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Demonstration of recovery from the potentially mutagenic effects of ultraviolet light by replication-inhibited Chinese hamster V79 cells
Mutation Research, 95 (1982) 493-503
493
Elsevier BiomedicalPress
Demonstration of recovery from the potentially mutagenic effects of ultraviolet light by replication-inhibited Chinese hamster V79 cells Donna S. Stone-Wolff and Toby G. Rossman * Department of Environmental Medicine, New York University School of Medicine, 550 First Avenue, New York, NY10016 (U.S.A.)
(Received I August 1981) (Revisionreceived 7 January 1982) (Accepted 10 January 1982)
Summary The effects of replication-inhibiting conditions on the ability of Chinese hamster (V79) cells to recover from the potentially mutagenic effects of ultraviolet (UV) light were investigated. V79 cells were synchronized by a new technique using a low concentration of hydroxyurea (0.2 mM), which provided a mildly-toxic, nonmutagenic method for producing large quantities of synchronized cells needed for these studies. The protocol developed for this study involved UV-irradiation of synchronized V79 cells which were blocked at the G 1 / S boundary. Following UV-irradiation, the cells were either allowed to enter S phase immediately or were blocked for increasing periods of time by the addition of more hydroxyurea. The former cells contained the highest frequencies of ouabain-resistant mutants, while cells whose replication was blocked following UV-irradiation showed decreasing mutation frequencies with respect to time. Therefore, V79 cells are able to demonstrate liquid holding recovery from potentially mutagenic UV-lesions. Since the UV-induced mutation frequency was reduced by almost 50% following 6 h of 'liquid holding', the mutagenic lesions seem to be removed at a faster rate than has previously been reported for the removal of pyrimidine dimers from these cells.
When UV-irradiated E. coli B was incubated in the dark in a suspension of buffer or saline, an increase in viability was observed (Hollaender and Curtis, 1935; Hollaender and Claus, 1937; Roberts and Aldous, 1949; Charles and Zimmerman, * To whom all correspondenceshould be sent. 0027-5107/82/0000-0000/$02.75 © ElsevierBiomedicalPress
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1956). This phenomenon has been called 'liquid holding recovery' or LHR (Castellani et al., 1964; Jagger et al., 1964) and is one of the first pieces of evidence for the existence of a DNA-repair mechanism in E. coll. According to the model described by Ganeson and Smith (1969), liquid-holding recovery involves the repair of pyrimidine dimers exclusively by a uvr+-dependent excision-repair mechanism, since the delay of DNA replication due to the incubation of the cells in buffer does not allow post-replication repair to occur. LHR was also demonstrated in yeast for a variety of mutagenic treatments (Patrick et al., 1961). The increased survival of Saccharomyces cerevisiae held in buffer after UV-irradiation is correlated with decreased UV-mutagenesis and intergenic recombination (Parry and Cox, 1968; Parry and Parry, 1972). Evidence for a similar response in mammalian cells was demonstrated in densityinhibited human diploid fibroblasts after X-irradiation (Simons, 1979; Little, 1973), UV-irradiation (Simons, 1979; Maher et al., 1979) or treatment with chemical carcinogens (Yang et al., 1980; Heflich et al., 1980). Recovery from potentially lethal damage was also demonstrated in density-inhibited Chinese hamster (HA-I) cells (Hahn et al., 1973; Hahn, 1975). On the other hand, the UV-induced mutation frequency and survival of excision-deficient xeroderma pigmentosum (XP12BE) cells did not change with time in confluence (Maher et al., 1979). These results demonstrate that excision repair is responsible for recovery from potentially lethal damage in human cells. The demonstration that LHR phenomenon in all of these different cell types indicates that excision repair of UV-photoproducts is a universal mechanism. However, in mammalian cells, excision-repair capacities vary with respect to species. Rodent cells resemble XP cells in having low levels of excision repair of UV-induced damage (Ahmed and Setlow, 1977; Cleaver, 1970; Meyn et al., 1974; Setlow et al., 1972), yet they are at least as resistant to UV as are normal human cells capable of high levels of dimer excision. We recently reported results of experiments designed to determine whether UV-mutagenesis in Chinese hamster cells is mediated by an 'inducible' error-prone repair system. Post-irradiation treatment with the translation inhibitors cycloheximide or puromycin decreased the UV-mutation frequency (Stone-Wolff and Rossman, 1981a) One possible explanation for this effect is that since both cycloheximide and puromycin inhibit DNA synthesis, more time may have been available for the limited excision-repair processes in these cells to remove UV dimers or other photoproducts before misreplication of unexcised lesions could lead to increased mutagenesis. These results bear some similarities to the decrease in UV-mutagenesis observed in density-inhibited human fibroblasts. Therefore, it was of interest to determine whether replication-inhibiting conditions would reduce UV-mutagenesis in V79 cells. Since V79 cells exhibit poor contact inhibition, a different protocol involving the use of hydroxyurea to block post-irradiation DNA replication was used. Under the experimental conditions of density-inhibition used in mammalian recovery experiments, the time periods reported for recovery from potentially lethal damage are inaccurate because they do not take into account the time necessary for
