[CANCER RESEARCH 27 Part 1, 1907-1914, October 1967]

Deoxycytidylate Deaminase Activity of Simian Virus 40-infectedCell Cultures1

SAUL KIT, R. A. de TORRES,2 AND DEL R. DUBBS

Division of Biochemical Virology, Baylor University College of Medicine, Houston, Texas 77025

SUMMARYDeoxycytidine-5'-triphosphate (dCTP) stabilizes deoxy-

cytidylate deaminase activity. Enzyme extracts prepared withbuffer containing dCTP are 2 to 6 times as active as those madewithout dCTP. Even when extracted in the presence of dCTP,the deoxycytidylate deaminase levels vary considerably fromcell cultures of different origin. Monkey and human cells exhibitabout twice the activity of LM(TK~) mouse fibroblast cells, and

about 20 and 40 times the activity of confluent monolayercultures of mouse embryo and mouse kidney cells, respectively.

An apparent stimulation of deoxycytidylate deaminase activity is observed following herpes simplex infection of LM(TK~) cells, when dCTP is omitted from the enzyme extractionbuffer. However, extracts from uninfected LM(TK~) cells

prepared with dCTP have as much activity as those from herpessimplex-infected cells. Uninfected LM(TK~) and monkey kid

ney cell extracts with dCTP also exhibit as much activity as dothose from vaccinia- and Simian Virus 40 (SV40)-infectedcultures, respectively.

Extracts prepared with dCTP from vaccinia- or herpes simplex-infected mouse embryo and mouse kidney cultures have nomore than 1.7 times the deoxycytidylate deaminase activity ofuninfected cell extracts; however, thymidine kinase activity ofvirus-infected cell extracts is greatly increased over that of uninfected cell extracts.

Either productive-polyoma virus infection or abortive-SV40infection of mouse kidney cells result in stimulations of bothdeoxycytidylate deaminase and thymidine kinase activities.These activities remain elevated in SV40-transformed mousekidney cell cultures. Puromycin, but not 1-0-D-arabinofuranosyl-cytosine, inhibits the induction of deoxycytidylate deaminase inSV40-infected mouse kidney cultures.

INTRODUCTION

The enzyme deoxycytidylate deaminase increases significantlyfollowing infection of Escherichia coli by T-even phages and ofBacillus subtilis by phages containing hydroxymethyl deoxy-uridylate in place of thymidylate in their DNA (5, 9, 23-26).

'This investigation was aided by grants from the National Science Foundation (GB 5917), the American Cancer Society (E 291),the Robert A. Welch Foundation (Q163), and by ÃœSPHS GrantsCA-OC656, l-KG-AI-2352, and 5-K3-CA-25, 797.

1 Postdoctoral Fellow of the Consejo Nacional de Investigaci

ones Científicas y Técnicas(Argentina).Received January 13, 1907; accepted June 30, 1967.

There is evidence that the production of this enzyme is controlledby phage genes. The capacity of T2 phage to induce deoxycytidylate deaminase is impaired by ultraviolet light, althoughthe enzyme-inducing capacity is more resistant to UV than isinfectivity (9). Mutant strains of T4 phage have been isolatedwhich are defective in deoxycytidylate deaminase-inducingactivity (6). Moreover, T4 amber mutants induce significantlyhigher levels of deoxycytidylate deaminase than wild-tyi«phageand extended enzyme synthesis is observed with the "DO" class

ot amber mutants (29).The finding that deoxycytidylate deaminase activity is in

duced by DNA-containing bacteriophages raises the questionwhether this enzyme also increases in animal cells infected withDNA-containing viruses. An increase of deoxycytidylate deaminase activity was not observed in monkey kidney cell culturesinfected with simian adenovirus SV15 (17). However, it has beenreported that this enzyme is augmented after productive infection of mouse kidney cultures with polyoma virus or of monkeykidney cultures with SV40 (3, 7, 8). Studies from our laboratoryhave confirmed the enhancement of deoxycytidylate deaminaseactivity in ]x>lyoma virus-infected mouse kidney cultures. Contrary to the report of Hatanaka and Dulbecco (8), however, wehave not observed an enzyme increase in SV40-infected monkeykidney cultures. The enzyme does increase in mouse kidney cultures abortively infected with SV40 and is elevated in SV40-transformed mouse kidney cells. Experiments with SV40 andalso with two other DNA-containing animal viruses, vacciniaand herpes simplex, are described in this paper. Preliminaryreports of these findings have been presented (10, 16, 19).

MATERIALS AND METHODSCell Lines. Monolayer cultures of LM(TK~), CV-1, mKS,

and primary cultures of mouse kidney, mouse embryo, andGMK3 cells were propagated as described previously (11, 13, 14).

