sucrose and 0.04 M dGMP -clUMP Z nicotinamide; 15 ml. of 0.~ M Tris:HC1, pH 8; i;q 500 and 10 ml. of 0.154 M KC1. Each solution with the I-- 0 exception of the MgC12 was made up in 0.~ M 13:: 400 Tris:HC1 buffer and adjusted to pH 8.0. Incuba- 13_ tions were carried out at pH 7.~ in open test tubes (.9 300 at 38 ~ C. without shaking; 0.5-ml. aliquots were 13._ removed at 0, 15, 30, 45, and 60 minutes and :::) deproteinized by ~ ml. of 0.5 M HC104. The deg- "13 200 00 radation rate of each compound was dete~ained LI.I ._1 by measuring the nonvolatile radioactivity remain- 0 I00 ing in the deproteinized supernatant, after removal of perchlorate as above and plating on aluminum N 3 H-I M E E 3 M 5 D L planchets. 06 CT T 9 I U I H Glucose-6-phosphate dehydrogenase was assayed v 0,P . vu spectrophotometrically as described by Kornberg 3 TISSUE P and Horecker (9). CHART 1.--Deoxycytidylate deaminase activity of eleven RESULTS hepatomas and of liver and thymus in the rat. Each vertical line or point represents one animal. Key to symbols: Deoxycytidylate deaminase.--The deoxycytidyl- LIV -- Liver. ate dealninase of a number of primary and trans- THY = Thymus. plantable hepatomas in the rat was determined 51~23 = Morris hepatoma 51~23, by the procedure outlined in the methods. In ETP = Primary hepatomas induced by dietary ethioniue,~ addition, the enzyme was assayed in the liver and ET1 = First- and second-generation transplants of ETP. MDABP = Primary hepatomas induced by dietary 3'-Me- thymus of normal rats. The results of these assays DAB. are shown in Chart 1. With the exception of the DUN = Dunning L-C18 hepatoma. Dunning hepatoma (17), the Morris hepatoma 39~4A = Morris hepatoma 39~4A. 51f~3, and normal liver (10), all the tissues sur- H-1 = H4 hepatoma (described in text). 3683 = Morris hepatoma 8683. veyed possessed measurable deaminase activity. MCC = McCoy hepatoma, The low levels of enzyme shown in several of NOV = Novikoff hepatoma.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. 1~58 Cancer Research Vol. ~20, September 1960 cells (15), as well as a distinct change in mor- had the capacity to degrade thymine to CO~, phologic character. Whether or not this change it became of importance to determine whether in the enzymatic and morphologic character of or not this neoplasm had the capacity to convert the cells reflects a heritable change in the genetic thymidine-~-C ~4 to C~402. In view of the thymine structure of the primary neoplasm or the over- degradation by the tumor, such an experiment growth of one cell type will be discussed in a would indicate the presence or absence of thy- subsequent paper (15). midine phosphorylase, which had previously been Thymine degradation.--In contrast to the de- shown to be present in low amounts in the Dun- oxycytidylate deaminase activities of normal and ning hepatoma and absent or of very low activity mahgnant tissues demonstrated in Chart 1, in the Novikoff hepatoma (8). In Table 1 are the capacity of these tissues to degrade thymine- the results of assays for thymidine catabolism 3-C 14 to C140~ is extremely limited. As seen in in the liver and Morris hepatoma 5133. Of the Table 1, the liver and the Morris hepatoma 5138 four tumors tested only one showed any appreci- have about the same capacity to degrade thymine. able capacity to degrade thymidine to CO:. The tu- Of several other normal tissues studied--heart, mor which showed thymidine breakdown showed kidney, testis, brain, thymus, spleen, and skeletal little or no necrosis on gross examination. Thus, muscle--only the kidney had any capacity to before any definite statement can be made with

TABLE 1 PYRIMIDINE CATABOLISM AND GLUCOSE-6-PttOSPItATE DEHYDROGENASE IN MORRIS HEPATt)5.IA NO. 51`23 AND IN LIVER FROM CONTROL AND TUMOR-BEARING ANIMALS All data given as gmoles per gram protein per hour

