Deoxycytidylate Deaminase Activity of Simian Virus 40-Infected Cell Cultures1

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

Deoxycytidylate Deaminase Activity of Simian Virus 40-infected Cell Cultures1

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

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

SUMMARY There is evidence that the production of this enzyme is controlled Deoxycytidine-5'-triphosphate (dCTP) stabilizes deoxy- by phage genes. The capacity of T2 phage to induce deoxy cytidylate deaminase is impaired by ultraviolet light, although cytidylate deaminase activity. Enzyme extracts prepared with the enzyme-inducing capacity is more resistant to UV than is buffer containing dCTP are 2 to 6 times as active as those made infectivity (9). Mutant strains of T4 phage have been isolated without dCTP. Even when extracted in the presence of dCTP, which are defective in deoxycytidylate deaminase-inducing the deoxycytidylate deaminase levels vary considerably from activity (6). Moreover, T4 amber mutants induce significantly cell cultures of different origin. Monkey and human cells exhibit about twice the activity of LM(TK~) mouse fibroblast cells, and higher levels of deoxycytidylate deaminase than wild-tyiÂ«phage and extended enzyme synthesis is observed with the "DO" class about 20 and 40 times the activity of confluent monolayer ot amber mutants (29). cultures of mouse embryo and mouse kidney cells, respectively. The finding that deoxycytidylate deaminase activity is in An apparent stimulation of deoxycytidylate deaminase ac duced by DNA-containing bacteriophages raises the question tivity is observed following herpes simplex infection of LM (TK~) cells, when dCTP is omitted from the enzyme extraction whether this enzyme also increases in animal cells infected with buffer. However, extracts from uninfected LM(TK~) cells DNA-containing viruses. An increase of deoxycytidylate deami nase activity was not observed in monkey kidney cell cultures prepared with dCTP have as much activity as those from herpes simplex-infected cells. Uninfected LM(TK~) and monkey kid infected with simian adenovirus SV15 (17). However, it has been reported that this enzyme is augmented after productive infec ney cell extracts with dCTP also exhibit as much activity as do tion of mouse kidney cultures with polyoma virus or of monkey those from vaccinia- and Simian Virus 40 (SV40)-infected kidney cultures with SV40 (3, 7, 8). Studies from our laboratory cultures, respectively. have confirmed the enhancement of deoxycytidylate deaminase Extracts prepared with dCTP from vaccinia- or herpes simplex- activity in ]x>lyoma virus-infected mouse kidney cultures. Con infected mouse embryo and mouse kidney cultures have no trary to the report of Hatanaka and Dulbecco (8), however, we more than 1.7 times the deoxycytidylate deaminase activity of have not observed an enzyme increase in SV40-infected monkey uninfected cell extracts; however, thymidine kinase activity of kidney cultures. The enzyme does increase in mouse kidney cul virus-infected cell extracts is greatly increased over that of un tures abortively infected with SV40 and is elevated in SV40- infected cell extracts. transformed mouse kidney cells. Experiments with SV40 and Either productive-polyoma virus infection or abortive-SV40 also with two other DNA-containing animal viruses, vaccinia infection of mouse kidney cells result in stimulations of both and herpes simplex, are described in this paper. Preliminary deoxycytidylate deaminase and thymidine kinase activities. reports of these findings have been presented (10, 16, 19). These activities remain elevated in SV40-transformed mouse kidney cell cultures. Puromycin, but not 1-0-D-arabinofuranosylcytosine, inhibits the induction of deoxycytidylate deaminase in MATERIALS AND METHODS SV40-infected mouse kidney cultures. Cell Lines. Monolayer cultures of LM(TK~), CV-1, mKS, and primary cultures of mouse kidney, mouse embryo, and INTRODUCTION GMK3 cells were propagated as described previously (11, 13, 14). The enzyme deoxycytidylate deaminase increases significantly The CV-1 cells are an established strain of GMK cells. The following infection of Escherichia coli by T-even phages and of mKS cells are SV40-transformed mouse kidney cells (15). Sub- Bacillus subtilis by phages containing hydroxymethyl deoxy- lines mKS-1, mKS-4, mKS-11, mKS-17, mKS-A, and mKS-B uridylate in place of thymidylate in their DNA (5, 9, 23-26). were obtained from confluent primary cultures of mouse kidney cells which had been inoculated with 150 PFU per cell of SV40. 'This investigation was aided by grants from the National Sci ence Foundation (GB 5917), the American Cancer Society (E 291), ' Abbreviations : SV40, Simian Virus 40: dCMP, deoxycytidylate; the Robert A. Welch Foundation (Q163), and by ÃœSPHS Grants GMK, green monkey kidney; PFU, plaque-forming units; dCTP, CA-OC656, l-KG-AI-2352, and 5-K3-CA-25, 797. deoxycytidine-5'-triphosphate; dllMP, deoxyuridine-5'-monophos- 1 Postdoctoral Fellow of the Consejo Nacional de Investigaci phate; ara-C, l-/3-D-arabinofuranosylcytosine; dTTP, deoxythy- ones CientÃficas y TÃ©cnicas(Argentina). midine-5'- triphosphate; dTMP, deoxythymidine-5'-monophos- Received January 13, 1907; accepted June 30, 1967. phate; Tris, tris(hydroxymethyl)aminomethane.