The CV-1 cells are an established strain of GMK cells. ThemKS cells are SV40-transformed mouse kidney cells (15). Sub-lines mKS-1, mKS-4, mKS-11, mKS-17, mKS-A, and mKS-Bwere obtained from confluent primary cultures of mouse kidneycells which had been inoculated with 150 PFU per cell of SV40.

' Abbreviations : SV40, Simian Virus 40: dCMP, deoxycytidylate;GMK, green monkey kidney; PFU, plaque-forming units; dCTP,deoxycytidine-5'-triphosphate; dllMP, deoxyuridine-5'-monophos-phate; ara-C, l-/3-D-arabinofuranosylcytosine; dTTP, deoxythy-midine-5'- triphosphate; dTMP, deoxythymidine-5'-monophos-

phate; Tris, tris(hydroxymethyl)aminomethane.

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Saul Kit, R. A. de Torres, and Del R. Ditbbs

The medium was replaced every 3 to 4 days and at 1, 4, 11, 17,22, and 30 days after SV40 inoculation, respectively, two primaryinfected cultures were trypsinized and subcultured. The cultureswere passaged again 10 to 23 days later after the monolayers hadbecome confluent or large colonies had formed. Monolayer cultures of mKS cells were propagated in R5a medium containing0.5% lactalbumin hydrolysate and 10% calf serum (1, 2). Thecells grew to populations of 10-15 million per 55 sq cm and, after the second passage, were subcultured at 3- to 4-day intervals. The mKS cells contained the SV40-tumor antigen, but notdetectable SV40-capsid antigen or virus particles. Extracts ofmKS cells did not yield infectious virus and plaques were notobserved when mKS cells were plated on CV-1 cell monolayers.SV40 virus was recovered, however, when mKS cells were grownin mixed cultures with CV-1 cells. Equal numbers of freshly trypsinized mKS and CV-1 cells were mixed and 2 X IO6 cells wereplated in 8-oz prescription bottles. After 8 days of incubation,the cells and medium from each mixed culture were harvested,transferred to Lusteroid tubes, treated for 1 min at 4°Cwith a

Raytheon sonic oscillator at 10 kc, and assayed on CV-1 monolayers for infectious virus. Infectious SV40 was recovered from 9 to10 strains tested in amounts ranging from 200 to 10,000 PFUper mixed culture.

Viruses. Clonal strain 307L of SV40 was grown and assayedin CV-1 cells as previously described (14). The propagation andassay of vaccinia and herpes simplex viruses have also beenreported (1, 2).

Enzyme Assays. Thymidine kinase was determined by themethod of Kit and Dubbs (12). The dCMP deaminase wasmeasured by modifications of the isotopie method of Maley andMaley (22). Each tube contained in a total volume of 0.2 ml thefollowing substances at the indicated final concentrations: 2.5mM dCMP-I4C (about 700,000 cpm/Mmole) or dCMP-3H (about2.5 X IO6 cpm/Mmole), 0.17 HIMdCTP, 27.2 mM potassiumfluoride, 2.5 HIMMgClz, 79 mM potassium chloride, 80 mM Tris-HC1 buffer (pH 8.0 at 25°C),5.25 mM 2-mercaptoethanol, and

enzyme. In the case of the mouse kidney cells, the enzyme extractconsisted of about 400-1000 ¿igof protein. For all other cellextracts, about 100-300 jugof protein per tube were used.

Enzyme extracts were usually prepared by sonication of 30-50

million cells sus[)ended in buffer containing 150 mM potassiumchloride, 10 mM 2-mercaptoethanol, 0.31 ÕÕIMdCTP, 1.25 mMMgCl2, and 10 HIMTris-HCl buffer (pH 8.0 at 25°C).In a few

experiments, however, sodium phosphate buffer (pH 7.3) wassubstituted for Tris-HCl buffer in the enzyme extraction solution.The crude extracts were centrifuged for 1 hr at 4°Ceither at

30,000 X g or at 105,000 X g and the supernatant employed forassay at three protein concentrations. Zero time and zeroenzyme blanks were included with the enzyme assays. Theenzyme reaction was terminated after either 10 or 20 min by theaddition of trichloroacetic acid to a final concentration of 5%.The dUMP, dCMP, and nucleosides were routinely separated bychromatography on DE-81 diethylaminoethyl cellulose paper(0.4 meq/gm) with 4 Mformic acid and 0.1 Mammonium formateas the solvent, and the amount of radioactive dUMP formed wasdetermined as described previously (19). A second solventsystem was used to study the conversion of dCMP-14C to

deoxycytidine and deoxyuridine. This latter solvent consisted of0.01 Mformic acid.

Protein was measured by the method of Lowry et al. (20). Theenzyme reaction was linear with protein concentration and withtime under the conditions routinely used.

