Archives of Virology
Archives of Virology 53, 255--259 (1977)
(C) by Spriugcr-Verlag 1977
Cytosine-Arabinoside Does Not Inhibit Incorporation of 3H-Thymidine in Herpes Simplex Virus Transformed Cells Brief Report By J. ROUBAL
Department Of Experimental Virology, Institute of Sera and Vaccines, Prague, Czechoslovakia With 2 Figures Accepted October 25, 1976
Smnmary The incorporation of SH-thymidine into hamster cells transformed by herpes simplex viruses was found to l~e resistent to the action of cytosine arabinoside. In the last few years cells of animal and human origin wore transformed i~7~vitro b y herpes simplex viruses (HSV) (for references see 17). Several authors demonstrated the presence of virus sl3ecific antigens or virus specific nucleic acid sequences in these cells (4, 13) or obtained an indirect, evidence on the presence of virus specific functions in them (2, 12). We have followed the effect of cytosine arabinoside tara-C), an inhibitor of DNA synthesis, on the incorporation of 3H-thymidine into the aeidopreeipitable fractions of hamster cells transformed either spontaneously or b y the action of different viruses. H E F cell line was derived from hamster embryo cultures. Initially the H E F cells exhibited contact inhibition at a saturation density of 10 a celts/cm 2. At that, time, inoculation of 106 cells did not induce tmnours in hamsters. After the 100th passage the cells lost contact inhibition and acquired oneogenie potential. TR-37 cells were derived from H E F cells by transformation with UV-irradiated HSV-2 (14). 333-8-9 and HASI/HSV-1 cell lines transformed by HSV-2 and HSV-1, respectively, in the laboratory of Dr. F. t{app (Hershey, U.S.A.) were kindly provided by Dr. L. Thiry (Bi~ssells, Belgium). TUB cells were derived in our laboratory from ~ h.amster tumour induced by SV40 transformed H50 cells (1). Ara-C (Upjohn Co., 2Kiehigan) at indicated concentrations was added to the sub-confluent cultures one hour prior to the addition of SH-thymidine (20 Ci/m;~, Institute for Research, Production and Application of Radioisotopes, Czechoslovakia) at the final concentration of 5 ~Ci/ml. After one hour labelling period
256
J. ROUBAL:
the cells were frozen (--70 ° C) and thawed. The cell lysis was completed by the addition of concentrated buffer to obtain a final concentration of 0.01 M tris-HC1 (pH 7.5), 0.005 M E D T A and 0.2 per cent sarcosytsulphate. The samples were then treated for 18 hours with prcincubated pronase (Koch & Light, England) at the final concentration of 1 mg/ml. After addition of the same volume of 10 per cent trichloroaeetic acid (TCA) and incubation at 4°C for at least 30 minutes, the precipitates were collected onto Synpor 6 filters (VCHZ Synthesia UhNn5ves, Czechoslovakia), washed twice with 5 per cent TCA, once with 96 per cent ethanol and dried. The radioactivity was determined as described previously (10). The rate of 3H-thymidine incorporation in the presence of ara-C was expressed as the percentage of incorporation into the respective cell type in the absence of the drug. The effect of 20 ag of ara-C/ml is shown in Table 1. As can be seen the incorporation into H E F and TUB cells was strongly inhibited in the presence of ara-C. On the other hand, the incorporation of the label into cells transformed with HSV was resistant to the action of the drug. I n fact, more label was incorporated into the treated than into the control cells. Table 1. E]jeet o] ara-C on aH-thymidine incorporation into acidoprecipitable ]ractions o] di]]erent hamster trans]ormed cellsa
Cells
Transforming agent
cpm of aN thymidine incorporated per culture Ara-C absent
Ara-C present c
Percentage of incorporation in Ara-C presence s
HE:F T:R-37 333-8-9 HAM/HSV-1 TUB
unknown HSV-2 I-ISV-2 HSV-1 SV 40
9,117 10,078 6,120 18,132 19,896
418 14,099 7,345 25,090 1,191
4.6 139.9 120.0 138.4 6.0
