[CANCER RESEARCH 29, 40—54, January 1969] Enzymes of Thymidine Triphosphate Synthesis in Selected Morris H epatomas 12 ThomasW.Sneider,Van R. Potter,and HaroldP. Morris3 McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin 53706 SUMMARY phosphates, dATP4, dGTP, dCTP, and dTFP. Since thymine is the only base unique to DNA, a study of the control of path The activities of certain enzymes catalyzing reactions leading ways leading to the synthesis of dTTP might lead to an under to the synthesis of thymidine triphosphate were analyzed in standing of the control of DNA synthesis (52). Chart 1 illus vitro in minimal deviation type Morris hepatomas of normal trates these latter pathways as they may appear in various karyotype as well as in Novikoff and Reuber H-35 cells from combinations in mammalian tissues. tissue culture and in Ehrlich ascites cells. Most Morris hepa That the activities of the enzymes catalyzing these reactions tomas had normal or near-normal levels of deoxycytidine are rigidly controlled and, to some extent, correlatable with monophosphate deaminase, while Novikoff and Ehrlich cells growth and cell division, is illustrated by the following data. and rat thymus and bone marrow showed extreme elevations The activities of the ribonucleotide reductase(s), dCMP deam in activity of this enzyme. The Novikoff, H-35, and Ehrlich inase, dTMP synthetasc, and TdR and dTMP kinases, are high ascites cells, and all of the Morris hepatomas except 9633, had in embryonic and newborn rat liver but decrease with increas elevated deoxythymidine monophosphate (dTMP) synthetase ing age, such that normal adult rat liver has barely detectable activity. All of the hepatomas and tissue culture material cx levels of activities of these enzymes (T. Sneider, unpublished hibited elevations in deoxythymidine (TdR) kinase and pos data) (24, 27). If, however, an adult rat is subjected to partial sibly dTMP kinase activities. Hepatomas 5123C, 7800, and hepatectomy, the activities of all of the aforementioned en 9618A possessed the ability to degrade thymine at lesser rates zymes rise in an ordered manner prior to the onset of DNA than normal adult rat liver, while hepatoma 9633 and Novi synthesis and cell division (2, 13, 14, 21—24, 26, 27). if the koff and H-35 cells from tissue culture apparently lacked posthepatectomy increase in dCMP deaminase (14, 26) and thymine degradative ability. With the exception of dTMP TdR kinase (26) is prevented by actinomycin D, the subse synthetase, the activities of these enzymes in normal karyo quent onset of DNA synthesis and cell division is also delayed. type Morris hepatomas showed no quantitative relationship It is therefore evident that the control(s) of the activities of with growth rate. Among the hepatomas and enzymes re the enzymes of the dTTP pathway is at least temporally re ported, TdR kinase was the only enzyme with consistently lated to the mechanism(s) controlling DNA synthesis and cell elevated activity. These results and certain unpublished data division. Although the available evidence is only suggestive, it are discussed with respect to possible alternative routes to is possible that the factors that control dTTP pool size could deoxythymidine triphosphate synthesis and the chemotherapy partially govern the onset and rate of DNA synthesis or could of cancer. be clues to the primary triggering mechanism(s). INTRODUCTION A prerequisite for the synthesis of DNA and subsequent cell division is the synthesis of the four deoxyribonucleoside tri 4Abbreviations used in this paper are: dATP, dGTP, dCTP, and dTTP, the triphosphates of deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine; dUMP, dCMP, and dTMP, the monophosphates of deoxyuridine, deoxycytidine, and deoxythymidine; dTDP, the diphos. 1This work was supported in part by Grant No. P-448 from the phate of deoxythymidine; UdR, CdR, and TdR, deoxyuridine, deoxy American Cancer Society, by USPHS Grant No. TO1-CA-5002 from the cytidine, and deoxythymidine; DEAE-, diethylaminoethyl-; F3TdR, National Cancer Institute, and by USPHS Grant No. CA-10729. A pre 5-trifluoromethyl-2'-deoxyuridine; 5-FUdR, 5-fiuoro-2'.deoxyuridine; liminary report was given before the American Association for Cancer 3-PGA, 3-phosphoglyceric acid. The trivial names of, and the reactions Research in Chicago, Illinois, in April 1967 (50). catalyzed by, the enzymes mentioned in this paper are: ribonucleotide 2Thispaperis the fIfth in aseriesentitledTheComparativeEnzymol. reductase(s), purine or pyrimidine ribonucleoside diphosphate to og@yand Cell Origin of Rat Hepatomas. deoxyribonucleoside diphosphate; deoxycytidylate deaminase, dCMP 3Laboratory of Biochemistry, National Cancer Insitute, NIH, Bethes to dUMP; thymidylate synthetase, dUMP to dTMP; thymidine kinase, da, Maryland 20014. Present address: Department of Biochemistry, TdR to dTMP; thymidylate kinase(s), dTMP to dTDP and dTTP; College of Medicine, Howard University, Washington, D. C. 20001. Sup thymine “reductase,―thymine to dihydrothymine, which is subse ported in part by USPHS Grant No. CA-10729. quently converted to 13-ureidoisobutyric acid (which then yields f@ Received February 23, 1968; accepted September 5, 1968 aminoisobutyric acid plus CO2). 