The Comparative Enzymology and Cell Origin of Rat Hepatomas III

The Comparative Enzymology and Cell Origin of Rat Hepatomas III. Some Enzymes of Amino Acid Metabolism*

HENRYC. PITOT,CARLPERAINO,!RICHARDH. BOTTOMLEY,ÃŽANDHAROLDP. MORRIS

(McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin; and the National Cancer Institute, National Institutes of Health, Bethesda, Maryland)

SUMMARY The activities of several enzymes of amino acid metabolism including tryptophan pyrrolase, threonine and serine dehydrase, proline oxidase, pyrroline-5-carboxylate reductase, tyrosine a-ketoglutarate transaminase, and histidase were measured in host livers and a number of minimal-deviation hepatomas. In contrast to previous reports on more anaplastic hepatic neoplasms, the tumors reported here possessed substantial amounts of many of these enzymes. However, all the tumors studied appeared to be unique in that no two possessed an identical enzyme pattern. The significance of a sec ondary site active in amino acid metabolism in the host-tumor relationship of rats bear ing minimal-deviation hepatomas is discussed.

The discovery of a morphologically (10) and interest to see the extent of these aberrations in enzymatically (12, 16) highly differentiated, sta related hepatomas and in other enzyme systems ble, transplantable hepatocellular carcinoma in the exhibiting control mechanisms. The subject of this Buffalo strain of rat has required a re-evaluation of paper is the assay of some of these enzymes in a some of our previous concepts of hepatic carci no- series of hepatomas closely related to the 5123 genesis and cancer. In studies thus far (14) on the or so-called "minimal-deviation hepatomas" (15). Morris Hepatoma 5123, few if any enzymes have The study of some of the factors which control the been "deleted" in the tumor when it is compared amount of these enzymes in vivowill be the subject with normal or regenerating rat liver. In addition, of future papers. Aisenberg (3) has shown that this neoplasm pos sesses essentially no aerobic glycolysis; however, MATERIALS AND METHODS Boxer and Devlin have indicated that some elec Most of the neoplasms that were used in these tron transport mechanisms in the tumor are prob studies, including Hepatomas 5123, 7316, 7793, ably abnormal (4). 7794, 7795, 7800, and the Reuber H-35 (17), were In contrast to the marked similarities between inoculated into rats of the Buffalo strain at the the tumor and liver, we have reported (13, 14) National Cancer Institute and after 1-2 months definite aberrations in some mechanisms control shipped to the McArdle Laboratory where they ling the amount of certain enzymes in the 5123 were used in the experiments reported here. Hepa hepatoma. Since most of these enzymes were in tomas 5123, 7777, 7793, 7794, 7795, and 7800 were volved in amino acid metabolism, it became of induced by N-(2-fluorenyl)phthalamic acid (10). * This work was supported in part by a grant (No. C-646) Hepatoma 7316 was induced by feeding 2,4,6- from the National Cancer Institute, National Institutes of trimethylaniline. Transplantation of all these neo Health, U.S. Public Health Service. plasms except the 7777 was initiated at the Na t Postdoctoral fellow (#13,911), National Cancer Institute, tional Cancer Institute, where they were induced. United States Public Health Service. The animal bearing the primary 7777 tumor was Ã•Postdoctoral fellow (#13,610), National Cancer Institute, sent to the McArdle Laboratory where the tumor United States Public Health Service. line was started in Buffalo strain rats obtained Received for publication July 16, 196-2. from the National Cancer Institute. The hepatoma 135

