Enzymes of Thymidine Triphosphate Synthesis in Selected Morris Epatomas

[CANCER RESEARCH 29, 40â€”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 All of the hepatomas and tissue culture material cx levels of activities of these enzymes (T. Sneider, unpublished hibited elevations in deoxythymidine (TdR) kinase and pos data) (24, 27).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, 21â€”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 â€œreductase,â€•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

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 dCMPâ€”*dU@Pâ€”@dTP@P@Â±dTDP@Â±dTTPâ€”.DNAT al. (31) have performed karyotype analyses of most of the Morris hepatomas and found that seven hepatomasâ€”7794A, CdRâ€”Ã˜ UdR Td 7800, 9108, 9098, 9121, 9618A, and 9633â€”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@Â±BUIBâ€”+BAIBâ€”â€”+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 â€œminimaldeviationâ€• 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 (35â€”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 (200â€”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 (â€œ8+16â€•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. Ehrlich ascites cells, grown intra adult rat liver. Bresnick et al. (3) found that a large number of peritoneally in mice, were obtained from Dr. Charles Heidel Morris hepatomas, including hepatomas 5123, 7800, and berger of McArdle Laboratory. All three cell types were har 7794A, as well as Reuber hepatoma H-35, all had elevated vested during the periods of high cell proliferation. The meth TdR kinase activities compared to normal adult liver. Ono et od of harvesting resulted in the equivalent of three washes a!. (32, 33) and Potter et al. (40) have demonstrated that cer with isotonic KC1. tam hepatomas, such as Morris hepatoma 5123 and its sublines (A, B, C, and D), Morris hepatomas 7800 and 7316A, and Preparation of Cell Extracts Reuber H-35, resemble normal adult liver in that they have the @ ability to degrade thymine to CO2 whereas Novikoff cells Both tumor-bearing rats and normal adult rats were sacri apparently lack this catabolic pathway. ficed by decapitation. Livers were excised, rinsed in iced iso From this brief resume, it is evident that only four slowly tonic saline solution, and placed in ice until homogenization. growing â€œminimal deviationâ€• hepatomas, Morris hepatomas Tumors were excised from the femoral musculature and simi

JANUARY 1969 41

Table1Time

(mo.) No. of ofChromosomeC1-lepatomaGencrationaHost of sacrificeAverage monthsb betweenNumber ratSexpostinoculationtransplantationstransfersnumber5123C62BuffaloF1.52.06180â€”997794

A (3) 7800182742â€•90988A 28 (1)Buffalo BuffaloM F2.5 2.34.0 2.617 CM3.03.7,3.6791089 x CF2.52.9,4.0842C91219(2)A x CM2.03.7,1.8842â€•9618(2)A x A3BuffaloM4.15.7242196335 (2)BuffaloM2.74.74421 â€œNumbersin parentheses refer to 1st, 2nd, or 3rd subtransfer in that generation. bFfrst value in all cases based on number of transfers noted in adjacent column. Second value, where noted, represents the time between transplants for the last three transfers. Data are from H. P. Morris (unpublished) and Morris and Wagner (28). CData from Nowell et al. (31). d@nor chromosome alterations noted. eNowell et a!. (31) indicate that this hepatoma has shifted to the aneuploid state. 1Apparently normal chromosome complement and karyotype. larly treated. Before homogenizing the hepatomas, connective of incubation, 0.2-ml aliquots of the reaction mixture were tissue and areas of necrosis and hemorrhage were dissected out withdrawn and pipetted into centrifuge tubes placed in a boil and discarded. Both the livers and hepatomas were finely ing water bath. The reaction was stopped by 2 minutes of this minced with scissors prior to homogenization. After addition of heat treatment. Each tube was acidified by the addition of sufficient 0.2 M Tris-Cl buffer (pH 8.0): 1 X i0@ M dithi 0.05 ml of 90% formic acid. After a low-speed centrifugation othreitol to make 20% (w/v) homogenates, the tissues were to remove the protein coagulum, the supernatants were de homogenized by means of the Polytron Kinematic Hi-frequen canted and stored at â€”30Â°Cuntil further analysis. After cy Processer (Kinematic GMBH, Lucerne, Switzerland). The streaking an aliquot of 0.05 ml of each acidified supernatant homogenates were centrifuged in a Beckman model L2 prepar onto 1 x 2.5 inch strips of Whatman No. 1 filter paper, the ative ultracentrifuge at 104,000 X g for 180 minutes. The clear strips were dried under infrared lamps and placed around the supernatants (termed 53 or soluble cell fraction) were de inside of glass liquid scintillation vials. Then 15 nil of a liquid canted by pipet. Assays for thymidylate synthetase and deoxy scintillant [4 gm 2,5-diphenyloxazole and 50 mg 1,4-bis-2-(4- cytidylate deaminase were performed immediately, whereas methyl-5-phenyloxazolyl)-benzene brought to a volume of aliquots of soluble fractions were frozen and stored at â€”30 C 1000 ml with reagent grade toluene] were added, and the vials for later analysis of TdR and dTMP kinase and thymine cata were counted in a Packard Tri-Carb liquid scintillation spec bolic activities. Preliminary studies showed that storage at trometer. The total counts per reaction mixture were plotted, â€”30Â°Cfor two weeks resulted in no loss of activity of the versus the time of incubation. The rate of decrease of counts aforementioned enzymes. Longer periods of storage led to in per reaction mixture was graphically obtained. The amount of creasingly lower dTMP kinase and thymine catabolic activities, thymine degraded to CO2 was calculated as while TdR kinase activity was stable for several months. In (decrease in total c@m/reaction mix/60 mm (1000) some cases (noted in the charts), dTMP kinase and thymine (cpm/mj.@mole thymme) (mg 53 protein/reaction mix) catabolic activities could not be determined, since the soluble fractions were frozen for one month. Generally, however, the which yields the millimicromoles of thymine degraded to CO2 frozen soluble fractions were analyzed after only 1â€”3days of per gram of 53 protein per hour. These values, however, are storage at â€”30Â°C. minimal estimates of thymine degradative capacity since dilu tion of the labeled precursor by endogenous thymine would Thymidine Degradation lower the specific activity of the thymine. The ability of tissue extracts to degrade thymine to CO2 was measured by the methods of Canellakis et a!. (5) and Potter et Thymidine Kinase Assay a!. (40) modified as indicated. The reaction mixture contained Thymidine kinase activities were determined by the methods 0.3 @.zmoleof triphosphopyridine nucleotide; 1.8 @molesof of Ives et al. (15), Potter et al. (39), and Gebert (8). The glucose-6-phosphate; 9.3 j.zmoles of Mg@; 20.9 @.zmolesof nic reaction mixture consisted of 0.2 ml of a cofactor solution otinamide; 0.22 enzyme unit of glucose-6-phosphate dehydro containing 18 @imolcs of Na@ATP4H2O, 15 j.zmoles of 3- genase; 60 @zmolesof Tris Cl buffer, pH 8.0; 0.03 pmole of phosphoglyceric acid (sodium salt), and 18 j.zmoles of dithiothreitol; and 0.1 @zmoleof thymine-2-'4C; in a total vol MgCl2 .6H@; 0.6 ml of Tris-Cl buffer, 100 @zmoles,pH 8.0; 0.4 ume of 1.0 ml. The specific activity of the thymine prepara ml of TdR-2-'4C, 0.2 pmole, about 4 @icuries;0.8 ml of soluble tions used ranged from 12,398 to 15,898 cpm/m@zmole. The fraction made in 0.2 M Tris-Cl buffer (pH 8.0): 1 X iO@ M cofactor solution was placed in a 12-nil conical centrifuge tube dithiothreitol. The total volume of the complete incubation in a volume of 0.7 ml and prewarmed to 37Â°C.Then 0.3 ml of mixture was 2.0 ml. The concentrations of the various constit soluble fraction was added, and the tube contents mixed and uents were: ATP, 9 mM ; 3-PGA, 7.5 mM ; Mg@ 9 mM ; Tris-Cl, replaced in the 37Â°C water bath. At 0, 10, 20, and 30 minutes 65 mr@i;TdR, 0.1 mM . Most preparations of TdR-2-' 4C had a