:Results of a representative experiment are given in the Table 1 b The average percentages of control incorporations as determined from several repeated experiments were: 3.8± 1.3 for HEF ( 13 experiments), 159.1 _4_-21.2 for TR-37 (12 experiments), 136.0-4= 15.6 for 333-8-9 (10 experiments), 152.44-24.4 for HAM/HSV-1 (5 experiments) and 5.04-2.6 for TUB (6 experiments) o Concentration of ara-C was 20 ~zg/ml As indicated in Figure 1 the incorporation into H E F cells was readily inhibited already at the concentration of 5 ~xg of ara-C/ml, while the two HSV-2 transformed cell lines incorporated 3H-thymidine still at the concentration of 40 ~xg of ara-C/ml. Figure 2 shows that the prolongation of the incubation of the cells with ara-C prior to the addition of the label did not reduce its incorporation into HSV-2 transformed cells. Several mechanisms can be considered in explaining the resistence of HSV transformed cells to ara-C. At first it was thought that the effect observed might be due to the non-penetrability for the drug induced by membrane changes associated with the transformation by HSV. However, in the subsequent double label experiment the ratios between the amounts of 3H-ara-C (The Radiochemical Centre Amersham, England; 15 Ci/m~; used at the concentration t .6 × 10-2 ~xg/ml)
Effect, of Ara-C on Thymidine Uptake by HSV-Transformed Cells
257
and 14C-thymidine (IRPAR, Czechoslovakia; 53 mCi/mM; used at the concentration 0.2 ~Ci/ml) incorporated into acidopreeipitable fractions were similar for M1 cell lines examined (0.81, 0.82, 0.87, 0.92, 0.96 for H E F , TR-37, HAM/HSV-1, 333-8-9 and TUB, respectively). I n addition, the incorporation of 3H-ara-C into the aeidoprecipitable fraction of both drug-resistant and drug-sensitive cells with similar efficiencies made it unlikely that the effect observed migh~ be due to the absence, or low activity, of deoxycytidine kinase (3, 9) in HSV transformed cells. Indeed, the phosphorylation of ara-C to at least a monophosphate was demonstrated (Z.;~VADA and ROUBAL, unpublished observation). I t is another possibility t h a t the observed effect might be due to the resistance of the DNA polymerase of I~SV-transformed cells to the action of an ara-C metabolite. I t should be recalled that ara-CTP was described to inhibit selectively several DNA polymerases (7, 8, t5, 18). Recently Mi2J~LE~ e t a l . (t6) have reported that HSV-1 specific DNA polymerase was resistant i n v i t r o to the action of ara-CTP while polymerases from non-infected B H K cells were inhibited in its presence. This raises the possibility 200
,,,
,
,
, ,
,
,
~
,,,-
180 .~ 160
4
4
12o
6o\
.=- 100
80
40
20
HEF 7
0
?
,
10
* .....
20 30 JJq ARA-c/ml
4,0
Fig. 1. Influence of different concentrations of ara-C on incorporation of ~H-thymidine into different hamster cell lines
.=o.140
TR-3F
o....C__
~120 o
•s 80
=-. ..... &
o ,l~-~
~
o
333.8°9
40 =o HEF
00
"2-
1
Addition
233 o¢
•
,,0
4
5
~-~hyrn]d[oe(HRS)
Fig. 2. Effect of prolonged incubation in the presence of ara-C on incorporation of aI-I-thymidine into different hamster cell lines. Ara-C was used at the concentration of 20 ~zg/mt
258
J. RO~BAL:
that HSV-transformed cells might contain HSV specific DNA polymerase the behavior of which resembles more the potymerase in the in. vitro than in the in vivo system because the synthesis of HSV DNA in the infected cells was described to be sensitive to the action of the drug (6). The results of DUFF and MAo (5) demonstrating the sensitivity of DNA polymerases from HSV transformed cells to the action of phosphonoacetic acid are in line with this hypothesis. However, it is still possible that the resistance of ItSV transformed cells to ara-C is of another nature involving, e.g., a modification of deoxycytidine metabolism similar to that recently described by JA~IESO~ and Su]3Ax-SI4am,]~ (l l) for HSV infected cells. Also ara-C deamination might be differently effective in the various cell lines and might, be responsible for the present phenomenon. Further work to elucidate it is in progress.
Aeknowledgment The excellent technical assistance of Mr. J. Vejsada is greatly appreciated.