Experiment Normal l~idney Normal liver Morris hepatoma 51~3 Host liver

Thymine Catabolism, Exp. 1 `20, 28, 18, ~0 Thymine Catabolism, Exp. 2* 2.4, 2.0, ~.7 18, 21, `20 Uracil Catabolism, Exp. `2 1.9, 1.6, 1.9 18, 19, `20 Thymine Catabolism, Exp. 3* 11, 11, `21, 9 17, `2`2, 20 Uracil Catabolism, Exp. 8 0, 7, 19, 0 16, 19, 15 Thymidine Catabolism, Exp. "~ 0, 0, 8, 0 9, 21, 15 G-6-P Dehydrogenase, Exp. 3 124, 107, 120, 105 "213, 191,113, `267 73, 100, 63

* Within each experiment, each value represents an individual rat, and the values are given in the same order for each type of enzyme assay to permit comparison of relative values for the different enzymes for each individual animal. The other hepatomas mentioned in Chart 1 were also studied in terms of thymine catabolism, but the rate of breakdown was ~nsignificant unde. the same conditions used to study the Morris hepatoma 5123. convert thymine (carbon 3) to CO2. Our data regard to the catabolism of thymidine by Morris on liver support the work of Fink et al. (6), hepatoma 51~3, the effect of the physiologic state who showed that thymine and, to a much greater of the tissue and the fate of the deoxyribose moiety extent, dihydrothymine gave rise to jS-amino- must be studied further. isobutyric acid when incubated with slices of rat Table 1 also shows the results of testing the liver in 95 per cent 02-5 per cent CO2. Recently, conversion of uracil-3-C 14 to C1402 in liver, kidney, Canellakis (3) demonstrated that extracts of ace- and Morris hepatoma 5133. Again, two of the tone powders of rat liver fortified with reduced tumors showed no capacity to degrade uracil, pyridine nucleotides would liberate C~402 when whereas at least one, which could also degrade incubated with uracil-~-C ~4 or thymine-2-C 14. In thymidine, showed appreciable uracil-catabolizing view of the work reported here it appears that the ability. However, all these same neoplasms had liver and kidney (=1/10 as much as liver) are the capacity to convert thymine-3-C 14 to C~402. the only organs in the normal rat capable of Whether or not there are two distinct enzymes, thymine degradation. Thus, the Morris hepatoma one specific for thymine, the other for uracil, 51~3 is more similar to a liver cell with respect as these data would suggest, is at present under to this enzymatic property than are any of the investigation in this laboratory. other tumors tested, including the Dunning hepatoma which had previously been shown to resemble DISCUSSION liver in many respects (16). The present work was concerned chiefly with The conversion of thymidine-2-C TM and uracil- a preliminary survey to ascertain whether there 2-C 14 to C1402.--Since the Morris hepatoma 513.3 might be a transplantable strain of rat hepatoma

cells that could be considered more closely related as studied here involves three steps. The first to parenchymal liver cells than the Dunning L-C18 step is probably rate-limiting, with no accumula- hepatoma, which had previously been shown to tion of intermediates prior to the loss of CO~. be closer to parenchymal liver cells than to the (7). The reactions are: Novikoff hepatoma (13, 16, 17). It appears that 1. Thymine -}- TPNH ~ dihydrothymine q- the survey was fortunate enough to include the TPN. Morris hepatoma No. 51s which on the basis 5~. Dihydrothymine ~ fl-ureidoisobutyrie acid. of our present data is unique among rat hepatomas 3. fl-ureidoisobutyric acid --+ fl-aminoisobutyric in the degree to which it approaches the enzyme acid q- CO2 if- NHa. pattern of normal liver (Table r Further supporting data are provided elsewhere (15). ENZYMATIC MARKERS In the present study the deamination of deoxycytidylate and the catabolism of thymine have D+T + (EMBRYONIC LIVER) been emphasized for the reason that, as shown BILE DUCT D+T § D-T + ADULT LIVER in Table ~, on the basis of these two enzymes EPITHELIUM alone a fairly sharp line can be drawn between - T 1- X normal rat liver and most other tissues, including NOVIKOFF D+T -' D-T + MORRIS IIEPATOMA thymus, Novikoff hepatoma, and Dunning hepa- HEPATOMA toma, but not between normal liver and Morris