42 CANCER RESEARCH VOL.29

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. dTTPSynthesisinMorrisHepatomas specific activity of about 30,000 cpm/m@imole when counted kinase may not always be measured under conditions of sub in the system described below. The various cofactors were strate saturation. In the present studies, thymidylate kinase placed in a conical 12-nd centrifuge tube and prewarmed in a activities were calculated from plots of the amount of dTDP + 37Â°C water-bath. The soluble fraction was then added and dTTP versus incubation time of the reaction mixture. In most mixed with the tube contents, a zero-time aliquot taken, and cases, a constant rate of production of dTDP + dTI'P was the tube replaced in the 37Â°Cwater bath. Further aliquots (of observed after 60 minutes of incubation. This rate was taken approximately 0.2 ml each) were removed after 10, 20, 30, 60, as a measure of dTMP-kinase activity. In certain instances, 90, and 120 minutes of incubation. Each timed aliquot was however, the rate of appearance of dTDP + dTFP continued to immediately pipetted into centrifuge tubes placed in a boiling increase even after 120 minutes of incubation (Chart 2). water bath, and the reaction was terminated by 2 minutes of Thymidylate kinase activities in these extracts were inexactly such heat treatment. After removal of the protein coagulum estimated by employing a rate determined from the 90 and by low-speed centrifugation, the supernatants were removed 120 minute time points. Although dTMP kinase activities de and stored at â€”30Â°Cuntil chromatographic analyses could be termined in this manner may be gross underestimations, they made (usually within 2 days). At that time, 0.025 ml of each may be used to determine if a given hepatoma possesses elevat timed aliquot was streaked onto 1 x 22.5 inch strips of DEAE ed dTMP kinase activity compared to normal or host livers modified chromatography paper (DE-81). Unlabeled dTMP (whichactivitieswerebasedonaconstantrateof production and TdR (1 @moleeach) were also streaked onto the origin of of dTDP + dTTP). More precise determinations of thymidylate each strip. The strips were clipped onto glass frames, placed in kinase activities would require the use of the actual substrate a chromatography cabinet and developed by descending chro (dTMP-2-'4 C) in the in vitro assay system. Hepatomas 9618A matography with one of several solvent pairs. The time re and 9633 will be reanalyzed in this manner. The method used quired for the solvent front to travel about 20 inches was in the present paper, however, suffices to give semiquantitative usually 3.5â€”4.5 hours. Since the ion exchange capacity of the data for most of the hepatomas analyzed. DEAE paper was found to vary drastically (even within the same lot of paper), the solvent pair that would effectively Thymidylate Synthetase Assay separate TdR from dTMP from dTDP-dTTP had to be deter mined for any given set of DEAE strips. The solvents ranged Thymidylate synthetase activity was assayed by modifica from 4 N HCOOH:0.1 M NH4HCOO to 0.1 N HCOOH:0.1 M tions of the technics of Hartmann and Heidelberger (1 1). The NH4HCOO. The TdR and dTMP regions were located under reaction mixture contained 50 @molesof phosphate buffer, pH UV light, cut out, and placed around the inside of glass liquid 6.8 ; 15 @.Lmolesof sodium fluoride; 4 @.zmolesof formaldehyde; scintillation vials. Due to the variability of the DEAE paper, 1 j.@mole of d,l-tetrahydrofolic acid; and 60â€”80 millimicro dTDP and dITP did not always remain at the origin. It was moles of deoxyuridine-5'-monophosphate-6-3H. Control therefore necessary to divide the strip between the origin and studies using crude enzyme from regenerating rat liver showed the beginning of the dTMP region into 2- to 2.5-inch sections that (a) doubling the concentration of the labeled substrate and measure the radioactivity present in all sections of the resulted in no increase in measured enzyme activity, and (b) strip. The cpm/reaction mixture for TdR, dTMP + dTDP + the rate of reaction remained linear over a 30-minute period dTTP, and dTDP + dTFP were then plotted versus the time of with either level of substrate. Hence, the lower level of sub incubation. Since thymidylate kinase and nucleoside diphos strate was used in these studies. Various preparations of tn phokinase were present in the crude cell extracts, the total tiated deoxyuridylic acid were used in these experiments. The amount of TdR phosphorylated by thymidine kinase was ob specific activities of the dUMP-3H varied from preparation to tamed by adding the radioactivity present in the dTMP, dTDP, preparation (16,000â€”50,000 dpm/millimicromole). The vari and d'Vi'P regions of each strip. Moreover, dTTP formed in the ous cofactors were added to conical 1 2-ml centrifuge tubes in reaction mixture is known to inhibit TdR kinase activity (15). a volume of 0.3 ml. The volume of soluble fraction per reac Hence, the initial rate of appearance of dTMP + dTDP + dTTP tion vessel was 0.2 ml, which usually contained about 5 mg of was taken as a measure of TdR kinase activity. Results are protein. The total volume of the reaction mixture was 0.5 ml expressed as nanomoles of TdR phosphorylated per gram of in all cases. supernatant protein per hour. The rate of appearance of dTDP Incubations were carried out for 30 minutes at 37Â°C. The + dTTP was taken as a measure of thymidylate k.inase activity. reaction was stopped by immersing the tubes in a boiling water It should be pointed out immediately that this method pro bath for 3 minutes. After cooling the tubes in an ice bath, 1 vides only the crudest quantitation of thymidylate kinase ac mg of crude Crotalus adamanteus venom in a volume of 0.1 ml tivity. First, the de novo synthesis of dTMP may be sufficient was added to each tube to dephosphorylate the nucleotides, ly great in some of the hepatomas to significantly dilute the and a second 37Â°C incubation was performed for 30 minutes. labeled dTMP formed via TdR kinase. Hence, use of the specif The reaction was again stopped by heat, the tubes cooled in an ic activity of the labeled TdR to calculate the amount of ice bath, and 5 .zmoles each ofunlabeled UdR and TdR added dTDP-dTTP formed can result in an underestimation of dTMP to each tube. The reaction mixtures were then desalted by kinase activity. Secondly, the labeled substrate for thymidy passage through small individual columns (10 mm diameter) late kinase (dTMP) is produced via the action of TdR kinase. containing an 8-mm layer of Dowex-1-formate X 12 (200â€”400 The concentration of dTMP depends on both the activity of mesh) over an 8-mm layer of Dowex-50W-H@ X 10 (200â€”400 TdR kinase in any given tissue extract used and the time of mesh). Each reaction tube was washed five times with 0.5 ml incubation. The possibility therefore exists that thymidylate of water. The adsorption eluate, as well as the tube washes,

JANUARY 1969 43

PHOSPHORYLATION OF TdR IN: ADULT RAT LIVER NOVIKOFFt.c. CELLS 0 9618A OR 9633 TMP+TDP+TTP-@_ â€”â€” â€”

TdR ,g/S/' +I I P

5. 5. TMP+TDP+TTP@.@

\%%%@@ ,pTNP -@- @ TMP+TDP+TTP-@ â€” â€” â€˜â€”@.. TMP@ KJ2030 60 90 20 0 KD20 3040 60 80 100 120 0102030 6Ã³ 90 20 MINUTES OF INCUBATION MINUTES OF INCUBATION MINUTES OF INCUBATION