Experiments with crude and with centrifuged extracts demonstrated that all of the deoxycytidylate deaminase activity waspresent in the supernatant fraction of the centrifuged extracts.

Experiments by Maley and Maley (21) have shown that 2-mercaptoethanol protects and activates deoxycytidylate deaminase. To learn whether optimal 2-mercaptoethanol concentrations were employed in the assays reported here, the concentration of this chemical in the enzyme extraction solution was variedfrom 3 mM to 30 nun (1.6 to 16 mM final concentration duringenzyme assay). With extracts prepared from either uninfectedmouse kidney cells or from cells 44 hr after infection, the deoxycytidylate deaminase activity was about the same at each of the2-mercaptoethanol concentrations studied. Either the standard2-mercaptoethanol concentration of 10 mM in the extractionbuffer or a concentration of 3 mil was used in the present experiments.

Materials. Deoxyuridine-3H was purchased from New England Nuclear Corporation, Boston, Mass., and dCMP-3H anddCMP-uC from either New England Nuclear Corporation or

Schwarz BioResearch, Inc., Orangeburg, New York. Puromycinand ara-C were obtained from Nutritional Biochemical Corporation, Cleveland, Ohio, and dCTP from Calbiochem, Los Angeles,California.

RESULTS

Deoxycytidylate Deaminase Activity of Tissue CultureCell Lines. The deoxycytidylate deaminase activity variesgreatly in different tissue culture lines (Table 1). The activity isremarkably high in confluent monolayer cultures of monkeykidney (GMK and CV-1) and human [ HeLa, HeLa (BU-100) ]cells but is very low in confluent primary cultures of mouse kidney and mouse embryo cells. The LM(TK~) mouse fibroblast

cells have about half the activity of CV-1 cell lines.Effect of dCTP on Deoxycytidylate Deaminase Activity.

dCTP is required for maximal deoxycytidylate deaminase activity (21, 22, 28, 29). Normally, the enzyme is relatively unstable,but it can be stabilized and activated by dCTP. Table 2 illus-

TABLE 1Deoxycytidylate (dCMP) Deaminase Activities of Confluent

Monolayer Cultures of Various Cell LinesThe enzyme extraction solution contained 0.313 mM deoxy-

cytidine-5'-t riphosphate.

CelllineGMKCV-1HeLaHeLa

(BU-100)LMLM(TK-)LM(TK~)

(suspension)Mouse

embryoMousekidneydCMP

deaminase activity (Amólesdeoxyuridine-5'-monophosphate formed/i<gprotein in 10 min at38°C)50138832831211919618711-352-15

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Deoxycytidylate Deaminase Activity of SV 40-infected Cells

trates experiments with SV40-infected monkey kidney cells andwith herpes simplex-infected LM(TK~) mouse fibroblast cells.

In all instances, the enzyme was extracted and assayed in thepresence of 2-mercaptoethanol, but either with or without dCTP.Three points are worthy of emphasis. First, deoxycytidylatedeaminase activity greatly exceeds thymidine kinase activity(see also Charts 1 and 2). Second, enzyme extracts prepared andassayed in the absence of dCTP exhibit much less deoxycytidylate deaminase activity than those prepared with buffercontaining dCTP. The dCTP is specific for deoxycytidylate deaminase; it does not enhance thymidine kinase activity. Third,the deoxycytidylate deaminase activities of uninfected cell extracts prepared without dCTP vary considerably from experiment to experiment. Enzyme extracts prepared in the absenceof dCTP from cultures infected for 6 hr with herpes simplex virusmay show higher activities than those from uninfected cultures(Table 2, Experiments II, III). Extracts prepared without dCTPfrom cultures infected for about 41 hr with SV40 may have somewhat lower activities than those from uninfected cultures (Table2, Experiment I). However, enzyme extracts prepared from uninfected cells with buffer containing dCTP are as high in deoxycytidylate deaminase activity as those from either SV40-infectedor from herpes simplex-infected cells. One possible interpretationof these results is that deoxycytidylate deaminase may be modified intracellularly by virus infection. When the virus-infected

TABLE 2Effect of dCTP' in Enzyme Extraction Buffer on the dCMP

Deaminase and Thymidine Kinase Activities of Uninfectedand Virus-infected Cell Cultures0

ExperimentIIIIIICells andvirusUninfected

GMKSV40-infectedGMKUninfected

LM(TK-)Herpes

simplex-infectedLM(TK-)Uninfected

LM(TR-)Herpes

simplex -infected LM(TK-)Hr

post-infection4166—

~dCMP

deaminaseactivity*Enzyme

extractionbufferNo

dCTP5738539892192With;dCTPd394383187150178177Thymidine

kinaseactivity"Enzyme

extractionbufferNo

dCTP1.07.10.0228.00.0211.4WithdCTPJ0.55.20.0424.40.046.2

0 Abbreviations : dCTP, deoxycytidine-5'-triphosphate; dCMP,

deoxycytidylate; GMK, green monkey kidney; PFU, plaque-forming units; SV40, Simian Virus 40.

