[CANCER RESEARCH 29, 40—54, January 1969]

phosphates, dATP4, dGTP, dCTP, and dTFP. Since thymine isthe only base unique to DNA, a study of the control of pathways leading to the synthesis of dTTP might lead to an understanding of the control of DNA synthesis (52). Chart 1 illustrates these latter pathways as they may appear in variouscombinations in mammalian tissues.

That the activities of the enzymes catalyzing these reactionsare rigidly controlled and, to some extent, correlatable withgrowth and cell division, is illustrated by the following data.The activities of the ribonucleotide reductase(s), dCMP deaminase, dTMP synthetasc, and TdR and dTMP kinases, are highin embryonic and newborn rat liver but decrease with increasing age, such that normal adult rat liver has barely detectablelevels of activities of these enzymes (T. Sneider, unpublisheddata) (24, 27). If, however, an adult rat is subjected to partialhepatectomy, the activities of all of the aforementioned enzymes rise in an ordered manner prior to the onset of DNAsynthesis and cell division (2, 13, 14, 21—24, 26, 27). if theposthepatectomy increase in dCMP deaminase (14, 26) andTdR kinase (26) is prevented by actinomycin D, the subsequent onset of DNA synthesis and cell division is also delayed.It is therefore evident that the control(s) of the activities ofthe enzymes of the dTTP pathway is at least temporally related to the mechanism(s) controlling DNA synthesis and celldivision. Although the available evidence is only suggestive, itis possible that the factors that control dTTP pool size couldpartially govern the onset and rate of DNA synthesis or couldbe clues to the primary triggering mechanism(s).

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 ofdeoxyuridine, deoxycytidine, and deoxythymidine; dTDP, the diphos.phate of deoxythymidine; UdR, CdR, and TdR, deoxyuridine, deoxycytidine, and deoxythymidine; DEAE-, diethylaminoethyl-; F3TdR,5-trifluoromethyl-2'-deoxyuridine; 5-FUdR, 5-fiuoro-2'.deoxyuridine;3-PGA, 3-phosphoglyceric acid. The trivial names of, and the reactionscatalyzed by, the enzymes mentioned in this paper are: ribonucleotidereductase(s), purine or pyrimidine ribonucleoside diphosphate todeoxyribonucleoside diphosphate; deoxycytidylate deaminase, dCMPto dUMP; thymidylate synthetase, dUMP to dTMP; thymidine kinase,TdR to dTMP; thymidylate kinase(s), dTMP to dTDP and dTTP;thymine “reductase,―thymine to dihydrothymine, which is subsequently converted to 13-ureidoisobutyric acid (which then yields f@aminoisobutyric acid plus CO2).

40 CANCER RESEARCH VOL.29

Enzymes of Thymidine Triphosphate Synthesis in Selected MorrisH 12epatomas

ThomasW. Sneider,Van R. Potter,andHaroldP. Morris3McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY

The activities of certain enzymes catalyzing reactions leadingto the synthesis of thymidine triphosphate were analyzed invitro in minimal deviation type Morris hepatomas of normal

karyotype as well as in Novikoff and Reuber H-35 cells fromtissue culture and in Ehrlich ascites cells. Most Morris hepatomas had normal or near-normal levels of deoxycytidinemonophosphate deaminase, while Novikoff and Ehrlich cellsand rat thymus and bone marrow showed extreme elevationsin activity of this enzyme. The Novikoff, H-35, and Ehrlichascites cells, and all of the Morris hepatomas except 9633, hadelevated deoxythymidine monophosphate (dTMP) synthetaseactivity. All of the hepatomas and tissue culture material cxhibited elevations in deoxythymidine (TdR) kinase and possibly dTMP kinase activities. Hepatomas 5123C, 7800, and9618A possessed the ability to degrade thymine at lesser ratesthan normal adult rat liver, while hepatoma 9633 and Novikoff and H-35 cells from tissue culture apparently lackedthymine degradative ability. With the exception of dTMPsynthetase, the activities of these enzymes in normal karyotype Morris hepatomas showed no quantitative relationshipwith growth rate. Among the hepatomas and enzymes reported, TdR kinase was the only enzyme with consistentlyelevated activity. These results and certain unpublished dataare discussed with respect to possible alternative routes todeoxythymidine triphosphate synthesis and the chemotherapyof cancer.

INTRODUCTION

A prerequisite for the synthesis of DNA and subsequent celldivision is the synthesis of the four deoxyribonucleoside tri

1This work was supported in part by Grant No. P-448 from theAmerican Cancer Society, by USPHS Grant No. TO1-CA-5002 from theNational Cancer Institute, and by USPHS Grant No. CA-10729. A preliminary report was given before the American Association for CancerResearch in Chicago, Illinois, in April 1967 (50).

2Thispaperis the fIfth in aseriesentitledTheComparativeEnzymol.og@yand Cell Origin of Rat Hepatomas.

3Laboratory of Biochemistry, National Cancer Insitute, NIH, Bethesda, Maryland 20014. Present address: Department of Biochemistry,College of Medicine, Howard University, Washington, D. C. 20001. Supported in part by USPHS Grant No. CA-10729.

Received February 23, 1968; accepted September 5, 1968

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

dlTP Synthesis in Morris Hepatomas

5123, 7800, and 7794A and Reuber H-35, have beenextensively studied with respect to the enzymes of the dTTPpathway. More recently developed Morris hepatomas, such as9098,9108,9121,9618A,and9633,arebothslowlygrowingand histologically similar to normal rat hepatocytes. Nowell etal. (31) have performed karyotype analyses of most of theMorris hepatomas and found that seven hepatomas—7794A,7800, 9108, 9098, 9121, 9618A, and 9633—all had forty-twochromosomes, which is the normal diploid chromosome complement for rat. Of these seven hepatomas with a normal chromosome number, hepatomas 9618A and 9633 exhibited completely normal chromosome morphology. The remaining fivehepatomas had minor changes in chromosomal morphology,such as abnormal satellites on certain chromosomes or abnormal arm lengths of other chromosomes.

In the present study, the thymidylate synthetase, deoxycytidylate deaminase, thymidine and thymidylate kinases, andthymine catabolic activities of solid hepatomas 5123C, 7794A,7800, 9098, 9108, 9121, 9618A, and 9633 were analyzed. Forcomparative purposes, analyses were performed on host liversfrom the tumor-bearing rats, normal adult rat liver, Novikoffhepatoma cells from tissue culture, Reuber H-35 cells fromtissue culture, and Ehrlich ascites cells.

MATERIALS AND METHODS

The solid hepatomas used in these studies (see Table 1 forpertinent data) were produced by Dr. H. P. Morris of theNational Cancer Institute, Bethesda, Maryland. All of the hepatomas were grown bilaterally in the femoral musculature oftheir respective host rats. Tumor-bearing rats were housed twoto a cage in a room of controlled temperature and humidity.Fluorescent lighting in the animal room was automatically regulated to provide 12 hours of light from 9 P.M. to 9 A.M. and12 hours of darkness from 9 A.M . to 9 P.M .; 12, 30 and 60%protein diets (prepared by General Biochemical Co., ChagrinFalls, Ohio) were made available to the rats only during petiods of darkness from 9 A.M . to 5 P.M . (53). Nontumor-bearingrats (200—250 gui) obtained from Holtzman Co. (Madison,Wisconsin) were similarly fed a 30% protein diet for 8 hoursout of every 24 hours (“8+ 16―regimen) (53).

Novikoff hepatoma cells (line Ni-Si) were grown in suspension culture as previously described (29) and were harvestedby centrifugation. Reuber H-35 hepatoma cells were grown asmonolayers in T-flasks as previously noted (34, 42). Cells werescraped into the medium with a rubber-tipped glass rod andcollected by centrifugation. Ehrlich ascites cells, grown intraperitoneally in mice, were obtained from Dr. Charles Heidelberger of McArdle Laboratory. All three cell types were harvested during the periods of high cell proliferation. The method of harvesting resulted in the equivalent of three washeswith isotonic KC1.

Preparation of Cell Extracts

Both tumor-bearing rats and normal adult rats were sacrificed by decapitation. Livers were excised, rinsed in iced isotonic saline solution, and placed in ice until homogenization.Tumors were excised from the femoral musculature and simi

JANUARY 1969 41

p 5-Me-dCMP

@ CDP UDPI 4'I dCDP dUDPL4@@ -

dCMP—* dU@P—@dTP@P@±dTDP@±dTTP—.DNAT

CdR—Ø UdR Td

4@tDHT@±BUIB—+BAIB——+CO2

Chart 1. Schematic representation of possible pathways of dTTPsynthesis. The relative importance of the various alternative pathways isas yet unknown for any specific cell type (52). The many interlockingfeedback controls within and affecting this scheme (15, 52) are toocomplex to be included in this chart or to be considered in the text.Abbreviations: CDP and dCDP, cytidine and deoxycytidinediphosphate; 5-Me-dCMP, 5-methyldeoxycytidine monophosphate; U,T, and DHT, uracil, thymine, and dihydrothymine; DNA-T, thymine inpolymeric DNA; $3UIB and aAIB, @3-ureido-and i3-amino-isobutyric acid.See Footnote 4 for other abbreviations.