Chart 2. Patterns of phosphorylation of TdR in soluble cell extracts of normal adult rat liver, Novikoff hepatoma cells from tissue culture, and solid Morris hepatomas numbered 9618A and 9633. See Chart 1 for abbreviations.

were collected in 12-ml centrifuge tubes that were attached to strips to a glass frame and equilibrating for 60 minutes in the each column. Each column was further washed with water closed chromatography cabinet, tertiary butanol: 2-butanone: until a total volume of 10â€”11 ml was collected. The column water:ammonium hydroxide solvent (40:30:20:10) (7) was eluate tubes were then evaporated to dryness and the residue added to the troughs, and the strips were developed for 24â€”36 redissolved in a volume of 0.5 ml of water. A 0.05-ml aliquot hours or until the solvent front had traveled approximately 20 was then spotted onto an 18 x 22.5 inch sheet of Whatman inches. After air-drying the strips, the UdR and TdR regions No. 1 paper, and chromatography and counting were carried were located by their UV absorption. The UdR and TdR spots out as described below. Control experiments indicated that from each reaction mixture were cut out and placed in sepa virtually 100% of the UdR and TdR present in the reaction rate scintillation vials. Following addition of 0.5 ml of anhy mixture could be recovered in the redissolved column eluate drous methanol the vial was capped, gently agitated, and then and that the snake venom treatment resulted in dephosphory allowed to stand at room temperature for 30 minutes before lation of all of the dUMP and dTMP to UdR and TdR. Later, adding 10 ml of liquid scintillation fluid. The scintillator solu however, incomplete recovery of the total radioactivity in the tion consisted of naphthalene, 80 gm ; 2,5-diphenyloxazole, 5 system from the columns was sometimes observed and was due gm; alpha-naphthylphenyloxazole, 50 mg; dissolved in 1 liter to incomplete breakdown of the deoxynibonucleotides to of a mixture of 5 parts xylene, 5 parts dioxane, and 3 parts deoxyribonucleosides. The incomplete breakdown to deoxy ethanol (17). The radioactivity present in each vial was deter nibonucleosides was due to both variations in the crude snake mined in a Packard Tri-Carb liquid scintillation spectrometer. venoms employed and the inhibitory effect of the phosphate Control experiments showed that (a) the methanol treatment buffer in the reaction mixture. In order to circumvent this elutes all of the UdR and TdR from the paper, (b) the use of problem, the following procedure has been employed: the automatic external standardization with such a counting sys heat-stopped reaction mixture was reincubated in the presence tem was valid, and (c) the efficiency of counting in such a of 2.0 mg of crude Crotalus adamanteus venom for 30 minutes system was approximately 23%. The completeness of the at 37Â°C, followed by addition of 5 pmoles each of unlabeled snake venom treatment was estimated by calculating the per UdR and TdR in a combined volume of 0.1 ml. After mixing centage of the total activity represented in the UdR and TdR and centrifuging out the protein coagulum, two 0.05-mi all bands. In most instances, the venom treatment resulted in quots of the supernatant were removed from each tube. One about 60% breakdown. Hence, the UdR and TdR activities aliquot was pipetted into a glass vial, the scintillator described were corrected to values that would obtain if the breakdown below added, and the total tritium activity determined. The had been complete. The completeness of dephosphorylation second aliquot was streaked onto a 1 x 22.5 inch strip of was individually determined for each reaction mixture. The Whatman No. 1 chromatography paper. After clipping the validity of this correction rests upon the observations that (a)

44 CANCER RESEARCH VOL.29

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. d77'P Synthesis in Morris Hepatomas no other deoxyribonucleosides were found on the chromato again heat treated at 100Â°C for 3 minutes and immediately grams and (b) the deoxyribonucleoside monophosphates re chilled at 0Â°C. Chromatography was carried out as described maining at the origin of the chromatogram accounted for the below. The results, illustrated in Chart 3, show that the residu remainder of the radioactivity in the aliquot analyzed. Hence, al deamination depends upon (a) the source of crude enzyme, all of the nucleosides produced by the venom treatment were (b) the temperature at which the heat pretreatment is per accounted for, and the efficiency of breakdown could be cal formed, and (c) the time for which the crude enzyme is culated. Enzyme specific activity is expressed in this paper as heated. If the heat-treated complete reaction mixture was held millimicromoles of dTMP formed per gram of supernatant pro at 0Â°Cuntil the supernatant was decanted and frozen, no re tein per hour of incubation at 37Â°Cand is calculated from the sidual deamination was observed. Under these conditions, line experimentally determined specific activity of the labeled pre arity of the reaction with time of incubation and quantity of cursor. Implicit in this calculation is the assumption that the enzyme were noted. Hence, all enzyme assays were performed endogenous pool size of dUMP is negligible; this is suggested in this way. No further attempt was made to clarify the nature by the work of Schneider and Brownell (48). While this as and mechanism(s) of the residual deamination. sumption is valid for nonproliferating tissues, it may not hold Chromatographic separation of dCMP from dUMP was for tissues exhibiting extensive DNA synthesis. Therefore, carried out in two ways. In early experiments, 100 @ilof reac values for thymidylate synthetase activities in proliferating tis tion mixture were streaked onto the origin of a 1 x 22.5 inch sues should be considered as minimal estimates. Under our strip of DEAE-substituted chromatography paper (DE-81, conditions, the assay is linear both with time of incubation Whatman Ltd.). Unlabeled dCMP and dUMP (1 @.zmoleeach) and amount of crude enzyme. The lower limit of sensitivity of were also streaked onto the origin. After clipping to glass the assay was approximately 20 millimicromoles per gram of frames, the strips were developed by descending chromatog soluble fraction protein per hour (about 0.05â€”0.1 millimicro raphy with 1.0 N formic acid, in 0.1 M ammonium formate. mole of dTMP formed per reaction vessel per 30 minutes). The usual running time for a 20-inch solvent front was 3.5â€”4 Duplicate assays were performed on all samples and generally hours. After air-drying the strips on the frames, the dCMP and agreed to within 5%. dUMP regions (RF's of 0.8 and 0.4 respectively) were located by their UV absorption, cut out, and placed in separate glass Deoxycytidylate Deaminase Assay scintillation vials. After adding 15 ml of liquid scintillation

Deoxycytidylate deaminase activity was assayed by modifi cations of the technics of Maley and Maley (25). The reaction mixtures contained 25 @.zmolesTris-Cl buffer (pH 8.0); 1 @zmole EFFECT OF PRE-INCUBATIOP4AT VARIOUS TEMPERATURES ON of MgCl2 ; 15 @.zmolesof NaF; 0.04 pmole of dCTP; and 1 DEOXYCYTIDYLATE DEAMINASE ftCTIVITY IN CELL EXTRACTS FROM: pmole of dCMP-2-'4C. The specffic activities of the deoxycy tidylic acid-14C used in these studies varied from 16,000 to NORMALADULT RAT LIVER RAT LIVER 24 HOURSAFTER PARTIAL HEPATECTOMY 94,000 cpm4zmole. The various components were added to conical 12-mi centrifuge tubes in a volume of 0.2 ml. General ly, 0.3 ml of soluble fraction, containing about 8 mg of pro z tein, was added to each reaction vessel. Soluble fractions of z tissues containing elevated dCMP deaminase activity were di luted prior to analysis. The final volume of reaction mixture was 0.5 ml in all cases. Incubations were carried out for 30 minutes at 37Â°C. The reaction was stopped by immersing the tubes in a boiling water bath for 3 minutes. After removal of the protein coagulum by centrifugation, the supernatants were decanted and stored at â€”20Â°Cuntil chromatographic analyses could be performed. Early studies of the deamination of dCMP with time of incuba tion indicated that the rate of reaction was not linear with time. Further experiments demonstrated that this nonlinearity was only apparent. Heat treatment of the reaction vessel did not completely stop the deamination reaction. If the heated reaction vessel was allowed to stand at room temperature, sig nificant quantities of dCMP were further deaminated. Further more, the extent of this residual deamination depended upon several factors. Crude enzyme from 48-hour posthepatectomy 2 4 6 rat liver and from normal rat liver were heat-treated at several MINUTES OF PRE-INCUBATION temperatures for vanioi..@lengths of time in the absence of Chart 3. Deoxycytidylate deaminase activity (as a percent of the reaction mixture components. The various cofactors were then nonpreincubated samples) of soluble cell extracts from normal adult rat added to each tube, and deaminase activity was assayed as liver or 24-hour postpartial hepatectomy rat liver after periods of pre noted previously. After the incubation period, all tubes were incubation for the times and temperatures noted.