OCTOBER 1967 1!K)7

The medium was replaced every 3 to 4 days and at 1, 4, 11, 17, Protein was measured by the method of Lowry et al. (20). The 22, and 30 days after SV40 inoculation, respectively, two primary enzyme reaction was linear with protein concentration and with infected cultures were trypsinized and subcultured. The cultures time under the conditions routinely used. were passaged again 10 to 23 days later after the monolayers had Experiments with crude and with centrifuged extracts demon become confluent or large colonies had formed. Monolayer cul strated that all of the deoxycytidylate deaminase activity was tures of mKS cells were propagated in R5a medium containing present in the supernatant fraction of the centrifuged extracts. 0.5% lactalbumin hydrolysate and 10% calf serum (1, 2). The Experiments by Maley and Maley (21) have shown that 2- cells grew to populations of 10-15 million per 55 sq cm and, af mercaptoethanol protects and activates deoxycytidylate deami ter the second passage, were subcultured at 3- to 4-day inter nase. To learn whether optimal 2-mercaptoethanol concentra vals. The mKS cells contained the SV40-tumor antigen, but not tions were employed in the assays reported here, the concentra detectable SV40-capsid antigen or virus particles. Extracts of tion of this chemical in the enzyme extraction solution was varied mKS cells did not yield infectious virus and plaques were not from 3 mM to 30 nun (1.6 to 16 mM final concentration during observed when mKS cells were plated on CV-1 cell monolayers. enzyme assay). With extracts prepared from either uninfected SV40 virus was recovered, however, when mKS cells were grown mouse kidney cells or from cells 44 hr after infection, the deoxy in mixed cultures with CV-1 cells. Equal numbers of freshly tryp cytidylate deaminase activity was about the same at each of the sinized mKS and CV-1 cells were mixed and 2 X IO6 cells were 2-mercaptoethanol concentrations studied. Either the standard plated in 8-oz prescription bottles. After 8 days of incubation, 2-mercaptoethanol concentration of 10 mM in the extraction the cells and medium from each mixed culture were harvested, buffer or a concentration of 3 mil was used in the present experi transferred to Lusteroid tubes, treated for 1 min at 4Â°Cwith a ments. Raytheon sonic oscillator at 10 kc, and assayed on CV-1 monolay Materials. Deoxyuridine-3H was purchased from New Eng ers for infectious virus. Infectious SV40 was recovered from 9 to land Nuclear Corporation, Boston, Mass., and dCMP-3H and 10 strains tested in amounts ranging from 200 to 10,000 PFU dCMP-uC from either New England Nuclear Corporation or per mixed culture. Schwarz BioResearch, Inc., Orangeburg, New York. Puromycin Viruses. Clonal strain 307L of SV40 was grown and assayed and ara-C were obtained from Nutritional Biochemical Corpora in CV-1 cells as previously described (14). The propagation and tion, Cleveland, Ohio, and dCTP from Calbiochem, Los Angeles, assay of vaccinia and herpes simplex viruses have also been California. reported (1, 2). Enzyme Assays. Thymidine kinase was determined by the RESULTS method of Kit and Dubbs (12). The dCMP deaminase was Deoxycytidylate Deaminase Activity of Tissue Culture measured by modifications of the isotopie method of Maley and Cell Lines. The deoxycytidylate deaminase activity varies Maley (22). Each tube contained in a total volume of 0.2 ml the greatly in different tissue culture lines (Table 1). The activity is following substances at the indicated final concentrations: 2.5 mM dCMP-I4C (about 700,000 cpm/Mmole) or dCMP-3H (about remarkably high in confluent monolayer cultures of monkey 2.5 X IO6 cpm/Mmole), 0.17 HIMdCTP, 27.2 mM potassium kidney (GMK and CV-1) and human [ HeLa, HeLa (BU-100) ] cells but is very low in confluent primary cultures of mouse kid fluoride, 2.5 HIMMgClz, 79 mM potassium chloride, 80 mM Tris- ney and mouse embryo cells. The LM(TK~) mouse fibroblast HC1 buffer (pH 8.0 at 25Â°C),5.25 mM 2-mercaptoethanol, and cells have about half the activity of CV-1 cell lines. enzyme. In the case of the mouse kidney cells, the enzyme extract consisted of about 400-1000 Â¿igof protein. For all other cell Effect of dCTP on Deoxycytidylate Deaminase Activity. extracts, about 100-300 jugof protein per tube were used. dCTP is required for maximal deoxycytidylate deaminase activ Enzyme extracts were usually prepared by sonication of 30-50 ity (21, 22, 28, 29). Normally, the enzyme is relatively unstable, but it can be stabilized and activated by dCTP. Table 2 illus- million cells sus[)ended in buffer containing 150 mM potassium chloride, 10 mM 2-mercaptoethanol, 0.31 Ã•Ã•IMdCTP,1.25 mM MgCl2, and 10 HIMTris-HCl buffer (pH 8.0 at 25Â°C).In a few TABLE 1 experiments, however, sodium phosphate buffer (pH 7.3) was Deoxycytidylate (dCMP) Deaminase Activities of Confluent substituted for Tris-HCl buffer in the enzyme extraction solution. Monolayer Cultures of Various Cell Lines The crude extracts were centrifuged for 1 hr at 4Â°Ceither at The enzyme extraction solution contained 0.313 mM deoxycytidine-5'-t riphosphate. 30,000 X g or at 105,000 X g and the supernatant employed for assay at three protein concentrations. Zero time and zero deaminase activity (AmÃ³les lineGMKCV-1HeLaHeLaCell deoxyuridine-5'-monophosphate formed/ enzyme blanks were included with the enzyme assays. The i