Although the activities of the dTI'P-synthesizing enzymesare controlled to a point of minimal activity in normal adultrat liver, little is known of the status of this pathway in cancerous liver cells that are morphologically and biochemicallysimilar to normal rat liver cells. Such cells are available in theform of the “minimaldeviation― hepatomas developed by H.P. Morris of the National Cancer Institute on the basis ofbiochemical findings first from this laboratory (35—38, 40)and subsequently by many other cooperating groups. Potter etal. (40), Pitot and Potter (35), and Maley and Maley (22) haveshown that dCMP deaminase activity was extremely low innormal adult rat liver, Dunning hepatoma L-C18, and MorrisHepatoma 5123 relative to Novikoff hepatoma or thymus tissue. Roth et al. (45), using a different technic, reported detectable but equivalent levels of dCMP deaminase activity in normal rat liver, Dunning L-C18, Reuber H-35, and Morris Hepatomas 5123, 7800, and 7794A. Maley and Maley (22) havealso shown that dTMP synthetase activity was very low inadult rat liver but elevated substantially in rat liver 24 hoursposthepatectomy and in Morris Hepatoma 5123. Bukovskyand Roth (4), studying Morris hepatoma 5123, and Potter eta!. (39) and Gebert (8), studying Morris hepatomas 7793 and

5123, and 7794A and 7800 respectively, found that all of thehepatomas phosphorylated TdR at rates greater than normaladult rat liver. Bresnick et al. (3) found that a large number ofMorris hepatomas, including hepatomas 5123, 7800, and7794A, as well as Reuber hepatoma H-35, all had elevatedTdR kinase activities compared to normal adult liver. Ono eta!. (32, 33) and Potter et al. (40) have demonstrated that cer

tam hepatomas, such as Morris hepatoma 5123 and its sublines(A, B, C, and D), Morris hepatomas 7800 and 7316A, andReuber H-35, resemble normal adult liver in that they have theability to degrade thymine to CO2@ whereas Novikoff cellsapparently lack this catabolic pathway.

From this brief resume, it is evident that only four slowlygrowing “minimal deviation― hepatomas, Morris hepatomas

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

Table1Time

(mo.)of sacrificeAverage

No. ofmonthsb betweenNumberofChromosomeC1-lepatomaGencrationaHost

ratSexpostinoculationtransplantationstransfersnumber5123C62BuffaloF1.52.06180—997794

A780018

(3)28 (1)Buffalo BuffaloM F2.5 2.34.0 2.6172742―90988A

xCM3.03.7,3.6791089

(2)A xCF2.52.9,4.0842C91219

(2)A xCM2.03.7,1.8842―9618A3BuffaloM4.15.7242196335

(2)BuffaloM2.74.74421

Thomas W. Sneider, Van R. Potter, and Harold P. Morris

“Numbersin 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, connectivetissue and areas of necrosis and hemorrhage were dissected outand discarded. Both the livers and hepatomas were finelyminced with scissors prior to homogenization. After addition ofsufficient 0.2 M Tris-Cl buffer (pH 8.0): 1 X i0@ M dithiothreitol to make 20% (w/v) homogenates, the tissues werehomogenized by means of the Polytron Kinematic Hi-frequency Processer (Kinematic GMBH, Lucerne, Switzerland). Thehomogenates were centrifuged in a Beckman model L2 preparative ultracentrifuge at 104,000 X g for 180 minutes. The clearsupernatants (termed 53 or soluble cell fraction) were decanted by pipet. Assays for thymidylate synthetase and deoxycytidylate deaminase were performed immediately, whereasaliquots of soluble fractions were frozen and stored at —30 Cfor later analysis of TdR and dTMP kinase and thymine catabolic activities. Preliminary studies showed that storage at—30°Cfor two weeks resulted in no loss of activity of theaforementioned enzymes. Longer periods of storage led to increasingly lower dTMP kinase and thymine catabolic activities,while TdR kinase activity was stable for several months. Insome cases (noted in the charts), dTMP kinase and thyminecatabolic activities could not be determined, since the solublefractions were frozen for one month. Generally, however, thefrozen soluble fractions were analyzed after only 1—3days ofstorage at —30°C.

Thymidine Degradation

The ability of tissue extracts to degrade thymine to CO2 wasmeasured by the methods of Canellakis et a!. (5) and Potter eta!. (40) modified as indicated. The reaction mixture contained0.3 @.zmoleof triphosphopyridine nucleotide; 1.8 @molesofglucose-6-phosphate; 9.3 j.zmoles of Mg@; 20.9 @.zmolesof nicotinamide; 0.22 enzyme unit of glucose-6-phosphate dehydrogenase; 60 @zmolesof Tris Cl buffer, pH 8.0; 0.03 pmole ofdithiothreitol; and 0.1 @zmoleof thymine-2-'4C; in a total volume of 1 .0 ml. The specific activity of the thymine preparations used ranged from 12,398 to 15,898 cpm/m@zmole. Thecofactor solution was placed in a 12-nil conical centrifuge tubein a volume of 0.7 ml and prewarmed to 37°C.Then 0.3 ml ofsoluble fraction was added, and the tube contents mixed andreplaced in the 37°C water bath. At 0, 10, 20, and 30 minutes

of incubation, 0.2-ml aliquots of the reaction mixture werewithdrawn and pipetted into centrifuge tubes placed in a boil

ing water bath. The reaction was stopped by 2 minutes of thisheat treatment. Each tube was acidified by the addition of0.05 ml of 90% formic acid. After a low-speed centrifugationto remove the protein coagulum, the supernatants were decanted and stored at —30°Cuntil further analysis. Afterstreaking an aliquot of 0.05 ml of each acidified supernatantonto 1 x 2.5 inch strips of Whatman No. 1 filter paper, thestrips were dried under infrared lamps and placed around theinside of glass liquid scintillation vials. Then 15 nil of a liquidscintillant [4 gm 2,5-diphenyloxazole and 50 mg 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene brought to a volume of1000 ml with reagent grade toluene] were added, and the vialswere counted in a Packard Tri-Carb liquid scintillation spectrometer. The total counts per reaction mixture were plotted,versus the time of incubation. The rate of decrease of countsper reaction mixture was graphically obtained. The amount ofthymine degraded to CO2 was calculated as

(decrease in total c@m/reaction mix/60 mm (1000)(cpm/mj.@mole thymme) (mg 53 protein/reaction mix)

which yields the millimicromoles of thymine degraded to CO2per gram of 53 protein per hour. These values, however, areminimal estimates of thymine degradative capacity since dilution of the labeled precursor by endogenous thymine wouldlower the specific activity of the thymine.

Thymidine Kinase Assay

Thymidine kinase activities were determined by the methodsof Ives et al. (15), Potter et al. (39), and Gebert (8). Thereaction mixture consisted of 0.2 ml of a cofactor solutioncontaining 18 @imolcs of Na@ATP4H2O, 15 j.zmoles of 3-phosphoglyceric acid (sodium salt), and 18 j.zmoles ofMgCl2 .6H@; 0.6 ml of Tris-Cl buffer, 100 @zmoles,pH 8.0; 0.4ml of TdR-2-'4C, 0.2 pmole, about 4 @icuries;0.8 ml of solublefraction made in 0.2 M Tris-Cl buffer (pH 8.0): 1 X iO@ Mdithiothreitol. The total volume of the complete incubationmixture was 2.0 ml. The concentrations of the various constituents were: ATP, 9 mM ; 3-PGA, 7.5 mM ; Mg@ 9 mM ; Tris-Cl,65 mr@i; TdR, 0.1 mM . Most preparations of TdR-2-' 4C had a

CANCER RESEARCH VOL.2942

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

dTTPSynthesisinMorrisHepatomasspecific activity of about 30,000 cpm/m@imole when countedin the system described below. The various cofactors wereplaced in a conical 12-nd centrifuge tube and prewarmed in a37°C water-bath. The soluble fraction was then added andmixed with the tube contents, a zero-time aliquot taken, andthe tube replaced in the 37°Cwater bath. Further aliquots (ofapproximately 0.2 ml each) were removed after 10, 20, 30, 60,90, and 120 minutes of incubation. Each timed aliquot wasimmediately pipetted into centrifuge tubes placed in a boilingwater bath, and the reaction was terminated by 2 minutes ofsuch heat treatment. After removal of the protein coagulumby low-speed centrifugation, the supernatants were removedand stored at —30°Cuntil chromatographic analyses could bemade (usually within 2 days). At that time, 0.025 ml of eachtimed aliquot was streaked onto 1 x 22.5 inch strips of DEAEmodified chromatography paper (DE-81). Unlabeled dTMPand TdR (1 @moleeach) were also streaked onto the origin ofeach strip. The strips were clipped onto glass frames, placed ina chromatography cabinet and developed by descending chromatography with one of several solvent pairs. The time required for the solvent front to travel about 20 inches wasusually 3.5—4.5 hours. Since the ion exchange capacity of theDEAE paper was found to vary drastically (even within thesame lot of paper), the solvent pair that would effectivelyseparate TdR from dTMP from dTDP-dTTP had to be determined for any given set of DEAE strips. The solvents rangedfrom 4 N HCOOH:0.1 M NH4HCOO to 0.1 N HCOOH:0.1 MNH4HCOO. The TdR and dTMP regions were located underUV light, cut out, and placed around the inside of glass liquidscintillation vials. Due to the variability of the DEAE paper,dTDP and dITP did not always remain at the origin. It wastherefore necessary to divide the strip between the origin andthe beginning of the dTMP region into 2- to 2.5-inch sectionsand measure the radioactivity present in all sections of thestrip. The cpm/reaction mixture for TdR, dTMP + dTDP +dTTP, and dTDP + dTFP were then plotted versus the time ofincubation. Since thymidylate kinase and nucleoside diphosphokinase were present in the crude cell extracts, the totalamount of TdR phosphorylated by thymidine kinase was obtamed by adding the radioactivity present in the dTMP, dTDP,and d'Vi'P regions of each strip. Moreover, dTTP formed in thereaction mixture is known to inhibit TdR kinase activity (15).Hence, the initial rate of appearance of dTMP + dTDP + dTTPwas taken as a measure of TdR kinase activity. Results areexpressed as nanomoles of TdR phosphorylated per gram ofsupernatant protein per hour. The rate of appearance of dTDP+ dTTP was taken as a measure of thymidylate k.inase activity.