JANUARY 1969 45

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. Thomas W Sneider, Van R. Potter, and Harold P. Morris solution [4 gm 2,5-diphenyloxazole and 50 mg 1,4-bis-2- deaminase activities must be considered as minimal estimates. (4-methyl-5-phenyloxazolyl)-benzene brought to a volume of The effect of pool size, however, is unlikely to be so great as 1000 ml with reagent grade toluene] to each vial, the amount to reduce the amount of labeled dUMP formed to the level of of labeled nucleotide was determined in a Packard Tri-Carb detection of the assay. Reinforcing this opinion is the fact that liquid scintillation spectrometer. Preliminary experiments Novikoff hepatoma cells have high levels of ribotide reduc demonstrated that (a) this method of counting was linear with tase(s) (27), yet they also demonstrate very high dCMP deam respect to the amount of labeled material on the paper versus inase activity when assayed with the present method. The ef the count rate observed, (b) the orientation of the paper in the fect of dilution of the labeled precursor is evidently offset by vial was irrelevant to the linearity of counting, and (c) the the greater rate of deamination of dCMP. absolute efficiency of counting in this system was about 71%. The lower limit of sensitivity of the assay was approximately The above method of chromatography of dCMP and dUMP 500 millimicromoles dCMP deaminated per gram of soluble was especially satisfactory in that it was very rapid and gave fraction protein per hour (about 2 millimicromoles per reac excellent separations of precursor from product. In the course tion vessel per 30 minutes) in most experiments. All assays of the present studies, different lots of DEAE-substituted were done in duplicate and results agreed to within 5%. paper had to be employed. We were completely unable to reproduce the dCMP and dUMP separation with these latter Determination of Proteins batches of paper. We were also unable to affect this separation by using both a wide range of eluants and prewashed and/or The amount of protein present in the soluble fraction of pretreated paper. We therefore modified the aforementioned tissue homogenates was determined by the Lowry et aL (20) method as follows: After the initial incubation and heat treat Folin-Ciocalteu phenol reagent method. A single lot of crystal ment, an aliquot of the reaction mixture supernatant was incu line bovine serum albumin was employed as a reference stan bated at 37Â°C for 30 minutes with 0.5 mg of crude Crotalus dard throughout these studies. This method, as performed in adamanteus venom. The reaction was terminated by heat and a 100-,.zl aliquot was streaked onto 1 x 22.5 inch strips of our laboratory, gave a linear plot of O.D.7oom,, versus protein concentration over the range of 5â€”90 @gofprotein. Hence the Whatman No. 1 filter paper. After adding 1 .zmole each soluble fractions of 20% (w/v) homogenates were diluted of unlabeled UdR and CdR to the origin, the strips were 200-fold prior to protein analysis. The miniscule amount of clipped to glass frames and developed by descending chroma Cleland's reagent in the highly diluted supernatant fractions tography with tertiary butanol: 2-butanone:water: formic acid did not interfere with the protein determination. (44:44:11:26) (7). The solvent front travels about 20 inches in 24 hours. In this system the mononucleotides remain at the origin. CdR has an RF of about 0.2 and UdR has an RF of Reagents about 0.5. The UdR and CdR regions were located by their UV absorption, cut out, and placed in glass scintillation vials. Tris buffers were made with heavy metal-free â€œAâ€•grade Counting was carried out as above. Preliminary studies showed tris-(hydroxymethyl)-aminomethane from Calbiochem (Los that the incubation with crude snake venom degraded most of Angeles, Calif.). Cleland's reagent (dithiothreitol) was obtained the dCMP and dUMP to CdR and UdR. In all experiments, from the same source. d,l-Tetrahydrofolic acid was purchased however, the radioactivity remaining at the origin was mea from Nutritional Biochemical Corp. (Cleveland, Ohio), stored sured and the percent breakdown of deoxyribonucleotides to in the dark in the presence of 1.0 M mercaptoethanol, and deoxyribonucleosides was calculated. The corresponding de sealed under nitrogen. Tritiated dUMP was obtained from two oxyribonucleoside counting data were then corrected for any sources. In early experiments, dUMP-3H was prepared from incomplete breakdown. Furthermore, the presence of fluoride dCMP-3H by nitrous acid deamination (1 1) and subsequently in the original reaction mixture prevented breakdown of the purified by Dowex-l-formate ion-exchange chromatography. dCMP to CdR during the course of the first incubation. Hence, Later experiments utilized dUMP-6-3H, which became avail the UdR noted after the incubation with snake venom was a able from Schwarz BioResearch (Orangeburg, N. Y.). result of dCMP deaminase activity and not of CdR deaminase dCMP-2-'4 C, TdR-2-'4 C, and thymine-2-14 C were also ob activity. Since no exogenous energy source or source of tamed from this source. Unlabeled deoxyribonucleosides and methylenetetrahydrofolic acid has been supplied in this assay, deoxyribonucleotides were obtained from Sigma Chemical it is unlikely that dTMP or its di- and triphosphate could be Corp. (St. Louis, Mo.) and Calbiochem. Crotalus adamanteus formed. In fact, pilot studies of tissues with high dTMP venom was purchased from the Ross Allen Reptile Farm (Sara synthetase activities show no conversion of labeled dUMP sota Springs, Fla.) and from the Sigma Chemical Corp. Crystal formed via the deaminase reaction to labeled dTMP. line bovine serum albumin was obtained from Sigma Chemical Calculation of dCMP deaminase activity was similar to that Co. Naphthalene was purchased from Matheson Scientific Co. noted for dTMP synthetase. The specific activity of the precur (Elk Grove Village, Ill.) and dioxane and xylene from Fisher sor was determined by UV analysis and by counting an aliquot Scientific Co. (Chicago, Ill.). Alphanaphthylphenyloxazole, of the precursor on Whatman No. 1 filter paper. The enzyme 2,5-diphenyloxazole, and 1,4-bis-2-(4-methyl-5-phenyloxa activities in this paper are expressed as millimicromoles of zolyl)-benzene were all products of Parkard Instrument Co. dCMP deaminated per gram of soluble fraction protein per (La Grange, Ill.). DEAE-modified cellulose chromatography hour. It should be noted that the above calculation ignores the paper (DE-81) was obtained from Reeve Angel Co. (Clifton, N. size of the endogenous dCMP pool. Thus, values for dCMP J.). Allother chemicalswereof reagentgradeor better.