bExperiment I: GMK cell cultures (13.8 X 10" cells/culture)

were inoculated with 14 PFU/cell SV40. Experiments II and III:LM(TK-) cell cultures (11.4 X IO6 cells/culture) were inoculated

with 5 PFU/cell herpes simplex virus.c Amóles deoxyuridine-5'-monophosphate formed per jug pro

tein in 10 min at 38°C.d Enzyme extraction buffer contained 0.313 HIM dCTP. The

final concentration of dCTP in the enzyme assay was 0.17 mw.

cells are disrupted to release the enzyme, either an "apparent"

stimulation or an inhibition may be observed. If the virus infection tends to stabilize deoxycytidylate deaminase, a stimulationwill be observed; the reverse will be seen if the virus infectionreduces the stability of the enzyme.

In further experiments, the concentration of dCTP in theenzyme extraction solution was decreased to 0.15 HIMor increasedto 0.45 niM. At each of the dCTP concentrations studied, SV40induced thymidine kinase but not deoxycytidylate deaminaseactivity in monkey kidney cell cultures.

The experiments of Table 2 with SV40-infected GMK cell cultures are contrary to those of Hatanaka and Dulbecco (8) whoreported approximately an 8-fold increase in deoxycytidylatedeaminase activity in SV40-infected GAIK cell cultures. In viewof this discrepancy, the SV40 monkey kidney cell system wasinvestigated in more detail.

First the metabolic state of the GMK cells was considered.Experiment I of Table 3 depicts an experiment in which 13-day-old primary GMK cell cultures were infected with SV40. Thesecultures had been in the stationary phase of the growth cyclefor at least 3 days. The deoxycytidylate deaminase activity 7,10, and 12 days after seeding the cultures was essentially the sameas that of the experimental period from Days 13 to 15. Therewas no enhancement by SV40 of deoxycytidylate deaminaseactivity over the time jieriod of 16.5 to 44 hr after infection. Atthese times, substantial increases in thymidine kinase and DNApolymerase activities have previously been observed and the incorporation of labeled thymidine or deoxyadenosine into DNAwas greatly stimulated (10, 14-16). Indeed, when the same extracts used to measure deoxycytidylate deaminase activity wereused to measure thymidine kinase activity, a marked stimulationof the latter enzyme activity was observed.

Table 3, Experiment II, shows that SV40-infection also did notenhance the deoxycytidylate deaminase activity of the CV-Iestablished line of GMK cells.

Second, the possibility that a microsomal inhibitor of deoxycytidylate deaminase (4) was affecting the ex]>eriments was considered. The enzyme extracts were therefore centrifuged at higherspeeds using the Spinco Model L-2 ultracentrifuge to removemore completely the microsomal fraction. Table 3, ExperimentIII, shows that this did not alter the ex]>erimental findings.

Third, enzyme extracts were prepared with phosphate buffer,pH 7.3, instead of Tris-HCl buffer, pH 8.O. The former bufferhad been employed by Hatanaka and Dulbecco (8). Table 3,Experiment III, shows that SV40 infection did not induce deoxycytidylate deaminase activity with these changed experimentalconditions.

Fourth, possible interference in the assay by nucleotidase enzymes was ruled out. It was observed with either Tris-HCl orphosphate extracts of uninfected cells or SV40-infected cells thatless than 0.5% of the dCMP-14C was converted to radioactive

deoxyuridine and deoxycytidine. However, 7 to 20% ofthe dCMP-14C was converted to labeled dUMP, depending on the

enzyme concentration used in the assay.Effect of Polyoma Virus Infection on Deoxycytidylate

Deaminase Activities of Murine Cell Cultures. In contrastto monkey kidney cell cultures, the levels of deoxycytidylate deaminase are very low in confluent monolayer cultures of mousekidney and mouse embryo cells (Table 1). These low activities are

OCTOBER 1967 1909

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Saul Kit, R. A. de Torres, and Del R. Dubbs

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SV40-INFECTED DEAMINASE

NONINFECTED DEAMINASE

NONINFECTED KINASE

IO 20 30 40HOURS AFTER SV40

50INFECTION

2.5 ±>

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1.5 2

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CHART1. Kinetics of deoxycytidylate deaminase and thymidine kinase formation following infection of confluent monolayer culturesof mouse kidney cells (4.1 X IO6cells/culture) with 257 plaque-form ing units/cell of Simian Virus 40 (SV40). Thymidine kinase activity:timóles deoxyuridine-5'-monophosphate (dUMP) formed per^g protein in 10 min at 38°C;deoxycytidylate deaminase activity: CiñiólesdUMP formed per ¡tgprotein in 20 min at 38°C.