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the cells to re-attach and enter S phase after they are replated at lower density. In order to avoid this problem, our replication-inhibiting protocol involves the initial synchronization of V79 cells at the G 1 / S boundary so that the cells will enter S phase immediately upon removal of the block. After synchronization, the cells are UV-irradiated and DNA replication is subsequently blocked by hydroxyurea for increasing periods of time to determine the effects of post-irradiation inhibition of DNA synthesis on the UV-mutation frequency. Various protocols have been described for the synchronization of mammalian cells (reviewed by Mitchison, 1971; Stubblefield, 1968). Some of these include: the use of a double thymidine block (Xeros, 1962; Bostock et al., 1971); collection of cells in mitosis by mitotic shake-off (Terasima and Tolmach, 1961, 1963; Peterson et al., 1968) or by centrifugal elutriation (Meistrich et al., 1977a, b; Gohde et al., 1979); and the use of inhibitors of DNA synthesis such as hydroxyurea (Erickson, 1966; Sinclair, 1965, 1967; Meyn et al., 1973). The synchronization technique needed for these experiments must be relatively non-toxic, non-mutagenic and able to yield large quantities of cells needed for mutagenesis. Synchronization by mitotic shake-off or centrifugal elutriation provide excellent degrees of synchrony but require specialized equipment to obtain synchronized cells in high enough yield to perform mutagenesis analysis (Burki and Aebersold, 1978; Keng et al., 1980). The double thymidine block and hydroxyurea techniques provide synchronized cells in high yield. However, it has been demonstrated that treatments with concentrations of thymidine used in synchronization protocols were mutagenic to both Chinese hamster ovary and V79 cells, whereas similar treatments with synchronizing concentrations of hydroxyurea were not mutagenic but produced a large amount of toxicity (Bradley and Sharkey, 1978; Huberman and Heidelberger, 1972; Stone-Wolff and Rossman, 1981b). Reported here is a synchronization technique using a low concentration of hydroxyurea which meets the criteria of being mildly toxic, non-mutagenic and able to produce at least partially synchronized cells at high yield. The optimal conditions of this technique and the effects of DNA replication-inhibiting conditions on the UV-induced mutation frequency of V79 cells are described.
Materials and methods Cell culture
A Chinese hamster cell line, V79, strain number 743-3-6, was obtained from Dr. Dennis Yep, University of Michigan. Cells were grown in Ham's F12 medium (GIBCO) containing 5% bobby calf serum (GIBCO), 50 #g/ml gentamicin (Schering Corp.) and 2 # g / m l fungizone (Squibb), at 37°C in an atmosphere of 5% CO 2. The cells were recloned in our laboratory, and a clone which had a low spontaneous mutation frequency was isolated and stored frozen in liquid nitrogen. New batches of cells are thawed every 6-8 weeks to insure low spontaneous mutation frequencies.
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Animal cell mutagenesis under replication-inhibiting conditions Animal cell mutagenesis experiments were performed using ouabain-resistance as a genetic marker as described by Chang et al. (1978). Survival dishes were seeded at a density of 300 cells/60-mm dish in triplicate. Mutagenesis dishes were seeded at a density of 4 × 105 cells/100-mm dish, and 10 dishes per condition were used. 4h after attachment, the cells were synchronized by incubation in 0.2 mM hydroxyurea (HU). After an incubation period of 16 h, the HU was removed and the cells were washed and immediately irradiated in 5 ml of Earle's balanced salt solution (EBSS) with a G1578 germicidal lamp at a distance of 50 cm, resulting in a fluence rate of 0.295 J/mZ/sec. Immediately following irradiation, growth medium with or without 0.2 mM HU was added to the cells for increasing durations as described in the protocols accompanying Table 1. The HU was removed and the ceils were washed with EBSS and replaced with fresh growth medium for an expression period of 48 h, at which time, the growth medium was replaced by selective medium containing 1 mM ouabain. Survival dishes were stained after 7 days, and mutagenesis dishes after 14 days, with a medium change after day 7. The mutation frequency was calculated by dividing the number of ouabain-resistant clones by the number of surviving clones, calculated from the survival dishes. Measurement of DNA synthesis Cells were seeded at a density of 105 cells/60-mm dish. After attachment, medium with or without 0.2 mM HU was added for different periods of time as described in the protocols accompanying the figures. The cells were pulse-labeled for I h in medium containing [Me-3H]thymidine (New England Nuclear, spec. act., 55.2 Ci/mmole) which was used at a final concentration of 5/iCi/ml, either in the presence of HU or after its removal. The cells were lysed by the addition of 0.2 ml of a 0.5% solution of sodium dodecyl sulfate (SDS) in EBSS, and the lysate was transferred into a tube containing 1.3 ml of EBSS. An equal volume of cold 10% TCA was added to precipitate the nucleic acids and proteins. Following a 15-rain incubation period in an ice bath, the TCA precipitates were collected on 0.45-/t Millipore filters, washed, dried, immersed in butyl PPD toluene scintillation mixture (New England Nuclear) and counted on a Packard scintillation counter. Mitotic index Cells were seeded at a density of 103 ceUs/25-mm dish. After attachment, growth media with or without hydroxyurea was added for different periods of time. After removal of the HU block, the cells were washed twice with EBSS and replaced with growth media containing colcemid (GIBCO) at a final concentration of 0.5 # g / m l for successive periods of 1 h, after which time, the cells were fixed and stained with Giemsa stain. The mitotic index was determined microscopically and expressed as the number of mitotic figures per 100 cells. Chemicals Hydroxyurea and ouabain were purchased from Sigma Chemical Company, St. Louis, MI.