190S CANCER RESEARCH VOL. 27

trates experiments with SV40-infected monkey kidney cells and cells are disrupted to release the enzyme, either an "apparent" with herpes simplex-infected LM(TK~) mouse fibroblast cells. stimulation or an inhibition may be observed. If the virus infec In all instances, the enzyme was extracted and assayed in the tion tends to stabilize deoxycytidylate deaminase, a stimulation presence of 2-mercaptoethanol, but either with or without dCTP. will be observed; the reverse will be seen if the virus infection Three points are worthy of emphasis. First, deoxycytidylate reduces the stability of the enzyme. deaminase activity greatly exceeds thymidine kinase activity In further experiments, the concentration of dCTP in the (see also Charts 1 and 2). Second, enzyme extracts prepared and enzyme extraction solution was decreased to 0.15 HIMor increased assayed in the absence of dCTP exhibit much less deoxy to 0.45 niM. At each of the dCTP concentrations studied, SV40 cytidylate deaminase activity than those prepared with buffer induced thymidine kinase but not deoxycytidylate deaminase containing dCTP. The dCTP is specific for deoxycytidylate de activity in monkey kidney cell cultures. aminase; it does not enhance thymidine kinase activity. Third, The experiments of Table 2 with SV40-infected GMK cell cul the deoxycytidylate deaminase activities of uninfected cell ex tures are contrary to those of Hatanaka and Dulbecco (8) who tracts prepared without dCTP vary considerably from experi reported approximately an 8-fold increase in deoxycytidylate ment to experiment. Enzyme extracts prepared in the absence deaminase activity in SV40-infected GAIK cell cultures. In view of dCTP from cultures infected for 6 hr with herpes simplex virus of this discrepancy, the SV40 monkey kidney cell system was may show higher activities than those from uninfected cultures investigated in more detail. (Table 2, Experiments II, III). Extracts prepared without dCTP First the metabolic state of the GMK cells was considered. from cultures infected for about 41 hr with SV40 may have some Experiment I of Table 3 depicts an experiment in which 13-day- what lower activities than those from uninfected cultures (Table old primary GMK cell cultures were infected with SV40. These 2, Experiment I). However, enzyme extracts prepared from un cultures had been in the stationary phase of the growth cycle infected cells with buffer containing dCTP are as high in deoxy for at least 3 days. The deoxycytidylate deaminase activity 7, cytidylate deaminase activity as those from either SV40-infected 10, and 12 days after seeding the cultures was essentially the same or from herpes simplex-infected cells. One possible interpretation as that of the experimental period from Days 13 to 15. There of these results is that deoxycytidylate deaminase may be modi was no enhancement by SV40 of deoxycytidylate deaminase fied intracellularly by virus infection. When the virus-infected activity over the time jieriod of 16.5 to 44 hr after infection. At these times, substantial increases in thymidine kinase and DNA polymerase activities have previously been observed and the in TABLE 2 Effect of dCTP' in Enzyme Extraction Buffer on the dCMP corporation of labeled thymidine or deoxyadenosine into DNA was greatly stimulated (10, 14-16). Indeed, when the same ex Deaminase and Thymidine Kinase Activities of Uninfected and Virus-infected Cell Cultures0 tracts used to measure deoxycytidylate deaminase activity were used to measure thymidine kinase activity, a marked stimulation of the latter enzyme activity was observed. deaminase kinase activity*Enzyme activity"Enzyme Table 3, Experiment II, shows that SV40-infection also did not

post- enhance the deoxycytidylate deaminase activity of the CV-I Experi mentIIIIIICellsvirusUninfectedand infec established line of GMK cells. tion4166â€” extraction extraction bufferNo bufferNo Second, the possibility that a microsomal inhibitor of deoxy cytidylate deaminase (4) was affecting the ex]>eriments was con dCTP5738539892192With;dCTPd394383187150178177ThymidinedCTP1.07.10.0228.00.0211.4WithdCTPJ0.55.20.0424.40.046.2sidered. The enzyme extracts were therefore centrifuged at higher speeds using the Spinco Model L-2 ultracentrifuge to remove GMKSV40-infected more completely the microsomal fraction. Table 3, Experiment GMKUninfected III, shows that this did not alter the ex]>erimental findings. LM(TK-)Herpes Third, enzyme extracts were prepared with phosphate buffer, pH 7.3, instead of Tris-HCl buffer, pH 8.O. The former buffer simplex-in fectedLM(TK-)Uninfected had been employed by Hatanaka and Dulbecco (8). Table 3, Experiment III, shows that SV40 infection did not induce deoxy LM(TR-)Herpes cytidylate deaminase activity with these changed experimental simplex -in conditions. fected LM(TK-)Hr Fourth, possible interference in the assay by nucleotidase en ~dCMP zymes was ruled out. It was observed with either Tris-HCl or 0 Abbreviations : dCTP, deoxycytidine-5'-triphosphate; dCMP, phosphate extracts of uninfected cells or SV40-infected cells that deoxycytidylate; GMK, green monkey kidney; PFU, plaque- less than 0.5% of the dCMP-14C was converted to radioactive forming units; SV40, Simian Virus 40. deoxyuridine and deoxycytidine. However, 7 to 20% of bExperiment I: GMK cell cultures (13.8 X 10" cells/culture) the dCMP-14C was converted to labeled dUMP, depending on the were inoculated with 14 PFU/cell SV40. Experiments II and III: LM(TK-) cell cultures (11.4 X IO6 cells/culture) were inoculated enzyme concentration used in the assay. with 5 PFU/cell herpes simplex virus. Effect of Polyoma Virus Infection on Deoxycytidylate c AmÃ³les deoxyuridine-5'-monophosphate formed per jug pro Deaminase Activities of Murine Cell Cultures. In contrast tein in 10 min at 38Â°C. to monkey kidney cell cultures, the levels of deoxycytidylate de d Enzyme extraction buffer contained 0.313 HIM dCTP. The aminase are very low in confluent monolayer cultures of mouse final concentration of dCTP in the enzyme assay was 0.17 mw. kidney and mouse embryo cells (Table 1). These low activities are

