Action spectrum for the in vitro induction of simian virus 40 by ultraviolet radiation

Mutation Research, 95 (1982) 95-103

95

Elsevier Biomedical Press

Action spectrum for the in vitro induction of simian virus 40 by ultraviolet radiation 1 Thomas P. Coohill 1, Sharon P. Moore, Daniel J. Knauer 2, Dennis G. Fry 3, Timothy J. Eichenbrenner and Larry E. Bockstahler Departments of Biology and Physics, Western Kentucky University ( T.P.C., S.P.M., T.J.E.), Bowling Green, Kentucky 42101; Department of Biology, University of Louisville (S.P.M.), Louisville, Kentucky 40208; and Bureau of Radiological Health, Food and Drug Administration (L.E.B.), Rockville, Maryland 20857 (U.S.A.)

(Received 30 September 1981) (Revision received25 January 1982) (Accepted I l February 1982)

Summary A line of simian virus 40-transformed hamster kidney cells was exposed to ultraviolet radiation at eleven different wavelengths in the region 238-302 nm. An action spectrum derived from the resulting exposure-response curves for the induction of simian virus 40 from these cells exhibits a broad peak in the region 260-270 n m suggesting D N A as the major chromophore for this response. This conclusion is consistent with results obtained by other investigators who have noted viral induction by a number of DNA-damaging agents.

Cells of many animal species contain endogenous viral genetic information. When such ceils are exposed to certain chemical and physical agents, viral induction can occur with the release of infectious virus a n d / o r expression of viral gene products (Bocksta~.er and Hellman, 1979). Viral activation in mammalian cells is analogous to prophage induction in lysogenic bacteria, which is an indicator of 'SOS induction' (Radman, 1980). Another property of 'SOS repair' is mutagenesis, which, if present I To whom requests for reprints should be addressed. 2 Present address: Department of Medical Microbiology, University of California, Irvine, California 92664, U.S.A. 3 Present address: Department of Microbiology, Medical College of Virginia, Richmond, Virginia 23219, U.S.A. Abbreoiations: PBS, Dulbecco's phosphate-buffered saline; PFU, plaque forming units; SV40, simian

virus 40. 0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press

96 in mammalian cells, may lead to carcinogenesis (Witkin, 1976). In addition, some inducible mammalian viruses have been demonstrated to be oncogenic or associated with oncogenesis. In recent years there has been speculation regarding possible relationships between induction of latent viruses and oncogenesis (Bockstahler and Hellman, 1979; Hirsch and Black, 1974; Todaro and Huebner, 1972). It has also been suggested that the only relationship may be that these phenomena represent independent by-products of altered cell regulatory functions (Hirsch and Black, 1974). Further basic information about latent virus induction is needed to establish possible relationships between viral induction, 'SOS induction' and carcinogenesis in mammalian systems. In 1954 Franklin (1954) published an action spectrum for the induction of the latent bacteriophage lambda from the bacterium Escherichia coli Kl2 by ultraviolet radiation in the wavelength region 240-300 nm. He interpreted his results to indicate that nucleic acid was the major chromophore for this means of viral activation. Parallels have been drawn between lysogenic bacteria and inducible lines of viral-transformed cells (Black, 1968; Burns and Black, 1969; Fogel, 1972; Fogel and Sachs, 1970; Rothschild and Black, 1970). Several chemical and physical agents which damage host cell DNA have been shown to induce virus from both lysogenic bacteria and certain virogenic mammalian cells (Fogel and Sachs, 1970; Kaplan et al., 1972, 1975). Accordingly, we have conducted experiments to determine if the action spectrum for viral induction by UV radiation in a viral-transformed mammalian cell line also indicated nucleic acid as the target chromophore. These experiments were made possible by the establishment of an SV40-transformed hamster cell clone, clone E, by Kaplan et al. (1975) that produces a relatively large yield of infectious SV40 when irradiated with UV. In this paper, we report a UV action spectrum for SV40 induction from these cells, and compare it to the absorption spectrum for DNA and to the action spectrum for production of thymine dimers in mammalian cell DNA (Doniger et al., 1981). To our knowledge this is the first reported action spectrum for virus induction in mammalian cells.