It should be pointed out immediately that this method provides only the crudest quantitation of thymidylate kinase activity. First, the de novo synthesis of dTMP may be sufficiently great in some of the hepatomas to significantly dilute thelabeled dTMP formed via TdR kinase. Hence, use of the specific activity of the labeled TdR to calculate the amount ofdTDP-dTTP formed can result in an underestimation of dTMPkinase activity. Secondly, the labeled substrate for thymidylate kinase (dTMP) is produced via the action of TdR kinase.The concentration of dTMP depends on both the activity ofTdR kinase in any given tissue extract used and the time ofincubation. The possibility therefore exists that thymidylate

kinase may not always be measured under conditions of substrate saturation. In the present studies, thymidylate kinaseactivities were calculated from plots of the amount of dTDP +dTTP versus incubation time of the reaction mixture. In mostcases, a constant rate of production of dTDP + dTI'P wasobserved after 60 minutes of incubation. This rate was takenas a measure of dTMP-kinase activity. In certain instances,however, the rate of appearance of dTDP + dTFP continued toincrease even after 120 minutes of incubation (Chart 2).Thymidylate kinase activities in these extracts were inexactlyestimated by employing a rate determined from the 90 and120 minute time points. Although dTMP kinase activities determined in this manner may be gross underestimations, theymay be used to determine if a given hepatoma possesses elevated dTMP kinase activity compared to normal or host livers(whichactivitieswerebasedonaconstantrateof productionof dTDP + dTTP). More precise determinations of thymidylatekinase activities would require the use of the actual substrate(dTMP-2-'4 C) in the in vitro assay system. Hepatomas 9618Aand 9633 will be reanalyzed in this manner. The method usedin the present paper, however, suffices to give semiquantitativedata for most of the hepatomas analyzed.

Thymidylate Synthetase Assay

Thymidylate synthetase activity was assayed by modifications of the technics of Hartmann and Heidelberger (1 1). Thereaction mixture contained 50 @molesof phosphate buffer, pH6.8 ; 15 @.Lmolesof sodium fluoride; 4 @.zmolesof formaldehyde;1 j.@mole of d,l-tetrahydrofolic acid; and 60—80 millimicromoles of deoxyuridine-5'-monophosphate-6-3H. Controlstudies using crude enzyme from regenerating rat liver showedthat (a) doubling the concentration of the labeled substrateresulted in no increase in measured enzyme activity, and (b)the rate of reaction remained linear over a 30-minute periodwith either level of substrate. Hence, the lower level of substrate was used in these studies. Various preparations of tntiated deoxyuridylic acid were used in these experiments. Thespecific activities of the dUMP-3H varied from preparation topreparation (16,000—50,000 dpm/millimicromole). The various cofactors were added to conical 1 2-ml centrifuge tubes ina volume of 0.3 ml. The volume of soluble fraction per reaction vessel was 0.2 ml, which usually contained about 5 mg ofprotein. The total volume of the reaction mixture was 0.5 mlin all cases.

Incubations were carried out for 30 minutes at 37°C. Thereaction was stopped by immersing the tubes in a boiling waterbath for 3 minutes. After cooling the tubes in an ice bath, 1mg of crude Crotalus adamanteus venom in a volume of 0.1 mlwas added to each tube to dephosphorylate the nucleotides,and a second 37°C incubation was performed for 30 minutes.The reaction was again stopped by heat, the tubes cooled in anice bath, and 5 .zmoles each ofunlabeled UdR and TdR addedto each tube. The reaction mixtures were then desalted bypassage through small individual columns (10 mm diameter)containing an 8-mm layer of Dowex-1-formate X 12 (200—400mesh) over an 8-mm layer of Dowex-50W-H@ X 10 (200—400mesh). Each reaction tube was washed five times with 0.5 mlof water. The adsorption eluate, as well as the tube washes,

43JANUARY 1969

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

TMP+TDP+TTP-@ —

20

Thomas W. Sneider, Van R. Potter, and Harold P. Morris

PHOSPHORYLATION OF TdR IN:

ADULT RAT LIVER

KJ2030 60 90MINUTES OF INCUBATION

NOVIKOFFt.c. CELLS

5.5.

\%%%@@ ,pTNP

—@0 KD20 3040 60 80 100 120

MINUTES OF INCUBATION

9618A OR 9633

TdR

TMP+TDP+TTP@.@

-@-‘—@.. TMP@

0102030 6ó 90MINUTES OF INCUBATION

0TMP+TDP+TTP-@_ ——

—

,g/S/' +I I P

20

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

were collected in 12-ml centrifuge tubes that were attached toeach column. Each column was further washed with wateruntil a total volume of 10—11 ml was collected. The columneluate tubes were then evaporated to dryness and the residueredissolved in a volume of 0.5 ml of water. A 0.05-ml aliquotwas then spotted onto an 18 x 22.5 inch sheet of WhatmanNo. 1 paper, and chromatography and counting were carriedout as described below. Control experiments indicated thatvirtually 100% of the UdR and TdR present in the reactionmixture could be recovered in the redissolved column eluateand that the snake venom treatment resulted in dephosphorylation of all of the dUMP and dTMP to UdR and TdR. Later,however, incomplete recovery of the total radioactivity in thesystem from the columns was sometimes observed and was dueto incomplete breakdown of the deoxynibonucleotides todeoxyribonucleosides. The incomplete breakdown to deoxynibonucleosides was due to both variations in the crude snakevenoms employed and the inhibitory effect of the phosphatebuffer in the reaction mixture. In order to circumvent thisproblem, the following procedure has been employed: theheat-stopped reaction mixture was reincubated in the presenceof 2.0 mg of crude Crotalus adamanteus venom for 30 minutesat 37°C, followed by addition of 5 pmoles each of unlabeledUdR and TdR in a combined volume of 0.1 ml. After mixingand centrifuging out the protein coagulum, two 0.05-mi allquots of the supernatant were removed from each tube. Onealiquot was pipetted into a glass vial, the scintillator describedbelow added, and the total tritium activity determined. Thesecond aliquot was streaked onto a 1 x 22.5 inch strip ofWhatman No. 1 chromatography paper. After clipping the

strips to a glass frame and equilibrating for 60 minutes in theclosed chromatography cabinet, tertiary butanol: 2-butanone:water:ammonium hydroxide solvent (40:30:20:10) (7) wasadded to the troughs, and the strips were developed for 24—36hours or until the solvent front had traveled approximately 20inches. After air-drying the strips, the UdR and TdR regionswere located by their UV absorption. The UdR and TdR spotsfrom each reaction mixture were cut out and placed in separate scintillation vials. Following addition of 0.5 ml of anhydrous methanol the vial was capped, gently agitated, and thenallowed to stand at room temperature for 30 minutes beforeadding 10 ml of liquid scintillation fluid. The scintillator solution consisted of naphthalene, 80 gm ; 2,5-diphenyloxazole, 5gm; alpha-naphthylphenyloxazole, 50 mg; dissolved in 1 literof a mixture of 5 parts xylene, 5 parts dioxane, and 3 partsethanol (17). The radioactivity present in each vial was determined in a Packard Tri-Carb liquid scintillation spectrometer.Control experiments showed that (a) the methanol treatmentelutes all of the UdR and TdR from the paper, (b) the use ofautomatic external standardization with such a counting system was valid, and (c) the efficiency of counting in such asystem was approximately 23%. The completeness of thesnake venom treatment was estimated by calculating the percentage of the total activity represented in the UdR and TdRbands. In most instances, the venom treatment resulted inabout 60% breakdown. Hence, the UdR and TdR activitieswere corrected to values that would obtain if the breakdownhad been complete. The completeness of dephosphorylationwas individually determined for each reaction mixture. Thevalidity of this correction rests upon the observations that (a)

44 CANCER RESEARCH VOL.29

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

d77'P Synthesis in Morris Hepatomas

no other deoxyribonucleosides were found on the chromatograms and (b) the deoxyribonucleoside monophosphates remaining at the origin of the chromatogram accounted for the

remainder of the radioactivity in the aliquot analyzed. Hence,all of the nucleosides produced by the venom treatment wereaccounted for, and the efficiency of breakdown could be calculated. Enzyme specific activity is expressed in this paper asmillimicromoles of dTMP formed per gram of supernatant protein per hour of incubation at 37°Cand is calculated from theexperimentally determined specific activity of the labeled precursor. Implicit in this calculation is the assumption that theendogenous pool size of dUMP is negligible; this is suggestedby the work of Schneider and Brownell (48). While this assumption is valid for nonproliferating tissues, it may not holdfor tissues exhibiting extensive DNA synthesis. Therefore,values for thymidylate synthetase activities in proliferating tissues should be considered as minimal estimates. Under ourconditions, the assay is linear both with time of incubationand amount of crude enzyme. The lower limit of sensitivity ofthe assay was approximately 20 millimicromoles per gram ofsoluble fraction protein per hour (about 0.05—0.1 millimicromole of dTMP formed per reaction vessel per 30 minutes).Duplicate assays were performed on all samples and generallyagreed to within 5%.