11-35 was obtained from Dr. Reuber by one of us scribed (11) on a fraction of the 83 which precipi (H.P.M.) at the ninth generation and maintained tated between 0.4 and 0.6 saturation with ammoni by transplantation in ACI strain brown rats. This um sulfate. Most of the other assay procedures tumor was originally induced by N-2-fluorenyldi- used were modifications of those previously em acetamide in the inbred A X C strain rat by a ployed and are given in detail here. technic slightly different from that used for the Tryptophan pyrrolase.â€”Theprocedure used was development of the other minimal-deviation hepa- a modification of that described by de Castro et al. tomas.1 In addition, two hepatomas, the 5123 H-2 (6) using the coupling procedure of Brown and and the II-4, were derived from the parent tumors, Price (5). Aliquots (0.3 and 0.6 ml. of a 20 per cent the Morris Hepatoma 5123 and the Reuber Hepa- homogenate) were incubated in 10-ml. Erlenmeyer toma H-35, respectively, by Mr. Paul Morse in flasks containing 0.3 ml. of 0.03 M L-tryptophan, our laboratory through the explantation of the 1.0 ml. trishydroxyamino methane (Tris) buffer, parent tissue to tissue culture. After growth of pH 7.5, 0.02 ml. of .025 per cent hematin, and these tissues in vitro in T-60 and T-30 culture water to make 4.0 ml. At 10 minutes and again at flasks for periods up to 6 months, the cultured cells 70 minutes after the start of the incubation, 1.0- (ca. K^-IO7 cells) were inoculated into suitable ml. aliquots were removed and added to small hosts, and the neoplasms obtained have been tubes containing 1.0 ml. of 8 per cent trichloro- maintained by transplantation in vivo since that acetic acid (TCA). Previous work with host liver time.2 and Hepatoma 5123 had demonstrated a linear The tumor-bearing animals were maintained in reaction rate during this time period. After com suspended wire-bottomed cages in our laboratory pletion of the incubation, the precipitated protein with laboratory chow and water given ad libitum. was centrifuged down, and an aliquot of the clear In some cases animals with large tumors were giv supernatant (0.1-1.5 ml.) was added to test en a 1 per cent solution of NaCl to drink instead tubes and the volume made to 2.0 ml. with 4 per of water. For the examination of tissues and their cent TCA when necessary. Distilled water (1.0 ml.) preparation for enzyme assay the tumor-bearing was added, after which procedure 0.2 ml. of 0.25 rats were killed by cervical dislocation, and the per cent NaNC>2(freshly prepared) was introduced tumor and liver were immediately removed and and the tube mixed well. Exactly 2 minutes later immersed in ice-cold 0.154 MKC1. Fibro-fatty tis 0.2 ml. of 10 per cent NH4 SO3NH2 was added, the sue and necrotic tumor were carefully dissected tubes were shaken and left to stand another 2 away from the grossly viable neoplastic tissue. minutes. At the end of this time 0.2 ml. of 0.25 per Portions of this tissue were taken for histologie cent N-1-Naphthylethylene diamine dihydrochlo- examination, fixed in Bouin's fluid, embedded, sec ride (prepared fresh every 2 weeks and stored in tioned, stained, and examined microscopically. the dark) was added, and the contents of the tubes The gross viability of the tissue was thus verified mixed well. After 30 minutes the optical density by histologie examination. The tissue to be used at 550 m/Â¿wasdetermined with a Beckman model for enzyme assay was homogenized in 4 volumes DU spectrophotometer. The addition of the last of 0.154 M KC1 with the use of an Ultraturrax three reagents was greatly facilitated by automatic homogenizer (Janke und Kunkel KG., Staufen, pipettes. A standard solution of kynurenine was Germany). Assays for tryptophan pyrrolase, thre- run with each set of determinations. Under the onine and serine dehydrase, and proline oxidase conditions reported here 0.02 /miÃ³lesofkynurenine were performed on the whole homogenate, whereas produces an optical density of about 0.20. The tyrosine a-ketoglutarate transaminase and histi- anthranilic acid-dye diazotate has essentially the dase activities were measured in the supernatant same extinction as that of kynurenine under these (S3) obtained from the whole homogenate by high conditions. Tryptophan and many of its other speed centrifugation at 100,000 X g for 1 hour in metabolites with the exception of indole give es a Spinco Model L. Pyrroline-5-carboxylate reduc- sentially no color under these conditions. Beer's tase was assayed by a procedure previously de- law is obeyed up to 0.1 /Â¿moleofkynurenine. 1H. P. Morris and B. P. Wagner, unpublished results. Tkreonine and serine dehydrase.â€”Threonine and These data were presented in part at the VIII International serine dehydrase activities are measured by a mod Cancer Congress in Moscow, July, 1962. Further details will ification of the procedure described by Sayre et al. be published later. (18). The reaction mixture contained the follow 2A more detailed account of the morphology, cultural ing: 0.1 or 0.2 ml. of the homogenate in 0.154 M conditions, and enzymology of the 5123-H-a and the H-4 cell KC1; 0.1 ml. of 0.5 M L-threonine or L-serine; 1.0 lines and hepatomas derived therefrom will be reported at a ml. of 0.1 M KH2PO4 buffer, pH 7.4; 0.1 ml. of later date. The authors are grateful to Mr. Morse for per 1 X 10~4Mpyridoxal phosphate: and distilled HjO mission to discuss part of this work here.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. PiTOT et al.â€”Enzymes of Amino Acid Metabolism 137 to a total volume of 2.0 ml. Pyridoxal phosphate maximal rate of change in O.D. noted. In this and was found to stimulate the reaction in both normal all enzyme assays, the rate of the reaction was liver and hepatoma by approximately 100 per cent. proportional to the volume of homogenate, 83, or The reaction was carried out in 25-ml. Erlenmeyer protein fraction. flasks at 37Â°C.for 30 minutes with shaking. En A'-Pyrroline-S-carboxylate was prepared and pu zyme activity was stopped and protein precipi rified by the method of Strecker (19). All other tated by the addition of 2 ml. of 10 per cent TCA. amino acids used as substrates and pyridoxal-5- Suitable aliquots were then taken and assayed for phosphate were obtained from Calbiochem. Am keto acid production by the method of Friedemann monium sulfamate, mercaptoethanol, and N-l- and Haugen (7). Results are expressed as /Â¿moles Naphthylethylene diamine HC1 were products of of alphaketobutyrate (or pyruvate) produced/gm. the Matheson, Coleman, and Bell Company. Di- liver/hour. phosphopyridine nucleotide (DPN), cytochrome TABLE1 TRYPTOPHANPYRROLASEACTIVITIESOFSOMEMINIMAL-DEVIATIONHEPATOMAS*