51~13 hepatoma. D-T- DUNNING HEPATOMA The deamination of dCMP is a reaction that is presumably brought about by a single enzyme, CHART ~.--Possible origins of hepatoma strains in terms of deletion theory and assays for dCMP deaminase (=D) and and no coenzymes are known for the reaction thymine reductase (= T). Deletion theory implies loss of at present. Our technics measure both the disap- enzyme-forming systems for enzymes symbolized by X that pearance of the substrate and the accumulation differentiates the Morris hepatoma 51~t3 from parenchymal of the product, which is dUMP. This compound liver cells. This chart summarizes certain relationships between rat hepatomas and possible cells from which they might is an important intermediate, since the dUMP originate but does not imply a direct genetic relationship lmr is converted to TTP via TMP and TDP and finally does it imply single-step mutations. TABLE ~. When thymine-~-C TM is the sebstrate, C'402 ENZYMATIC MARKERS FOR RAT LIVER is formed, and can be determined directly or by AND I-IEPATOMA STRAINS difference. No information is provided by this assay as to further catabolic steps. The enzyme dCMP Thymine Tissue catalyzing the first step can be referred to as deaminase reductase thymine reductase in preference to the term di- Embryonic rat liver + hydrothymine dehydrogenase, since the reverse Normal adult rat liver + reaction is probably of little significance. Thymine Adult rat liver with proliferating bile duct epithelium* + + catabolism is considered to be a useful marker Morris hepatoma 51~8 + for liver cells, and the ability of the 51~3 hepatoma Novikoff hepatoma + to degrade this compound suggests that this tumor Dunning hepatoma L-C18 Thymus + is derived from a liver cell. However, we do not Most other normal tissues know whether bile duct cells have the enzyme or not, and it is only because of the absence * Animals fed 8'-methyl-4-dimethylaminoazobenzene (18). of dCMP deaminase that the further inference Data on thymine reductase to be published elsewhere. All (-) values indicate presence of enzyme is "question- is drawn that the 51e8 is derived from a parenchy- able"; (+) values do not imply quantities are identical. mal liver cell. The findings can be summarized in terms of incorporated into DNA. Its absence from the the deletion theory (18, 19) by a diagram as in Morris 51~3 and Dunning hepatomas implies that Chart e, in which the various hepatomas are labeled these cells obtain dUMP by other means, or that according to the presence or absence of certain their requirements for the enzyme are so small enzymes in a manner analogous to the designation that we are misled as to the meaning of the results of microbial mutants. Just as a T- bacterial of the assays. However, the assays probably do mutant is understood to lack an enzyme or en- yield values that are proportional to the amount zymes necessary for the synthesis of a thymine of enzyme in the fractions tested. derivative, we propose to label a tumor strain In the case of thymine catabolism, the reaction according to whether it lacks a given enzyme

