The results indicate that these drugs act at different levels to modify the permissiveness of cells to simian virus 40. 5-Iodo-2'-deoxyuridine (IUdR) and mitomy-.
JOURNAL OF VIROLOGY, Oct. 1980, 0022-538X/80/10-0295/03$02.00/0
Vol. 36, No. 1
p. 295-297
Simian Virus 40-Chinese Hamster Kidney Cell Interaction V. Cooperative Effect of 5-Iodo-2'-Deoxyuridine and Mitomycin C in the Enhancement of Virus Replication in Infected Cells HORACIO G. SUAREZ,* CHRISTIAN LAVIALLE,
AND ROLANDO CASSINGENA Institut de Recherches Scientifiques sur le Cancer, 94800, Villejuif, France
A cooperative effect of 5-iodo-2'-deoxyuridine and mitomycin C which amplified (10- to 50-fold) their stimulating effect on simian virus 40 replication was obtained in infected Chinese hamster kidney cells. The results indicate that these drugs act at different levels to modify the permissiveness of cells to simian virus 40.
inoculum (4). SV40 DNA was prepared from virus strain SVLP as described previously (4, 11). Four sets of cultures were handled: (i) without addition of IUdR or MC, (ii) with addition of IUdR only, (iii) with addition of MC only, and (iv) with addition of IUdR plus MC (Fig. 1). All experiments were done under subdued light. Figure 1 shows a kinetic study of infectious virus production in control and drug-treated SV40 DNA-infected cells. Compared with untreated cultures, the increase in virus production after IUdR or MC treatment varied from 10- to 50-fold, depending on the experiment. The rise in virus titers started earlier in IUdR-pretreated cultures. The IUdR-plus-MC pretreatment reproducibily amplified (10- to 50-fold) the stimulating effect of IUdR or MC alone. For further analysis of the nature of the IUdRcycle, before T-antigen synthesis. Experiments plus-MC-promoted activation of SV40 synthesis performed to determine whether IUdR affects in infected CHK clone 10 cells, infectious-center adsorption, penetration, or decapsidation of experiments were performed. Table 1 shows SV40 indicated that the analog acts between the that: (i) IUdR treatment increased the number arrival of virus DNA in the nucleus and the of virus-producing cells by a factor of 2; (ii) MC antigen production (11, 12). MC enhancement of treatment enhanced about three times the numcell permissiveness may occur at a later step, ber of infectious centers; (iii) the number of cells intermediate between T-antigen production and treated with IUdR plus MC forming infectious centers was approximately four- to fivefold SV40 DNA replication (4). Having observed the high efficiency of IUdR greater than that in control cells. However, this and MC in enhancing the permissiveness to increase did not fully account for the enhanceSV40 in different cellular systems (4, 11, 12), we ment of the total virus yield (10- to 50-fold for examined their combined effect on virus repli- IUdR and MC; 100- to 500-fold for IUdR plus cation on previously characterized semipermis- MC) and indicated that there was an increase in sive Chinese hamster kidney cells. Upon infec- the virus burst size, which could be estimated tion with SV40 or virus DNA, Chinese hamster from the ratio of the final virus yields to the kidney cells supported virus DNA and virus respective number of virus-producing cells (Tasynthesis at a low level; this cell system was ble 1). Indirect immunofluorescence was used to dedefined as semipermissive (4). CHK clone 10 is derived from an established line of Chinese ham- termine the effect of IUdR plus MC on the ster kidney cells (5). For demonstration of infec- induction of SV40-specific T- and V-antigens in tious SV40 synthesis, these cells had to be in- samples of the same CHK clone 10 cell suspenfected with virus DNA to overcome their limited sions used for the infectious-center assay. Alsusceptibility to virus particles and to avoid though IUdR and IUdR-plus-MC treatment masking of the virus progeny by the input virus caused a similar increase of T-antigen synthesis,
5-Iodo-2'-deoxyuridine (IUdR) and mitomycin C (MC) are inducers of virus production in simian virus 40 (SV40)- and polyoma virustransformed cells (2, 6-9). The precise mechanisms underlying the induction event are at present unknown. It has also been reported that the pretreatment of cells with IUdR or MC may augment their permissiveness toward the replication in lytic infection of SV40 (4, 11, 12). These two phenomena may be connected, chemical treatment of cells containing integrated proviruses stimulating permissiveness of the cell and allowing fuller expression of virus functions including autonomous replication of the virus genome (after excision) and the production of mature virus. IUdR appears to stimulate virus replication by a different mechanism compared with MC. IUdR acts at an early step of the virus
295
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in control and treated cultures. This observation, previously reported in infected or transformed Chinese hamster kidney cultures (4, 8, 12) treated or not treated with IUdR or MC, suggests some block at maturation of the virus which cannot be completely overcome by the drugs. The presence of V-antigen implies the synthesis of virus DNA, but clearly many Vantigen-positive cells do not produce sufficient quantities of infectious virus to give an infectious center.