HepatomaReuber liver2.4

H-35Morris &9cf (3.9,3.3,2.9,2.8, (0.6,0.6,0.3,0.3, 0.9)t.i(2.3,1.6, 1 4, 0.15)0.8(0.8,0.3, 0.2,0.2,

7800Morris &9cf 23, 2.2, 2.0, 0.4, 0.2, 0.2, 3.3, 2.2,1.1)2.7 0.2, 0.2,0.1)1.4

7793Morris &9cT9o" (3.5, 3 5,3.1,0.7)2.5 (2.1, 1.4,1.1,0.9)0.2 7794Morris (3.8, 2.5,1.2)2.6(3. (0.2,0.2, 0.1)0.3 7795Morris Â«,2.4,2.3)2.1 (0.4,0.8,0.3)0 7316Normal &9cf 15 (3.0,2.2, 1.9, 1.8, (0.2,0.2,0.15, 0.1, 1.5, 1.5)2.5 0.10,0.10)

liverGeneration11-133-52-42-4a2-3SexcT& 9Host (3.0, 2.8, 2.5, 2.3, 2.0)Neoplasm0.4

* All units are in AmÃ³leskynurenine/hr/gm tissue. No difference was noted with the gen eration number of the tumor or the sex of the host. Each value in parentheses is from a sin gle animal; the average is given above them.

Proline oxidase.â€”Theprocedure for the assay of C, and adenosine monophosphate (AMP) for use this enzyme complex has been previously reported in the proline oxidase assay were products of the (11). Sigma Company. Hemin was a product of L. Light Tyrosine a-ketoglutarate transaminase.â€”This en & Co., Ltd., Colnbrook, England; 0.025 per cent zyme was assayed by a procedure described pre hematin solutions were prepared fresh weekly by viously (13). dissolving 25 mg. hemin in 100 cc. distilled HaO Histidase.â€”This enzyme was assayed by a mod made slightly alkaline with NaOH. The solution ification of the procedure of Tabor and Mehler was stored at â€”15Â°C. (20) ; 0.02 or 0.04 ml. of the S3was added to quartz cuvettes of 1.0 cm. light path containing 5 /Â¿moles RESULTS of mercaptoethanol, 10 /Â¿molesof L-histidine, 30 Tryptophan pyrrolase.â€”-Thetryptophan pyrro- /Â¿molesof L-histidine, and 30 /Â¿molesof sodium lase activities of six minimal-deviation hepatomas pyrophosphate, pH 8.6, in a final volume of 3.0 ml. are seen in Table 1. As seen from the range of The blank cuvette contained no enzyme. Read individual values, the enzyme level in the host ings were taken every 2-3 minutes at 277 m/Â¿at liver varies from 1.0 to 4.0 units. Studies in this 24Â°C. This was continued for 30 min., and the laboratory have shown that the tryptophan pyr-