Deoxycytidylate Deaminase Assay

Deoxycytidylate deaminase activity was assayed by modifications of the technics of Maley and Maley (25). The reactionmixtures contained 25 @.zmolesTris-Cl buffer (pH 8.0); 1 @zmoleof MgCl2 ; 15 @.zmolesof NaF; 0.04 pmole of dCTP; and 1pmole of dCMP-2-'4C. The specffic activities of the deoxycytidylic acid-14C used in these studies varied from 16,000 to94,000 cpm4zmole. The various components were added toconical 12-mi centrifuge tubes in a volume of 0.2 ml. Generally, 0.3 ml of soluble fraction, containing about 8 mg of protein, was added to each reaction vessel. Soluble fractions oftissues containing elevated dCMP deaminase activity were diluted prior to analysis. The final volume of reaction mixturewas 0.5 ml in all cases.

Incubations were carried out for 30 minutes at 37°C. Thereaction was stopped by immersing the tubes in a boiling waterbath for 3 minutes. After removal of the protein coagulum bycentrifugation, the supernatants were decanted and stored at—20°Cuntil chromatographic analyses could be performed.Early studies of the deamination of dCMP with time of incubation indicated that the rate of reaction was not linear withtime. Further experiments demonstrated that this nonlinearitywas only apparent. Heat treatment of the reaction vessel didnot completely stop the deamination reaction. If the heatedreaction vessel was allowed to stand at room temperature, significant quantities of dCMP were further deaminated. Furthermore, the extent of this residual deamination depended uponseveral factors. Crude enzyme from 48-hour posthepatectomyrat liver and from normal rat liver were heat-treated at severaltemperatures for vanioi..@lengths of time in the absence ofreaction mixture components. The various cofactors were thenadded to each tube, and deaminase activity was assayed asnoted previously. After the incubation period, all tubes were

again heat treated at 100°C for 3 minutes and immediatelychilled at 0°C. Chromatography was carried out as describedbelow. The results, illustrated in Chart 3, show that the residual deamination depends upon (a) the source of crude enzyme,(b) the temperature at which the heat pretreatment is performed, and (c) the time for which the crude enzyme isheated. If the heat-treated complete reaction mixture was heldat 0°Cuntil the supernatant was decanted and frozen, no residual deamination was observed. Under these conditions, linearity of the reaction with time of incubation and quantity ofenzyme were noted. Hence, all enzyme assays were performedin this way. No further attempt was made to clarify the natureand mechanism(s) of the residual deamination.

Chromatographic separation of dCMP from dUMP wascarried out in two ways. In early experiments, 100 @ilof reaction mixture were streaked onto the origin of a 1 x 22.5 inchstrip of DEAE-substituted chromatography paper (DE-81,Whatman Ltd.). Unlabeled dCMP and dUMP (1 @.zmoleeach)were also streaked onto the origin. After clipping to glassframes, the strips were developed by descending chromatography with 1.0 N formic acid, in 0.1 M ammonium formate.The usual running time for a 20-inch solvent front was 3.5—4hours. After air-drying the strips on the frames, the dCMP anddUMP regions (RF's of 0.8 and 0.4 respectively) were locatedby their UV absorption, cut out, and placed in separate glassscintillation vials. After adding 15 ml of liquid scintillation

EFFECT OF PRE-INCUBATIOP4AT VARIOUS TEMPERATURES ONDEOXYCYTIDYLATE DEAMINASE ftCTIVITY IN CELL EXTRACTS FROM:

zz

NORMALADULT RAT LIVER RAT LIVER 24 HOURSAFTERPARTIAL HEPATECTOMY

Chart 3. Deoxycytidylate deaminase activity (as a percent of thenonpreincubated samples) of soluble cell extracts from normal adult ratliver or 24-hour postpartial hepatectomy rat liver after periods of preincubation for the times and temperatures noted.

2 4 6

MINUTES OF PRE-INCUBATION

JANUARY 1969 45

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

Thomas W Sneider, Van R. Potter, and Harold P. Morris

solution [4 gm 2,5-diphenyloxazole and 50 mg 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene brought to a volume of1000 ml with reagent grade toluene] to each vial, the amountof labeled nucleotide was determined in a Packard Tri-Carbliquid scintillation spectrometer. Preliminary experimentsdemonstrated that (a) this method of counting was linear withrespect to the amount of labeled material on the paper versusthe count rate observed, (b) the orientation of the paper in thevial was irrelevant to the linearity of counting, and (c) theabsolute efficiency of counting in this system was about 71%.

The above method of chromatography of dCMP and dUMPwas especially satisfactory in that it was very rapid and gaveexcellent separations of precursor from product. In the courseof the present studies, different lots of DEAE-substitutedpaper had to be employed. We were completely unable toreproduce the dCMP and dUMP separation with these latterbatches of paper. We were also unable to affect this separationby using both a wide range of eluants and prewashed and/orpretreated paper. We therefore modified the aforementionedmethod as follows: After the initial incubation and heat treatment, an aliquot of the reaction mixture supernatant was incubated at 37°C for 30 minutes with 0.5 mg of crude Crotalusadamanteus venom. The reaction was terminated by heat and a100-,.zl aliquot was streaked onto 1 x 22.5 inch strips ofWhatman No. 1 filter paper. After adding 1 .zmole eachof unlabeled UdR and CdR to the origin, the strips wereclipped to glass frames and developed by descending chromatography with tertiary butanol: 2-butanone:water: formic acid(44:44:11:26) (7). The solvent front travels about 20 inchesin 24 hours. In this system the mononucleotides remain at theorigin. CdR has an RF of about 0.2 and UdR has an RF ofabout 0.5. The UdR and CdR regions were located by theirUV absorption, cut out, and placed in glass scintillation vials.Counting was carried out as above. Preliminary studies showedthat the incubation with crude snake venom degraded most ofthe dCMP and dUMP to CdR and UdR. In all experiments,however, the radioactivity remaining at the origin was measured and the percent breakdown of deoxyribonucleotides todeoxyribonucleosides was calculated. The corresponding deoxyribonucleoside counting data were then corrected for anyincomplete breakdown. Furthermore, the presence of fluoridein the original reaction mixture prevented breakdown of thedCMP to CdR during the course of the first incubation. Hence,the UdR noted after the incubation with snake venom was aresult of dCMP deaminase activity and not of CdR deaminaseactivity. Since no exogenous energy source or source ofmethylenetetrahydrofolic acid has been supplied in this assay,it is unlikely that dTMP or its di- and triphosphate could beformed. In fact, pilot studies of tissues with high dTMPsynthetase activities show no conversion of labeled dUMPformed via the deaminase reaction to labeled dTMP.

Calculation of dCMP deaminase activity was similar to thatnoted for dTMP synthetase. The specific activity of the precursor was determined by UV analysis and by counting an aliquotof the precursor on Whatman No. 1 filter paper. The enzymeactivities in this paper are expressed as millimicromoles ofdCMP deaminated per gram of soluble fraction protein perhour. It should be noted that the above calculation ignores thesize of the endogenous dCMP pool. Thus, values for dCMP

deaminase activities must be considered as minimal estimates.The effect of pool size, however, is unlikely to be so great asto reduce the amount of labeled dUMP formed to the level ofdetection of the assay. Reinforcing this opinion is the fact thatNovikoff hepatoma cells have high levels of ribotide reductase(s) (27), yet they also demonstrate very high dCMP deaminase activity when assayed with the present method. The effect of dilution of the labeled precursor is evidently offset bythe greater rate of deamination of dCMP.

The lower limit of sensitivity of the assay was approximately500 millimicromoles dCMP deaminated per gram of solublefraction protein per hour (about 2 millimicromoles per reaction vessel per 30 minutes) in most experiments. All assayswere done in duplicate and results agreed to within 5%.

Determination of Proteins

The amount of protein present in the soluble fraction oftissue homogenates was determined by the Lowry et aL (20)Folin-Ciocalteu phenol reagent method. A single lot of crystalline bovine serum albumin was employed as a reference standard throughout these studies. This method, as performed inour laboratory, gave a linear plot of O.D.7oom,, versus proteinconcentration over the range of 5—90 @gof protein. Hence thesoluble fractions of 20% (w/v) homogenates were diluted200-fold prior to protein analysis. The miniscule amount ofCleland's reagent in the highly diluted supernatant fractionsdid not interfere with the protein determination.

Reagents

Tris buffers were made with heavy metal-free “A―gradetris-(hydroxymethyl)-aminomethane from Calbiochem (LosAngeles, Calif.). Cleland's reagent (dithiothreitol) was obtainedfrom the same source. d,l-Tetrahydrofolic acid was purchasedfrom Nutritional Biochemical Corp. (Cleveland, Ohio), storedin the dark in the presence of 1.0 M mercaptoethanol, andsealed under nitrogen. Tritiated dUMP was obtained from twosources. In early experiments, dUMP-3H was prepared fromdCMP-3H by nitrous acid deamination (1 1) and subsequentlypurified by Dowex-l-formate ion-exchange chromatography.Later experiments utilized dUMP-6-3H, which became available from Schwarz BioResearch (Orangeburg, N. Y.).dCMP-2-'4 C, TdR-2-'4 C, and thymine-2-14 C were also obtamed from this source. Unlabeled deoxyribonucleosides anddeoxyribonucleotides were obtained from Sigma ChemicalCorp. (St. Louis, Mo.) and Calbiochem. Crotalus adamanteusvenom was purchased from the Ross Allen Reptile Farm (Sarasota Springs, Fla.) and from the Sigma Chemical Corp. Crystalline bovine serum albumin was obtained from Sigma ChemicalCo. Naphthalene was purchased from Matheson Scientific Co.(Elk Grove Village, Ill.) and dioxane and xylene from FisherScientific Co. (Chicago, Ill.). Alphanaphthylphenyloxazole,2,5-diphenyloxazole, and 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene were all products of Parkard Instrument Co.(La Grange, Ill.). DEAE-modified cellulose chromatographypaper (DE-81) was obtained from Reeve Angel Co. (Clifton, N.J.). Allother chemicalswereof reagentgradeor better.