497 Results
Synchronization of V79 cells with a low concentration of hydroxyurea (HU) Fig. 1 is a dose-response curve representing the effects of 16-h exposure to various concentrations of hydroxyurea on the clonal survival of V79 cells. A 16-h treatment with 2 mM H U was very toxic, yielding a cloning efficiency of only 20% of control cells. This was surprising in light of the fact that many cell synchronization protocols use 2 mM H U as a minimum concentration. A 16-h treatment with 0.2 m M H U was only mildly toxic, yielding a cloning efficiency of 80% of control cells. It was of interest to determine whether this concentration of hydroxyurea could be substituted for the more toxic one in the synchronization technique. Fig. 2 demonstrates that 0.2 m M H U can inhibit up to 95% of D N A synthesis of control cells within the first 4 h. A complete reversal of this inhibition occurs immediately upon removal of the HU. Fig. 3a shows the rate of incorporation of [ 3H]thymidine into cold-acid-precipitable material at 1-h intervals following release from a single 16-h block with 0.2 mM HU. The rate of D N A synthesis increases immediately and reaches a maximum level between 1 and 2 h after removal of the H U block. The length of this S phase is approx. 3.25 h which was estimated by measuring the time interval between the end of the H U block and the point at which the labeling index had fallen to half the maximum (see Fig. 3a). This parasynchronous wave of D N A synthesis is followed by a wave of mitosis which peaks 4-5 h following the removal of the H U block. A second wave of DNA synthesis begins 6 - 7 h after the removal of the H U block. It has been demonstrated that the use of a double block improves the synchrony caused by excess thymidine (Peterson and Anderson, 1964). The rationale is that a single thymidine treatment results in some cells being blocked within S phase (Puck, 1964). If the block is lifted and the cells are resuspended in growth medium for a period of 5 - 6 h (a length of time longer than S phase), the blocked cells will progress through S phase but will be caught at the G 1 / S boundary in the next cycle by a second block of thymidine. It was of interest to see whether a similar double block protocol with H U would improve the synchrony of V79 cells. Cells were treated for 16 h with 0.2 mM hydroxyurea, the H U was removed and the cells were washed and incubated in fresh growth medium for 6 h. A second 16 h treatment with 0.2 mM H U was given and the rate of [3H]thymidine incorporation at 1-h intervals was determined following removal of the second block. The effects of a double H U block on the rates of [3H]thymidine incorporation and mitotic index are shown in Fig. 3b. The double hydroxyurea block yields similar results with respect to synchrony as those obtained following a single H U block. The rate of DNA synthesis increases immediately and reaches a peak between 1 and 2 h after removal of the second H U block, as was seen after removal of the first H U block. The length of S phase is also the same as after a single H U treatment. A wave of mitosis occurs following S which also reaches a maximum 4-5 h after the removal of the HU. However, the maximum mitotic index was increased from 27% after a single block to 32% after a double block. The second wave of DNA synthesis starts 8-9 h after removal of the second H U block. This is approx. 2 h later than the start of the second cycle after a single
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Fig. 1. Effects of hydroxyurea on clonal survival of V79 cells. Cells were incubated with different concentrations of hydroxyureafor 16 h. The hydroxyureawas then removedand the cells were incubated with growth medium for a period of 1 week or until colonies became macroscopic. Fig. 2. The inhibition and recoveryof DNA synthesis in V79 cell treated with 0.2 mM hydroxyurea.V79 cells (10~ cells/dish) were given 1-h pulses of [3H]thymidinein the presence of 0.2 mM HU (added at time zero and after its removal).