rolase activity of livers of tumor-free rats of the tivity of the tumor is within the normal range Buffalo strain is about 2.5. The lower values in found for host livers. In fact, in one animal the some host livers may reflect the pathologic changes level in the neoplasm was higher than that of the seen in some, especially fatty metamorphosis. On host liver, possibly because of a marked fatty the other hand, the great majority of tumors stud metamorphosis of the latter. ied possessed levels of the enzyme between 0.1 and In several cases of low tumor levels of trypto 0.3 units. Such a level is readily measurable with phan pyrrolase, the effect of the addition of higher the assay procedure described in this paper. The or lower levels of hematin than those described in tryptophan pyrrolase activity of the 5123, Dun the assay, graded levels of ascorbic acid (10~2- ning L-C18, and some primary hepatomas (12) is 10~6M), and of mixing tumor and liver homog- in this same range. The exception to this rule is the enates was studied. In no case was any activation Morris Hepatoma 7793, in which the enzyme ac of the tumor enzyme obtained, although high con- TABLE2 DEHYDRASEACTIVITIESOFSOMEMINIMAL-DEVIATIONHEPATOMAS*

H EP ATOMAGENERATIONSEXHOST LIVERTcMORThreonine

dehydrase5123

H-2Reuber &90"rf1 (34, 32, 8, 5, 5,0)31 (1334, 1198, 424, 374, 370, 234)0 H-35Morris (18, 38, 36)4 (0, 0,0)32(48, 7316Morris &9c? (4, 6, 3, 4)20 48, 20,la)295 7793Morris &9

dehydraseMorris

7794Morris 47, 43,18)9 (3, 3, 0, 0)7 7795Morris (10, 9,9)5(5, (9, 7, 5)17 7800Normal &9

* All values are in Â¿tmolesaketo acid/hr/gm tissue. As in Table 1 no consistent effect of the sex of the host or the generation number of the tumor was noted. The zero values indicate that no enzyme activity can be detected by the measurements employed.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. PiTOT et al.â€”Enzymes of Amino Acid Metabolism 139 centrations of any one or all three activators were tions to this do occur, as seen in the table. Pre inhibitory. liminary studies by one of us (C.P.) indicate that Threonine and serine dehydrase.â€”InTable 2 are high threonine dehydrase in the tumor is asso listed the activities of threonine and serine dehy ciated with a significant lowering (co. 50 per cent) drase in a number of minimal-deviation hepa- of the plasma threonine of the tumor-bearing rat. tomas. In most cases, the enzyme levels in the host Thus, as has been previously suggested (14), thre liver are somewhat lower than those found in nor onine dehydrase levels of host liver may be sig mal liver of tumor-free rats. The general excep nificantly affected by the presence of this enzyme, tions to this appear to be in the Reuber H-35 and which normally occurs only in hepatic tissue, at a Morris 7794 and 7777 hepatomas, in which the distant siteâ€”i.e., the transplanted neoplasm. Fur threonine dehydrase is very low or negligible. A thermore, this effect may be mediated through the high level of threonine dehydrase in the neoplasm plasma level of threonine. â€”e.g., Morris 7793 and 5123/H-2 as well as the Table 2 also depicts the serine dehydrase activi Morris 5123 (14)â€”is usually accompanied by a ties in three of these hepatomas. Since this enzyme low level of this enzyme in the host liver. Excep activity closely parallels that of threonine dehy-