46 CANCER RESEARCH VOL.29

96I8A 9633 7794A 9108 9098 7800 5I23C 9121 H-35tc NOVIIÃ˜FEHRLICH t.c. ASCITES

N@6 99 1218 514642402525 55 99 2323 2 6 2 @@@ EJ HOSTLIVER HEPATOMA Chart 4. Levels of deoxycytidylate deaminase activity in rat liver, solid Morris hepatomas, and selected tissue culture (t.c.) and ascites material. In this, and the remaining charts, numerals along the abscissa indicate the number of individual liver or hepatoma samples analyzed or the number of experiments performed with tissue culture or ascites material. Open bars represent mean values obtained with normal adult liver or host livers; closed bars represent mean tumor values. The line above and below the mean values indicates the standard error of the mean. Note that Charts 4â€”7 have one or more breaks in the ordinates. The hepatomas values are arranged from left to right in order of decreasing transplantation time (cf. Table 1). In the case ofdCMP deaminase activity in Novikoff cell extracts, the value ofthe ordinate should be multiplied by a factor of 102.

RESULTS tamed undetectable levels of dCMP deaminase activity if the soluble cell fractions were prepared from cells homogenized in The activities of the enzymes catalyzing the synthesis of buffer containing no dCTP. If homogenization was carried out thymidine triphosphate in the normal and tumor tissues noted in buffer 0.5 to 1.0 mM in dCTP, high levels of deaminase are illustrated in Charts 4 through 8 . Although the rats bearing activity were measurable (900- and 202-fold in the two cell the hepatomas were fed diets containing 12, 30, or 60% pro types respectively compared to adult liver). Reuber H-35 hepa tein and were sacrificed at various times of day, in only one toma cells, grown as monolayers in tissue culture, showed no instance (hepatoma 9633) was there any effect of these van detectable deaminase activity regardless of the presence of 0.5 ables on the activities of any of the enzymes analyzed (cf 39, or 1.0 mM dCTP in the homogenizing medium. The presence 43). Therefore enzyme data from all animals, regardless of the or absence of dTCP in the homogenizing medium did not af protein composition of the diet or the time of killing, were fect deaminase activity of normal liver, hepatomas 7800 and averaged to yield the values recorded in the charts. 9618A, or their host livers, while hepatoma 9633 showed only Enzyme activities in the various tumor tissues are compared slight increases in dCMP deaminase activity when the homog with values obtained from analysis of livers of adult Holtzman enizing medium was 0.5 mM in dCTP. The remainder of the rats and of the host rats. Since unpublished data from the hepatomas in Chart 4 were homogenized in media that did not authors' laboratory and information in the literature (cf 8, contain dCTP. 39) indicate that livers from adult rats of several strains all have low levels of activities for the enzymes studied here, no analy dTMP Synthetase ses were made of livers from nontumor-bearing AxC or Buffalo With the exception of hepatoma 9633, all of the hepatomas strain rats. analyzed exhibited elevated dTMP synthetase activities (Chart dCMP Deaminase 5). Hepatoma 5123C had 2050% of the adult liver value; 7800, 1500%; 7794A, 940%; 9098, 1370%; 9108, 1160%; 9121, Many of the hepatomas exhibited dCMP deaminase activities 4150%; 9618A, 350%; Novikoff cells from tissue culture, equivalent to the low levels noted in normal adult rat liver 2740%; H-35 hepatoma cells from tissue culture, 340%; and (Chart 4). Hepatoma 9098 had reduced deaminase activity Ehrlich ascites cells, 2060%. Experiments with Novikoff hepa (21% of host liver) while hepatomas 5123, 9121, and 9633 toma cells from suspension culture indicated that dTMP had slightly elevated levels of enzyme activity compared to synthetase activity rapidly decayed during homogenization host liver (178%, 214%, and 139% respectively). Novikoff hep and preparation of the soluble cell fraction (T. Sneider, unpub atoma cells from tissue culture and Ehrlich ascites cells con lished data). These studies indicated that dTMP synthetase

JANUARY 1969 47

THYMIDYLATE SYNTHETASE NOVIKOFFEHRIJCH 96I8A 9633 7794A 9108 9098 7800 5123C 912I H-35t. C. tc. â€¢@ASCITES

@I5 5I46@40@25 55 99 2323 2 6 2 @ EJ aHOSTLIVER@â€¢:HEF*TOMA

Chart 5. Levels of thymidylate synthetase activity in liver, solid Morris hepatomas, and selected tissue culture (t.c.) and ascites material. Remainder as in Chart 4.

TdR KINASE ADULT H-35 N@1IKOFF LIVER 96I8A 963@3 7794A 9108 9098 7800 5I23C 9121 t.c. t.c. 450O@ 44500

33500 3@

JO 500 @Oo0@

.@,250( j20@ E 150( I0O( 50(

N@6 3 8 99471413495599 99 2 6 @ EJ HOSTUVER @@HEPAT0MA

Chart 6. Levels of thymidine (TdR) kinase activity in liver, solid Morris hepatomas, and selected tissue culture (t.c.) material. The value above the data for hepatoma 9121 represents the standard error of the mean which, due to the breaks in the ordinate, could not be indicated in the usual manner. Remainder as in Chart 4.

48 CANCER RESEARCH VOL.29

d7TP Synthesis in Moms Hepatomas

THYMIDYLATE KINASE CS) @ I 96I8A96339108909878005123C IO300F IO2OOJ@ :@ 490c1 Â±761 3 ..... 2480 :@ !:@@% Â±227

@400 @ @: -

@ Ji: â€” Nu6 38 1114 I4I2 49 55 99 2 6 @ E:J HOSTLIVER@â€¢:â€¢HEPATOMA Chart 7. Levels of deoxythymidine monophosphate kinase activities in normal adult rat liver, solid Morris hepatomas, and selected tissue (t.c.) culture material. Dashed bars and asterisks for hepatomas 9618A and 9633 indicate inability to assign exact values for technical reasons (see Methods and Results sections for further details). Remainder as in Charts 4 and 6.

THYMINE DEGRADATION activity could be preserved during the preparation of the sol H-35 NOVIKOFF @@@ ADULT 9633 7800 5I23C tâ€¢ @. uble cell fraction by utilizing very concentrated homogenates. LIVER In view of these studies, hepatoma 9633 was renalyzed. Sol uble cell fractions were prepared from concentrated homogen ates of 9633. Even under these conditions no dTMP synthetase activity was detectable.

TdR Kinase and dTMP Kinase 0

All of the hepatomas studied showed elevated levels of TdR z w kinase (Chart 6). Hepatoma 5123C had 2680% of the adult I- liver value; 7800, 1470%; 7794A, 450%, 9098, 300%; 9108, 0 680%; 9121,4100%; 9618A, 765%; 9633, 1090%; Novilcoff cells from tissue culture, 17,830%; and H-35 hepatoma cells from tissue culture, 19,550%. of six Morris hepatoma lines E analyzed (Chart 7), hepatomas 5123C, 7800, 9098, and 9108 -5.. showed elevated dTMP kinase activities (8130%, 1510%, 850%, and 720% respectively compared to host liver values). E The soluble cell fractions from hepatomas 7794A and 9121 had been stored at â€”30Â°Cfor approximately one month. Hence, dTMP kinase activities were not determined for these samples. Novikoff and H-35 cells from tissue culture also had high dTMP kinase activities (7100% and 10,280% compared to adult liver values). Although hepatomas 9618A and 9633 pos 38 99 55 99 2 6 sessed dTMP kinase activity, quantitation of the levels of such @ activity was not possible. In analyzing the four aforemen 0 HOST @:aHEPATOMA tioned Morris hepatomas as well as the tissue culture material, Chart 8. Thymine-2-14C degradation to 14C02 by adult rat liver, a constant rate of production of dTDP and dTTP was generally solid Morris hepatomas, and selected tissue culture (t.c.) material. Re noted after 30 to 60 minutes of incubation of reaction mix mainder as in Chart 4.