H U block and is in better agreement with published values for the V79 cell cycle (Robbins and Scharff, 1967). Therefore, these differences imply that the double block showed a small improvement in the level of synchrony in V79 cells over the single block. Although the number of cells, concentration of [3H]thymidine, and labeling conditions were the same in both synchronization treatments, the amount of [3 H]thymidine incorporation after the double block equalled approximately one half of the rate of [3H]thymidine incorporation observed following the single block. One possible explanation for this difference is that the d o u b l e block caused increased toxicity in V79 cells. A comparison of the effects of a double verses a single H U block on clonal survival of V79 cells showed that a single 16-h treatment with 0.2 m M H U resulted in a survival of 88% of untreated cells, while a double treatment resulted in 54.4% of control survival. Since the double block showed increased toxicity without significantly improving the level of synchrony in the first S phase, the single 16-h treatment was employed in the following experiments.
Effects of replication-inhibiting conditions on UV-mutagenesis in synchronized V79 cells Following a 16-h treatment with 0.2 mM HU, V79 cells were washed with buffer and UV-irradiated as described in the Methods section. Post-irradiation D N A synthesis was blocked with 0.2 mM H U for different durations from 0 to 10h. Table 1 shows the effects of these 'hquid-holding' conditions on UV-mutagenesis.
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Fig. 3a. Cell synchronization after a single treatment with 0.2 mM hydroxyurea. The effects of a single 16-h block with 0.2 mM HU on DNA synthesis (0) and mitotic index (dk) are demonstrated here. V79 cells were given I-h pulses of [3H]thymidine at various times after the removal of the block. Mitotic indices were calculated each hour after the removal of the HU block as described in the Methods section. Fig. 3b. Cell synchronization after a double treatment with 0.2 mM hydroxyurea. The effects of a double HU block on DNA synthesis (Q) and mitotic index (&) are demonstrated here. V79 cells were given 1-h pulses of [3H]thymidine at various times after the removal of the second 16-h HU block. Mitotic indices were calculated each hour after the removal of the block as described in the Methods section.
Treatments in which cells were allowed to enter S phase immediately after UV (Table 1, 0 h) demonstrated the highest UV-mutation frequencies. Cells whose replication was blocked following UV-irradiation demonstrated gradually decreasing mutation frequencies with respect to the amount of time that DNA synthesis remained inhibited. For example, in Expt. 1, the UV-induced mutation frequency was reduced by almost 50% when the cells were held for 6 h. Therefore, V79 cells are able to demonstrate replication-inhibited recovery from potentially mutagenic UV lesions.
Discussion
Chinese hamster cells were synchronized by a new technique using a low concentration of hydroxyurea which provides a mildly-toxic, non-mutagenic method of producing large quantities of synchronized cells needed for studying mutagenesis. Cells treated with 0.2 mM HU for 16 h demonstrate a parasynchronous wave of DNA synthesis which reaches a maximum between 1 and 2 h after removal of the
500 TABLE ! EFFECTS OF REPLICATION-INHIBITING CONDITIONS ON THE U V - I N D U C E D MUTATION F R E Q U E N C Y IN V79 CELLS a Duration of postirradiation inhibition of DNA synthesis by 0.2 mM H U
Survival (% control)
Total number of Oua r mutants
Mutation frequency (mutants/106 survivors)
Expt. 1 (a) Unirradiated control b (b) Oh (c) 3 h (d) 6 h
100.00 88.00 70.80 67.10
3 183 133 72
1.84 127.97 115.70 66.10
Expt. 2 (a) Unirradiated control b (b) Oh (c) 2 h (d) 4 h (e) 6 h (f) 8 h (g) 10 h
100.00 103.10 107.10 111.40 81.70 72.00 62.90
5 186 153 133 90 70 60
2.80 100.54 79.69 73.89 68.18 60.34 58.82
a All UV-irradiations were performed at 3.5 J/m 2, b Unirradiated controls were blocked for 16 h with 0.2 mM HU, washed with buffer, given a mock UV-irradiation treatment.