TABLE 3 PHOLINEOXIDASEANDPYHHOLINE-S-CARBOXYLATEREDUCTASE ACTIVITIESOFSOMEMINIMAL-DEVIATIONHEPATOMAS

HEP A.TO MAGENERATIONSEXHOST LIVERTUMOR

Proline oxidase*

Morris5123Morris &9e? 137,179)177(152, 10,12)14(16,

7800Morris &9

7798Morris &9

7316Reuber &9d" (174, 182, 175,190)174 38)11(10,57, 64, H-35Normal &9d1 (186, 165,170)206 15, 9)

liver24-263-52-42-311-12cf & 9165(178, (113, 227, 278)9(5,

PyrroIine-5-carboxylate reducÃaset

Morris5123Morris &9t?9a"a"cf 195)140(120,193, 85, 133, (450, 670,580)158(150,

7800Morris 160)138 165)124

7793Morris (140, 135)163 (115, 132)57

7316Reuber (157,169)150(146, (52, 61)168 H-35Normal 153)181 (160, 175)

liver24-26322<7 & 9150(145, (145, 182, 216)567

* All values are expressed in AmÃ³lesO2/hr/gm tissue (see Table 2). t All values are expressed in jumÃ³lesDPNH2/hr/gm tissue (see Table 2).

drase, only the assays depicted here are reported. 7316 about one-third of the activity of the host Proline oxidase and pyrroline-5-carboxylafe re liver. Studies in this laboratory indicate that tu ducÃ•ase.â€”Theactivities of the enzymes immediate mors having high reducÃase and negligible oxidase ly involved with proline degradation and syn activities accumulate considerable free intracellu- thesis are seen in Table 3. Both the synthetic (re- lar proline, up to 30 times the normal level in some ductase) and degradative (oxidase) enzymes in the instances (11). host liver are essentially unaffected by the presence Tyrosine a-ketoglutarate transaminase.â€”The ma of the tumor as seen in comparison with the en jority of the tumors assayed for this enzyme pos zyme levels in normal liver. However, the oxidase sessed high levels of activity (Table 4), ranging up activities of the 5123, 7800, and H-35 hepatomas to 10 times that found in the host liver. Such a are very low and can only be measured with cer pattern was also reported for this enzyme in the tainty by first isolating mitochondria from the neo 5123 (13). However, as with all the other enzymes plasm and then assaying a concentrated suspen studied, exceptions were found in Hepatomas 7795 sion of these organdÃes. On the other hand, the and 7316 wherein the tyrosine transaminase ac-

TABLE 4 TYROSINEÂ«I-KETOGLUTARATETRANSAMIXASEACTIVITIES OFSOMEMINIMAL-DEVIATIONHEPATOMAS*

HepatomaReuber liver30 H-35Morris &9rf (50, 31, 26, 26,15)39 (585, 374, 270, 263, 176)212 7800Morris &9

* All values are in Â¿irnolesp-hydroxyphenylpyruvate/hr/grn tissue. oxidase levels in Hepatomas 7793 and 7316 are tivities of the neoplasms were within the ranges more readily measured, although still considerably seen in the host liver. It is of interest that these lower than that of the comparable host liver. The two tumors differed markedly in their morphology low levels of the mitochondrial enzyme in Hepa from the neoplasms found to have high trans toma 5123 probably indicate a specific change in aminase activities. This will be discussed more the individual mitochondrion rather than a de fully in a future publication. crease in their number, since the mitochondrial Histidase.â€”Five minimal-deviation hepatomas enzymes, choline oxidase (12) and succinoxidase,3 were assayed for histidase activity. Although the measured on the same homogenate is normal. numbers employed are too small to draw any ma In contrast to the lowered proline oxidase levels jor conclusion, it would appear that the level of of all the neoplasms, the pyrroline-5-carboxylate this enzyme in the neoplasms studied was consid reductase activities of the tumors varied consider erably lower than that found in host livers. It is ably. The level in the Morris 5123 was three- to of interest that other workers have noted an effect fourfold higher in the tumor than the liver. How of sex on the level of the enzyme in liver (2). In ever, the 7800, 7793, and H-35 hepatomas possessed our small series, the histidase activity of livers and essentially normal levels of the enzyme and the tumors has shown no consistent variation between 3H. C. Pilot, unpublished data. males and females. The low levels in the livers