The proportion of SV40 DNA-infected cells TABLE 1. Effect of IUdR, MC, and IUdR-plus-MC treatment on SV40 burst size and number of virusproducing cellsa ec-
Maimum virUs yield
Expt
Virus burst size (PFU/
tiOU
centers
(PFU/106
per
cells)
cel)
1.
Control IUdR MC IUdR plus MC
HOURS ATER INFECTION
FIG. 1. CHK clone 10 cells suspended in Eagle minimal essential medium with 10% calf serum were seeded in 25-cm2 flasks in the presence or absence of 100 pg of IUdR per ml and incubated at 37°C. After 4 days the medium was removed, and after three washings with serum-free medium the cultures were exposed to 2 pg of MC per ml. After 3 h of incubation at 37°C, the medium was removed and the cultures were rinsed; after 2 h in drug-free medium, cultures were infected with SV40 DNA at a multiplicity of infection of 1 PFU per cell as previously described (4). At designated times after infection, control and treated cultures were harvested by freezing at -20'C, cell lysates were prepared by three cycles of freezing and thawing followed by sonication, and virus yields were determined by plaque titration on CVI monolayers (4, 11). Symbols: 0, control; A, IUdR treated; 0, MC treated; 0, IUdR plus MC treated.
MC did not significantly change the proportion of positive cells up to 4 days after infection with virus DNA (Table 2). These results indicate that IUdR and MC act at different levels of the virus cycle. The IUdR-plus-MC enhancement of the percentage of positive cells reflects the stimulating action of the thymidine analog before the antigen synthesis. The proportion of cells synthesizing V-antigen was increased in IUdR, MC-, or IUdR-plus-MC-treated cells by levels which are in good agreement with the increase in the number of infectious centers (Table 1). Both data indicate an enhancement of the mean level of permissiveness of the cell population. However, more (about 100-fold) V-antigen-positive cells than infectious centers were present
90
49 97 157 230
3,100 2,900 75,000
1.8 32.0 18.5 326.0
2.
Control 125 53 2.3 IUdR 89 2,980 33.5 MC 3,870 149 26.0 IUdR plus MC 69,000 209 330.0 a Control and treated CHK clone 10 celi cultures were infected with SV40 DNA as described in the legend to Fig. 1. At 2 h after infection, cells were trypsinized and plated with CV1 cells for infectiouscenter determination. To determine the virus burst per cell, fractions of 10 CHK clone 10 cells from same suspensions as those used for the infectious-center assay were seeded in 25-cm2 flasks, and the kinetics of virus production was followed over a period of 7 days. TABLE 2. Effect of IUdR, MC, and IUdR-plus-MC treatment on SV40 T- and V-antigen synthesis in SV40 DNA-infected CHK clone 10 celisa % of cells positive for: Expt
T-antigen at time postinfection (h): 24
48
72
96
V-antigen at time postinfection (h): 24
48 72 96
Control 4.4 6.2 6.8 9 0.01 0.2 0.5 1.0 IUdR 11.8 22.0 31.0 31.0 0.3 0.6 1.6 2.5 MC 4.2 6.6 6.9 11.2 0.1 0.8 2.0 3.2 IUdR plus MC 12.1 18.0 28.2 36.0 0.5 2.4 6.0 8.0 a Samples of the same CHK clone 10 cell suspensions as those used in experiment 1 (Table 1) were seeded on cover slips, fixed at the times indicated, and stained for SV40 T- and V-antigens by indirect immunofluorescence. Percentage of positive cells was obtained by counting 2,000 cells.