40 CANCER RESEARCH VOL.29 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. dlTP Synthesis in Morris Hepatomas p 5-Me-dCMP 5123, 7800, and 7794A and Reuber H-35, have been extensively studied with respect to the enzymes of the dTTP @ CDP UDP pathway. More recently developed Morris hepatomas, such as I 4' 9098,9108,9121,9618A, and 9633,arebothslowlygrowing L4@@ I dCDP dUDP - and histologically similar to normal rat hepatocytes. Nowell et dCMP—*dU@P—@dTP@P@±dTDP@±dTTP—.DNAT al. (31) have performed karyotype analyses of most of the Morris hepatomas and found that seven hepatomas—7794A, CdR—Ø UdR Td 7800, 9108, 9098, 9121, 9618A, and 9633—all had forty-two chromosomes, which is the normal diploid chromosome com plement for rat. Of these seven hepatomas with a normal chro 4@t mosome number, hepatomas 9618A and 9633 exhibited com DHT@±BUIB—+BAIB——+CO2 pletely normal chromosome morphology. The remaining five Chart 1. Schematic representation of possible pathways of dTTP hepatomas had minor changes in chromosomal morphology, synthesis. The relative importance of the various alternative pathways is such as abnormal satellites on certain chromosomes or abnor as yet unknown for any specific cell type (52). The many interlocking mal arm lengths of other chromosomes. feedback controls within and affecting this scheme (15, 52) are too In the present study, the thymidylate synthetase, deoxycy complex to be included in this chart or to be considered in the text. tidylate deaminase, thymidine and thymidylate kinases, and Abbreviations: CDP and dCDP, cytidine and deoxycytidine diphosphate; 5-Me-dCMP, 5-methyldeoxycytidine monophosphate; U, thymine catabolic activities of solid hepatomas 5123C, 7794A, T, and DHT, uracil, thymine, and dihydrothymine; DNA-T, thymine in 7800, 9098, 9108, 9121, 9618A, and 9633 were analyzed. For polymeric DNA; $3UIB and aAIB, @3-ureido-and i3-amino-isobutyric acid. comparative purposes, analyses were performed on host livers See Footnote 4 for other abbreviations. from the tumor-bearing rats, normal adult rat liver, Novikoff hepatoma cells from tissue culture, Reuber H-35 cells from tissue culture, and Ehrlich ascites cells. Although the activities of the dTI'P-synthesizing enzymes are controlled to a point of minimal activity in normal adult MATERIALS AND METHODS rat liver, little is known of the status of this pathway in can cerous liver cells that are morphologically and biochemically The solid hepatomas used in these studies (see Table 1 for similar to normal rat liver cells. Such cells are available in the pertinent data) were produced by Dr. H. P. Morris of the form of the “minimaldeviation― hepatomas developed by H. National Cancer Institute, Bethesda, Maryland. All of the hep P. Morris of the National Cancer Institute on the basis of atomas were grown bilaterally in the femoral musculature of biochemical findings first from this laboratory (35—38, 40) their respective host rats. Tumor-bearing rats were housed two and subsequently by many other cooperating groups. Potter et to a cage in a room of controlled temperature and humidity. al. (40), Pitot and Potter (35), and Maley and Maley (22) have Fluorescent lighting in the animal room was automatically reg shown that dCMP deaminase activity was extremely low in ulated to provide 12 hours of light from 9 P.M. to 9 A.M. and normal adult rat liver, Dunning hepatoma L-C18, and Morris 12 hours of darkness from 9 A.M . to 9 P.M .; 12, 30 and 60% Hepatoma 5123 relative to Novikoff hepatoma or thymus tis protein diets (prepared by General Biochemical Co., Chagrin sue. Roth et al. (45), using a different technic, reported detec Falls, Ohio) were made available to the rats only during peti table but equivalent levels of dCMP deaminase activity in nor ods of darkness from 9 A.M . to 5 P.M . (53). Nontumor-bearing mal rat liver, Dunning L-C18, Reuber H-35, and Morris Hepa rats (200—250 gui) obtained from Holtzman Co. (Madison, tomas 5123, 7800, and 7794A. Maley and Maley (22) have Wisconsin) were similarly fed a 30% protein diet for 8 hours also shown that dTMP synthetase activity was very low in out of every 24 hours (“8+16―regimen) (53). adult rat liver but elevated substantially in rat liver 24 hours Novikoff hepatoma cells (line Ni-Si) were grown in suspen posthepatectomy and in Morris Hepatoma 5123. Bukovsky sion culture as previously described (29) and were harvested and Roth (4), studying Morris hepatoma 5123, and Potter et by centrifugation. Reuber H-35 hepatoma cells were grown as a!. (39) and Gebert (8), studying Morris hepatomas 7793 and monolayers in T-flasks as previously noted (34, 42). Cells were 5123, and 7794A and 7800 respectively, found that all of the scraped into the medium with a rubber-tipped glass rod and hepatomas phosphorylated TdR at rates greater than normal collected by centrifugation.