found despite the addition of dCTP to the enzyme extractionbuffer. The data of Table 4 confirm the findings of Dulbecco andcoworkers (3, 7) that the low activities of murine cell cultures areenhanced after polyoma virus infection. It is to emphasized,however, that even after deoxycytidylate deaminase is inducedby polyoma virus, the enzyme activity is only a fraction of thatobserved with monkey, HeLa, or LM(TK~) mouse fibroblast

cultures.Effect of Abortive-SV40 Infection on the Enzyme Activ

ities of Mouse Kidney Cells. SV40 undergoes an abortive infection in mouse kidney cultures; the SV40-tumor antigen and

enzymes of DNA metabolism are induced although capsid proteins and infectious virus are not made in most cells (10, 15, 16).Chart 1 shows that deoxycytidylate deaminase activity is increased following abortive-SV40 infection of mouse kidney cell

cultures. The increase is detected at about 25 to 30 hr, severalhours after the induction of thymidine kinase is detected. At 40hr, the deoxycytidylate deaminase activity of SV40-infected cul

tures is about three times greater than that of uninfected cellcultures. Chart 2 depicts an experiment in which the deoxycytidylate deaminase and thymidine kinase activities of mousekidney cultures were studied from 26 to 72 hr after SV40 infection. The deoxycytidylate deaminase activity reached a maximum at about 56 hr and remained elevated through 72 hr. At72 hr, the activity of SV40-infected cultures was about four timesthat of uninfected cultures. The thymidine kinase of SV40-in-

fected cultures was eleven times greater than that of uninfectedcultures at 56 hr but declined considerably between 56 and 72 hr.

The deoxycytidylate deaminase activity of SV40-infectedmouse kidney cultures was enhanced in extracts prepared in Tris-

HC1 buffer or phosphate buffer and with the supernatant fractionof extracts centrifuged at either 30,000 X g or 105,000 X g

With the uninfected or SV40-infected mouse kidney cultures,less than 0.2% of the dCMP-14C was converted to deoxyuridine.About 0.7-1% was dephosphorylated to deoxycytidine underconditions where about 4-10% of the dCMP-14C was deaminated

to dUMP.Effect of Puromycin on the Stimulation of Deoxycytidy

late Deaminase Activity by SV40. To learn whether concurrentprotein synthesis was needed for the stimulation of deoxycytidylate deaminase activity by SV40, mouse kidney cultures weretreated with puromycin. Table 5 shows that deoxycytidylatedeaminase activity increased about threefold at 38 hr after SV40infection in cultures not treated with puromycin. Puromycin(10~5 M) treatment from 20 to 38 hr prevented this increase.

Effect of ara-C Treatment on the Induction of Deoxy

cytidylate Deaminase Activity by SV40. As previously observed (15), SV40 infection stimulates the incorporation of tri-tiated thymidine into DNA of mouse kidney cultures. ara-C is

a potent inhibitor of DNA biosynthesis in a variety of cell systems(13, 18). Although, 15 pg/ml ara-C inhibited the incorporationof tritiated thymidine into DNA of either SV40-infected or uninfected mouse kidney cell cultures by 90-99%, the drug had no

effect on the induction of either deoxycytidylate deaminase orthymidine kinase in infected cells (Table 6). These and previousexperiments (10, 14, 15) demonstrate that DNA synthesis is notrequired for enzyme induction after either productive- orabortive-SV40 infections.

Enzyme Activities of SV40-traiisformed Mouse KidneyCell Lines. It has previously been shown that mouse kidney celllines transformed by SV40 exhibit elevated levels of thymidinekinase, dTMP kinase, and DNA polymerase activities (10, 15,16). Table 7 shows that the deoxycytidylate deaminase activitiesof SV40-transformed cell lines are also elevated. In the case of

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SV40-INFECTEDKINASE

Deoxycytidylate Deaminase Activity of SV40-infectcd Cells

4.5

4.0

3.5 >_

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SV40-INFECTEDDEAMINASE

NON INFECTEDDEAMINASE

NON INFECTEDKINASEMEDIA

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0 c2.5

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20 30 40 50 60 70 80HOURS AFTER SV40 INFECTION

CHART2. Activities of deoxycytidylate deaminase and thymidine kinase of Simian Virus40 (SV40)-infected mouse kidney cell culturo«26 to 72 hr after virus infection. Cultures (5.5 X IO6cells/culture) were inoculated with 2G2plaque-forming units/cell of SV40; the virusadsorption time was 2 hr. At the end of 2 hr and again at 24 hr the media of "mock-infected" control cultures and SV40-infected cultureswere changed. Thymidine kinase activity :/inmoles deoxyuridine-5'-monophosphate (dUMP) formed per^g of protein in 10 min at 38°C;deoxycytidylate deaminase activity: «uniólesdUMP formed per iig protein in 20 min at 38°C.