that can be quantitated in an enzyme assay, and views are not excluded by the data, and we have to begin by designating dCMP deaminase as D, encouraged several other investigators to carry and thymine reductase as T, as shown in Chart out as many and as diverse a series of biochemical e. Obviously, the interpretations are tentative, tests on the 51~3 hepatoma as possible. but the chart helps to explain the nature of the The most positive result in the present report problem and the available data. It should be is the documentation of extreme diversity among understood that since a (q-) designation is assigned various representatives of a single type of experi- for any amount of enzyme above the "question- mental cancer, namely, the rat hepatoma, and able" level on the basis of current assay procedures the finding that out of a series of hepatomas only the presence of a detectable amount of enzyme one possessed a pattern resembling liver. With implies the presence of the corresponding EFS. the two enzyme parameters employed here, three However, the presence of the EFS does not control of the four possible patterns were found, namely the absolute amount of enzyme that is present D-T- (Dunning), D+T - (Novikoff), and D-T + owing to feedback considerations (19). We have (Morris 51~3). This result demands that we exer- to assign D- to any tissue in which the amount cise extreme caution in the interpretation of the of enzyme is below the limits of available assay Greenstein Hypothesis of Convergence (of tumor procedures, even though the corresponding EFS enzyme patterns to a single type). Convergence might be present. A given EFS should be assumed may be a phenomenon brought about by the use negative until experimental conditions necessary of animal tumors that have been transplanted to induce the enzyme in readily demonstrable in noninbred strains for many generations. It amounts can be found. is probably no coincidence that the two hepatomas In examining Chart ~ or the data summarized that we find to most closely resemble normal liver in Table ~ it is at once apparent that the loss cells in our limited survey have been tumors of thymine reductase is not a necessary deletion that originated and were transplanted in highly for the conversion of a normal liver cell to a inbred strains of rats: The Dunning is carried hepatoma cell, since No. 51~8 contains the enzyme. in the Fischer strain, and the Morris 51~3 is However, taken as a whole, the findings are in carried in the Buffalo strain. line with the earlier suggestion (19) that such A final word needs to be said about the use of a change may be "cancer promoting" in the sense the enzyme assays to label hepatoma strains as of increased rate of cell division, since this hepa- D+T - etc. It is realized that we are dealing toma grows extremely slowly (le). Whereas the with tissues not composed or derived from single difference between a parenchymal liver cell and cells, and attempts are being made to obtain the 51e3 hepatoma does not emerge from the hepatomas based on clones derived from cultures study of dCMP deaminase and thymine reductase, of the hepatoma cells in vitro. Studies by Prof. marked differences have been seen in the case H. E. Swim 4 in the McArdle Memorial Laboratory of several amino acid-catabolizing enzymes (15). have proved the feasibility of this approach with In the present work several specimens were found the Dunning and the Novikoff hepatomas, and to lack uracil reductase, but since other specimens work with the Morris 51~3 has begun. If the contained the enzyme the data indicate that loss various hepatoma strains can be cloned and grown of the enzyme is not required for carcinogenesis. in reasonable quantity, tests for the presence or Preliminary tests indicate the presence of low absence of a specific EFS can be made by conven- levels of thymidine kinase and thymidylate kinase, tional microbiological technics. enzymes for which the corresponding enzyme- forming systems are thus present in hepatoma REFERENCES 51~t8. The inducibility of these enzymes (1, 8) 1. BOLLUM, F.; ANDEREGG, J. W.; ~':[cELYA, A. B.; and suggests that the EFS is suppressed in adult POTTER, V. R. Nucleic Acid Metabolism in Regenerating Rat Liver VII. Effect of X-radiation on Enzymes of DNA rat liver cells until some event such as partial Synthesis. Cancer Research, 20:138-43, 1960. hepateetomy alters the balance of inducers and 2. CANELLAKIS, E. S. Pyrimidine Metabolism I. Enzymatic suppressors, and the slow rate of growth shown Pathways of Uracil and Thymine Degradation. J. Biol. by the 51~3 suggests that the Tdr and TMP Chem., 221:315-~2, 1958. kinases may still be subject to considerable sup- 3. DEVERDIER, C. H., and POTTER, V. R. Alternative Path- ways of Thymine and Uracil Metabolism in the Liver and pression at the EFS level. Hepatoma. J. Nat. Cancer Inst., 24:13-~29, 1960. Although our emphasis in the present report 4. DUNNING, W. F.; CURTIS, M. R.; and MEUN, M. E. The has been oriented toward the pyrimidine pathways Effect of Added Dietary Tryp0tphane on the Occurrence leading to DNA and the discussion has been in 4 On leave from the Department of Microbiology, Western terms of deletion, we must emphasize that other Reserve University Medical School, Cleveland, Ohio.