JANUARY 1969 49

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. Thomas W. Sneider, Van R. Potter, and Harold P. Moms tures (Chart 2). Incubations of soluble cell fractions from hep difficult to demonstrate in hepatomas 9618A and 9633. Hepa atomas 9618A and 9633 led to production of labeled dTDP tomas 5123C and 7800, which could be classified as more and dTTP only after 90 and 120 minutes (Chart 2). Inasmuch deviated from hepatocytes on the basis of TdR and dTMP Id as the rate of production of labeled dTDP + dTTP continually nases and dTMP synthetase, were shown to be similar to adult increased, it was not possible to obtain an accurate rate mea liver, in that they possessed the ability to catabolize thymine surement for the synthesis of dTDP plus dTTP. The amount of and apparently lacked dCMP deaminase activity. Hepatoma thymidine di- and triphosphate present after 120 minutes of 9633 is similar to adult liver in that it has normal low levels of incubation was much greater (400â€”500%) than that noted in dCMP deaminase and dTMP synthetase but differs from adult adult liver or host liver reaction vessels after 120 minutes of liver since it possesses elevated TdR and dTMP kinases and incubation. This latter observation would indicate that the lacks thymine catabolic activity. dTMP kinase activities of hepatomas 9618A and 9633 were While, in general, the 3 enzymes involving thymidylic path greater than adult or host liver, and, if incubation times had ways are strikingly increased in comparison with normal liver, been extended, a linear rate of production of dTDP plus dTTP and indeed from 10 to 40-fold in many cases, there is a failure would have been observed. to demonstrate coordinate increases, especially among the hep atomas that are less than 10-fold higher than normal liver. The Thymine Degradation most consistent increase in enzyme activity in the hepatomas studied thus far appears to be thymidine kinase, which was Four Morris hepatomas (5123C, 7800, 9618A, and 9633) as elevated in all hepatomas herein reported and in a series of well as Novikoff and H-35 tissue culture cells were assayed for earlier Morris hepatomas studied by Bresnick et a!. (3). Ex their ability to degrade thymine-2-'4 C to 14C02 (Chart 8). cluding 5123C, which cannot be considered as minimal devia Hepatomas 5123C, 7800, and 9618A catabolized thymine at tion because of karyotype findings (31), the hepatomas with rates lower than normal adult or host livers (54%, 47%, and 42 chromosomes have elevations of TdR kinase between 3-fold 16% respectively compared to host liver). Thymine catabolic and 40-fold over normal liver. The failure of this enzyme to be activity in both the host livers and the hepatomas of rats bear repressed under conditions that repress its synthesis in the cells ing hepatoma 7800 was grossly elevated compared to normal of homologous origin may be the best indication of malignan adult rat liver. This finding is at present unexplained. Reanaly cy so far available in hepatomas, but the quantitative propor sin of a later generation of hepatoma 7800 (sacrificed earlier tionality with respect to growth cannot be demonstrated when after transplantation than the first group) showed normal 1ev the hepatomas with abnormal chromosome numbers are cx els of degradative ability in the host livers. The hepatomas cluded (cf. 19). Nevertheless, the consistent elevation in again showed approximately 50% less catabolic activity than TdR kinase is more striking than that for any other enzyme or the host liver. Hepatoma 9633 as well as Novikoff and H-35 enzyme system studied thus far, including glycolysis (cf. 41). cells from tissue culture carried out no measurable degradation Moreover, the present results seem to indicate that the genes of thymine to CO2. Hepatomas 7794A, 9098, 9108, and 9121 controlling the synthesis of these dTTP-synthesizing enzymes were not assayed for thymine degradative ability. may not all be coordinately expressed. For example, hepatoma cells of a given strain might be synthesizing large amounts of DISCUSSION dTMP synthetase while expression of the genes for dCMP de aminase is nil. Although this conclusion is only tentative at The results reported above represent an attempt to deter this time (see Addendum), it seems likely that coordinate cx mine if the activities of enzymes catalyzing the synthesis of pression, if it exists in this system, may apply to alternative dTrP exhibit a characteristic pattern among hepatomas of the pathways leading to dTTP rather than to the entire constella minimal deviation type. It was also of interest to determine if tion of possible pathways (Chart 1). the activities of these enzymes, which are all under stringent The present data, as well as the two conclusions just dis control in normal adult rat liver, were proportionately or dis cussed, are of immediate significance with respect to (a) the proportionately expressed within a given hepatoma strain. Re dTrP pathway, (b) the chemotherapy of cancer, and (c) the duced to their simplest form, these objectives can be expressed concept of the minimal deviation hepatoma. The low levels of in the following questions: Are there enzymatic changes on dCMP deaminase in all of the Morris hepatomas analyzed make the dTTP pathway that can characterize malignancy? Are the the contribution of the dCMP to dUMP step in dTTP synthesis activities of the enzymes of the dTFP pathway coordinately slightly suspect as an obligatory pathway. Either the low level expressed? Only qualified answers to these questions can be of dCMP deaminase activity is sufficient to ensure an adequate given at the present time. pool of dUMP for methylation to dTMP or the preferred path Deoxycytidylate deaminase activity, which is apparently cor for the synthesis of dUMP is via reduction of uridine diphos related with DNA synthesis in fetal and regenerating adult rat phate. Recent work of Crone and Itzhaki (6) supports earlier liver and in certain cell types (35, 40), is not significantly data from this laboratory (12) that the regenerating rat liver elevated in any hepatomas of the minimal deviation type but is utilizes the path UDP to dUDP to dUMP to dTMP in preference grossly elevated in Novikoff cells from tissue culture and Ehr to CDP to dCDP to dCMP to dUMP to dTMP (See Chart 1). lich ascites cells. This enzyme, however, may not be obligatory The contribution of dCMP to dUMP was held to be less impor for DNA synthesis (6, 12), and its activity may reflect differ tant in the synthesis of dTMP. What role, therefore, can the ences in cell type (35). Thymidylate synthetase activity is dc dCMP deaminase assume in the dTTP pathway? Our analyses vated in most hepatomas analyzed but is apparently low or of Novikoff cells and Ehrlich ascites cells, as well as rat thymus

