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VIROLOGY

(1975)

Cell Cycle Dependence Transformed JOAN Department

C. KAPLAN,’

of Simian

Hamster LAWRENCE

Virus 40 Induction

Cells by Ultraviolet F. KLEINMAN,

AND

from

Irradiation PAUL

H. BLACK

of Medicine, Massachusetts General Hospital, and Departments of Microbiology and Molecular Genetics and Medicitze, Harvard Medical School Boston, Massachusetts 02124 Accepted June 16, 1975 The induction of infectious simian virus 40 (SV40) was studied in synchronized cultures of SV40-transformed hamster cells. Cells were synchronized with a double excess thymidine block and induced by mitomycin C treatment or uv irradiation at hourly intervals after release from the block. Infectious virus was measured in cell-free extracts at ‘2 hr after initiating induction. The results indicated that virus activation by these agents was restricted to certain phases of the cell cycle. Mitomycin C was most effective if it was applied to cells 4 hr after the peak of DNA synthesis, With uv radiation, virus production was maximal if cells were irradiated l-2 hr after the peak of DNA synthesis during the late SC, phase of the cell cycle. The most sensitive period of the cell cycle for virus activation by uv is the one in which eukaryotic cells are most resistant to uv radiation, as measured by enhanced survival after irradiation. Therefore, it is possible that cellular DNA repair processes are more active during this interval, promoting DNA strand break formation and viral genome excision. INTRODUCTION

In the preceding paper it was demonstrated that clones of simian virus 40 (SV40) -transformed hamster cells could be induced to produce infectious virus by treatment with a number of chemical and physical agents, such as mitomycin C or uv irradiation, which cause strand breakage in DNA (Kaplan et al., 1975). A direct relationship was established between the dose of inducing agent and virus yield which suggested that DNA strand break formation may be related to virus induction. We next considered the influence of the physiological state of the host cell on virus induction and whether virus activation could be correlated with a particular phase of the cell cycle. Therefore, induction studies were carried out on synchronized populations of transformed cells. It has been shown that the replication of SV4O in permissive monkey cells is linked I Send reprint requests to J.C.K., Infectious Disease Unit, Massachusetts General Hospital, Boston. Mass. 02114.

215 Copyright All rights

0 1975 by Academic Press, of reproduction in any form

Inc. reserved

to the cell cycle (Pages et al., 1973). Hampar et al. have reported that there is cell cycle dependence for the activation of EB viral antigens. In the latter study, synchronized human lymphoblastoid cell lines were exposed to a number of nucleoside analogs, including IdU, and viral antigen production was observed only if the drug was applied to cells early in the S period (Hampar et al., 1973, 1974). Similarly, the activation of endogenous RNA tumor viruses by IdU or cycloheximide has been shown to be related to particular phases of the cell cycle (Greenberger and Aaronson, 1975). In this report, we present results concerning the induction of infectious virus from synchronized cultures of a clone of transformed hamster cells by mitomycin C or uv irradiation. Our studies indicate that virus activation by these agents is restricted to certain phases of the cell cycle. In the case of uv irradiation we were able to show that infectious SV40 was preferentially activated from cells which were in the

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late S-G, interval at the time of irradiation. Studies of synchronized cells which identify a sensitive phase for the induction of virus should provide information about the mechanism of virus activation and increase our understanding of the early events in virus induction. MATERIALS