OCTOBER 1967 1909

> > 60r SV40-INFECTED KINASE T

g uj 50 SV40- 2.5 Â± INFECTED DEAMINASE > z i 40 2.0 < tu u (O o 30 1.5 2 ÃœJ I- 20 1.0 UJ NONINFECTED DEAMINASE IO 0.5 O > X NONINFECTED KINASE o 0.0 IO 20 30 40 50 HOURS AFTER SV40 INFECTION CHART1. Kinetics of deoxycytidylate deaminase and thymidine kinase formation following infection of confluent monolayer cultures of mouse kidney cells (4.1 X IO6cells/culture) with 257 plaque-form ing units/cell of Simian Virus 40 (SV40). Thymidine kinase activity: timÃ³les deoxyuridine-5'-monophosphate (dUMP) formed per^g protein in 10 min at 38Â°C;deoxycytidylate deaminase activity: CiÃ±iÃ³les dUMP formed per Â¡tgprotein in 20 min at 38Â°C. found despite the addition of dCTP to the enzyme extraction With the uninfected or SV40-infected mouse kidney cultures, buffer. The data of Table 4 confirm the findings of Dulbecco and less than 0.2% of the dCMP-14C was converted to deoxyuridine. coworkers (3, 7) that the low activities of murine cell cultures are About 0.7-1% was dephosphorylated to deoxycytidine under enhanced after polyoma virus infection. It is to emphasized, conditions where about 4-10% of the dCMP-14C was deaminated however, that even after deoxycytidylate deaminase is induced to dUMP. by polyoma virus, the enzyme activity is only a fraction of that Effect of Puromycin on the Stimulation of Deoxycytidy observed with monkey, HeLa, or LM(TK~) mouse fibroblast late Deaminase Activity by SV40. To learn whether concurrent cultures. protein synthesis was needed for the stimulation of deoxycy Effect of Abortive-SV40 Infection on the Enzyme Activ tidylate deaminase activity by SV40, mouse kidney cultures were ities of Mouse Kidney Cells. SV40 undergoes an abortive in treated with puromycin. Table 5 shows that deoxycytidylate fection in mouse kidney cultures; the SV40-tumor antigen and deaminase activity increased about threefold at 38 hr after SV40 enzymes of DNA metabolism are induced although capsid pro infection in cultures not treated with puromycin. Puromycin teins and infectious virus are not made in most cells (10, 15, 16). (10~5 M) treatment from 20 to 38 hr prevented this increase. Chart 1 shows that deoxycytidylate deaminase activity is in Effect of ara-C Treatment on the Induction of Deoxy creased following abortive-SV40 infection of mouse kidney cell cytidylate Deaminase Activity by SV40. As previously ob cultures. The increase is detected at about 25 to 30 hr, several served (15), SV40 infection stimulates the incorporation of tri- hours after the induction of thymidine kinase is detected. At 40 tiated thymidine into DNA of mouse kidney cultures. ara-C is hr, the deoxycytidylate deaminase activity of SV40-infected cul a potent inhibitor of DNA biosynthesis in a variety of cell systems tures is about three times greater than that of uninfected cell (13, 18). Although, 15 pg/ml ara-C inhibited the incorporation cultures. Chart 2 depicts an experiment in which the deoxycy of tritiated thymidine into DNA of either SV40-infected or un tidylate deaminase and thymidine kinase activities of mouse infected mouse kidney cell cultures by 90-99%, the drug had no kidney cultures were studied from 26 to 72 hr after SV40 infec effect on the induction of either deoxycytidylate deaminase or tion. The deoxycytidylate deaminase activity reached a maxi thymidine kinase in infected cells (Table 6). These and previous mum at about 56 hr and remained elevated through 72 hr. At experiments (10, 14, 15) demonstrate that DNA synthesis is not 72 hr, the activity of SV40-infected cultures was about four times required for enzyme induction after either productive- or that of uninfected cultures. The thymidine kinase of SV40-in- abortive-SV40 infections. fected cultures was eleven times greater than that of uninfected Enzyme Activities of SV40-traiisformed Mouse Kidney cultures at 56 hr but declined considerably between 56 and 72 hr. Cell Lines. It has previously been shown that mouse kidney cell The deoxycytidylate deaminase activity of SV40-infected lines transformed by SV40 exhibit elevated levels of thymidine mouse kidney cultures was enhanced in extracts prepared in Tris- kinase, dTMP kinase, and DNA polymerase activities (10, 15, HC1 buffer or phosphate buffer and with the supernatant fraction 16). Table 7 shows that the deoxycytidylate deaminase activities of extracts centrifuged at either 30,000 X g or 105,000 X g of SV40-transformed cell lines are also elevated. In the case of