References i. ASHKENAZY, A., I~[ELNICK, J. L.: Tumorogenicity of simian papovavirus SV4O and of virus transformed cells. J. Natl. Cancer Inst. 30, 1227--1265 (1963). 2. BLACKLOW, N. R.: Potentiation of an adenovirus associated virus by herpes simplex virus type 2--transformed cells. J. Natl. Cancer Inst. 54, 241--244 (1975). 3. CH~, 3/i. Y., FISCHE~, G. A. : Colr~parative studies of leukemic cells sensitive and resistant to cytosine arabinoside. Biochem. Pharmaeol. 14, 333--341 (1965). 4. COLLARD, W., THO]aNTOI% H., G~EEN, M. : Cells transformed by human herpesvirus type 2 transcribe virus specific I~NA sequences shared by herpesvirus type 1 and 2. Nature (New Biol.) 243, 264---266 (1973). 5. DUFF, l~. G., MAo, J. C. H. : Inhibition by phosphonoacetate of cells transformed by herpes simplex virus. Symposium on Ilerpesviruses (Abstracts of Papers), Libliee 1976, 55, Praha, Czeehosl. Soc. Microbiol. Czechosl. Acad. Sei. 1976. 6. FALSE, ]2)., IIE[C_KE, B., B£SSLER, R.: The effect of arabino-furanosyl-eytosine upon the synthesis of herpesvirus hominis. Arch. ges. Virusforsch. 39, 48--62 (1972). 7. F~j:a:rH, J. J., COI4EN, S. S. : Inhibition of mammalian DNA polymerase by the 5'-triphosphate of I-~-D arabinofuranosylcytosine and 5'-triphosphate of 9-~-D arabinofuranosyladenine. Cancer Res. 28, 2061--2067 (1968).
8. GRAmxsI, F. L., ~VHITYIO:aE,G. F. : Studies in mouse L-cells on the incorporation of 1- [-D-arabinofuranosylcytosine into DNA and on inhibition of DNA polymerase by 1-~-D~arabinofuranosyl-cytosine 5'-triphosphate. Cancer Res. 30, 2636--2644 (1970). 9. HAY, J., PERERA, P. A. J., MOI~RISON, J. M., GENT~Y, G. A., SUBAK-SIIAI~PE, J. ~. : I-Ierpes virus specified proteins. In: WOLSTENI~OLME, G. E. W., O'CoNNOr, M. (eds.), Strategy of the Viral Genome (A Ciba Foundation Symposium), 355--372. Edinburgh-London : Churchill Livingstone 197 I. I0. I-II1~SCg, I., VONI4A, V. : II.ibonueleotides linked to I)NA of herpes simplex virus type I. J. Virol. 13, 1162---1168 (1974). ii. JANIESO:N, A. T., SUBAK-S~ARP]~, J. H.: Herpes simplex virus specified deoxypyrimidine kinase and the uptake of exogenous nueleosides by infected cells. J. ten. Virol. 31, 303---314 (1976). 12. KIMU]aA, S., ESPARZA, J., BENYESI[-MELNICK, M., SCJ:~AFFEI% P. A.: Enhanced replication of temperature-sensitive mutants of herpes simplex virus type 2 (IISV-2) at, the non-permissive temperature in cells transformed by HSV-2. Intervirology 3, 162--169 (1974).
Effect of Ara-C on Thymidine Uptake by HSV-Transformed Cells
259
13. KBEISELBURD, E., GAGE, L. P., W'lgISSBACH,A. : Presence of a herpes simplex virus 14.
t5. 16.
17. 18.
D N A fragment in an L~eell clone obtained after infection with irradiated herpes simplex virus 1. J. mol. Biol. 97, 533--537 (1975). Ku~ov£, L., VO~'~:A, V., B~OU~K, J. : Increased oneogenieity and synthesis of herpesvirus antigens in hamster cells exposed to herpes simplex type 2 virus. J. Natl. Cancer Inst. 5@, 759--766 (1973). MO~PARLER, 1%. L. : Effect of cytosine arabinoside-5'-triphosphate on mammalia.n DNA polymerase. Bioehem. biophys. R.es. Commun. 34, 465--471 (1969). M~Ls~R, W. E. G., FALKE, D., ZA:~N, R. If. : DNA-dependent D N A polymerase pattern in noninfeeted and herpesvirus infected rabbit kidney cells. Arch. ges. Virusforsch. 42, 278--284 (1973). l~aPP, F,, R~:D, C. : Experimental evidence for the oncogenic potential of herpes simplex virus. Cancer Res. 36, 800--806 (t976). TUONINEN, F. W., KENNE¥, F. T. : Inhibition of l~NA-direeted DNA polymerase from raucher leukemia virus by the 5'-triphosphate of cytosine arabinoside. Bioehem. biophys. I%es. Commun. 48, 1469--t475 (t972).