AND

METHODS

Cells. A cloned line of SV40-transformed hamster cells, THK22E Cl l-1, AP, (clone E) was grown in Eagle’s minimum essential medium (Gibco) with four fold the usual concentration of vitamins and essential amino acids (MEM x 4), 10% fetal calf serum (Gibco), glutamine (2 mM), penicillin (250 units/ml) and streptomycin sulfate (250 pg/ml) (maintenance medium). Cell synchronization. Cell cultures were synchronized at the beginning of the S period by the double excess thymidine block procedure (Pages et al., 1973). Cells were seeded at 1 x lo5 cells per 60-mm petri dish (Falcon) and maintained for 48 hr without a medium change before being treated with the first thymidine block. This procedure extends the generation time of these cells to 24 hr. Once fresh medium is returned to the cultures, their generation time decreases from 24 to 10 hr. After 48 hr, the maintenance medium was removed and medium containing 2.5 mM thymidine was added. After 16 hr, this medium was removed, the monolayers were washed with phosphate-buffered saline, pH 7.2 (PBS), and medium containing 10m5M deoxycytidine (unblocking medium) was added for 8 hr. At this time the medium was removed and the cells received the second thymidine block (2.5 mM thymidine in maintenance medium) for 16 hr. At the end of this time period, cells were released from the second block by removal of the medium, washed three times with PBS, and incubated in unblocking medium. Autoradiography for cell cycle analysis. Autoradiography was performed as described by Little (1970), with minor modifications. Clone E cells were grown on glass coverslips in 60-mm petri dishes and exposed to [methyI-SH]dT (5 &i/ml; specific activity, 20 Ci/mmol; New England

AND BLACK

Nuclear, NET-027E) for 60 min at the indicated times. Then the coverslips were washed in Dulbecco’s Tris buffer, pH 7.4, and fixed in cold 5% trichloroacetic acid (TCA) followed by 954 ethanol. The coverslips were air dried, attached to glass slides, prestained with phloxine B solution (2.5%), dipped in Kodak NTB nuclear track emulsion, exposed for 7 days at 4”, and finally developed in Kodak Dektol developer and Kodak fixer. The coverslips were stained through the emulsion with Harris-Lillie acetic acid hematoxylin and eosin (Fisher Scientific Co.) and attached with the emulsion side down to clean glass slides with Permount. Cells containing labeled nuclei were counted in 20-40 random 45x fields containing a total of 800-2000 cells. Measurement of DNA synthesis. Subconfluent cultures of clone E, in 60.mm plastic petri dishes, were exposed to [methyl-3H]dT (5 pCi/ml; specific activity, 20 New England Nuclear, Ci/mmol; NET-027E) for 30 min. At the end of the labeling period, the medium was withdrawn and the cultures were washed three times each with PBS and cold 5% TCA. Then the cells were dissolved by incubation at 37” in 1.5 ml of 0.5 N NaOH for 20 min. Aliquots (0.3 ml) of this solution were neutralized with 0.05 ml of 3 N HCl and the DNA precipitated by the addition of an equal volume of cold 10% TCA. The precipitates were collected by filtration onto Whatman GF/C glass-fiber disks, dried and counted in 10 ml of Omnifluor-toluene (New England Nuclear Corp.). Determination of protein concentration. Total protein was determined in aliquots of the alkaline monolayer solution (described above) by the Lowry procedure with minor modifications (Lowry et al., 1951). The bovine serum albumin standards were dissolved in 0.5 N NaOH and the copperrtartrate reagent containing 3% Na,CO, was dissolved in distilled H,O instead of in alkali. Virus induction by mitomycin C. Mitomycin C in unblocking medium (1.0 pg/ml) was applied to replicate cultures at hourly intervals following release of the cells from the second thymidine block.

CELL CYCLE

DEPENDENCE

After 8 hr of exposure, mitomycin C-containing medium was removed, the cells were washed once with PBS, and fresh maintenance medium was added for another 64 hr. Then the cultures were harvested and cell-free extracts were prepared as described (Kaplan et al. 1975). Virus induction by uu irradiation. The uv source was a mercury vapor 5-bulb G.E. germicidal lamp providing a 90% output at 2539 A with a constant fluence of 4.5 ergs/mm2/sec. Irradiation was carried out on replicate cultures at hourly intervals following release of the cells from the second thymidine block. The medium was removed and saved. Then the cultures were washed once with PBS and the cells were exposed to 100 ergs/mm* of uv irradiation at room temperature (24’) in an elapsed time of 22.2 sec. Following irradiation, the conserved medium was returned immediately to the cultures which were subsequently incubated for 3 days. Then the cultures were harvested and processed as described above. Assay of SV40 infectiuity. Cell-free extracts were tested for SV40 infectivity on TC-7 monkey kidney cells by a plaque assay which has been described (Kaplan et al., 1975). Virus yield was expressed as plaque forming units (PFU) per milligram of protein at the corresponding time points.