Materials and methods Cell cultures. Clone E cells, an SV40-transformed line of weanling inbred Syrian hamster kidney cells (Kaplan et al., 1975), were obtained from J.C. Kaplan and P.H. Black of Harvard Medical School. These cells were maintained in Eagle's Minimum Essential Medium (Grand Island Biological Co., Grand Island, NY) supplemented with four-fold the usual concentration of vitamins and essential amino acids and the following components perl:10% fetal bovine serum, penicillin (140000 units). Fungizone (35 #g), streptomycin (140000 units) and a buffer system consisting of 3.57 g N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonicacid, 3.44 g N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonicacid, 3.14 g morpholinopropanesulfonic acid and 2.0 g sodium bicarbonate, pH 7.2. CV-1P cells, a highly contact-inhibited clone of African green monkey kidney cells, were used to assay SV40. The cells were grown in 1 × Dulbecco's Modified

97

Eagle's Medium (Grand Island Biological Co.) supplemented with 10% fetal bovine serum with the same antibiotics and buffers as those used above for clone E cells. Irradiation of clone E cells. Clone E cells were trypsinized at confluence and passaged into 60 mm tissue culture dishes (Falcon Plastics, Oxnard, CA) at a density of 8 × 105 cells/dish. Cells were incubated at 37 ° for 24h before irradiation. Immediately prior to irradiation the medium was removed from each dish and the cells were rinsed twice with PBS. The final rinse was followed by a 2-ml PBS overlay which remained on the cells during irradiation. A complete description of the radiation source, dispersion system and method of radiation measurement is contained in Coohill et al. (1977). Briefly, radiation from a 2.5-kW high pressure mercury-xenon lamp (929 B Hanovia Lamp, Newark, N J) was passed through two grating monochromators (Schoeffel Inst., Westwood, N J) to obtain sufficient spectral separation. All experiments were conducted using 4-mm slits which gave a half-band width of 6.0 nm. Exposure rates were measured with a calibrated UV-sensitive photodiode (CaI-UV, United Detector Technology, Inc., Santa Monica, CA). Cells were irradiated in horizontally oriented open petri dishes which were rotated at 0.5 rev/sec during irradiation. Reciprocity of time and intensity was tested over a factor of at least three at several wavelengths and no dependence of viral induction on intensity was observed. Immediately following irradiation the PBS overlay was removed and replaced with 4 ml of fresh medium. Control (unirradiated) monolayers were treated the same as the irradiated cultures. All cells were incubated at 37 ° for 96 h at which time they were removed from the incubator and placed at - 4 0 ° until harvesting. During each experiment one plate of cells was trypsinized and the cells were counted on a hemacytometer to determine the number of cells irradiated per dish. SV40 harvest. Induced SV40 was harvested by removing both cells and media from the dishes after they had been frozen and then thawed. This suspension was centrifuged at 200 × g for 10 min to pellet the cells. The supernatant was removed and the pellet sonified to release any intracellular virions. Following sonification, the sonicated pellets were recombined with their respective supernatants and stored at 40 ° until assayed. Plaque assay. CV-IP cells used for the plaque assay were trypsinized at confluence and passaged into 60-mm tissue culture dishes at a density of 3-6 ( × 105) cells/dish. When the cells formed confluent monolayers, the growth medium was removed from the dishes and the monolayers were infected with different dilutions of the harvested virus. Infection was carried out at 37 ° for 2 h with constant rocking. Two types of controls were used with the plaque assay: stock SV40 of known titer and uninfected monolayers. Following the 2-h infection period, the virus suspension was removed from the CV-IP cells, and the cells were fed with 10 ml of plaquing medium consisting of equal volumes of 1% Sea-Kem Agarose (Microbiological Associates Bioproducts, Bethesda, MD) and 2 × Minimum Essential Medium supplemented with 4% fetal bovine serum, antibiotics, buffers, and 1 0 - g M dexamethasone. As soon as the agarose hardened, plates were transferred to 37 ° and incubated for 7-10 days. Beginning the 7th day after infection, monolayers were checked for plaques. If -

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plaques were visible, monolayers were stained with 0.02% neutral red, and plaques were counted 2 days after staining. Data from a preliminary assay were used to conduct a final assay in which viral dilutions were adjusted to give 10-40 plaques/dish. These numbers are within the linear response range for experiments with SV40. The resulting numbers of plaques were expressed as P F U / 1 0 6 clone E cells. Chromosome counting. Clone E cells were fixed and stained for chromosome counts by a method similar to that of Pollack and Pfeiffer (1971). Results Fig. 1 presents the average response curves for the induction by UV radiation of SV40 from clone E cells at 11 separate wavelengths. Each curve represents the