Cell Line mKS-B, the deoxycytidylate deaminase activity wasalmost as great as that of LM mouse fibroblast cells.

Effect of Vaccinia and Herpes Simplex Infections on Enzymes of Murine Cell Cultures. Although herpes simplex virusdoes not stimulate deoxycytidylate deaminase activity of LM(TK~) cell cultures (Table 2), it was of interest to learn whether

the virus would induce the enzyme in mouse embryo and mousekidney cultures. Table 8 shows that herpes simplex infectionenhanced the deoxycytidylate deaminase activity of the mousecell cultures by only about 1.7 times, although the thymidinekinase activity increased 7 to 9 times.

The data of Table 9 indicate that vaccinia infection fails tostimulate deoxycytidylate deaminase activity in LM(TK~),

mouse embryo, or mouse kidney cell cultures. With each of themurine cell cultures, and induction of thymidine kinase activitywas observed.

DISCUSSION

Studies by Maley and Maley (21, 22) and Scarano et al. (27,28) have shown that the control of deoxycytidylate deaminase

activity is complex. In the absence of stabilizing compounds, theenzyme is rapidly converted to an inactive state even at 0-5°C.Magnesium ions, 2-mercaptoethanol, and dCTP stabilize andactivate the enzyme while dTTP is a potent feedback inhibitor.It is apparent that intracellular events leading to the formationor breakdown of dCTP and dTTP or to changes in sulfhydrylconcentrations will affect enzyme activit3r. Disruption of cellsand dilution of deoxycytidylate deaminase in buffer solution mayproduce not only suboptimal conditions for enzyme activity butmight also lead to the activation of deoxycytidine triphosphataseactivity and reduction of the dCTP concentration (4). Moreover,a natural inhibitor of the enzyme has been described in the micro-somal fraction of cell extracts (4). In view of the properties ofdeoxycytidylate deaminase, caution is indicated in interpretingresults of experiments with virus-infected cells.

The experiments depicted in Table 2 show that the deoxycytidylate deaminase extracted from herpes simplex-infected cellswith buffer containing 2-mercaptoethanol, but without dCTP,may be inactivated less than similar enzyme preparations fromuninfected cells. As the SV40 experiment of Table 2 illustrates,the opposite result may also be obtained. It has been our experi-

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Saul Kit, R. A. de Torres, and Del R. Dubbs

TABLE 3dCMP' Deaminase Activities of SV'40-infected GMK and CV-1

Cell Cultures Measured at Various Times after Virus Infectionb

ExperimentiiiinHostcellsGMKCV-1CV-1ExtractionbufferTris-HCl,pH

8.0Tris-HCl,pH

8.0Tris-HCl,pHS.OPhosphate,pH

7.3Extractcentrifuged

for 1 hrat:30,000

Xg30,000

Xg105,000

Xg105,000

X gH

r afterSV40

infection16.524313844182941.51928414719284147dCMPdeaminaseactivitydeoxyuridine-5'-monophosphateformed//igproteinin10min at38°C)Unin

fected514544560504535498510565463464430406367349337315Infected590545599443519572541700508452468435393398376330

" Abbreviations: dCMP, deoxycytidylate; GMK, green monkey

kidney; Tris, tris(hydroxymethyl)aminomethane, SV40, SimianVirus 40.

6Stationary-phase monolayer cultures (9-11 X IO6 cells/

culture) were inoculated in Experiments I, II, and III, respectively, with 108, 32, and 75 plaque-forming units/cell of SV40.Enzyme extracts were prepared with buffer containing 0.313 mMdeoxy cy tidine-5'-t riphosphat e.

enee that the deoxycytidylate deaminase activity of a given tissueis extremely variable unless precautions are taken to protect theenzyme prior to assay. When extracts are prepared in the absenceof activators, the addition of these activators to the enzyme assaytubes may not suffice to restore all the activity.

Previous studies indicate that simian adenovirus SV15 doesnot increase the deoxycytidylate deaminase activity of monkeykidney cells (17). The present data fail to show a clear-cut induction of this enzyme by herpes simplex or vaccinia viruses.Moreover, contrary to the report of Hatanaka and Dulbecco (8),an increase of this enzyme in SV40-infected monkey kidney cellswas not observed. However, all four classes of viruses greatlystimulated thymidine kinase activity.