block, followed by a wave of mitosis which peaks 4 - 5 h following removal of the H U block. The S period which immediately follows the release from the single H U block in these experiments is shorter than the S period reported by Robbins and Scharff (1967) for the same cell line. This could be the result of incomplete inhibition of D N A synthesis by hydroxyurea. Fig. 3 shows that a small amount of [3H]thymidine incorporation still occurs in the presence of 0.2 mM H U even after a 16-h treatment. Since many of the cells have already synthesized some D N A during the block, completion of D N A synthesis after the block requires less time than a normal S period. Bostock et al. (1971) observed this same phenomenon in Chinese hamster ovary cells synchronized with 2 mM thymidine. A double block with 0.2 mM H U had no effect on this foreshortened S period. The double H U block delayed the onset of the second wave of D N A synthesis by 2 h as compared to the single block. This may be an indication that the double block was more inhibitory with respect to allowing cells to progress through S phase. However, a double block with H U was too toxic for use in these experiments. Although, the synchronization after a 16-h treatment with 0.2 mM H U is only partial, its low toxicity and lack of mutagenicity, makes it a useful technique in cases where large quantities of synchronized cells are needed for mutagenesis analysis. . We previously demonstrated that post-irradiation treatment of V79 cells with the translation inhibitors puromycin or cycloheximide decreased the UV-mutation
501 frequency, and proposed that this decrease was due to their inhibitory effects on DNA synthesis rather than their inhibition of de novo protein synthesis (Stone-Wolff and Rossman, t981a). Inhibition of post-irradiation DNA synthesis may have allowed more time for the limited excision repair processes in these cells to remove dimers or other photoproducts before the misreplication of unexcised photoproducts could lead to increased mutagenesis. These results bear some similarities to the decrease in UV-mutagenesis observed in density-inhibited human fibroblasts, a phenomenon similar to 'liquid-holding recovery' in bacteria. In order to see whether this interpretation of the cycloheximide and puromycin effects was correct, the effects of replication-inhibiting conditions on UV-mutagenesis in Chinese hamster cells were examined. The protocol developed for this study involved UV-irradiation of synchronized V79 cells which were blocked at the G 1 / S boundary. Immediately following UV-irradiation, the cells were either allowed to enter S phase or remained blocked for increasing periods of time by the addition of hydroxyurea. Cells which were allowed to enter S phase immediately following UV-irradiation contained the highest frequencies of ouabain-resistant mutants, while cells whose replication was blocked following UV-irradiation showed decreasing mutation frequencies with respect to time. Therefore, a delay in post-irradiation DNA synthesis by cycloheximide, puromycin or hydroxyurea results in a decrease in the UV-induced mutation frequency. These results imply that the reduction in UV-mutagenesis following replication-inhibiting conditions results from the increased time available for the limited excision-repair processes in these cells to remove UV lesions before the misreplication of unexcised lesions can lead to mutagenesis. It has been demonstrated that complete excision repair of pyrimidine dimers caused by UV light requires at least 24h for human cells (Rauth, 1970; Cleaver, 1974; Regan and Setlow, 1974) and much longer for Chinese hamster cells (Meyn et al., 1974). According to Yarosh and Setlow (1981), V79 cells remove less than 15% of the dimers induced by 10 J / m 2 of UV within 9 h. Our results demonstrating that inhibition of post-irradiation DNA synthesis for a period of 6 h in V79 cells causes a 50% reduction in the mutation frequency induced by 3.5 J / m 2 of UV light, might be interpreted in 2 ways. First, the rate of excision repair in these cells may be faster at non-lethal UV fluences, and may even approach human excision-repair rates. This would imply that the differences between human and rodent excision-repair capacities may be due to different saturation levels rather than to any specific deficiency in the rodent cell excision-repair system. A second interpretation of our data may be that mutagenesis induced by UV light in V79 cells is dependent upon a combination of dimer and non-dimer photoproducts whose composite rates of excision repair approaches a half-time of 6 h.
Acknowledgements This study was funded by Grant No. CA19421 (National Cancer Institute) and is part of Center programs supported by Grant ES-00260 from the National Institute of Environmental Health Sciences and by Grant CA 13343 from the National Cancer Institute.