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Fig. 1. Induction of simian virus 40 from irradiated clone E cells by monochromatic ultraviolet radiation, The abscissa is the exposure of U V to the cells at the stated wavelength in J/m2; the ordinate is th( number of plaque forming units of SV40 induced per 10 4 irradiated clone E cells. U V wavelengths as indicated. Each point represents an averaged value of the results of at least three experiments.

99 averaged data from at least three separate experiments. In each experiment three separate cell monolayers were irradiated at each exposure; consequently each datum point represents the average response of at least nine monolayers of cells. The levels of induction varied somewhat from experiment to experiment even for the same UV wavelength but were generally comparable to those obtained by other investigators (Bockstahler and Cantwell, 1979; Kaplan et al., 1975), i.e. two to three orders of magnitude above spontaneous background levels. Rothschild and Black (1970) also reported variability in their induction levels and attributed it to unknown differences in the physiologic state of the cells from experiment to experiment. Routine chromosome counts revealed that our clone E cells contained chromosome numbers within the range of that which had been previously reported (Kaplan et al., 1975). In each figure the best approximate straight line was drawn for the initial induction response. This essentially exponential rise in induction level with UV exposure has been reported by others (Bockstahler and Cantwell, 1979; Kaplan et al., 1975). All the reported curves subsequently reached a maximum induction level and then began to decrease for radiation exposures above this peak value. Several points should be made about the shape and consistency of these curves. First, the level of spontaneous induction (background level, no UV exposure to the cells) varied considerably from day to day in most experiments. This variation markedly affected the slope of the initial response curves and thus appeared to change the efficiency of response. However, it should be noted that in those experiments where the spontaneous induction level was high (greater than 10 PFU/106 cells), the peak response was often higher than for those experiments in which the spontaneous induction level was low (less than 10 PFU/106 cells). Second, the maximum induction level varied by as much as a factor of six from experiment to experiment. Third, the most consistent parameter at any wavelength was the exposure required to elicit a peak response. This peak response is regarded as the point above which additional UV radiation begins to adversely affect those cellular functions necessary for viral production. Accordingly, we compiled a list of exposure to peak values for the curves in Fig. 1 and report them in Table 1. In addition the values for the slope of the initial response curves are included even though in view of the above comments they are probably less reliable for comparison purposes between wavelengths than are the exposure to peak values. Regardless of the level of spontaneous induction or the amount of maximum induction, the exposure to peak values varied no more than --- 16% at any wavelength whereas the values of the slope varied by as much as ---25% (Table 1). Since biological responses to light are normally considered to be the result of the number of photons absorbed by the sample rather than the total energy absorbed, both the exposure to peak and the slope values reported in Table 1 were quantum corrected (Jagger, 1967). An action spectrum derived from the data in Fig. 1 and Table 1 is presented in Fig. 2. Both the reciprocal of the exposure required to elicit a maximum induction level at each wavelength and values for the slope of the initial rise of the induction curves for each wavelength are represented in Fig. 2. Also included in this figure are the normalized data for the production of pyrimidine dimers by UV radiation of Syrian hamster embryo cells (Doniger et al., 1981).

100 TABLE 1 I N D U C T I O N O F SV40 F R O M C L O N E E CELLS BY U L T R A V I O L E T R A D I A T I O N Exposure to peak, reciprocal of exposure to peak, and slope from induction curves in Fig. 1. Wavelength (nm)

Exposure to peak (Ep) J / m E with per cent standard errors

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Slope b (QC) with per cent standard errors

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0.067 0.102 0.100

'0.095 + 24% 0.092-+ 10% 0.100± 13%

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0.099 0.086 0.021

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0.005 0.001

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101

In order to determine whether the breadth of the peak in Fig. 2 was an artifact resulting from daily variation, we conducted an experiment in which cells from the same stock culture Were exposed on the same day to levels of radiation which usually elicit maximum induction at wavelengths 254, 260, 265, 270 and 280 nm. The results of that experiment indicated that the relative response of the cells irradiated with these wavelengths was as shown in Fig. 2. On either side of a broad peak the amount of Viral induction falls off rapidly and parallels the wavelength response of dimer formation (Doniger et al., 1981).