of ~-Acetylaminofluorine-induced Liver and Bladder Can- 13. NOVIKOFF, A. B. A Transplantable Liver Tumor In- cer in Rats. Cancer Research, 10:454-59, 1950. duced by l-Dimethylaminoazobenzene. Cancer Research, 5. FARBER, E. Carcinoma of the Liver in Rats fed Ethionine. 17:1010-~7, 1957. A.M.A. Arch. Path., 69.:445-53, 1956. 14. ----. Enzyme Localization in Tumor Cells. In: T. C. 6. FINK, R. 3;[.; FINK, K.; and HENDERSON, R. B./~-Amino Hsty (ed.), Cell Physiology of Neoplasia. Austin: Univer- Acid Formation by Tissue Slices Incubated with Pyrim- sity of Texas Press, 1960. idines. J. Biol. Chem., 201:349-55, 1953. 15. PITOT, H. C. The Comparative Enzymology and Cell 7. FRITZSON, P. The Catabolism of C~4-1abeled Uracil, Di- Origin of Rat Hepatomas. II. Glutamate Dehydrogenase, hydrouracil, and ~-Ureidoproprionic Acid in Rat Liver Choline Oxidase, and Glucose-6-phosphatase. Cancer Re- Slices. J. Biol. Chem., 226:~e3-~8, 1957. search, 20:1~6~, 1960. 8. HIATT, H. H., and BOJARSKI, T. B. Stimulation of Thy- 1~. PITOT, H. C.; FOrIN, C. H.; CLARK, W. H., JR.; and midylate Kinase Activity in Rat Tissues by Thymidine I~ARBER, E. A Comparative Biochemical and Morphologic Administration. Biochem. & Biophys. Res. Comm., 2: Study of Some Transplanted and Primary Rat IIepatomas. 35-40, 1960. Proc. Am. Assoc. Cancer Research, 3:5~, 1959. 9. KoRNnERG, A., and HORECKER, B. L. Glucose-6-phosphate 17. PITOT, H. C., and POTTER, V. n. An Enzymic Study on Dehydrogenase. In: S. COLOWICK and N. H. KAPLAN. the Cellular Origin of the Dunning and the Novikoff Methods in Enzymology, 1:3e3. New York: Academic Hepatomas in the Rat. Biochim. et Biophys. Acta, 40: Press, 1955. 537-39, 1960. 10. MALEY, G. F., and MALEY, F. Nucleotide Conversions 18. POTTER, V. R. The Present Status of the Deletion Hy- in Embryonic and Neoplastic Tissues. I. The Conversion pothesis. U. Mich. M. Bull., 23:401-1% 1957. of Deoxycytidylic Acid to Deoxyuridylic Acid and Thy- 19. ------. The Biochemical Approach to the Cancer Problem. midylic Acid J. Biol. Chem., 234:~975-80, 1960. Fed. Proc., 17 : 691-97, 1958. 11. McCoY, T. A., and MAXWELL, M. Some Nutritional As- ~0. POTTER, V. R.; PITOT, H. C.; MCELYA, A. B.; and MORSE, pects of a 3'-Methyl-4-dimethylaminoazobenzene-Induced A. B. Alternative Pathways for Biosynthesis of Thymidylic Hepatoma in Vitro. J. Nat. Cancer Inst., 23:385-93, 1959. Acid. Fed. Proc., 19:31~, 1960. 1~2. MORRIS, H. P. ; SIDRANSKY,H.; WAGNER, B. P. ; and DYER, ~1. POTTER, V. R.; PITOT, H. C.; and ONO, T. Pathways of H. M. Some Characteristics of Transplantable Rat Hepa- Thymidylate Synthesis and Degradation in Normal, Re- toma No. 51~23 Induced by Ingestion of N-(~-fluorenyl) generating, and Preneoplastic Rat Livers and in Hepa- phthalamic acid. Cancer Research, 20:1~5~-54, 1960. tomas. Proc. Am. Assoc. Cancer Research, 3:14~, 1960.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. The Comparative Enzymology and Cell Origin of Rat Hepatomas I. Deoxycytidylate Deaminase and Thymine Degradation

Van R. Potter, Henry C. Pitot, Tetsuo Ono, et al.

Cancer Res 1960;20:1255-1261.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/20/8/1255

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/20/8/1255. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.