of some animals may reflect certain pathologic stimuli. The combination of these factors appears changes such as fatty metamorphosis. to have profound effects on the host as evidenced by emaciation and fatty metamorphosis of the liv DISCUSSION er in some animals while the tumor is still com In 1958 Auerbach and Waisman (2) reported paratively small in size. Animals maintained on the absence of many enzymes of amino acid me high protein diets appear to support tumor growth tabolism in the Novikoff hepatoma. Later studies to a greater degree than those fed laboratory ra showed, however, that the Dunning L-C18 hepa tion. As suggested before (14), and in part possibly toma possessed activities of at least two enzymes as a consequence of the unresponsiveness of the involved in amino acid metabolism which were re tumor metabolism, the latter may compete with portedly absent in the Novikoff (12). It is apparent the liver for host nitrogen. Such a suggestion is now that some hepatomas may possess a number if not incompatible with the studies of Mider (9), not most of the spÃ©cifieenzymesof amino acid syn LePage etal. (8), and others that neoplasms in gener thesis and degradation which are normally found al compete strongly for host nitrogen. In the case of almost exclusively in the liver. Quantitatively, the the minimal-deviation hepatomas, the competition levels of a number of these enzymes in minimal- appears not to result from the demand of a rapidly TABLE5 HISTIDASEACTIVITIESOFSOMEMINIMAL-DEVIATIONHEPATOMAS*

HepatomaMorris liver(28.(12.(8.8(25.(21.17.9 51285123-H-2Reuber &9c? 1, 16.2, 16.0,16.4)9.1 9,4.4,3.2, 1.8,1.0)1.3 &9c?d"ef 4,8.8,6.0)7.6 6, 1.0)2.5 H-35Morris ,6.4)17.9 8,2.8,1.8)1.54, 7316Morris 1, 14.8,13.7)18.9 1.6,0.4)3.7 7777 1, 15.6) 0, 1.3) NormalliverNormal cf9Host 24.076.8Neoplasm(5(1(2(2(63.3 liverGeneration27-2831Sexrf1

' All values are in /imolesurocanate/hr/gm tissue (seeTable 2). deviation hepatomas may differ markedly from growing cell population, but rather may be a con the amount of the corresponding enzymes in host sequence of the abnormal levels and stability of and in normal liver. In some cases this variation the enzyme population of the tumor cells. The appears to be the result of some abnormal control lowered plasma threonine in rats bearing tumors mechanism within the neoplastic cell itself (14). with highly active threonine dehydrase might sub In other instances, the reason is not yet clearâ€”e.g., stantiate this hypothesis. proline oxidase, histidase, and pyrroline-5-carbox- Finally, it is of interest that the enzyme pattern ylate reductase. of each of the minimal-deviation hepatomas stud The control of amino acid metabolism, both syn ied is different in one respect or another. This is thesis and degradation, as well as the maintenance outlined in Table 6. Only the 5123 and 7793 have of plasma amino acid levels are primarily hepatic been found thus far to have exceedingly high thre functions. The interesting phenomenon of having onine dehydrase activities. It is of interest that two primary sites of amino acid metabolism is ex these two tumors were induced in females, whereas emplified by rats bearing transplanted minimal- the 7800 was induced in a male by the same agent. deviation hepatomas. However, it would appear The Reuber H-35 hepatoma was also induced in from previous studies (14) that, in contrast to a male but in a different strain and by slightly amino acid metabolism in the liver which responds different means (17). The 7800 and H-35 are most to dietary demands, the tumor or second site of alike in these enzymes, but the latter produces bile amino acid metabolism is unresponsive to such whereas the former does not. In studies to be re-