Discussion

The action spectrum for SV40 induction from clone E cells (Fig. 2) peaks in the region 260-270 nm and parallels the action spectrum for pyrimidine dimer formation in hamster cells (Doniger et al., 1981). It also somewhat resembles the general shape of the action spectrum for mutagenesis in mouse cells (Jacobson et al.; 1981). Our action spectrum is consistent with the hypothesis that DNA is the major target molecule for viral induction and suggests that induction by UV may be a direct or indirect consequence of DNA structural damage (Bockstahler and Hellman, 1979). Our peak is considerably broader than that reported by Franklin (1954) for the induction of bacteriophage lambda from Escherichia coli. Similar broad peaks have been reported for action spectra dealing with other mammalian cell functions such as cell death (Rothman and Setlow, 1979; Todd et al., 1968), mutation (Jacobson et al., 1981), cell transformation (Doniger et al., 1981), cellular capacity for viral growth (Coohill et al., 1977), and cellular reactivation of UV-damaged virus (Coohill et al., 1978).11 The mechanism of viral induction from transformed mammalian cells is not yet clearly understood. In agreement with the results reported here, other authors have published results suggesting a DNA dependence for this effect. Several agents which produce DNA damage or influence DNA repair have been reported to increase induction from cells transformed by DNA viruses. These include mitomycin C (Burns and Black, 1969; Fogel and Sachs, 1970; Kaplan et al., 1975; Morris et al., 1977; Rothschild and Black, 1970), UV radiation (Kaplan et al., 1975; Morris et al., 1977; Rothschild and Black, 1970), X-rays (Rothschild and Black, 1970), substitution of DNA base analogs (Fogel and Sachs, 1970; Morris et al., 1977), ,/-radiation (Kaplan et al., 1975), proflavine plus visible light (Bockstahler and Cantwell, 1979), and bromodeoxyuridine plus visible light (Fogel, 1973; Kaplan et al., 1975). In addition, Rakusanova et al. (1976) reported evidence that excision of the viral genome from its integrated state in the host cell DNA is an early step in induction. Whether excision occurs as a direct result of the inducing agent or by repair systems is unknown (Fogel, 1972). DNA damage can lead to the production of single-strand breaks by DNA repair processes. The breaks may, in turn, facilitate the expression of the viral genome (Radman, 1980). In addition, Zamansky et al. (1976) have shown that caffeine, a chemical known to interfere with the filling of the gaps formed during post-UV DNA repair, stimulated the UV induction of SV40 from clone E cells.

102 I n conclusion, the action spectrum reported here for SV40 i n d u c t i o n from a m a m m a l i a n cell line b y U V agrees with the work of other investigators suggesting that D N A is the m a j o r target molecule for this effect.

Acknowledgements The authors wish to express appreciation to C.D. Lytle for suggesting the project a n d for advice, to Y.J. Stifel for p e r f o r m i n g the c h r o m o s o m e counts, to L.F. K l e i n m a n of Boston U n i v e r s i t y a n d G.B. Z a m a n s k y a n d J.C. K a p l a n of H a r v a r d Medical School for valuable discussions a n d to colleagues of the Bureau of Radiological Health for critical review of the manuscript. This work was supported b y C o n t r a c t No. 232-78-6018 from the F o o d a n d D r u g A d m i n i s t r a t i o n .