50 CANCER RESEARCH VOL.29

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. dlTP Synthesis in Morris Hepatomas and bone marrow, are intimately related to this question. hepatoma might, therefore, be expected to be insensitive to (Thymus and marrow dCMP deaminase show 18,000% and 5-FIJdR but sensitive to agents designed to inhibit the 5- 4000%, and dTMP synthetase 170% and 400%, respectively of methyl-dCMP to dTMP step. Another tumor treated with adult liver levels.) These cells exhibit enormous levels of dCMP 5-FUdR might be insensitive because it can obtain enough deaminase activity yet show relatively moderate elevations in TdR from dying or dead host cells to overcome the dUMP to dTMP synthetase activity; this pattern is the reverse of that dTMP block. Succinctly stated, effective cancer chemotherapy seen in most of the Morris hepatomas examined. An ad hoc in this area must be predicated upon an effort to block all explanation of these results might be that, in tissues exhibiting routes to dTMP synthesis inasmuch as a given tumor cell line either pattern, the preferred route of dTMP synthesis is via can potentially â€œbreakoutâ€• of its uniquely preferred route to reduction of the ribose moiety of UDP. The elevationâ€”or lack dTMP synthesis and choose an alternative route. thereofâ€”of dCMP deaminase activity might then reflect the Finally, the relationship of the present results to the minimal status of an ancillary route to dTMP synthesis via deamination deviation hepatoma concept must be considered. This concept of 5-methyl-dCMP. It is interesting to note that 5-methyl classed transplantable rat hepatomas into broad categories on deoxycytidine has been found in several rat tissues (51). Nishi the basis of their relative morphologic and biochemical similar haa et al. (30) and Geraci et a!. (10) have demonstrated that ity to normal rat liver (36, 38). It was recognized (31) that partially purified deoxycytidylate deaminase catalyzed the those hepatomas first found to be in the minimal deviation deamination of 5-methyl-dCMP as well as dCMP. Evidence sup category might, in the future, be replaced by hepatomas that porting the existence of this alternative route to dTMP synthe deviated to an even lesser extent. The biochemical alterations sis has been gleaned from recent studies on sea urchin embryos possibly relevant to carcinogenesis might then be more easily by Bieiavsky and Leong (1), on a strain of neoplastic mouse identified and studied on the basis of an on-going process of cells in tissue culture by Price et al. (44) and on amethopterin selection of experimental material. The criteria to be met by a blocked Novikoff cells in tissue culture by Gentry et al. (9). hepatoma minimally deviating from liver were subsequently Moreover, Karlstrom and Larsson (16) recently found that as expanded by Potter and Watanabe (41)@ who emphasized the much as 75% of the dTTP formed in E. coil must have been need for comparisons with newborn rat liver (such compari. synthesized via deamination of 5-methyl-dCTP. Only 25% of sons in the case of the dTFP pathways have not yet been the dTIP was synthesized by methylation of dUMP. The completed in the authors' laboratory). The minimal deviation studies of Bieliavsky and Leong (1) and Karlstrom and Larsson concept, however, did not include the a priori expectation that (16) both indicate that S-adenosylmethionine serves as the only one or two patterns of enzyme activities and metabolic methyl donor in the conversion of dCMP to 5-methyl-dCMP. responses are present in all hepatomas. Hence, the absence of a The apparent lack of elevated dCMP deaminase activity in unique pattern of enzyme activities along the dTTP pathway Morris hepatomas might indicate absence or continued strin in the hepatomas examined in this studyâ€”as well as the varia gent regulation of the proposed ancillary pathway. A totally tions noted by Potter et al. (43) with respect to carbohydrate different role for dCMP deaminase has recently been proposed and protein metabolismâ€”does not negate the minimal devia by Scarano et al. (46, 47) on the basis of a study of the tion concept. The present results do, however, qualify the con metabolic heterogeneity of thymine methyl groups in sea ur tention that individual enzymes of the dTI'P pathway are cor chin embryo DNA. In this model, the deamination of DNA-5- relatable with the growth rate of the tumor. On the basis of methyl-cytosine leads to base shifts which, in turn, constitute studies including poorly differentiated, rapidly growing hepa a molecular mechanism for differentiation. While no direct tomas, it has been stated (54) that thymidylate synthetase, evidence supports this most interesting hypothesis, such base deoxycytidylate deaminase, and thymidine and thymidylate shifts, if reversible, might be involved in initiation of transcrip kinases are all elevated in hepatomas to degrees reflecting the tion. If the latter is indeed the case, then cells such as Novi growth rates of the hepatomas. The catabolism of thymine to koffâ€”which must be transcribing at least those regions of their CO2 was said to inversely reflect the growth rate. In the pres genome concerned with DNA synthesis and cell division at a ent study, however, slowly growing, well-differentiated, and very high rateâ€”could be expected to have substantial activity normal karyotype hepatomas with different growth rates of enzymes capable of deaminating dCMP at the polymer or (Table1)wereanalyzed.Acomparisonoftheaveragetrans mononucleotide level. plantation times listed in Table 1 with the data shown in The present results, as well as the preceding comments, bear Charts 4 through 8 clearly illustrates that only dTMP synthe directly upon the problem of cancer chemotherapy. Che tase shows any correlation with growth rate (imperfectly motherapeutic agents, such as F3TdR, 5-FUdR, and ametho measured as transplantation time). The significance of this one pterin, have been developed to inhibit the dUMP to dTMP positive correlation with growth rate is not clear, since (a) the step. The efficacy of such agents depends on several factors, correlation could break down when more hepatomas of the among which must be included the development of resistance to the agent. In the case of 5-FUdR, a strain of Novikoff cells in tissue culture has been obtained that is resistant to 5-FUdR 5A recent suggestion that the minimal deviation label should be aban because of an apparent lack of TdR kinase (29). Hence, the doned (55) might well be considered in the context of the more recent 5-FUdR cannot be phosphorylated to the inhibitory form, data (41). We agree that the better collective label in many cases might 5-FdUMP. Insensitivity to chemotherapy might also be due to be â€œMorrishepatomas,â€•while specific hepatoma lines should be re the lack of the appropriate target molecule. Hepatoma 9633, ferred to by numbers and compared with each other according to the for example, appears to lack dTMP synthetase activity. This concept which, as Wu agrees, continues to be fruitful.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1969 American Association for Cancer Research. Thomas W. Sneider, Van R. Potter, and Harold P. Morris minimal deviation type are developed and analyzed, and (b) at 104,000 x g for 30 minutes. If a concentrated suspension of Novi alternative pathways to dTMP synthesis exist that can circum koff cells is homogenized and subsequently diluted with homogenizing vent the dUMP to dTMP step. If dTMP can be synthesized at medium, dTMP synthetase activity will be found in both homogenates and soluble cell fractions of only the more concentrated dilutions. Lit rates that do not limit growth via phosphorylation of TdR or tle or no enzyme activity is discernible in the homogenates and soluble via deamination of 5-methyl-dCMP, then a correlation of cell fractions from higher dilutions even though the sensitivity of the dTMP synthetase activity with growth rate in such tumors enzyme assay is such that enzyme activity proportional to the dilution assumes questionable significance. It is therefore felt that a would have been easily detectable. The addition of dATP, dGTP, dTl'P, priori predictions about the quantitative enzymatic potential dCTP, dUMP, dTMP, N5'10-methylenetetrahydrofolic acid (at levels of of the components of the dTTP pathway in hepatomas (and 0.5 mM) or of bovine serum albumin (20 mg/mi) to the homogenizing perhaps in any malignant tissue) cannot convincingly be made medium does not aid in retaining the enzyme activity of homogenates at this time. In conclusion, it must be reiterated that there are or soluble cell fractions of diluted suspensions of Novikoff cells. Pre several possible pathways for the synthesis of dTI'P and dCTP liminary experiments (T. W. Sneider and V. R. Potter, unpublished (Chart 1). The present data, as well as information akeady in data) have shown that use of a solution of high molecular weight (>10,000) components of soluble extracts from concentrated homoge the literature, reinforce the position that there must be many nates of Novikoff cells or 24-hour regenerating rat liver as a medium for patterns of enzyme activities compatible with the conversion homogenization makes it possible to detect dTMP synthetase activity in of sufficient amounts of uridylic acid to dTI'P and dCTP to dilute suspensions of Novikoff cells. A solution of the low molecular permit DNA synthesis. It is therefore important to analyze as weight (< 10,000) components of soluble extract from a concentrated many enzymes as possible along this pathway in a given tissue homogenate of 24-hour regenerating rate liver, when used as homoge and to search for new pathways. In this manner, some concept nizing medium, also enables detection of enzyme activity in dilute sus of possible preferred routes of synthesis of dTTP and dCTP in pensions of Novikoff cells. The high molecular weight (> 10,000) com individual tissues might be obtained. This same goal can be ponents of normal adult rat liver soluble cell extract and the low molec ular weight (< 10,000) components of Novikoff cells and normal adult approached by autoradiographic and biochemical analyses of rat liver soluble cell extracts have no such â€œprotectiveâ€•effect on the incorporation of various labeled precursors into DNA in enzyme activity. Further studies on these phenomena are continuing in vivo. Utilization of the autoradiographic methodology is virtu our laboratory. What has been learned thus far, however, dramatically ally mandatory inasmuch as the hepatomasâ€”while relatively demonstrates the need for continual reappraisal of both in vitro enzyme homogeneous in cell typeâ€”are heterogeneous with respect to assays and data garnered therefrom. In light of the â€œparadoxicalâ€• the cell cycle and probably with respect to various stages of phenomena described here, interpretations of and conclusions from maturation along lines of differentiation (41). Correlation of such data must be tentative. the data from the in vitro enzyme assays and the in vivo pre cursor incorporation analyses in the specific hepatoma lines ACKNOWLEDGMENTS could well yield very fundamental information about pyrimi dine deoxyribonucleotide synthesis per se and its relation to The authors wish to thank Mrs. Lynn Dean and Miss Stefanie Get the control of DNA synthesis, just as the use of E. coli mu finger for their capable technical assistance and Miss Joyce Becker for tants has proved useful in elucidating aspects of biochemical providing the tissue culture material. genetics. REFERENCES ADDENDUM 1. Bieliavsky, N., and Leong, G. F. The Effect of FUdR on Segments The present study was based upon enzyme assays of crude extracts of tion and Incorporation of 3H-Uracil in Amphibian Embryos. Expti. hepatomas. Thus, the possibility of uncontrolled inhibition or activa Cell Res., 47: 261â€”270,1967. tion or degradation, etc., of enzymes under our experimental condi 2. Bollum, F.J., and Potter, V. R. Nucleic Acid Metabolism in Regen tions must be admitted. For example, Novikoff and H-35 cells from crating Liver. VI. Soluble Enzymes Which Convert Thymidine to tissue culture, as well as rat thymus and 24-hour posthepatectomy rat Thymidine Phosphates and DNA. Cancer Res., 19: 561â€”565, liver, must be homogenized in media containing 0.5 to 1.0 mM dCTP to 1959. permit demonstrable activity for dCMP deaminase. Kit et aL (18) have 3. Bresnick,E., Thompson,U. B., Morris, H. P., and Liebeit, A. G. noted a similar phenomenon. If the homogenizing medium does not Inhibition of Thymidine Kinase Activity in Liver and Hepatomas contain dCTP, or if the dCTP is added to the assay mixture after the by TTP and dCTP. Biochem. Biophys. Res. Commun., 16: preparation of the soluble cell extract, little or no dCMP deaminase 278â€”284, 1964. activity is noted. The addition of dCTP to the homogenizing medium 4. Bukovsky,J., and Roth, J. A. Some Factors Affecting the Phospho has little or no effect on dCMP deaminase activity in normal adult rat rylation of Thymidine by Transplantable Rat Hepatomas. Cancer liver or on most of the hepatomas analyzed in the present study. A Res., 25: 358â€”364,1965. critical effect of homogenizing conditions also occurs in the analysis of 5. Canellakis, E. S. Pyrimidine Metabolism. I. Enzymatic Pathways of dTMP synthetase activity: depending upon the tissue analyzed, the Uracil and Thymine Degradation. J. Biol. Chem., 221: 315â€”322, presence or absence of synthetase activity is contingent upon the origi 1958. iial concentration of the homogenate. If Novikoff cells, H-35 cells, or 6. Crone, M., and Itzhaki, S. On the Relative Functioning of the Path chick embryo fibroblasts (all from tissue culture) are homogenized at ways for Formation of Thymidine Nucleotides in the Regenerating ratios less than one volume of cells to one volume of homogenizing Liver and Spleen of the Rat. Biochim. Biophys. Acts, 95: 8â€”13, medium, or if rat thymus or regenerating rat liver are prepared as less 1965. than 20â€”30%(w/v) homogenates, little or no dTMP synthetase activity 7. Fink, K., and Adams, W. S. Paper Chromatographic Data for Pu can be found in either the homogenate (analyzed immediately after rifles, Pyrimidines and Derivatives in a Variety of Solvents. J. homogenization) or the supernatants of such homogenates centrifuged Chromatog., 22: 118â€”129,1966.