The lack of stimulation of dCMP deaminase activity in SV40-infected monkey kidney cultures described in this study can beattributable to the fact that the cellular levels of this enzyme arealready very high in uninfected cells. The dCMP deaminaseactivities reported by Hatanaka and Dulbecco (8) were expressedin undefined arbitrary enzyme units, and the experimental con

ditions were somewhat different so that comparisons with thepresent data are difficult to make. We believe that the discrepancy between their results and ours is most likely due to thefailure to activate fully their deoxycytidylate deaminase fromuninfected GMK cells at the enzyme dilutions that they employed.

In contrast to monkey kidney cells, mouse kidney cells exhibit

TABLE 4Effect of Polyoma Virus Infection on the dCMP" Deaminase Activity

of Murine Cell Cultures

ExperimentiiiniIVvHostcellsMouse

embryoMouse

embryoMouse

kidneyMouse

kidneyMouse

kidneyHr

afterinfection18

284125

48.54248dCMP

deaminase activity^(¿i/imolesdeoxyuridine-5'-monophosphate formed/jigprotein in 10 min at38°C)Uninfected29.6

35.113.04.8

2.62.63.1Polyoraa

virus-infected48.1

67.442.125.5

61.113.221.6

" Abbreviations: dCMP, deoxycytidylate; PFU, plaque-form

ing units.6Enzyme extraction buffer contained 0.313 mM deoxycytidine-

5'-triphosphate. In Experiments I and II, confluent mouse embryo cultures (14 X IO6 cells/culture) were inoculated with 206

and 50 PFU/cell polyoma virus, respectively. In Experiments IIIto V, confluent mouse kidney cultures (4-6 X IO6 cells/culture)were inoculated with about 160 PFU/cell polyoma virus.

TABLE 5Effect of Puromycin (10~&¡t)on the Stimulation of Deoxycytidylate

(dCMP) Deaminase Activity in Simian Virus 40 (SV40)-infected Mouse Kidney Cell Cultures"

Hr after infectionenzymeassayed022038»38Interval

of puromycintreatment (hr after

infection)NoneNoneNoneNone20

to 38dCMP

deaminase activity(Amóles deoxyuridine-5'-monophosphate formed/Mgprotein in 20 min at38°C)Uninfected12.317.111.98.64.4SV40-infected11.611.628.45.7

" Confluent mouse kidney cultures (5.7 X IO6 cells/culture)were inoculated with 196 plaque-forming units/cell SV40. Enzymeextracts were prepared in buffer containing 0.313 mM deoxycyti-dine-5'-triphosphate.

6Thymidine kinase activity of infected cultures was 6 timesgreater than that of uninfected cultures at 38 hr postinfection.

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DeoxycyíidylateDeaminase Activity of SV40-infected Cells

TABLE 6Effect of ara-C" •bon the Induction of Thymidine Kinase and dCMP

Deaminase Activities in SVJfi-infected Primary Mouse KidneyCell Cultures'

Hrs afterinfection38

45Thymidine

kinase activity(/immolesdUMP formed/jigprotein in 10min at38°C)UninfectedNo

ara-C0.17

0.23With

ara-C0.250.24SV40-infectedNoara-C1.551.20Withara-C1.50

1.44dCMP

deaminase activity(/jamólesdUMP formed/Mgprotein in 20min at38°C)UninfectedNo

ara-C27.5Withara-C18.522.9SV40-infectedNoara-C49.270.7Withara-C55.7

66.2

" Abbreviations: ara-C, l-/3-D-arabinofuranosylcytosine; dCMP,deoxycy tidy late; dUMP, deoxyuridine-5'-monophosphate; SV40,

Simian Virus 40.0 Abbreviations: ara-C, 1 - ß- D - arabinofuranosylcytosine;

dCMP, deoxycytidylate; dUMP, deoxyuridine-5'-monophosphate,

SV40, Simian Virus 40.6ara-C (15 jug/ml) was added to cultures 2 hr after infection.c Confluent 7-day-old cultures containing 5.5 X 10°cells/

culture were inoculated with 250 plaque-forming units/cell ofSV40. Enzyme extracts were prepared with buffer containing 0.313mm deoxycytidine-o'-triphosphate.