11I10 CANCER RESEARCH VOL. 27

90 SV40-INFECTED 4.5 KINASE 80 4.0 > K > 3.5 >_ U

u 60 3.0 i= in z i 50 2.50 c LÃ¼ LÃ¼ O SV40-INFECTED DEAMINASE LÃ¼ 40 2.0 ÃœJ o I 30 NON INFECTED .5 DEAMINASE K o 20 i.o x o o IO NON INFECTED 0.5 MEDIA KINASE CHANGED

20 30 40 50 60 70 80 HOURS AFTER SV40 INFECTION CHART2. Activities of deoxycytidylate deaminase and thymidine kinase of Simian Virus40 (SV40)-infected mouse kidney cell culturoÂ« 26 to 72 hr after virus infection. Cultures (5.5 X IO6cells/culture) were inoculated with 2G2plaque-forming units/cell of SV40; the virus adsorption 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 cultures were changed. Thymidine kinase activity :/inmoles deoxyuridine-5'-monophosphate (dUMP) formed per^g of protein in 10 min at 38Â°C; deoxycytidylate deaminase activity: Â«uniÃ³lesdUMP formed per iig protein in 20 min at 38Â°C.

Cell Line mKS-B, the deoxycytidylate deaminase activity was activity is complex. In the absence of stabilizing compounds, the almost as great as that of LM mouse fibroblast cells. enzyme is rapidly converted to an inactive state even at 0-5Â°C. Effect of Vaccinia and Herpes Simplex Infections on En Magnesium ions, 2-mercaptoethanol, and dCTP stabilize and zymes of Murine Cell Cultures. Although herpes simplex virus activate the enzyme while dTTP is a potent feedback inhibitor. does not stimulate deoxycytidylate deaminase activity of LM It is apparent that intracellular events leading to the formation (TK~) cell cultures (Table 2), it was of interest to learn whether or breakdown of dCTP and dTTP or to changes in sulfhydryl the virus would induce the enzyme in mouse embryo and mouse concentrations will affect enzyme activit3r. Disruption of cells kidney cultures. Table 8 shows that herpes simplex infection and dilution of deoxycytidylate deaminase in buffer solution may enhanced the deoxycytidylate deaminase activity of the mouse produce not only suboptimal conditions for enzyme activity but cell cultures by only about 1.7 times, although the thymidine might also lead to the activation of deoxycytidine triphosphatase kinase activity increased 7 to 9 times. activity and reduction of the dCTP concentration (4). Moreover, The data of Table 9 indicate that vaccinia infection fails to a natural inhibitor of the enzyme has been described in the micro- stimulate deoxycytidylate deaminase activity in LM(TK~), somal fraction of cell extracts (4). In view of the properties of mouse embryo, or mouse kidney cell cultures. With each of the deoxycytidylate deaminase, caution is indicated in interpreting murine cell cultures, and induction of thymidine kinase activity results of experiments with virus-infected cells. was observed. The experiments depicted in Table 2 show that the deoxycy tidylate deaminase extracted from herpes simplex-infected cells DISCUSSION with buffer containing 2-mercaptoethanol, but without dCTP, may be inactivated less than similar enzyme preparations from Studies by Maley and Maley (21, 22) and Scarano et al. (27, uninfected cells. As the SV40 experiment of Table 2 illustrates, 28) have shown that the control of deoxycytidylate deaminase the opposite result may also be obtained. It has been our experi-

OCTOBER 1967 1911

TABLE 3 ditions were somewhat different so that comparisons with the dCMP' Deaminase Activities of SV'40-infected GMK and CV-1 present data are difficult to make. We believe that the discrep Cell Cultures Measured at Various Times after Virus Infectionb ancy between their results and ours is most likely due to the failure to activate fully their deoxycytidylate deaminase from uninfected GMK cells at the enzyme dilutions that they em ployed. r after In contrast to monkey kidney cells, mouse kidney cells exhibit Experi formed//ig mentiiiinHostcellsGMKCV-1CV-1ExtractionbufferTris-HCl,pH SV40 for 1 hrat:30,000 infection16.524313844182941.51928414719284147dCMPdeaminaseactivitydeoxyuridine-5'-monophosphateproteinin10min at38Â°C)Unin TABLE 4 Effect of Polyoma Virus Infection on the dCMP" Deaminase Activity fected514544560504535498510565463464430406367349337315Infected590545599443519572541700508452468435393398376330of Murine Cell Cultures

deaminase activity^ Xg30,000 (Â¿i/imolesdeoxyuridine-5'- 8.0Tris-HCl,pH monophosphate formed/jig after 38Â°C)Uninfected29.6protein in 10 min at ExperimentiiiniIVvHostcellsMouse infection18