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OF SV40 INDUCTION

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Fro. 1. Induction of infectious SV4O from synchronized clone E cells by mitomycin C. Cultures were released from the second dT block at time zero. At hourly intervals after release, unblocking medium containing mitomycin C (1 pg/ml) was added to replicate cultures for an 8-hr period at 37”. Then the drug was removed, cultures were washed twice and incubated in fresh unblocking medium for 64 more hr. Virus titer is expressed as PFU per milligram of protein (0-O). The time course of DNA synthesis through the cell cycle was followed by measuring the uptake of [methyl-3H]dT into parallel untreated cultures after pulse-labeling for 30 min. [Methy/-3H]dT incorporation is expressed as counts per minute per milligram of protein (C-0).

the degree of cell synchrony averaged between 85 and 92% (data not shown). Since Induction of Virus from Synchronized Cells previous studies had indicated that an 8hr by Mitomycin C exposure of cells to mitomycin C at a Clone E cells were chosen for these concentration of 1 pg/ml caused the induction of enough virus for quantitative meaexperiments because little or no infectious virus could be detected in uninduced asyn- surements (Kaplan et al., 1975), these were used in the synchrony chronously growing cultures. The cells were conditions C was applied to synchronized at the G,-S border by a experiments. Mitomycin double excess thymidine block. After re- synchronized cultures at hourly intervals after release from the block and removed 8 lease from the second thymidine block, hr later. All cultures were harvested 72 hr [methyL3H]dT incorporation into DNA mitomycin C treatment. was followed, and typical results are shown after initiating in Fig. 1. With removal of the excess dT, Results (Fig. 1) indicate that the optimum C occurred the block in DNA synthesis was reversed inducing effect of mitomycin and the cells moved in a wave through S when cultures were treated during the phase. A synchronized burst of DNA syn- interval between 6 and 9 hr after release, C thesis was observed which peaked 4 hr after which was after the S phase. Mitomycin declined at later times. No release and lasted for approximately 6 hr. inducibility virus was activated in control synchronized As measured by autoradiographic analysis, RESULTS

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KAPLAN,

KLEINMAN

cells that had not been treated with mitomycin C, so thymidine itself did not activate virus. These results indicated that induction of virus by mitomycin C was dependent on the cells being in a particular phase of the cycle. However, it was difficult to determine the precise location of this sensitive phase because of uncertainty as to the rates of absorption of mitomycin C and conversion to a reactive form by these cells. It is also not known how much mitomycin C is washed out of the cells or how long it remains active. In order to delineate more precisely the most sensitive phase for virus induction, experiments were carried out using uv irradiation on synchronized cultures of clone E cells. With uv light, exposure times are short and DNA damage is produced immediately and directly. The nature of the DNA damage and its repair have both been well defined in molecular terms (Grossman. 1974). Induction of Infectious chronized Clone Irradiation

SV40 from SynE Cells by uu

It has been determined

that the produc-

AND BLACK

tion of virus after uv irradiation of clone E cells is proportional to uv dose and that maximum induction is achieved with a dose of 100 ergs/mm” (Kaplan et al., 1975). Therefore, after clone E cells were synchronized and released from the second thymidine block, cultures were irradiated with this dose of uv light (exposure time, 22.2 set) at hourly intervals during an 18-hr time period. After irradiation, unblocking medium was returned to the cells which were incubated for another 72 hr. Figure 2 shows the results of virus activation by uv radiation at various times after release. Cells irradiated during S phase exhibited little or no virus induction, but virus production started towards the end of S and reached a maximum l-2 hr after the peak of DNA synthesis. As observed in the mitomycin C experiments, excess thymidine itself did not activate detectable virus in control unirradiated synchronized cells. The degree of synchrony during the first phase of DNA synthesis gave an average of 90% as measured by autoradiography but was not as high in the second period (data not shown). In spite of the deterioration of synchrony, a second peak of virus activa( o-----o