Author's address : Dr. J. I~ou~AL, Department of Experimental Virology, Institute of Sera and Vaccines, W. Pieeka Str. 108, 10103 Prague 10, Czechoslovakia. t~eeeived August 23, 1976
Archives of Virology 53, 255--259 (1977)
(C) by Spriugcr-Verlag 1977
Cytosine-Arabinoside Does Not Inhibit Incorporation of 3H-Thymidine in Herpes Simplex Virus Transformed Cells Brief Report By J. ROUBAL
Department Of Experimental Virology, Institute of Sera and Vaccines, Prague, Czechoslovakia With 2 Figures Accepted October 25, 1976
Smnmary The incorporation of SH-thymidine into hamster cells transformed by herpes simplex viruses was found to l~e resistent to the action of cytosine arabinoside. In the last few years cells of animal and human origin wore transformed i~7~vitro b y herpes simplex viruses (HSV) (for references see 17). Several authors demonstrated the presence of virus sl3ecific antigens or virus specific nucleic acid sequences in these cells (4, 13) or obtained an indirect, evidence on the presence of virus specific functions in them (2, 12). We have followed the effect of cytosine arabinoside tara-C), an inhibitor of DNA synthesis, on the incorporation of 3H-thymidine into the aeidopreeipitable fractions of hamster cells transformed either spontaneously or b y the action of different viruses. H E F cell line was derived from hamster embryo cultures. Initially the H E F cells exhibited contact inhibition at a saturation density of 10 a celts/cm 2. At that, time, inoculation of 106 cells did not induce tmnours in hamsters. After the 100th passage the cells lost contact inhibition and acquired oneogenie potential. TR-37 cells were derived from H E F cells by transformation with UV-irradiated HSV-2 (14). 333-8-9 and HASI/HSV-1 cell lines transformed by HSV-2 and HSV-1, respectively, in the laboratory of Dr. F. t{app (Hershey, U.S.A.) were kindly provided by Dr. L. Thiry (Bi~ssells, Belgium). TUB cells were derived in our laboratory from ~ h.amster tumour induced by SV40 transformed H50 cells (1). Ara-C (Upjohn Co., 2Kiehigan) at indicated concentrations was added to the sub-confluent cultures one hour prior to the addition of SH-thymidine (20 Ci/m;~, Institute for Research, Production and Application of Radioisotopes, Czechoslovakia) at the final concentration of 5 ~Ci/ml. After one hour labelling period
256
J. ROUBAL:
the cells were frozen (--70 ° C) and thawed. The cell lysis was completed by the addition of concentrated buffer to obtain a final concentration of 0.01 M tris-HC1 (pH 7.5), 0.005 M E D T A and 0.2 per cent sarcosytsulphate. The samples were then treated for 18 hours with prcincubated pronase (Koch & Light, England) at the final concentration of 1 mg/ml. After addition of the same volume of 10 per cent trichloroaeetic acid (TCA) and incubation at 4°C for at least 30 minutes, the precipitates were collected onto Synpor 6 filters (VCHZ Synthesia UhNn5ves, Czechoslovakia), washed twice with 5 per cent TCA, once with 96 per cent ethanol and dried. The radioactivity was determined as described previously (10). The rate of 3H-thymidine incorporation in the presence of ara-C was expressed as the percentage of incorporation into the respective cell type in the absence of the drug. The effect of 20 ag of ara-C/ml is shown in Table 1. As can be seen the incorporation into H E F and TUB cells was strongly inhibited in the presence of ara-C. On the other hand, the incorporation of the label into cells transformed with HSV was resistant to the action of the drug. I n fact, more label was incorporated into the treated than into the control cells. Table 1. E]jeet o] ara-C on aH-thymidine incorporation into acidoprecipitable ]ractions o] di]]erent hamster trans]ormed cellsa
Cells
Transforming agent
cpm of aN thymidine incorporated per culture Ara-C absent
Ara-C present c
Percentage of incorporation in Ara-C presence s
HE:F T:R-37 333-8-9 HAM/HSV-1 TUB
unknown HSV-2 I-ISV-2 HSV-1 SV 40
9,117 10,078 6,120 18,132 19,896
418 14,099 7,345 25,090 1,191
4.6 139.9 120.0 138.4 6.0