46 CANCER RESEARCH VOL.29

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

d77'P Synthesis in Morris Hepatomas

CEOXYCYTIDYLATEDEAMINASE

EJ@ HOSTLIVER@@ HEPATOMAChart 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 numberof 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 4—7have 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.

96I8A 9633 7794A 9108 9098 7800 5I23C 9121 H-35 NOVIIØFEHRLICHtc t.c. ASCITES

N@6 99 1218 514642402525 55 99 2323 2 6 2

I

RESULTS

The activities of the enzymes catalyzing the synthesis ofthymidine triphosphate in the normal and tumor tissues notedare illustrated in Charts 4 through 8 . Although the rats bearingthe hepatomas were fed diets containing 12, 30, or 60% protein and were sacrificed at various times of day, in only oneinstance (hepatoma 9633) was there any effect of these vanables on the activities of any of the enzymes analyzed (cf 39,43). Therefore enzyme data from all animals, regardless of theprotein composition of the diet or the time of killing, wereaveraged to yield the values recorded in the charts.

Enzyme activities in the various tumor tissues are comparedwith values obtained from analysis of livers of adult Holtzmanrats and of the host rats. Since unpublished data from theauthors' laboratory and information in the literature (cf 8,39) indicate that livers from adult rats of several strains all havelow levels of activities for the enzymes studied here, no analyses were made of livers from nontumor-bearing AxC or Buffalostrain rats.

dCMP Deaminase

Many of the hepatomas exhibited dCMP deaminase activitiesequivalent to the low levels noted in normal adult rat liver(Chart 4). Hepatoma 9098 had reduced deaminase activity(21% of host liver) while hepatomas 5123, 9121, and 9633had slightly elevated levels of enzyme activity compared tohost liver (178%, 214%, and 139% respectively). Novikoff hepatoma cells from tissue culture and Ehrlich ascites cells con

tamed undetectable levels of dCMP deaminase activity if thesoluble cell fractions were prepared from cells homogenized inbuffer containing no dCTP. If homogenization was carried outin buffer 0.5 to 1.0 mM in dCTP, high levels of deaminaseactivity were measurable (900- and 202-fold in the two cell

types respectively compared to adult liver). Reuber H-35 hepatoma cells, grown as monolayers in tissue culture, showed nodetectable deaminase activity regardless of the presence of 0.5or 1.0 mM dCTP in the homogenizing medium. The presenceor absence of dTCP in the homogenizing medium did not affect deaminase activity of normal liver, hepatomas 7800 and9618A, or their host livers, while hepatoma 9633 showed onlyslight increases in dCMP deaminase activity when the homogenizing medium was 0.5 mM in dCTP. The remainder of thehepatomas in Chart 4 were homogenized in media that did notcontain dCTP.

dTMP Synthetase

With the exception of hepatoma 9633, all of the hepatomasanalyzed exhibited elevated dTMP synthetase activities (Chart5). Hepatoma 5123C had 2050% of the adult liver value; 7800,1500%; 7794A, 940%; 9098, 1370%; 9108, 1160%; 9121,

4150%; 9618A, 350%; Novikoff cells from tissue culture,2740%; H-35 hepatoma cells from tissue culture, 340%; and

Ehrlich ascites cells, 2060%. Experiments with Novikoff hepatoma cells from suspension culture indicated that dTMPsynthetase activity rapidly decayed during homogenizationand preparation of the soluble cell fraction (T. Sneider, unpublished data). These studies indicated that dTMP synthetase

JANUARY 1969 47

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

N@6 3 8 99471413495599 99 2 6

Thomas W. Sneider, Van R. Potter, and Harold P. Morris

THYMIDYLATE SYNTHETASE

EJ aHOSTLIVER @•:@ HEF*TOMA

NOVIKOFFEHRIJCHtc. •@ASCITES96I8A 9633 7794A 9108 9098 7800 5123C 912I H-35t. C.

@I5 5I46@40@25 55 99 2323 2 6 2

Chart 5. Levels of thymidylate synthetaseRemainder as in Chart 4.

activity in liver, solid Morris hepatomas, and selected tissue culture (t.c.) and ascites material.

TdR KINASEADULTLIVER 96I8A 963@3 7794A 9108 9098 7800 5I23C 9121

Chart 6. Levels of thymidine (TdR) kinase activity in liver, solid Morris hepatomas, and selected tissue culture (t.c.) material. The value abovethe 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 usualmanner. Remainder as in Chart 4.

H-35 N@1IKOFFt.c. t.c.

450O@

44500

33500

3@

JO 500

@Oo0@

.@,250(j20@E 150(

I0O(

50(

EJ@ HOSTUVER @@HEPAT0MA

48 CANCER RESEARCH VOL.29

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

d7TP Synthesis in Moms Hepatomas

THYMIDYLATE KINASE CS)

I@ 96I8A96339108909878005123C@IO300F

IO2OOJ@

:@490c1 ±761

3 .....

2480 :@@% ±227

!:@

@4@

@400

@:@ -@ Ji:@ —

Nu6 38 1114 I4I2 49 55 99 2 6

E:J@ HOSTLIVER @•:•HEPATOMAChart 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 (seeMethods and Results sections for further details). Remainder as in Charts 4 and 6.

THYMINE DEGRADATION

activity could be preserved during the preparation of the soluble cell fraction by utilizing very concentrated homogenates.In view of these studies, hepatoma 9633 was renalyzed. Soluble cell fractions were prepared from concentrated homogenates of 9633. Even under these conditions no dTMP synthetaseactivity was detectable.

TdR Kinase and dTMP Kinase

All of the hepatomas studied showed elevated levels of TdRkinase (Chart 6). Hepatoma 5123C had 2680% of the adultliver value; 7800, 1470%; 7794A, 450%, 9098, 300%; 9108,680%; 9121,4100%; 9618A, 765%; 9633,1090%; Novilcoffcells from tissue culture, 17,830%; and H-35 hepatoma cellsfrom tissue culture, 19,550%. of six Morris hepatoma linesanalyzed (Chart 7), hepatomas 5123C, 7800, 9098, and 9108showed elevated dTMP kinase activities (8130%, 1510%,850%, and 720% respectively compared to host liver values).The soluble cell fractions from hepatomas 7794A and 9121had been stored at —30°Cfor approximately one month.Hence, dTMP kinase activities were not determined for thesesamples. Novikoff and H-35 cells from tissue culture also hadhigh dTMP kinase activities (7100% and 10,280% compared toadult liver values). Although hepatomas 9618A and 9633 possessed dTMP kinase activity, quantitation of the levels of suchactivity was not possible. In analyzing the four aforementioned Morris hepatomas as well as the tissue culture material,a constant rate of production of dTDP and dTTP was generallynoted after 30 to 60 minutes of incubation of reaction mix

ADULTLIVER

H-35 NOVIKOFF9633 7800 5I23C t•@@ @.@

0

zwI-0

E-5..

E

38 99 55 99 2 6

0@ HOST @:a HEPATOMAChart 8. Thymine-2-14C degradation to 14C02 by adult rat liver,

solid Morris hepatomas, and selected tissue culture (t.c.) material. Remainder as in Chart 4.

JANUARY 1969 49

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

Thomas W. Sneider, Van R. Potter, and Harold P. Moms

tures (Chart 2). Incubations of soluble cell fractions from hepatomas 9618A and 9633 led to production of labeled dTDPand dTTP only after 90 and 120 minutes (Chart 2). Inasmuchas the rate of production of labeled dTDP + dTTP continuallyincreased, it was not possible to obtain an accurate rate measurement for the synthesis of dTDP plus dTTP. The amount ofthymidine di- and triphosphate present after 120 minutes ofincubation was much greater (400—500%) than that noted inadult liver or host liver reaction vessels after 120 minutes ofincubation. This latter observation would indicate that thedTMP kinase activities of hepatomas 9618A and 9633 weregreater than adult or host liver, and, if incubation times hadbeen extended, a linear rate of production of dTDP plus dTTPwould have been observed.

Thymine Degradation

Four Morris hepatomas (5123C, 7800, 9618A, and 9633) aswell as Novikoff and H-35 tissue culture cells were assayed fortheir ability to degrade thymine-2-'4 C to 14C02 (Chart 8).Hepatomas 5123C, 7800, and 9618A catabolized thymine atrates lower than normal adult or host livers (54%, 47%, and16% respectively compared to host liver). Thymine catabolicactivity in both the host livers and the hepatomas of rats bearing hepatoma 7800 was grossly elevated compared to normaladult rat liver. This finding is at present unexplained. Reanalysin of a later generation of hepatoma 7800 (sacrificed earlierafter transplantation than the first group) showed normal 1evels of degradative ability in the host livers. The hepatomasagain showed approximately 50% less catabolic activity thanthe host liver. Hepatoma 9633 as well as Novikoff and H-35cells from tissue culture carried out no measurable degradationof thymine to CO2. Hepatomas 7794A, 9098, 9108, and 9121were not assayed for thymine degradative ability.

DISCUSSION

The results reported above represent an attempt to determine if the activities of enzymes catalyzing the synthesis ofdTrP exhibit a characteristic pattern among hepatomas of theminimal deviation type. It was also of interest to determine ifthe activities of these enzymes, which are all under stringentcontrol in normal adult rat liver, were proportionately or disproportionately expressed within a given hepatoma strain. Reduced to their simplest form, these objectives can be expressedin the following questions: Are there enzymatic changes onthe dTTP pathway that can characterize malignancy? Are theactivities of the enzymes of the dTFP pathway coordinatelyexpressed? Only qualified answers to these questions can begiven at the present time.