VOL. 36, 1980
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synthesizing T-antigen was not significantly transformed cells increased their inducibility by changed by MC. This observation should be UV or X-ray irradiation. compared with the results obtained with IUdR, which enhances the growth of adenovirus 7 and The technical assistance of J. Stevenet and S. Estrade is SV40 upon treatment of cells semipermissive or gratefully acknowledged. permissive for these viruses (3, 10-12). In these two cases, it has been shown that IUdR pretreatLITERATURE CITED ment stimulates the percentage of T-antigensynthesizing cells and acts at a step preceding 1. Biczysko, W., D. Solter, M. Pienkowsky, and H. Kothe expression of early viral functions. Using prowsky. 1973. Interactions of early mouse embryos with oncogenic viruses simian virus 40 and polyoma. I. mouse embryo IUdR-pretreated SV40-infected Ultrastructural studies. J. Natl. Cancer Inst. 51:1945cells, we have recently shown that the analog 1954. enhances the viral early transcription (H. G. 2. Fogel, M. 1972. Induction of virus synthesis in polyomaSuiirez, M. Lange, and R. Cassingena, J. Gen. transformed cells by DNA antimetabolites and by irradiation after pre-treatment with 5-bromodeoxyuriVirol., in press). Data reported by Lavialle et al. dine. Virology 49:12-22. (4) suggest that MC acts between T-antigen 3. Jerkofsky, M., and F. Rapp. 1975. Stimulation of adesynthesis and SV40 replication, perhaps by trignovirus replication in simian cells in the absence of a gering the production of a cellular factor(s) conhelper virus by pretreatment of the cells with iododeoxyuridine. J. Virol. 15:253-258. cerned in the permissiveness of the cell. This C., A. G. Morris, H. G. Suarez, S. Estrade, hypothesis was strengthened by the observation 4. Lavialle, J. Stevenet, and R. Cassingena 1977. Simian virus that MC allows detectable expression of late 40-Chinese hamster kidney cell interaction. IV. Envirus functions in three lines of SV40-transhanced virus replication in infected cells upon treatment with Mitomycin C. J. Gen. Virol. 36:137-149. formed mouse cells (6), strongly suggesting that C., J. Stevenet, A. G. Morris, H. G. Suarez, these mouse cells contain genetic information 5. Lavialle, S. Estrade, J. C. Salomon, and R. Cassingena. 1975. for specific cellular functions required for the Simian virus 40-Chinese hamster kidney cell interaccompletion of the virus growth cycle. In this tion. I. Relationship of chromosome changes to transformation. Arch. Virol. 49:127-139. context, it is also interesting to recall observations of Biczysko et al. (1) showing that certain 6. Lavialle, C., H. G. Suarez, A. G. Morris, and R. Cassingena 1978. Induction of infectious virus DNA and cells of early mouse embryos are permissive for virus particles by mitomycin C in SV40-transformed SV40. MC treatment could thus derepress celmouse cells. J. Gen. Virol. 41:201-205. lular functions involved in permissiveness to 7. Morris, A. G., C. Lavialle, H. G. Suarez, and R. Cassingena. 1975. The induction of SV40-transformed SV40 and which are not expressed, or are parChinese hamster and mouse kidney cells by mitomycin tially expressed, at late stages of cellular differC. Intervirology 5:305-312. entiation leading to (i) synthesis of larger num- 8. Morris, A. G., C. Laviafle, H. G. Suarez, J. Stevenet, S. Estrade, and R. Ca8singena. 1977. Simian virus bers of virions in semipermissive, infected cells 40-Chinese hamster kidney cell interaction. III. Char(4; this paper) and (ii) more efficient replication acteristics of chemical induction in a clone of virogenic of the virus genome excised from the host cell transformed cells. J. Gen. Virol. 36:123-135. cells DNA in transformed (6, 9). 9. Rakusanova, T., J. C. Kaplan, W. P. Smales, and P. H. Black. 1976. Excision of viral DNA from host cell The results presented in this investigation DNA after induction of simian virus 40-transformed show a cooperative effect of IUdR and MC hamster cells. J. Virol. 19:279-285. which amplified their stimulating effect on SV40 10. Staal, S. P., and W. P. Rowe. 1975. Enhancement of replication in infected CHK clone 10 cells. They adenovirus infection in WI 38 and AGMK cells by pretreatment of cells with 5-iodo-2'-deoxyuridine. Virology confirm previous data (4, 11, 12) suggesting that 64:513-519. these chemical agents act at different leveLs to H. G., C. Lavialle, J. Stevenet, S. Estrade, A. modify the permissiveness of cells to SV40. They 11. Suarez, G. Morris, and R. Cassingena. 1977. Enhanced SV40 further suggest that SV40 induction in virusvirus replication in fully pennissive monkey kidney cells transformed cells may proceed through different pre-treated with 5-iodo-2'-deoxyuridine (IdUrd). J. Gen. pathways depending on the inducing agent em- 12. Virol. 37:569-584. Suarez, H. G., A. G. Morris, C. Laviafle, and R. Casployed. This latter supposition is consistent with singena. 1976. Enhanced SV40 virus replication in findings reported elsewhere (2) showing that Chinese hamster kidney cells pre-treated with 5-iodo2'-deoxyuridine. Arch. Virol. 50:249-253. bromodeoxyuridine pretreatment of polyoma-