:Results of a representative experiment are given in the Table 1 b The average percentages of control incorporations as determined from several repeated experiments were: 3.8± 1.3 for HEF ( 13 experiments), 159.1 _4_-21.2 for TR-37 (12 experiments), 136.0-4= 15.6 for 333-8-9 (10 experiments), 152.44-24.4 for HAM/HSV-1 (5 experiments) and 5.04-2.6 for TUB (6 experiments) o Concentration of ara-C was 20 ~zg/ml As indicated in Figure 1 the incorporation into H E F cells was readily inhibited already at the concentration of 5 ~xg of ara-C/ml, while the two HSV-2 transformed cell lines incorporated 3H-thymidine still at the concentration of 40 ~xg of ara-C/ml. Figure 2 shows that the prolongation of the incubation of the cells with ara-C prior to the addition of the label did not reduce its incorporation into HSV-2 transformed cells. Several mechanisms can be considered in explaining the resistence of HSV transformed cells to ara-C. At first it was thought that the effect observed might be due to the non-penetrability for the drug induced by membrane changes associated with the transformation by HSV. However, in the subsequent double label experiment the ratios between the amounts of 3H-ara-C (The Radiochemical Centre Amersham, England; 15 Ci/m~; used at the concentration t .6 × 10-2 ~xg/ml)
Effect, of Ara-C on Thymidine Uptake by HSV-Transformed Cells
257
and 14C-thymidine (IRPAR, Czechoslovakia; 53 mCi/mM; used at the concentration 0.2 ~Ci/ml) incorporated into acidopreeipitable fractions were similar for M1 cell lines examined (0.81, 0.82, 0.87, 0.92, 0.96 for H E F , TR-37, HAM/HSV-1, 333-8-9 and TUB, respectively). I n addition, the incorporation of 3H-ara-C into the aeidoprecipitable fraction of both drug-resistant and drug-sensitive cells with similar efficiencies made it unlikely that the effect observed migh~ be due to the absence, or low activity, of deoxycytidine kinase (3, 9) in HSV transformed cells. Indeed, the phosphorylation of ara-C to at least a monophosphate was demonstrated (Z.;~VADA and ROUBAL, unpublished observation). I t is another possibility t h a t the observed effect might be due to the resistance of the DNA polymerase of I~SV-transformed cells to the action of an ara-C metabolite. I t should be recalled that ara-CTP was described to inhibit selectively several DNA polymerases (7, 8, t5, 18). Recently Mi2J~LE~ e t a l . (t6) have reported that HSV-1 specific DNA polymerase was resistant i n v i t r o to the action of ara-CTP while polymerases from non-infected B H K cells were inhibited in its presence. This raises the possibility 200
,,,
,
,
, ,
,
,
~
,,,-
180 .~ 160
4
4
12o
6o\
.=- 100
80
40
20
HEF 7
0
?
,
10
* .....
20 30 JJq ARA-c/ml
4,0
Fig. 1. Influence of different concentrations of ara-C on incorporation of ~H-thymidine into different hamster cell lines
.=o.140
TR-3F
o....C__
~120 o
•s 80
=-. ..... &
o ,l~-~
~
o
333.8°9
40 =o HEF
00
"2-
1
Addition
233 o¢
•
,,0
4
5
~-~hyrn]d[oe(HRS)
Fig. 2. Effect of prolonged incubation in the presence of ara-C on incorporation of aI-I-thymidine into different hamster cell lines. Ara-C was used at the concentration of 20 ~zg/mt
258
J. RO~BAL:
that HSV-transformed cells might contain HSV specific DNA polymerase the behavior of which resembles more the potymerase in the in. vitro than in the in vivo system because the synthesis of HSV DNA in the infected cells was described to be sensitive to the action of the drug (6). The results of DUFF and MAo (5) demonstrating the sensitivity of DNA polymerases from HSV transformed cells to the action of phosphonoacetic acid are in line with this hypothesis. However, it is still possible that the resistance of ItSV transformed cells to ara-C is of another nature involving, e.g., a modification of deoxycytidine metabolism similar to that recently described by JA~IESO~ and Su]3Ax-SI4am,]~ (l l) for HSV infected cells. Also ara-C deamination might be differently effective in the various cell lines and might, be responsible for the present phenomenon. Further work to elucidate it is in progress.
Aeknowledgment The excellent technical assistance of Mr. J. Vejsada is greatly appreciated.