Deoxycytidylate deaminase activity, which is apparently correlated with DNA synthesis in fetal and regenerating adult ratliver and in certain cell types (35, 40), is not significantlyelevated in any hepatomas of the minimal deviation type but isgrossly elevated in Novikoff cells from tissue culture and Ehrlich ascites cells. This enzyme, however, may not be obligatoryfor DNA synthesis (6, 12), and its activity may reflect differences in cell type (35). Thymidylate synthetase activity is dcvated in most hepatomas analyzed but is apparently low or

difficult to demonstrate in hepatomas 9618A and 9633. Hepatomas 5123C and 7800, which could be classified as moredeviated from hepatocytes on the basis of TdR and dTMP Idnases and dTMP synthetase, were shown to be similar to adultliver, in that they possessed the ability to catabolize thymineand apparently lacked dCMP deaminase activity. Hepatoma9633 is similar to adult liver in that it has normal low levels ofdCMP deaminase and dTMP synthetase but differs from adultliver since it possesses elevated TdR and dTMP kinases andlacks thymine catabolic activity.

While, in general, the 3 enzymes involving thymidylic pathways are strikingly increased in comparison with normal liver,and indeed from 10 to 40-fold in many cases, there is a failureto demonstrate coordinate increases, especially among the hepatomas that are less than 10-fold higher than normal liver. Themost consistent increase in enzyme activity in the hepatomasstudied thus far appears to be thymidine kinase, which waselevated in all hepatomas herein reported and in a series ofearlier Morris hepatomas studied by Bresnick et a!. (3). Excluding 5123C, which cannot be considered as minimal deviation because of karyotype findings (31), the hepatomas with42 chromosomes have elevations of TdR kinase between 3-foldand 40-fold over normal liver. The failure of this enzyme to berepressed under conditions that repress its synthesis in the cellsof homologous origin may be the best indication of malignancy so far available in hepatomas, but the quantitative proportionality with respect to growth cannot be demonstrated whenthe hepatomas with abnormal chromosome numbers are cxcluded (cf. 19). Nevertheless, the consistent elevation inTdR kinase is more striking than that for any other enzyme orenzyme system studied thus far, including glycolysis (cf. 41).

Moreover, the present results seem to indicate that the genescontrolling the synthesis of these dTTP-synthesizing enzymesmay not all be coordinately expressed. For example, hepatomacells of a given strain might be synthesizing large amounts ofdTMP synthetase while expression of the genes for dCMP deaminase is nil. Although this conclusion is only tentative atthis time (see Addendum), it seems likely that coordinate cxpression, if it exists in this system, may apply to alternativepathways leading to dTTP rather than to the entire constellation of possible pathways (Chart 1).

The present data, as well as the two conclusions just discussed, are of immediate significance with respect to (a) thedTrP pathway, (b) the chemotherapy of cancer, and (c) theconcept of the minimal deviation hepatoma. The low levels ofdCMP deaminase in all of the Morris hepatomas analyzed makethe contribution of the dCMP to dUMP step in dTTP synthesisslightly suspect as an obligatory pathway. Either the low levelof dCMP deaminase activity is sufficient to ensure an adequatepool of dUMP for methylation to dTMP or the preferred pathfor the synthesis of dUMP is via reduction of uridine diphosphate. Recent work of Crone and Itzhaki (6) supports earlierdata from this laboratory (12) that the regenerating rat liverutilizes the path UDP to dUDP to dUMP to dTMP in preferenceto CDP to dCDP to dCMP to dUMP to dTMP (See Chart 1).The contribution of dCMP to dUMP was held to be less important in the synthesis of dTMP. What role, therefore, can thedCMP deaminase assume in the dTTP pathway? Our analysesof Novikoff cells and Ehrlich ascites cells, as well as rat thymus

50 CANCER RESEARCH VOL.29

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

dlTP Synthesis in Morris Hepatomas

and bone marrow, are intimately related to this question.(Thymus and marrow dCMP deaminase show 18,000% and4000%, and dTMP synthetase 170% and 400%, respectively ofadult liver levels.) These cells exhibit enormous levels of dCMPdeaminase activity yet show relatively moderate elevations indTMP synthetase activity; this pattern is the reverse of thatseen in most of the Morris hepatomas examined. An ad hocexplanation of these results might be that, in tissues exhibitingeither pattern, the preferred route of dTMP synthesis is viareduction of the ribose moiety of UDP. The elevation—or lackthereof—of dCMP deaminase activity might then reflect thestatus of an ancillary route to dTMP synthesis via deaminationof 5-methyl-dCMP. It is interesting to note that 5-methyldeoxycytidine has been found in several rat tissues (51). Nishihaa et al. (30) and Geraci et a!. (10) have demonstrated thatpartially purified deoxycytidylate deaminase catalyzed thedeamination of 5-methyl-dCMP as well as dCMP. Evidence supporting the existence of this alternative route to dTMP synthesis has been gleaned from recent studies on sea urchin embryosby Bieiavsky and Leong (1), on a strain of neoplastic mousecells in tissue culture by Price et al. (44) and on amethopterinblocked Novikoff cells in tissue culture by Gentry et al. (9).Moreover, Karlstrom and Larsson (16) recently found that asmuch as 75% of the dTTP formed in E. coil must have beensynthesized via deamination of 5-methyl-dCTP. Only 25% ofthe dTIP was synthesized by methylation of dUMP. Thestudies of Bieliavsky and Leong (1) and Karlstrom and Larsson(16) both indicate that S-adenosylmethionine serves as themethyl donor in the conversion of dCMP to 5-methyl-dCMP.The apparent lack of elevated dCMP deaminase activity inMorris hepatomas might indicate absence or continued stringent regulation of the proposed ancillary pathway. A totallydifferent role for dCMP deaminase has recently been proposedby Scarano et al. (46, 47) on the basis of a study of themetabolic heterogeneity of thymine methyl groups in sea urchin embryo DNA. In this model, the deamination of DNA-5-methyl-cytosine leads to base shifts which, in turn, constitutea molecular mechanism for differentiation. While no directevidence supports this most interesting hypothesis, such baseshifts, if reversible, might be involved in initiation of transcription. If the latter is indeed the case, then cells such as Novikoff—which must be transcribing at least those regions of theirgenome concerned with DNA synthesis and cell division at avery high rate—could be expected to have substantial activityof enzymes capable of deaminating dCMP at the polymer ormononucleotide level.

The present results, as well as the preceding comments, beardirectly upon the problem of cancer chemotherapy. Chemotherapeutic agents, such as F3TdR, 5-FUdR, and amethopterin, have been developed to inhibit the dUMP to dTMPstep. The efficacy of such agents depends on several factors,among which must be included the development of resistanceto the agent. In the case of 5-FUdR, a strain of Novikoff cellsin tissue culture has been obtained that is resistant to 5-FUdRbecause of an apparent lack of TdR kinase (29). Hence, the5-FUdR cannot be phosphorylated to the inhibitory form,5-FdUMP. Insensitivity to chemotherapy might also be due tothe lack of the appropriate target molecule. Hepatoma 9633,for example, appears to lack dTMP synthetase activity. This

hepatoma might, therefore, be expected to be insensitive to5-FIJdR but sensitive to agents designed to inhibit the 5-methyl-dCMP to dTMP step. Another tumor treated with5-FUdR might be insensitive because it can obtain enoughTdR from dying or dead host cells to overcome the dUMP todTMP block. Succinctly stated, effective cancer chemotherapyin this area must be predicated upon an effort to block allroutes to dTMP synthesis inasmuch as a given tumor cell linecan potentially “breakout― of its uniquely preferred route todTMP synthesis and choose an alternative route.

Finally, the relationship of the present results to the minimaldeviation hepatoma concept must be considered. This conceptclassed transplantable rat hepatomas into broad categories onthe basis of their relative morphologic and biochemical similarity to normal rat liver (36, 38). It was recognized (31) thatthose hepatomas first found to be in the minimal deviationcategory might, in the future, be replaced by hepatomas thatdeviated to an even lesser extent. The biochemical alterationspossibly relevant to carcinogenesis might then be more easilyidentified and studied on the basis of an on-going process ofselection of experimental material. The criteria to be met by ahepatoma minimally deviating from liver were subsequentlyexpanded by Potter and Watanabe (41)@ who emphasized theneed for comparisons with newborn rat liver (such compari.sons in the case of the dTFP pathways have not yet beencompleted in the authors' laboratory). The minimal deviationconcept, however, did not include the a priori expectation thatonly one or two patterns of enzyme activities and metabolicresponses are present in all hepatomas. Hence, the absence of aunique pattern of enzyme activities along the dTTP pathwayin the hepatomas examined in this study—as well as the variations noted by Potter et al. (43) with respect to carbohydrateand protein metabolism—does not negate the minimal deviation concept. The present results do, however, qualify the contention that individual enzymes of the dTI'P pathway are correlatable with the growth rate of the tumor. On the basis ofstudies including poorly differentiated, rapidly growing hepatomas, it has been stated (54) that thymidylate synthetase,deoxycytidylate deaminase, and thymidine and thymidylatekinases are all elevated in hepatomas to degrees reflecting thegrowth rates of the hepatomas. The catabolism of thymine toCO2 was said to inversely reflect the growth rate. In the present study, however, slowly growing, well-differentiated, andnormal karyotype hepatomas with different growth rates(Table1) wereanalyzed.A comparisonof theaveragetransplantation times listed in Table 1 with the data shown inCharts 4 through 8 clearly illustrates that only dTMP synthetase shows any correlation with growth rate (imperfectlymeasured as transplantation time). The significance of this onepositive correlation with growth rate is not clear, since (a) thecorrelation could break down when more hepatomas of the

5A recent suggestion that the minimal deviation label should be abandoned (55) might well be considered in the context of the more recentdata (41). We agree that the better collective label in many cases mightbe “Morrishepatomas,― while specific hepatoma lines should be referred to by numbers and compared with each other according to theconcept which, as Wu agrees, continues to be fruitful.