virus-infected48.1

Xg105,000 embryoMouse 8.0Tris-HCl,pHS.OPhosphate,pH 284125 35.113.04.8 67.442.125.5

embryoMouse Xg105,000 kidneyMouse 48.54248dCMP2.62.63.1Polyoraa61.113.221.6

kidneyMouse X gH 7.3Extractcentrifuged kidneyHr " Abbreviations: dCMP, deoxycytidylate; PFU, plaque-form ing units. " Abbreviations: dCMP, deoxycytidylate; GMK, green monkey 6Enzyme extraction buffer contained 0.313 mM deoxycytidine- kidney; Tris, tris(hydroxymethyl)aminomethane, SV40, Simian 5'-triphosphate. In Experiments I and II, confluent mouse em Virus 40. bryo cultures (14 X IO6 cells/culture) were inoculated with 206 6Stationary-phase monolayer cultures (9-11 X IO6 cells/ and 50 PFU/cell polyoma virus, respectively. In Experiments III culture) were inoculated in Experiments I, II, and III, respec to V, confluent mouse kidney cultures (4-6 X IO6 cells/culture) tively, with 108, 32, and 75 plaque-forming units/cell of SV40. were inoculated with about 160 PFU/cell polyoma virus. Enzyme extracts were prepared with buffer containing 0.313 mM deoxy cy tidine-5'-t riphosphat e. TABLE 5 Effect of Puromycin (10~&Â¡t)on the Stimulation of Deoxycytidylate enee that the deoxycytidylate deaminase activity of a given tissue (dCMP) Deaminase Activity in Simian Virus 40 (SV40)- is extremely variable unless precautions are taken to protect the infected Mouse Kidney Cell Cultures" enzyme prior to assay. When extracts are prepared in the absence deaminase activity of activators, the addition of these activators to the enzyme assay (AmÃ³les deoxyuridine-5'- tubes may not suffice to restore all the activity. of puromycin monophosphate formed/Mg Hr after infection treatment (hr after 38Â°C)Uninfected12.317.111.98.64.4SV40-infected11.611.628.45.7protein in 20 min at Previous studies indicate that simian adenovirus SV15 does enzymeassayed022038Â»38Intervalinfection)NoneNoneNoneNone20 not increase the deoxycytidylate deaminase activity of monkey kidney cells (17). The present data fail to show a clear-cut in duction 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 cells was not observed. However, all four classes of viruses greatly stimulated thymidine kinase activity. to 38dCMP The lack of stimulation of dCMP deaminase activity in SV40- " Confluent mouse kidney cultures (5.7 X IO6 cells/culture) infected monkey kidney cultures described in this study can be were inoculated with 196 plaque-forming units/cell SV40. Enzyme attributable to the fact that the cellular levels of this enzyme are extracts were prepared in buffer containing 0.313 mM deoxycyti- already very high in uninfected cells. The dCMP deaminase dine-5'-triphosphate. activities reported by Hatanaka and Dulbecco (8) were expressed 6Thymidine kinase activity of infected cultures was 6 times in undefined arbitrary enzyme units, and the experimental con greater than that of uninfected cultures at 38 hr postinfection.

1912 CANCER RESEARCH VOL. 27

TABLE 6 the formation of deoxycytidylate deaminase. It does not follow, Effect of ara-C" â€¢bon the Induction of Thymidine Kinase and dCMP that the same control mechanisms mediate the enzyme increase Deaminase Activities in SVJfi-infected Primary Mouse Kidney observed after either SV40 or polyoma virus infection of mouse Cell Cultures' kidney cells. Conclusions as to whether the mouse kidney enzyme is "derepressed" or whether new virus-specific enzymes are made kinase activity deaminase activity (/jamÃ³lesdUMPformed/Mg 38Â°C)UninfectedNo(/immolesdUMPprotein in 10min formed/jig at 38Â°C)UninfectedNoprotein in 20min at are premature at this time. It is significant that the enhanced deoxycytidylate deaminase levels persist after mouse kidney cells Hrs after infection38 are transformed by SV40. Elevated levels of thymidine kinase, DNA polymerase, and dTMP kinase have also been observed in ara-C0.17ara-C0.250.24SV40-infectedNoara-C1.551.20Withara-C1.50ara-C27.5Withara-C18.522.9SV40-infectedNoara-C49.270.7Withara-C55.7 TABLE 8 dCMP" Deaminase and Thymidine Kinase Activities of Herpes Simplex-infected Cell Culures^ 45Thymidine0.23With 1.44dCMP 66.2 deaminase kinase " Abbreviations: ara-C, l-/3-D-arabinofuranosylcytosine; dCMP, activity*1Uninfected3732251110HerpesactivityUninfected2.30.3Herpes deoxycy tidy late; dUMP, deoxyuridine-5'-monophosphate; SV40, HostcellsMouse after Simian Virus 40. infection3610.56gdCMP 0 Abbreviations: ara-C, 1 - ÃŸ- D - arabinofuranosylcytosine; simplex- simplex- dCMP, deoxycytidylate; dUMP, deoxyuridine-5'-monophosphate, infected4049401716Thymidineinfected21.02.1 SV40, Simian Virus 40. embryoMouse 6ara-C (15 jug/ml) was added to cultures 2 hr after infection. c Confluent 7-day-old cultures containing 5.5 X 10Â°cells/ culture were inoculated with 250 plaque-forming units/cell of SV40. Enzyme extracts were prepared with buffer containing 0.313 mm deoxycytidine-o'-triphosphate. kidneyHr