References i. ASHKENAZY, A., I~[ELNICK, J. L.: Tumorogenicity of simian papovavirus SV4O and of virus transformed cells. J. Natl. Cancer Inst. 30, 1227--1265 (1963). 2. BLACKLOW, N. R.: Potentiation of an adenovirus associated virus by herpes simplex virus type 2--transformed cells. J. Natl. Cancer Inst. 54, 241--244 (1975). 3. CH~, 3/i. Y., FISCHE~, G. A. : Colr~parative studies of leukemic cells sensitive and resistant to cytosine arabinoside. Biochem. Pharmaeol. 14, 333--341 (1965). 4. COLLARD, W., THO]aNTOI% H., G~EEN, M. : Cells transformed by human herpesvirus type 2 transcribe virus specific I~NA sequences shared by herpesvirus type 1 and 2. Nature (New Biol.) 243, 264---266 (1973). 5. DUFF, l~. G., MAo, J. C. H. : Inhibition by phosphonoacetate of cells transformed by herpes simplex virus. Symposium on Ilerpesviruses (Abstracts of Papers), Libliee 1976, 55, Praha, Czeehosl. Soc. Microbiol. Czechosl. Acad. Sei. 1976. 6. FALSE, ]2)., IIE[C_KE, B., B£SSLER, R.: The effect of arabino-furanosyl-eytosine upon the synthesis of herpesvirus hominis. Arch. ges. Virusforsch. 39, 48--62 (1972). 7. F~j:a:rH, J. J., COI4EN, S. S. : Inhibition of mammalian DNA polymerase by the 5'-triphosphate of I-~-D arabinofuranosylcytosine and 5'-triphosphate of 9-~-D arabinofuranosyladenine. Cancer Res. 28, 2061--2067 (1968).
8. GRAmxsI, F. L., ~VHITYIO:aE,G. F. : Studies in mouse L-cells on the incorporation of 1- [-D-arabinofuranosylcytosine into DNA and on inhibition of DNA polymerase by 1-~-D~arabinofuranosyl-cytosine 5'-triphosphate. Cancer Res. 30, 2636--2644 (1970). 9. HAY, J., PERERA, P. A. J., MOI~RISON, J. M., GENT~Y, G. A., SUBAK-SIIAI~PE, J. ~. : I-Ierpes virus specified proteins. In: WOLSTENI~OLME, G. E. W., O'CoNNOr, M. (eds.), Strategy of the Viral Genome (A Ciba Foundation Symposium), 355--372. Edinburgh-London : Churchill Livingstone 197 I. I0. I-II1~SCg, I., VONI4A, V. : II.ibonueleotides linked to I)NA of herpes simplex virus type I. J. Virol. 13, 1162---1168 (1974). ii. JANIESO:N, A. T., SUBAK-S~ARP]~, J. H.: Herpes simplex virus specified deoxypyrimidine kinase and the uptake of exogenous nueleosides by infected cells. J. ten. Virol. 31, 303---314 (1976). 12. KIMU]aA, S., ESPARZA, J., BENYESI[-MELNICK, M., SCJ:~AFFEI% P. A.: Enhanced replication of temperature-sensitive mutants of herpes simplex virus type 2 (IISV-2) at, the non-permissive temperature in cells transformed by HSV-2. Intervirology 3, 162--169 (1974).
Effect of Ara-C on Thymidine Uptake by HSV-Transformed Cells
259
13. KBEISELBURD, E., GAGE, L. P., W'lgISSBACH,A. : Presence of a herpes simplex virus 14.
t5. 16.
17. 18.
D N A fragment in an L~eell clone obtained after infection with irradiated herpes simplex virus 1. J. mol. Biol. 97, 533--537 (1975). Ku~ov£, L., VO~'~:A, V., B~OU~K, J. : Increased oneogenieity and synthesis of herpesvirus antigens in hamster cells exposed to herpes simplex type 2 virus. J. Natl. Cancer Inst. 5@, 759--766 (1973). MO~PARLER, 1%. L. : Effect of cytosine arabinoside-5'-triphosphate on mammalia.n DNA polymerase. Bioehem. biophys. R.es. Commun. 34, 465--471 (1969). M~Ls~R, W. E. G., FALKE, D., ZA:~N, R. If. : DNA-dependent D N A polymerase pattern in noninfeeted and herpesvirus infected rabbit kidney cells. Arch. ges. Virusforsch. 42, 278--284 (1973). l~aPP, F,, R~:D, C. : Experimental evidence for the oncogenic potential of herpes simplex virus. Cancer Res. 36, 800--806 (t976). TUONINEN, F. W., KENNE¥, F. T. : Inhibition of l~NA-direeted DNA polymerase from raucher leukemia virus by the 5'-triphosphate of cytosine arabinoside. Bioehem. biophys. I%es. Commun. 48, 1469--t475 (t972).
Author's address : Dr. J. I~ou~AL, Department of Experimental Virology, Institute of Sera and Vaccines, W. Pieeka Str. 108, 10103 Prague 10, Czechoslovakia. t~eeeived August 23, 1976