)

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‘~.lIL~--

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2

4

6

HOURS

8 AFTER

10

I2

14

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FIG. 2. Induction of infectious SV40 from synchronized clone E cells by uv irradiation. Cultures were released from the second dT block at time zero. At hourly intervals after release, replicate cultures were washed, irradiated with 100 ergs/mm* of uv (exposure time, 22.2 set), and after irradiation unblocking medium was added to the cultures which were harvested after 72 hr. Virus titer is expressed as PFU per milligram of protein (GO). DNA synthesis was determined by uptake of [methyl-3H]dT into parallel cultures which had been pulse-labeled for 30 min. [Methyl-3H]dT incorporation is expressed as counts per minute per milligram of protein (C-0).

CELL

CYCLE

DEPENDENCE

tion was observed which followed a second round of DNA synthesis. These results indicate that induction of SV4Q in synchronized clone E cells by uv irradiation occurs preferentially when cells are in the late S-G2 period of their cycle at the time of irradiation. DISCUSSION

We have demonstrated that mitomycin C or uv irradiation induces virus activation in THK22E Cl l-l, AP, (clone E) SV40-transformed hamster cells and that this induction shows cell cycle dependency. The effect of mitomycin C in inducing virus was maximal when it was applied to the cells after the S phase. However, it was difficult to determine precisely when it was acting because the length of time for absorption, conversion and maintenance in an active form are not known for these cells. Similar reservations can be made about other studies utilizing drugs to identify the optimal point in the cell cycle for virus induction. Therefore, synchronized cultures of clone E cells were also induced with uv irradiation to avoid the variables encountered when using mitomycin C. Since irradiation times were short and DNA damage was made directly, it was possible to determine accurately the period in the cell cycle in which the cells were most susceptible to virus activation. Induction of virus by uv light was most pronounced towards the end of S and reached a maximum when cells were irradiated l-2 hr beyond the peaks of DNA synthesis. Therefore, virus induction occurred optimally if cells were in or leaving the late S phase at the time of irradiation. After cell division the cells became more resistant to virus induction by uv radiation until they again reached the late S-G, period. Since we have shown that virus induction is directly proportional to uv dose (Kaplan et al., 1975), it is possible that the production of uv-induced lesions and their repair are also cell cycle dependent in these cells. With respect to the latter, it is of interest that the uv sensitivity of mammalian cells is dependent on the phase of the cycle that cells occupy at the time of irradiation. Reasons for cyclic fluctuations

OF SV40 INDUCTION

219

in sensitivity to uv irradiation are not understood but the phenomenon has been established for a number of cell types (Watanabe and Horikawa, 1973; Sinclair and Morton, 1965; Rauth and Whitmore, 1966; Djordjevic and Tolmach, 1967). Sensitivity to uv radiation increases late in G1, reaches a maximum in the middle of S and decreases through the remainder of the S and G, phases. The period of maximum resistance to uv (late S-G,) corresponds to our observed peaks of uv-induced virus production. Transient sensitivity to uv radiation could have several explanations. Cell cycle dependent changes in the conformation of DNA may make it more accessible to radiation. It has been observed that the amount of uv-induced thymine dimer formation in cellular DNA is related to cyclic variations in cell survival (Watanabe and Horikawa, 1974). Increased resistance to uv killing in late S-G, may reflect more efficient DNA repair going on in this interval which could possibly be involved with viral genome excision. Restriction of the susceptibility of clone E cells to uv or mitomycin C induction of virus to a limited period of the cell cycle may reflect requirements for the presence or absence of one or more cell factors that promote viral genome excision or replication and only appear at certain times. The nature of host cell events which stimulate genome excision or SV40 replication are not known, but there is undoubtedly strict cellular control over the process. The replication of SV40 is cell cycle dependent in monkey cultures (Pages et al., 1973). Apparently the cells must pass through a critical stage in late G, or early S in order for replication to be initiated. In our induction system, SV40 viral genomes may possibly be excised during the late S-G, phase and start replicating only after the cells have passed through the late G,-early S interval. Reasons for differences in the most sensitive phases of the cell cycle for activating oncogenic DNA viruses by either IdU (Hampar et al., 1973), mitomycin C or uv irradiation are not known but may reflect differences in the mechanism of action of