References Black, P.H. (1968) The oncogenic DNA viruses: a review of in vitro transformation studies, Annu. Rev. Microbiol., 22, 391-426. Bockstahler, L.E., and J.M. Cantwell (1979) Photodynamic induction of an oncogenic virus in vitro, Biophys. J., 25, 209-213. Bockstahler, L.E., and K.B. Hellman (1979) Induction of oncogenic viruses by light, Photochem. Photobiol., 30, 743-748. Burns, W.H., and P.H. Black (1969) Analysis of SV40-inducexttransformation of hamster kidney tissue in vitro, VI. Characteristics of mitomycin C induction, Virology, 39, 625-634. Coohill, T.P., S.P; Moore and S. Drake (1977) The wavelength dependence of ultraviolet inactivation of host capacity in a mammalian cell-virus system, Photochem. Photobiol., 26, 387-391. Coohill, T.P., L.C. James and S.P. Moore (1978) The wavelength dependence of ultraviolet enhanced reactivation in a mammalian cell-virus system, Photochem. Photobiol., 27, 725-730. Doniger, J., E.D. Jacobson, K. Kreli and J.A. DiPaolo (1981) Ultraviolet light action spectra for neoplastic transformation and lethality of Syrian hamster embryo ceils correlate with that for pyrimidine dimer formation in cellular DNA, Proc. Natl. Acad. Sci. (U.S.A.), 78, 2378-2382. Fogel, M. (1972) Induction of virus synthesis in polyoma-transformed cells bY DNA antimetabolites and by irradiation after pre-treatment with 5-bromodeoxyuridine,Virologyl 49, 12-22. Fogel, M. (1973) Induction of polyoma virus by fluorescent (visible) light in polyoma-transformed cells pretreated with 5-bromodeoxyuridine,Nature New Biol. (London), 241, 182-184. Fogel, M., and L. Sachs (1970) Induction of virus synthesis in polyoma transformed cells by ultraviolet light and mitomycin C, Virology,42, 251-256. Franklin, R. (1954) The action spectrum for the ultraviolet induction of lysis in Escherichia coli K-12, Biochim. Biophys. Acta, 13, 137-138. Hirsch, M.S., and P.H. Black (1974) Activation of mammalian leukemia viruses, Adv. Virus Res., 19, 265-313. Jacobson, E.D., K. Krell and M.J. Dempsey (1981) The wavelength dependence of ultraviolet light-induced cell killing and mutagencsis in L5178Y mouse lymphoma cells, Photochem. Photobiol., 33, 257-260. Jagger, J.J. (1967) Introduction to Research in Ultraviolet Photobiology, Prentice-Hall, Englewood Cliffs, N.J. Kaplan, J.C., S. Wilbert and P.h. Black (1972) Analysis of simian virus 40-induced transformation of hamster kidney tissue in vitro, VIII. Induction of infectious simian virus 40 from virogenic transformed hamster cells by amino acid deprivation or cycloheximidetreatment, J. Virol., 9, 448-453.

103 Kaplan, J.C., S.M. Wilbert, J.J. Collins, T. Rakusanova, G.B. Zamansky and P.H. Black (1975) Isolation of simian virus 40-transformed inbred hamster cell lines heterogeneous for virus induction by chemicals or radiation, Virology, 68, 200-214. Morris, A.G., C. Lavialle, H.G. Suarez, J. Stevenet, S. Estrade and R. Cassingena (1977) Simian virus -40-Chinese hamster kidney cell interaction, III. Characteristics of chemical induction in a clone of virogcnic transformed cells, J. Gen. Virol., 36, i 23-135. Pollack, R., and S. Pfeiffer (1971) Animal Cell Culture, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Radman, M. (1980) Is there SOS induction in mammalian cells? Photochem. Photobiol., 32, 823-830. Rakusanova, T., J.C. Kaplan, W.P. Smales and P.H. Black (1976) Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells, J. Virol., 19, 279-285. Rothman, R.H., and R.B. Setlow (1979) An action spectrum for cell killing and pyrimidine dimer formation in Chinese hamster V-79 cells, Photochem. Photobiol., 29, 57-61. Rothschild, H., and P.H. Black (1970) Analysis of SV40-induced transformation of hamster kidney tissue in vitro, VII. Induction of SV40 virus from transformed hamster cell clones by various agents, Virology, 42, 251-256. Todaro, G.J., and R.J. Huebner (1972) The viral oncogene hypothesis: New evidence, Proc. Natl. Acad. Sci. (U.S.A.), 69, 1009-1015. Todd, P., T.P. Coohill and J.A. Mahoney (1968) Responses of cultured Chinese hamster cells to ultraviolet light of different wavelengths, Radiation Res., 35, 390-400. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible repair in Escherichia coil, Bacteriol. Rev., 40, 869-907. Zamansky, G.B., L.F. Kleinman, J.B. Little, P.H. Black and J.C. Kaplan (1976) The effect of caffeine on the ultraviolet light induction of SV40 virus from transformed hamster cells, Virology, 72, 468-475.