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References Ahmed, F.E., and R.B. Setlow (1977) DNA repair in V79 cells treated with combinations of ultravioletradiation and N-acetoxy-2-acetylamino-fluorene, Cancer Res., 37, 3414-3419. Bostock, C.J., D.M. Prescott and J.B. Kirkpatrick (1971) An evaluation of the double thymidine block for synchronizing mammalian cells at the G I / S border, Exptl. Cell Res., 68, 163-168. Bradley, M.O., and N.A. Sharkey (1978) Mutagenicity of thymidine to cultured Chinese hamster cells, Nature (London), 274, 607-608. Burki, H.J., and P.M. Aebersold (1978) Bromodeoxyuridine-induced mutations in synchronous Chinese hamster cells: Temporal induction of 6-thioguanine and ouabain-resistance during DNA replication, Genetics, 90, 311-321. Castellani, A., J. Jagger and R.B. Setlow (1964) Overlap of photoreactivation and liquid holding recovery in Escherichia coil B, Science, 143, 1170-1171. Chang, C.C., J.E. Trosko and J. Akera (1978) Characterization of ultraviolet fight-induced ouabain-resistant mutations in Chinese hamster cells, Mutation Res., 51, 85-98. Charles, R.L., and L.N. Zimmerman (1956) Dark reactivation in ultraviolet-irradiated Escherichia coli, J. Bacteriol., 71,611-616. Cleaver, J.E. (1970) Repair replication in Chinese hamster cells after damage from ultraviolet light, Photochem. Photobiol., 12, 17-28. Cleaver, J.E. (1974) Repair processes for photochemical damage in mammalian cells, Adv. Radiat. Biol., 4, 1-75. Erikson, T. (1966) Partial synchronization of cell division in suspensions of Haplopappus gracilus, Physiol. Plant., 19, 900-910. Ganeson, A.K., and K.C. Smith (1968) Dark recovery processes in Escherichia coli irradiated with ultraviolet light, I Effect of rec- mutation on liquid holding recovery, J. Bacteriol., 96, 365-373. Ganeson, A.K., and K.C. Smith (1969) Dark recovery processes in Escherichia coli irradiated with ultraviolet light, II. Effect of uvr genes in liquid holding recovery, J. Bacteriol., 97, 1129-1133. Gohde, W., M.L. Meistrich, R.E. Meyn, J. Schumann, D. Johnston and B. Barlogie (1979) Cell-cycle phase dependence of drug-induced cycle progression delay, J. Histochem. Cytochem., 27, 470-473. Hahn, G.M. (1975) Repair (or recovery) effects in quiescent Chinese hamster cells: an attempt at classification, in: P.C. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, Plenum, New York, pp. 601-605. Hahn, G.M., M.A. Bagsliaw, R.G. Evans and L.F. Gordon (1973) Repair of potentially lethal lesions in X-irradiated, density-inhibited Chinese hamster cells: Metabolic effects and hypoxia, Radiat. Res., 55, 280-290. Heflich, R.H., R.M. Hazard, L. Lommel, J.D. Seribner, V.M. Maher and J.J. McCormick (1980) A comparison of the DNA binding; cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens, Chem.-Biol. Interact., 29, 43-56. Hollaender, A., and W. Claus (1937) An experimental study of the problem of mitogenic radiation, Bull. Natl. Res. Council, Natl. Acad. Sci. (U.S.A.), 100, 75-88. Hollaender, A., and J.T. Curtis (1935) Effect of sublethal doses of monochromatic ultraviolet-radiation on bacteria in liquid suspensions, Prec. Soc. Exptl. Biol. Med., 33, 61. Huberman, E., and C. Heidelberger (1972) The mutagenicity of mammalian cells to pyrimidine nucleotide analogs, Mutation Res., 14, 130-132. Jagger, J., W.C. Wise and R.S. Stafford (1964) Delay in growth and division induced by near ultravioletradiation in Escherichia coli B and its role in photoprotection and liquid holding recovery, Photochem. Photobiol., 3, 11-24. Keng, P.C., C.K.N. Li and K.T. Wheeler (1980) Synchronization of 9L rat brain tumor cells by centrifugal elutriation, Cell Biophys., 2, 191-206. Little, J.B. (1973) Factors influencing the repair of potentially lethal radiation damage in growth-inhibited human cells, Radiat. Res., 56, 320-333. Maher, V.M., D.J. Dorney, A.L. Mendrala, B. Konze-Thomas and J.J. McCormick (1979) DNA excision repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet-irradiation, Mutation Res., 62, 311-323.
503 Meyn, R.E., R.R. Hewitt and R.M. Humphrey (1973) Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine, Exptl. Cell Res., 82, 137-142. Mitchison, J.M. (1971) The Biology of the Cell Cycle, Cambridge University Press, London, 313 pp. Parry, J.M., and B;S. Cox (1968) The effects of dark holding and photoreactivation on ultraviolet light-induced mitotic recombination and survival in yeast, Genet. Res., 12, 187. Parry, J.M., and E.M. Parry (1972) The genetic implications of UV light exposure and liquid holding post-treatment in yeast, Saccharomyces cerevisiae, Genet. Res., 19, 1-16. Patrick, M.N., R.N. Haynes and P.B. Uritz (1961) Dark recovery phenomena in yeast, I. Comparative effects with various inactivating agents, Radiat. Res., 