TABLE 7 " Abbreviations: dCMP, deoxycytidylate; PFU, plaque- DeoxycyÃidylate (dCMP) Deaminase Activity of Simian Virus Jfl forming units. (8V40)-transformed Mouse Kidney Cell Lines 6The herpes simplex virus input multiplicities were 4 PFU/ cell and 15 PFU/eell, respectively, for mouse embryo and mouse deaminase activity kidney. passage (/inmolesdeoxyuridine-5'- CellsUninfected number1875793410dCMPmonophosphate formed//*g Â°Enzyme extraction buffer contained 0.313 IHMdeoxyeytidioe- protein in 10min at38Â°C)2-154648303593103415'-triphosphate. The dCMP deaminase and thymidine kinase activities are expressed as (iMniolesdeoxyuridine-S'-monophosphate kidneyTransformedmKS-1mKS-4mKS-11mKS-17mKS-BmKS-BÂ«mKS-ACellmouse formed/yug protein in 10 min at 38Â°C.

TABLE 9 DeoxycyÃidylate (dCMP) Deaminase and Thymidine Kinase Activities of Vaccinia-infected Cell Cultures"

deaminase kinase activity"Unin activity6Unin after HostcellsLM(TK-) infection5535.58dCMP 0 mKS-B cell line adapted to grow in medium containing 25 fected17221151313Vaccinia-infected1762516149Thymidinefected0.070.90.3Vaccinia-infected17.36.21.1 jig/ml bromodeoxyuridine. At the 34th passage, the cells exhibited mousefibroblastsMouse 6.5 complement-fixation units of SV40-tumor antigen per 10' cells and had moderate levels of thymidine kinase activity. embryoMouse very low deoxycytidylate deaminase activity even when extracts are prepared with buffer containing dCTP and 2-mercapto- kidneyHr ethanol. This low activity can be increased somewhat following either productive-polyoma virus or abortive-SV40 infection. Con comitant protein but not DNA synthesis is required for the papovavirus-induced increases (Tables 5, 6; Ref. 7). The 0 The vaccinia input multiplicity was 8-10 plaque-forming units /cell. Mouse kidney cultures contained 3 X IO6 cells/culture, requirement for protein synthesis could signify that new deoxy while mouse embryo and LM(TK~) cells had 19 X IO6 cells/cul cytidylate deaminase polypeptide chains are synthesized after ture. SV40 infection. However, the alternative possibility that con 6Enzyme extraction buffer contained 0.313 HIMdeoxycytidine- tinued protein synthesis is needed to stabilize the enzyme is not 5'-triphosphate. The dCMP deaminase and thymidine kinase excluded. activities expressed as /i/imoles deoxyuridine-5'-monophosphate There is abundant evidence that bacteriophage genes control formed per Â¿Â»gproteinin 10 min at 38Â°C.