TABLE 7Deoxycyíidylate (dCMP) Deaminase Activity of Simian Virus Jfl

(8V40)-transformed Mouse Kidney Cell Lines

CellsUninfected

mousekidneyTransformedmKS-1mKS-4mKS-11mKS-17mKS-BmKS-B«mKS-ACell

passagenumber1875793410dCMP

deaminase activity(/inmolesdeoxyuridine-5'-monophosphate formed//*gprotein in 10min at38°C)2-15464830359310341

0 mKS-B cell line adapted to grow in medium containing 25jig/ml bromodeoxyuridine. At the 34th passage, the cells exhibited6.5 complement-fixation units of SV40-tumor antigen per 10' cellsand had moderate levels of thymidine kinase activity.

very low deoxycytidylate deaminase activity even when extractsare prepared with buffer containing dCTP and 2-mercapto-ethanol. This low activity can be increased somewhat followingeither productive-polyoma virus or abortive-SV40 infection. Concomitant protein but not DNA synthesis is required forthe papovavirus-induced increases (Tables 5, 6; Ref. 7). Therequirement for protein synthesis could signify that new deoxycytidylate deaminase polypeptide chains are synthesized afterSV40 infection. However, the alternative possibility that continued protein synthesis is needed to stabilize the enzyme is notexcluded.

There is abundant evidence that bacteriophage genes control

the formation of deoxycytidylate deaminase. It does not follow,that the same control mechanisms mediate the enzyme increaseobserved after either SV40 or polyoma virus infection of mousekidney cells. Conclusions as to whether the mouse kidney enzymeis "derepressed" or whether new virus-specific enzymes are made

are premature at this time. It is significant that the enhanceddeoxycytidylate deaminase levels persist after mouse kidney cellsare transformed by SV40. Elevated levels of thymidine kinase,DNA polymerase, and dTMP kinase have also been observed in

TABLE 8dCMP" Deaminase and Thymidine Kinase Activities of Herpes

Simplex-infected Cell Culures^

HostcellsMouse

embryoMouse

kidneyHr

afterinfection3610.56gdCMP

deaminaseactivity*1Uninfected3732251110Herpes

simplex-infected4049401716Thymidine

kinaseactivityUninfected2.30.3Herpes

simplex-infected21.02.1

" Abbreviations: dCMP, deoxycytidylate; PFU, plaque-

forming units.6The herpes simplex virus input multiplicities were 4 PFU/

cell and 15 PFU/eell, respectively, for mouse embryo and mousekidney.

°Enzyme extraction buffer contained 0.313 IHMdeoxyeytidioe-5'-triphosphate. The dCMP deaminase and thymidine kinaseactivities are expressed as (iMniolesdeoxyuridine-S'-monophosphateformed/yug protein in 10 min at 38°C.

TABLE 9Deoxycyíidylate (dCMP) Deaminase and Thymidine Kinase

Activities of Vaccinia-infected Cell Cultures"

HostcellsLM(TK-)

mousefibroblastsMouse

embryoMouse

kidneyHr

afterinfection5535.58dCMP

deaminaseactivity"Unin

fected17221151313Vaccinia-infected1762516149Thymidine

kinaseactivity6Unin

fected0.070.90.3Vaccinia-infected17.36.21.1

0 The vaccinia input multiplicity was 8-10 plaque-forming units/cell. Mouse kidney cultures contained 3 X IO6 cells/culture,while mouse embryo and LM(TK~) cells had 19 X IO6 cells/cul

ture.6Enzyme extraction buffer contained 0.313 HIMdeoxycytidine-

5'-triphosphate. The dCMP deaminase and thymidine kinaseactivities expressed as /i/imoles deoxyuridine-5'-monophosphateformed per ¿»gprotein in 10 min at 38°C.

OCTOBER 1967 1913

on June 14, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Saul Kit, R. A. de Torres, and Del R. Dubbs

SV40-transformed mouse kidney cells (10, 15, 16). The higher"thermostat-setting" may be a factor in the vigorous DNA syn

thesis and growth of the transformed cells.

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3. Dulbecco, R., Hartwell, L. H., and Vogt, M. Induction ofCellular DNA Synthesis by Polyoma Virus. Proc. Nati.Acad. Sei. U. S., 53: 403^10, 1965."

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7. Hartwell, L. H., Vogt, M., and Dulbecco, R. Induction ofCellular DNA Synthesis by Polyoma Virus. II. Increase inthe Rate of Enzyme Synthesis After Infection with PolyomaVirus in Mouse Kidney Cells. Virology, 27: 262-272, 1965.

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16. Kit, S., Melnick, J. L., Dubbs, D. R., Piekarski, L. J., andTorres, R. A. de. Early Enzyme Synthesis in Cell CulturesInfected with Papovaviruses, SV40 and Polyoma. In: M.Pollard (ed.), Perspectives in Virology V., Virus-Directed HostResponse. Chapter 3, pp. 63-86. New York: Academic Press,Inc., 1967.

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1914 CANCER RESEARCH VOL. 27

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1967;27:1907-1914. Cancer Res   Saul Kit, R. A. de Torres and Del R. Dubbs  Cell CulturesDeoxycytidylate Deaminase Activity of Simian Virus 40-infected

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