JANUARY 1969 51

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

Thomas W. Sneider, Van R. Potter, and Harold P. Morris

at 104,000 x g for 30 minutes. If a concentrated suspension of Novikoff cells is homogenized and subsequently diluted with homogenizingmedium, dTMP synthetase activity will be found in both homogenatesand soluble cell fractions of only the more concentrated dilutions. Little or no enzyme activity is discernible in the homogenates and solublecell fractions from higher dilutions even though the sensitivity of theenzyme assay is such that enzyme activity proportional to the dilutionwould have been easily detectable. The addition of dATP, dGTP, dTl'P,dCTP, dUMP, dTMP, N5'10-methylenetetrahydrofolic acid (at levels of0.5 mM) or of bovine serum albumin (20 mg/mi) to the homogenizingmedium does not aid in retaining the enzyme activity of homogenatesor soluble cell fractions of diluted suspensions of Novikoff cells. Preliminary experiments (T. W. Sneider and V. R. Potter, unpublisheddata) have shown that use of a solution of high molecular weight( >10,000) components of soluble extracts from concentrated homogenates of Novikoff cells or 24-hour regenerating rat liver as a medium forhomogenization makes it possible to detect dTMP synthetase activity indilute suspensions of Novikoff cells. A solution of the low molecularweight (< 10,000) components of soluble extract from a concentratedhomogenate of 24-hour regenerating rate liver, when used as homogenizing medium, also enables detection of enzyme activity in dilute suspensions of Novikoff cells. The high molecular weight (> 10,000) components of normal adult rat liver soluble cell extract and the low molecular weight (< 10,000) components of Novikoff cells and normal adultrat liver soluble cell extracts have no such “protective―effect onenzyme activity. Further studies on these phenomena are continuing inour laboratory. What has been learned thus far, however, dramaticallydemonstrates the need for continual reappraisal of both in vitro enzymeassays and data garnered therefrom. In light of the “paradoxical―phenomena described here, interpretations of and conclusions fromsuch data must be tentative.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Lynn Dean and Miss Stefanie Getfinger for their capable technical assistance and Miss Joyce Becker forproviding the tissue culture material.

REFERENCES

1. Bieliavsky, N., and Leong, G. F. The Effect of FUdR on Segmentstion and Incorporation of 3H-Uracil in Amphibian Embryos. Expti.Cell Res., 47: 261—270,1967.

2. Bollum, F.J., and Potter, V. R. Nucleic Acid Metabolism in Regencrating Liver. VI. Soluble Enzymes Which Convert Thymidine toThymidine Phosphates and DNA. Cancer Res., 19: 561—565,1959.

3. Bresnick,E., Thompson,U. B., Morris, H. P., and Liebeit, A. G.Inhibition of Thymidine Kinase Activity in Liver and Hepatomasby TTP and dCTP. Biochem. Biophys. Res. Commun., 16:278—284, 1964.

4. Bukovsky,J., and Roth, J. A. Some Factors Affecting the Phosphorylation of Thymidine by Transplantable Rat Hepatomas. CancerRes., 25: 358—364,1965.

5. Canellakis, E. S. Pyrimidine Metabolism. I. Enzymatic Pathways ofUracil and Thymine Degradation. J. Biol. Chem., 221: 315—322,1958.

6. Crone, M., and Itzhaki, S. On the Relative Functioning of the Pathways for Formation of Thymidine Nucleotides in the RegeneratingLiver and Spleen of the Rat. Biochim. Biophys. Acts, 95: 8—13,1965.

7. Fink, K., and Adams, W. S. Paper Chromatographic Data for Purifles, Pyrimidines and Derivatives in a Variety of Solvents. J.Chromatog., 22: 118—129,1966.

minimal deviation type are developed and analyzed, and (b)alternative pathways to dTMP synthesis exist that can circumvent the dUMP to dTMP step. If dTMP can be synthesized atrates that do not limit growth via phosphorylation of TdR orvia deamination of 5-methyl-dCMP, then a correlation ofdTMP synthetase activity with growth rate in such tumorsassumes questionable significance. It is therefore felt that apriori predictions about the quantitative enzymatic potentialof the components of the dTTP pathway in hepatomas (andperhaps in any malignant tissue) cannot convincingly be madeat this time. In conclusion, it must be reiterated that there areseveral possible pathways for the synthesis of dTI'P and dCTP(Chart 1). The present data, as well as information akeady inthe literature, reinforce the position that there must be manypatterns of enzyme activities compatible with the conversionof sufficient amounts of uridylic acid to dTI'P and dCTP topermit DNA synthesis. It is therefore important to analyze asmany enzymes as possible along this pathway in a given tissueand to search for new pathways. In this manner, some conceptof possible preferred routes of synthesis of dTTP and dCTP inindividual tissues might be obtained. This same goal can beapproached by autoradiographic and biochemical analyses ofthe incorporation of various labeled precursors into DNA invivo. Utilization of the autoradiographic methodology is virtually mandatory inasmuch as the hepatomas—while relativelyhomogeneous in cell type—are heterogeneous with respect tothe cell cycle and probably with respect to various stages ofmaturation along lines of differentiation (41). Correlation ofthe data from the in vitro enzyme assays and the in vivo precursor incorporation analyses in the specific hepatoma linescould well yield very fundamental information about pyrimidine deoxyribonucleotide synthesis per se and its relation tothe control of DNA synthesis, just as the use of E. coli mutants has proved useful in elucidating aspects of biochemicalgenetics.

ADDENDUM

The present study was based upon enzyme assays of crude extracts ofhepatomas. Thus, the possibility of uncontrolled inhibition or activation or degradation, etc., of enzymes under our experimental conditions must be admitted. For example, Novikoff and H-35 cells fromtissue culture, as well as rat thymus and 24-hour posthepatectomy ratliver, must be homogenized in media containing 0.5 to 1.0 mM dCTP topermit demonstrable activity for dCMP deaminase. Kit et aL (18) havenoted a similar phenomenon. If the homogenizing medium does notcontain dCTP, or if the dCTP is added to the assay mixture after thepreparation of the soluble cell extract, little or no dCMP deaminaseactivity is noted. The addition of dCTP to the homogenizing mediumhas little or no effect on dCMP deaminase activity in normal adult ratliver or on most of the hepatomas analyzed in the present study. Acritical effect of homogenizing conditions also occurs in the analysis ofdTMP synthetase activity: depending upon the tissue analyzed, thepresence or absence of synthetase activity is contingent upon the origiiial concentration of the homogenate. If Novikoff cells, H-35 cells, orchick embryo fibroblasts (all from tissue culture) are homogenized atratios less than one volume of cells to one volume of homogenizingmedium, or if rat thymus or regenerating rat liver are prepared as lessthan 20—30%(w/v) homogenates, little or no dTMP synthetase activitycan be found in either the homogenate (analyzed immediately afterhomogenization) or the supernatants of such homogenates centrifuged

52 CANCER RESEARCH VOL.29

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

dTTPSynthesisinMorrisHepatomas

8. Gebert, R. A. Studies on the Mechanisms of Control of DNA andRNA Metabolism in Minimal Deviation Hepatomas and Normal RatLiver. Ph.D. Dissertation University of Wisconsin, Memorial Library, Madison, Wisconsin, 1966.

9. Gentry, G. A., Morse, P. A. , Ives, D. H., Gebert, R. A., and Potter,V. R. Pyrimidine Metabolism in Tissue Culture Cells Derived fromRat Hepatomas. II. Thymidine Uptake in Suspension Cultures Dcrived from the Novikoff Hepatoma. Cancer Res., 25: 509—516,1965.

10. Geraci, G., Rossi, M., and Scarano, E. Deoxycytidylate Aminohydrolase. I. Preparation and Properties of the Homogeneous Enzyme. Biochemistry, 6: 183—191, 1967.

11. Hartman, K-U., and Heidelberger, C. Studies on Fluorinated Pyrimi

dines. XIII. Inhibition of Thymidylate Synthetase. J. BioL Chem.,236: 3006—3013, 1961.

12. Hecht, L. I., and Potter, V. R. Nucleic Acid Metabolism in Regencrating Rat Liver. III. Intermediates in the Synthesis of DNA Pyrimidine Nucleotides. Cancer Res., 16: 999—1004, 1956.

13. Hiatt, H. H., andBojarski,T. B. StimulationofThymidylate KinaseActivity in Rat Tissues by Thymidine Administration. Biochem.Biophys. Res. Commun., 2: 35—39,1960.

14. Holtzer, R. L., Oda,A., and Chiga,M. Effect ofActinomycin D onDeoxycytidylate Deaminase Activity of Rat Liver after PartialHepatectomy. Lab. Invest., 13: 1514—1519, 1964.

15. Ives,D. H., Morse,P.A., and Potter, V. R. Feedbackinhibition ofThymidine Kinase by Thymidine Triphosphate. J. Biol. Chem.,238: 1467—1474, 1963.

16. Karlstrom, 0., and Larsson, A. Significance of Ribonucleotide Reduction in the Biosynthesis of Deoxyribonucleotides in Escherichiacoli. European J. Biochem., 3: 164—170, 1967.