21, 144-163. Peterson, D.F., and E.C. Anderson (1964) Quantity production of synchronized mammalian cells in suspension culture, Nature (London), 203, 642-643. Peterson, D.F., E.C. Anderson and R.A. Tobey (1968) Mitotic cells as a source of synchronized cultures, in: D.M. Prescott (Ed.), Methods in Cell Physiology, Vol.3, Academic Press, New York, pp. 347-370. Puck, T.T. (1964) Phasing, mitotic delay, and chromosomal aberrations in mammalian cells, Science, 144, 565-566. Rauth, A.M. (1970) Effects of ultraviolet light in mammalian cells in culture, Curr. Top. Rad. Res., 6, 195-248. Regan, J.D., and R.B. Setlow (1974) Two forms of repair in the DNA of human cells damaged by chemical carcinogens and mutagens, Cancer Res., 34, 3318-3325. Robbins, E., and M.D. Scharff (1967) The absence of a detectable GI phase in a cultured strain of Chinese hamster lung cell, J. Cell Biol., 34, 684-685. Roberts, R.B., and E. Aldous (1949) Recovery from ultraviolet-irradiation in Escherichia coil, J. Bacteriol., 57, 363-375. Setlow, R.B., J.D. Regan, and W.L. Carrier (1972) Different levels of excision repair in mammalian cell lines, Biophys. Soc. Ann. Meeting Abstr., 12, 19a. Simons, J.W.I.M. (1979) Development of a liquid holding technique for the study of DNA repair in human diploid fibroblasts, Mutation Res., 59, 273-283. Sinclair, W.K. (1965) Hydroxyurea, Differential lethal effects on cultured mammalian cells during the cell cycle, Science, 150, 1729-1731. Sjt~berg, B.M., P. Reichard, A. Graslund and E. Ehrenberg (1977) Nature of the free radical in ribonucleoside reductase from E. coil, J. Biol. Chem., 252, 536-541. Stone-Wolff, D.S., and T.G. Rossman (1981a) Effects of inhibitors of de novo protein synthesis on UV-mutagenesis in Chinese hamster cells: evidence against mutagenesis via inducible error-prone DNA repair, Mutation Res., 82, 147-157. Stone-Wolff, D.S., and T.G. Rossman (1981b) The mutagenicity of deoxynucleosides in Chinese hamster cells, 12th Annual Meeting, Environmental Mutagen Society, Abstr. No. Ca-2, p. 117. Stubblefield, E. (1968) Synchronization methods for mammalian cell culture, in: D.M. Prescott (Ed.) Methods in Cell Physiology, Vol. 3, Academic Press, New York, pp. 25-43. Terasima, T., and L.J. Tolmach (1961) Changes in X-ray sensitivity of HeLa ceils during the division cycle, Nature (London), 190, 1210-1211. Terasima, T., and L.J. Tolmach (1963) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells, Exptl. Cell Res., 30, 344-362. Timson, J. (I 975) Hydroxyurea, Mutation Res., 32, 115-132. Xeros, N. (1962) Deoxyriboside control and synchronization of mitosis, Nature (London), 194, 682-683. Yang, L.L., V.M. Maher and J.J. McCormick (1980) Error-free excision of the cytotoxic, mutagenic N2-deoxyguanosine DNA adduct formed in human fibroblasts by (±)-7[3,8a-dihydroxy-9a, lOaepoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, Proc. Natl. Acad. Sci. (U.S.A.), 77, 5933-5937. Yarosh, D.B., and R.B. Setlow (1981) Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus, Mol. Cell Biol., I, 237-244.
493
Elsevier BiomedicalPress
Demonstration of recovery from the potentially mutagenic effects of ultraviolet light by replication-inhibited Chinese hamster V79 cells Donna S. Stone-Wolff and Toby G. Rossman * Department of Environmental Medicine, New York University School of Medicine, 550 First Avenue, New York, NY10016 (U.S.A.)
(Received I August 1981) (Revisionreceived 7 January 1982) (Accepted 10 January 1982)
Summary The effects of replication-inhibiting conditions on the ability of Chinese hamster (V79) cells to recover from the potentially mutagenic effects of ultraviolet (UV) light were investigated. V79 cells were synchronized by a new technique using a low concentration of hydroxyurea (0.2 mM), which provided a mildly-toxic, nonmutagenic method for producing large quantities of synchronized cells needed for these studies. The protocol developed for this study involved UV-irradiation of synchronized V79 cells which were blocked at the G 1 / S boundary. Following UV-irradiation, the cells were either allowed to enter S phase immediately or were blocked for increasing periods of time by the addition of more hydroxyurea. The former cells contained the highest frequencies of ouabain-resistant mutants, while cells whose replication was blocked following UV-irradiation showed decreasing mutation frequencies with respect to time. Therefore, V79 cells are able to demonstrate liquid holding recovery from potentially mutagenic UV-lesions. Since the UV-induced mutation frequency was reduced by almost 50% following 6 h of 'liquid holding', the mutagenic lesions seem to be removed at a faster rate than has previously been reported for the removal of pyrimidine dimers from these cells.