OCTOBER 1967 1913

SV40-transformed mouse kidney cells (10, 15, 16). The higher 16. Kit, S., Melnick, J. L., Dubbs, D. R., Piekarski, L. J., and "thermostat-setting" may be a factor in the vigorous DNA syn Torres, R. A. de. Early Enzyme Synthesis in Cell Cultures thesis and growth of the transformed cells. Infected with Papovaviruses, SV40 and Polyoma. In: M. Pollard (ed.), Perspectives in Virology V., Virus-Directed Host Response. Chapter 3, pp. 63-86. New York: Academic Press, REFERENCES Inc., 1967. 1. Dubbs, D. R., and Kit, S. Isolation and Properties of Vaccinia 17. Kit, S., Piekarski, L. J., Dubbs, D. R., Torres, R. A. de, and Mutants Deficient in Thymidine Kinase-Inducing Activity. Anken, M. Enzyme Induction in Green Monkey Kidney Cul Virology, eu: 214-225, 1964. tures Infected with Simian Adenovirus. J. Virology, 1: 10-15, 2. Dubbs D. R., and Kit, S. The Effect of Temperature on In 1967. duction of Deoxythymidine Kinase Activity by Herpes Sim 18. Kit, S., Torres, R. A. de, and Dubbs, D. R. Arabinofuranosyl- plex Mutants. Virology, 25: 250-270, 1965. cytosine-induced Stimulation of Thymidine Kinase and 3. Dulbecco, R., Hartwell, L. H., and Vogt, M. Induction of Deoxycytidylic Deaminase Activities of Mammalian Cultures. Cancer Res., 26: 1859-1866, 1966. Acad.Cellular Sei. DNA U. S., Synthesis 53: 403^10, by 1965." Polyoma Virus. Proc. Nati. 19. Kit, S., Torres, R. A. de, and Dubbs, D. R. Induction of 4. Fiala, S., and Fiala, A. E. Deoxycytidylic Acid Deaminase Deoxycytidylate (dCMP) Deaminase Activity in SV40-In- in Ehrlich Ascites Tumor Cells. Cancer Res., 25: 922-932, fected Mouse Kidney Cultures. Proc. Am. Assoc. Cancer Res., 1965. 8: 37, 1967. 5. Fleming, W. H., and Bessman, M. J. The Enzymology of 20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, Virus-infected Bacteria. VIII. The Deoxycytidylate Deam R. J. Protein Measurement with Folin-Phenol Reagent. J. inase of TG-infected Escherichia coli. 3. Biol. Chem., 240: Biol. Chem., 193: 265-275, 1951. PC4108-PC4110, 1965. 21. Maley, F., and Maley, G. F. On the Mechanism of Feedback 6. Hull, D. H., and Tessman, I. T4 Mutants Unable to Induce Inhibition of Deoxycytidylate Deaminase by Deoxythymidine Deoxycytidylate Deaminase. Virology, 29: 339-345, 1966. Triphosphate. J. Biol. Chem., 204: PC3226-PC3227, 1965. 7. Hartwell, L. H., Vogt, M., and Dulbecco, R. Induction of 22. Maley, G. F., and Maley, F. The Purification and Properties Cellular DNA Synthesis by Polyoma Virus. II. Increase in of Deoxycytidylate Deaminase from Chick Embryo Extracts. the Rate of Enzyme Synthesis After Infection with Polyoma J. Biol. Chem., 2S9: 1168-1176, 1964. Virus in Mouse Kidney Cells. Virology, 27: 262-272, 1965. 23. Maley, G. F., and Male5', F. The Significance of the Substrate 8. Hatanaka, M., and Dulbecco, R. Induction of DNA Syn Specificity of T2r+-Induced Deoxycytidylate Deaminase. J. thesis by SV40. Proc. Nati. Acad. Sei. U. S., 56: 736-740, Biol. Chem., 241: 2176-2177, 1966. 1966. 24. PenÃ©,J. J., and Marmur, J. Deoxyribonucleic Acid Replica 9. Keck, K., Mahler, H. R., and Fraser, D. Syntheses of Deoxy- tion and Expression of Early and Late Bacteriophage Func cytidine-5'-Phosphate Deaminase in Escherichia coli Infected tions in Bacillus subtilis. J. Virol., I: 86-91, 1967. by T2 Bacteriophage. Arch. Biochem. Biophy., 86: 85-88, 25. Roscoe, D. H., and Tucker, R. G. The Biosynthesis of a Py- 1960. rimidine Replacing Thymine in Bacteriophage DNA. Bio 10. Kit, S. Induction of Enzymes of DNA Metabolism by Simian chem. Biophy. Res. Commun., 16: 106-111, 1964. Virus 40. In: Y. Ito (ed.), Symposium on Subviral Carcino- 26. Roscoe, D. H., and Tucker, R. G. The Biosynthesis of Hy- genesis, Nagoya, Japan, in press. droxymethyldeoxyuridylic Acid in Bacteriophage-Infected 11. Kit, S., and Dubbs, D. R. Biochemistry of Vaccinia-Infected Bacillus sublilis. Virology, 29: 157-166, 1966. Mouse Fibroblasts (Strain L-M). I. Effects on Nucleic Acid 27. Scarano, E., Bonaduce, L., and De Petrocellis, B. Amino- and Protein Synthesis. Virology, 18: 274-285, 1962. hydrolysis of 4-Aminopyrimidine Deoxyribonucleotides. III. Purification and Properties of 2'-Deoxyribosly 4-Aminopy- 12. Kit, S., and Dubbs, D. R. Properties of Deoxythymidine Kinase Partially Purified from Noninfected and Virus-In rimidone-2,5'-Phosphate Aminohydrolase from Monkey Liver. fected Mouse Fibroblast Cells. Virology, 26: 16-27, 1965. J. Biol. Chem., 237: 3742-3751, 1962. 13. Kit, S., Dubbs, D. R., and Frearson, P. M. Enzymes of 28. Scarano, E., Ceraci, G., Polzella, A., and Campanile, E. The Nucleic Acid Metabolism in Cells Infected with Polyoma Enzymatic Aminohydrolysis of 4-Aminopyrimidine Deoxy Virus. Cancer Res., 26: 638-646, 1966. ribonucleotides. IV. On the Possibility of the Occurrence of an Allosteric Site on 2'-Deoxyribosyl 4-Aminopyrimidone- 14. Kit, S., Dubbs, D. R., Frearson, P. M., and Melnick, J. L. Enzyme Induction in SV40-Infected Green Monkey Kidney 2,5'-Phosphate Aminohydrolase. J. Biol. Chem., 2S8: PC Cultures. Virology, 29: 69-83, 1966. 1556-PC1557, 1963. 15. Kit, S., Dubbs, D. R., Piekarski, L. J., Torres, R. A. de, and 29. Warner, H. R., and Lewis, N. The Synthesis of Deoxycytidyl Melnick, J. L. Acquisition of Enzyme Function by Mouse ate Deaminase and Dihydrofolate ReducÃase and Its Control Kidney Cells Abortively Infected with Papovavirus SV40. in Escherichia coli Infected with Bacteriophage T4 and T4 Proc. Nati. Acad. Sei. U. S., 56: 463-470, 1966. Amber Mutants. Virology, 29: 172-175, 1966.

1914 CANCER RESEARCH VOL. 27

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

Cancer Res 1967;27:1907-1914.

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