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these agents. IdU must be incorporated into DNA in order to induce which presumably explains the fact that induction of EB viral antigens by this agent occurred only if the drug was applied early in the S phase. However, both mitomycin C and uv irradiation act on preformed DNA, and the lesions they produce may not necessarily be repaired by the same processes (Cole, 1973; Kaplan et al., 1969). Since our data suggest a correlation between uv resistance and virus activation in transformed hamster cells, it is tempting to speculate that DNA repair processes and strand break formation, which could favor viral genome excision, are more active during the resistant phase. To evaluate fully the possible role of DNA repair enzymes in virus induction, we intend to study both DNA incision and viral genome excision as a function of the cell cycle. ACKNOWLEDGMENTS This investigation was supported by U.S.P.H.S. Grant No. CA-11126-07 from the National Cancer Institute. Joan C. Kaplan was the recipient of a Cancer Research Scholar Award from the American Cancer Society (Massachusetts Division). We thank Dr. John B. Little for providing radiation facilities and advice for these experiments. REFERENCES COLE, R. S. (1973). Repair of DNA containing interstrand crosslinks in E. cob. Sequential excision and recombination. Proc. Nat. Acad. Sci. USA 70, 1064-1068. DJORDJEVIC,B., and TOLMACH, L. J. (1967). Responses of synchronous populations of Hela cells to ultraviolet irradiation at selected stages of the generation cycle. Radiat. Res. 32, 327-346. GREENBERGER,J. D., and AARONSON, S. A. (1975). Cycloheximide induction of xenotropic type C virus from synchronized mouse cells: Metabolic requirements for virus activation. J. Viral. 15, 64-70. GROSSMAN,L. (1974). Enzymes involved in the repair of DNA. Advan. Radiat. Biol. 4, 77-129.

AND BLACK HAMPAR, B., DERC.E,J. G.. MARTOS, L. 34.. TN;AMETS. M. A., CHANC. S. Y., and CHAKHABAR.T\. d. (19713). Identification of a critical period during the S phase for activation of the Epstein--Barr virus by 5. iododeoxyuridine. Nature Neu, Biol. 244, 214-217. HAMPAR B., DERGE, J. G., and SHOWALTER, S. D. (1974). Enhanced activation of the repressed Epstein-Barr viral genome by inhibitors of DNA synthesis. Virology 58, 298X(01. KAPLAN, J. C.. KUSHNER, S. R.. and GROSSMAN, I,. (1969). Enzymatic repair of DNA. I. Purification of two enzymes involved in the excision of thymine dimers from ultraviolet-irradiated DNA. Proc. Nat. Acad. Sci. USA 63, 144-131. KAPLAN, ,J. C., WILBERT, S. M.. COLLINS, .J. J., RAKLSANOVA,T., ZAMANSKI.. G. B.. and BLACK, P. H. (1975). Isolation of simian virus 40.transformed kidney cell lines heterogeneous for virus induction by chemicals or radiation. Virology 00, 0CWx)O. LITTLE, J. B. (1970). Irradiation of primary human amnion cell cultures: Effects on DNA synthesis and progression through the cell cycle. Radiat. Res. 44, 674-699. LOWRY, H., ROSEBRO~~~.H,N. J., FARR. A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 26