ported later, the quantitative divergence in en The Determination of Keto Acids in Blood and Urine. J. zyme pattern noted here in these closely related Biol. Chem., 147:415-42, 1943. neoplasms may be largely eliminated by adrenalec- 8. LEPAGE, G. A.; POTTER, V. R.; BUSCH,H.; HEIDEL BERGER,C.; and HURLBERT,R. B. Growth of Carcinoma tomy of the host. This would suggest that many Implants in Fed and Fasted Rats. Cancer Res., 12:153-57, of these apparent differences in enzyme levels 1952. among closely related tumors are primarily the 9. MIDER, G. B. Some Aspects of Nitrogen and Energy result of a complicated host-tumor interaction, Metabolism in Cancerous Subjects: A Review.Cancer Res., 11:821-29, 1951. the mechanism of which remains to be elucidated. 10. MORRIS, H. P.; SIDRANSKY,H.; WAGNER,B. P.; and DYER,H. M. Some Characteristics of Transplantable Rat TABLE 6 Hepatoma No. 5123 Induced by Suggestion of N-(2- ENZYMESOFAMINOACIDMETABOLISMIN fluorenyl) phthalamic acid. Cancer Res., 20:1252-54,1960. MINIMAL-DEVIATIONHEPATOMAS* 11. PERAINO,C., and PITOT, H. C. Proline Synthesis and Degradation in a Minimal Deviation Hepatoma. Biochim. Biophys. Acta, 62:585-87, 1962. 5-carbox- 12. PITOT, H. C. The Comparative Enzymology and Cell TumorH-357800779373165123Tryp-tophan trans- Origin of Rat Hepatomas. II. Glutamate Dehydrogenase, dehydrase02929532500Tyrosineoxidase111485519Pyrroline-ylate pyrrolase0.40.31.40.20.2Threonineaminase33421227577300Proline Choline Oxidase, and Glucose-6-phosphatase. Cancer Res., reducÃase1681581245756720:1262-68, 1960. 13. PITOT,H. C., and MORRIS,H. P. Metabolic Adaptations in Rat Hepatomas. II. Tryptophan Pyrrolase and Tyrosine a-Ketoglutarate Transaminase. Cancer Res., 21:1009-14, 1961. 14. PITOT,H. C.; POTTER,V.R.; and MORRIS,H. P. Metabolic Adaptations in Rat Hepatomas. I. The Effect of Dietary * The values are averages of the values in Tables 1-5 or are Protein on Some Inducible Enzymes in Liver and Hepa taken from previous work as in the case of the 5123 (13, 14). toma 5123. Cancer Res., 21:1001-8, 1961. 15. POTTER,V. R. Transplantable Animal Cancer, the Pri REFERENCES mary Standard. Cancer Res., 21:1331-33, 1961. 1. AISENBBBO,A.C., and MORRIS,H. P. Energy Pathways 16. POTTER,V. R.; PITOT,H. C.; ONO,T.; and MORRIS,H. P. of Hepatoma No. 5123. Nature, 191:1314-15, 1961. The Comparative Enzymology and Cell Origin of Rat 2. AUERBACH,V. H., and WAISMAN,H. A. Amino Acid Hepatomas. I. Deoxycytidylate Deaminase and Thymine Metabolism of Novokoff Hepatoma. Cancer Res., 18:543- Degradation. Cancer Res., 20:1255-61, 1960. 47, 1958. 17. REUBER,M. D. A Transplantable Bile-Secreting Hepato- 3. . Tryptophan Peroxidase-Oxidase, Histidase, and eellular Carcinoma in the Rat. J. Nati. Cancer Inst., 26: Transaminase Activity in the Liver of the Developing 891-97, 1961. Rat. J. Biol. Chem., 234:304-6, 1959. 18. SAYRE,F. W.; JENSEN,D.; and GREENBERG,D.M. Sub 4. BOXER,G. E., and DEVLIN,T. M. Pathways of Intra- strate Induction of Threonine Dehydrase in Vim and in cellular Hydrogen Transport. Science, 134:1495-1501, Perfused Rat Livers. J. Biol. Chem., 219:111-17, 1956. 1961. 19. STRECKER,H. J. The Interconversion of Glutamic Acid 5. BROWN,R. R., and PRICE,J. M. Quantitative Studies on and Proline. II. The Preparation and Properties of A1- Metabolites of Tryptophan in the Urine of the Dog, Cat, pyrroline-5-carboxylic acid. J. Biol. Chem., 236:2045-50, Rat, and Man. J. Biol. Chem., 219:985-97, 1956. 1960. 6. DECASTRO,F. T.; BROWN,R. R.; and PRICE,J. M. The 20. TABOR,H., and MEHLER,A. H. Histidase and Urocanase. Intermediary Metabolism of Tryptophan by Cat and Rat In: S. P. COLOWICKandN. O. KAPLAN (eds.), Methods in Tissue Preparations. J. Biol. Chem., 228:777-84, 1957. Enzymology, 2:228-33, New York: Academic Press, 7. FRIEDEMANN,T.E., and HAUGEN,G. E. Pyruvic Acid. II. 1955.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. The Comparative Enzymology and Cell Origin of Rat Hepatomas: III. Some Enzymes of Amino Acid Metabolism

Henry C. Pitot, Carl Peraino, Richard H. Bottomley, et al.

Cancer Res 1963;23:135-142.

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