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 re- ductase, 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 ÕPostdoctoral 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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. 136 Cancer Research Vol. 23, January 1963 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.—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/¿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 /miólesofkynurenine 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 /¿moleofkynurenine. 1H. P. Morris and B. P. Wagner, unpublished results. Tkreonine and serine dehydrase.—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.—Enzymes of Amino Acid Metabolism 137 to a total volume of 2.0 ml.