17. Kinard, F. E. Liquid Scintillator for the Analysis of Tritium inWater. Rev. Sci. Instr., 28: 293—294,1957.

18. Kit, S., Piekarski, L. J., Dubbs, D. R., deTorres, R. A.,and Anken,M. Enzyme Induction in Green Monkey Kidney Cultures Infectedwith Simian Adenovirus. J. Virol., 1: 10—15, 1967.

19. Lea, M. A., Morris, H.P., and Weber, G. Comparative Biochemistryof Hepatomas. VI. Thymidine Incorporation into DNA as a Measure of Hepatoma Growth Rate. Cancer Res., 26: 465—469, 1966.

20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.Protein Measurement with the Folin Phenol Reagent. J. Biol.Chem., 193: 265—275,1951.

21. Maley,F., and Maley, G. F. NucleotideInterconversions.II. Elevation of Deoxycytidylate Deaminase and Thymidylate Synthetase inRegenerating Rat Liver. J. Biol. Chem., 235: 2968—2970, 1960.

22. Maley, F., and Maley, G. F. Nucleotide interconversions. IV. Activities of Deoxycytidylate Deaminase and Thymidylate Synthetasein Normal Rat Liver and Hepatomas. Cancer Res., 21: 1421—1428,1961.

23. Maley, F., and Maley, G. F. The Presence of Deoxycytidylate

Deaminase in Normal Adult Rat Liver. Biochim. Biophys. Acta,47: 181—183,1961.

24. Maley, G. F., and Maley, F. Nucleotide Interconversions in Embryonic and Neoplastic Tissues. I. The Conversion of DeoxycytidylicAcid to Deoxyuridylic Acid and Thymidylic Acid. J. BioL Chem.,234: 2975—2980, 1959.

25. Maley, G. F., and Maley, F. The Purification and Properties ofDeoxycytidylate Deaminase from Chick Embryo Extracts. J. Biol.Chem.,239: 1168—1176,1964.

26. Maley, G. F., Lorenson, M. G., and Maley, F. Inhibitors of ProteinSynthesis: Effect on the Levels of Deoxycytidylate Deaminase,Thymidylate Synthetase, and Thymidine Kinase in RegeneratingRat Liver. Biochem. Biophys. Res. Commun., 18: 364—370,1965.

27. Moore, E. C., and Huribert, R. B. Reduction of Cytidine Nucleotides to Deoxycytidine Nucleotides by Mammalian Enzymes.Biochim. Biophys. Acta, 55: 651—663, 1962.

28. Morris, H. P., and Wagner, B. P. Induction and Transplantation ofRat Hepatomas with Different Growth Rate (including “MinimalDeviation―Hepatomas). In: H. Busch (ed.), Methods in CancerResearch, Vol. 4, pp. 125—152. New York: Academic Press,1968.

29. Morse, P. A., and Potter, V. R. Pyrimidine Metabolism in TissueCulture Cells Derived from Rat Hepatomas. I. Suspension Cell Caltures Derived from the Novikoff Hepatoma. Cancer Res., 25:499—508,1965.

30. Nishihara, M., Chrambach, A., and Aposhian, H. V. The Deoxycytidylate Deaminase Found in Bacillus subtilis Infected withPhage SP8. Biochemistry, 6: 1877—1886, 1967.

31. Nowell, P. C., Morris, H.P., and Potter, V. R. Chromosomesof“MinimalDeviation― Hepatomas and Some Other TransplantableRat Tumors, Cancer Res., 27: 1565—1579, 1967.

32. Ono, T., Blair, D. G. R., Potter, V. R., and Morris, H. P. The Com

parative Enzymology and Cell Origin of Rat Hepatomas. IV. Pyrimidine Metabolism in Minimal Deviation Tumors. Cancer Res., 23:240—249,1963.

33. Ono, T., Potter, V. R., Pitot, H. C., and Morris, H. P. MetabolicAdaptations in Rat Hepatomas. III. Glucose-6-Phosphate Dehydrogenase and Pyrimidine Reductases. Cancer Res., 23: 385—391,1963.

34. Pitot, H. C., Peraino, C., Morse, P.A.,and Potter, V. R. Hepatomasin Tissue Culture Compared with Adapting Liver in vivo. Natl.Cancer Inst. Monograph, 13: 229—245,1964.

35. Pitot, H. C., and Potter, V. R. An Enzymic Study on the CellularOrigin of the Dunning and the Novikoff Hepatomas in the Rat.Biochim. Biophys. Acta, 40: 537—539,1960.

36. Potter, V. R. Transplantable Animal Cancer, the Primary Standard.Guest Editorial. Cancer Res., 21: 1331—1333, 1961.

37. Potter, V. R. Biochemical Perspectives in Cancer Research. CancerRes., 24: 1085—1098, 1964.

38. Potter, V. R. BiochemicalStudieson Minimal Deviation Hepatomas In: P. Emmelot and 0. Muhblock (eds.), Cellular ControlMechanisms and Cancer, pp. 190—210.Amsterdam: Elsevier Publashing Company, 1964.

39. Potter, V. R., Gebert, R. A., Pitot, H. C., Peraino, C., Lamar, C.,Lesher, S., and Morris, H. P. Systematic Oscillations in MetabolicActivity in Rat Liver and in Hepatomas. I. Morris Hepatoma No.7793. Cancer Res., 26: 1547—1560, 1966.

40. Potter, V. R., Pitot, H. C.,Ono,T., and Morris, H. P. The Comparative Enzymology and Cell Origin of Rat Hepatomas. I. Deoxycytidylate Deaminase and Thymine Degradation. Cancer Res., 20:1255—1261,1960.

41. Potter, V. R., and Watanabe,M. SomeBiochemicalEssentialsofMalignancy: The Challenge of Diversity. In: C. J. D. Zarafonetis(ed.), Proceedings of International Conference on LeukemiaLymphoma, pp. 33—46.Philadelphia: Lea and Febiger Pub. Co.,1968.

42. Potter, V. R., Watanabe, M., Becker, J. E., and Pitot, H. C. Hormonal Effects on Enzyme Activities in Tissue Culture and in WholeAnimals. Advan. Enzyme Regulation, 5: 303—316,1967.

43. Potter, V. R., Watanabe, M., Pitot, H.C.,and Morris, H. P. Systematic Oscillations in Rat Liver and Hepatomas. II. Survey of NormalDiploid and Related Strains. Cancer Res., in press, 1969.

44. Price, F. M., Rotherham,J., and Evans, V. J. Pyrimidine NucleosideRequirements of a Neoplastic C3H Mouse Cell Strain. J. Natl. Cancer Inst., 39: 529—538,1967.

45. Roth, J. S., Sheid, B., and Morris, H. P. Some Observations on theDeamination of Deoxynucleotides and Deoxynucleosides by Normal Rat Liver and Hepatomas. Cancer Res., 23: 454—461, 1963.

46. Scarano, E., laccarino, M., Grippo, P., and Parisi, E. The Heterogeneity of Thymine Methyl Group Origin in DNA Pyrimidine Isostichs of Developing Sea Urchin Embryos. Proc. Natl. Acad. ScL, U.

53JANUARY 1969

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

Thomas W. Sneider, Van R. Potter, andHaroldP. Morris

sues. Biochim. Biophys. Acta, 55: 550—551,1962.52. Sugino, Y. Metabolism of Deoxyribonucleotides. Ann. Report Inst.

Virus Res. (Kyoto University), 9: 1—29, 1966.

53. Watanabe, M., Potter, V. R., and Pitot, H. C. Systematic Oscillations in Tyrosine Transaminase and Other Metabolic Functions inLiver of Normal and Adrenalectomized Rats on Controlled FeedingSchedules. J. Nutrition, 95: 238—246,1968.

54. Weber, G. The Molecular Correlation Concept: Studies on theMetabolic Patterns of Hepatomas. Gann Monograph, 1: 151—178,1966.

55. Wu, C. “MinimalDeviation― Hepatomas: A Critical Review of theTerminology, Including a Commentary on the Correlation of Enzyme Activity with Growth Rate of Hepatomas. J. Nat!. CancerInst., 39: 1149—1154, 1967.

S., 57: 1394—1400, 1967.47. Scarano, E., laccarino, M., Grippo, P., and Winckelmans, D. On

Methylation of DNA during the Development of the Sea UrchinEmbryo. J. Mol. Biol., 14: 603—607,1965.

48. Schneider, W. C., and Brownell, L. W. Deoxyribosidic Compoundsin Regenerating Rat Liver. J. Nat!. Cancer Inst. 18: 579—586,1967.

49. Shonk, C. E., Morris, H. P., and Boxer, G. E. Patterns of GlycolyticEnzymes in Rat Liver and Hepatoma. Cancer Res., 25: 671—676,1965.

50. Sneider, T. W., and Potter, V. R. Thymidylate Synthetase and Related Enzymes in Rat Minimal Deviation Hepatomas. Proc. Am.Assoc. Cancer Res., 8: 61, 1967.

51. Soska, J., and Bezdek, M. Free 5-Methyldeoxycytidine in Rat Tis

54 CANCER RESEARCH VOL.29

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from

1969;29:40-54. Cancer Res   Thomas W. Sneider, Van R. Potter and Harold P. Morris  Morris HepatomasEnzymes of Thymidine Triphosphate Synthesis in Selected

  Updated version

  http://cancerres.aacrjournals.org/content/29/1/40

Access the most recent version of this article at:

   

   

   

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

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/29/1/40To request permission to re-use all or part of this article, use this link

Research. on December 31, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from