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8. Gebert, R. A. Studies on the Mechanisms of Control of DNA and 28. Morris, H. P., and Wagner, B. P. Induction and Transplantation of RNA Metabolism in Minimal Deviation Hepatomas and Normal Rat Rat Hepatomas with Different Growth Rate (including â€œMinimal Liver. Ph.D. Dissertation University of Wisconsin, Memorial Li Deviationâ€•Hepatomas). In: H. Busch (ed.), Methods in Cancer brary, Madison, Wisconsin, 1966. Research, Vol. 4, pp. 125â€”152. New York: Academic Press, 9. Gentry, G. A., Morse, P. A., Ives, D. H., Gebert, R. A., and Potter, 1968. V. R. Pyrimidine Metabolism in Tissue Culture Cells Derived from 29. Morse, P. A., and Potter, V. R. Pyrimidine Metabolism in Tissue Rat Hepatomas. II. Thymidine Uptake in Suspension Cultures Dc Culture Cells Derived from Rat Hepatomas. I. Suspension Cell Cal rived from the Novikoff Hepatoma. Cancer Res., 25: 509â€”516, tures Derived from the Novikoff Hepatoma. Cancer Res., 25: 1965. 499â€”508,1965. 10. Geraci, G., Rossi, M., and Scarano, E. 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Cancer Res., 16: 999â€”1004, 1956. idine Metabolism in Minimal Deviation Tumors. Cancer Res., 23: 13. Hiatt, H. H., andBojarski,T. B. StimulationofThymidylate Kinase 240â€”249,1963. Activity in Rat Tissues by Thymidine Administration. Biochem. 33. Ono, T., Potter, V. R., Pitot, H. C., and Morris, H. P. Metabolic Biophys. Res. Commun., 2: 35â€”39,1960. Adaptations in Rat Hepatomas. III. Glucose-6-Phosphate Dehy 14. Holtzer, R. L., Oda,A., and Chiga,M. Effect ofActinomycin D on drogenase and Pyrimidine Reductases. Cancer Res., 23: 385â€”391, Deoxycytidylate Deaminase Activity of Rat Liver after Partial 1963. Hepatectomy. Lab. Invest., 13: 1514â€”1519, 1964. 34. Pitot, H. C., Peraino, C., Morse, P.A.,and Potter, V. R. Hepatomas 15. Ives, D. H., Morse,P.A., and Potter, V. R. Feedbackinhibitionof in Tissue Culture Compared with Adapting Liver in vivo. Natl. Thymidine Kinase by Thymidine Triphosphate. J. Biol. Chem., Cancer Inst. Monograph, 13: 229â€”245,1964. 238: 1467â€”1474, 1963. 35. 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Muhblock (eds.), Cellular Control 19. Lea, M. A., Morris, H.P., and Weber, G. Comparative Biochemistry Mechanisms and Cancer, pp. 190â€”210.Amsterdam: Elsevier Pub of Hepatomas. VI. Thymidine Incorporation into DNA as a Mea lashing Company, 1964. sure of Hepatoma Growth Rate. Cancer Res., 26: 465â€”469, 1966. 39. Potter, V. R., Gebert, R. A., Pitot, H. C., Peraino, C., Lamar, C., 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Lesher, S., and Morris, H. P. Systematic Oscillations in Metabolic Protein Measurement with the Folin Phenol Reagent. J. Biol. Activity in Rat Liver and in Hepatomas. I. Morris Hepatoma No. Chem., 193: 265â€”275,1951. 7793. Cancer Res., 26: 1547â€”1560,1966. 21. Maley,F., and Maley, G. F. NucleotideInterconversions.II.Eleva 40. Potter, V. R., Pitot, H. C.,Ono,T., and Morris, H. P. The Compara tion of Deoxycytidylate Deaminase and Thymidylate Synthetase in tive Enzymology and Cell Origin of Rat Hepatomas. I. Deoxy Regenerating Rat Liver. J. 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Thomas W. Sneider, Van R. Potter and Harold P. Morris

Cancer Res 1969;29:40-54.

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