When UV-irradiated E. coli B was incubated in the dark in a suspension of buffer or saline, an increase in viability was observed (Hollaender and Curtis, 1935; Hollaender and Claus, 1937; Roberts and Aldous, 1949; Charles and Zimmerman, * To whom all correspondenceshould be sent. 0027-5107/82/0000-0000/$02.75 © ElsevierBiomedicalPress
494
1956). This phenomenon has been called 'liquid holding recovery' or LHR (Castellani et al., 1964; Jagger et al., 1964) and is one of the first pieces of evidence for the existence of a DNA-repair mechanism in E. coll. According to the model described by Ganeson and Smith (1969), liquid-holding recovery involves the repair of pyrimidine dimers exclusively by a uvr+-dependent excision-repair mechanism, since the delay of DNA replication due to the incubation of the cells in buffer does not allow post-replication repair to occur. LHR was also demonstrated in yeast for a variety of mutagenic treatments (Patrick et al., 1961). The increased survival of Saccharomyces cerevisiae held in buffer after UV-irradiation is correlated with decreased UV-mutagenesis and intergenic recombination (Parry and Cox, 1968; Parry and Parry, 1972). Evidence for a similar response in mammalian cells was demonstrated in densityinhibited human diploid fibroblasts after X-irradiation (Simons, 1979; Little, 1973), UV-irradiation (Simons, 1979; Maher et al., 1979) or treatment with chemical carcinogens (Yang et al., 1980; Heflich et al., 1980). Recovery from potentially lethal damage was also demonstrated in density-inhibited Chinese hamster (HA-I) cells (Hahn et al., 1973; Hahn, 1975). On the other hand, the UV-induced mutation frequency and survival of excision-deficient xeroderma pigmentosum (XP12BE) cells did not change with time in confluence (Maher et al., 1979). These results demonstrate that excision repair is responsible for recovery from potentially lethal damage in human cells. The demonstration that LHR phenomenon in all of these different cell types indicates that excision repair of UV-photoproducts is a universal mechanism. However, in mammalian cells, excision-repair capacities vary with respect to species. Rodent cells resemble XP cells in having low levels of excision repair of UV-induced damage (Ahmed and Setlow, 1977; Cleaver, 1970; Meyn et al., 1974; Setlow et al., 1972), yet they are at least as resistant to UV as are normal human cells capable of high levels of dimer excision. We recently reported results of experiments designed to determine whether UV-mutagenesis in Chinese hamster cells is mediated by an 'inducible' error-prone repair system. Post-irradiation treatment with the translation inhibitors cycloheximide or puromycin decreased the UV-mutation frequency (Stone-Wolff and Rossman, 1981a) One possible explanation for this effect is that since both cycloheximide and puromycin inhibit DNA synthesis, more time may have been available for the limited excision-repair processes in these cells to remove UV dimers or other photoproducts before misreplication of unexcised lesions could lead to increased mutagenesis. These results bear some similarities to the decrease in UV-mutagenesis observed in density-inhibited human fibroblasts. Therefore, it was of interest to determine whether replication-inhibiting conditions would reduce UV-mutagenesis in V79 cells. Since V79 cells exhibit poor contact inhibition, a different protocol involving the use of hydroxyurea to block post-irradiation DNA replication was used. Under the experimental conditions of density-inhibition used in mammalian recovery experiments, the time periods reported for recovery from potentially lethal damage are inaccurate because they do not take into account the time necessary for
495
the cells to re-attach and enter S phase after they are replated at lower density. In order to avoid this problem, our replication-inhibiting protocol involves the initial synchronization of V79 cells at the G 1 / S boundary so that the cells will enter S phase immediately upon removal of the block. After synchronization, the cells are UV-irradiated and DNA replication is subsequently blocked by hydroxyurea for increasing periods of time to determine the effects of post-irradiation inhibition of DNA synthesis on the UV-mutation frequency. Various protocols have been described for the synchronization of mammalian cells (reviewed by Mitchison, 1971; Stubblefield, 1968). Some of these include: the use of a double thymidine block (Xeros, 1962; Bostock et al., 1971); collection of cells in mitosis by mitotic shake-off (Terasima and Tolmach, 1961, 1963; Peterson et al., 1968) or by centrifugal elutriation (Meistrich et al., 1977a, b; Gohde et al., 1979); and the use of inhibitors of DNA synthesis such as hydroxyurea (Erickson, 1966; Sinclair, 1965, 1967; Meyn et al., 1973). The synchronization technique needed for these experiments must be relatively non-toxic, non-mutagenic and able to yield large quantities of cells needed for mutagenesis. Synchronization by mitotic shake-off or centrifugal elutriation provide excellent degrees of synchrony but require specialized equipment to obtain synchronized cells in high enough yield to perform mutagenesis analysis (Burki and Aebersold, 1978; Keng et al., 1980). The double thymidine block and hydroxyurea techniques provide synchronized cells in high yield. However, it has been demonstrated that treatments with concentrations of thymidine used in synchronization protocols were mutagenic to both Chinese hamster ovary and V79 cells, whereas similar treatments with synchronizing concentrations of hydroxyurea were not mutagenic but produced a large amount of toxicity (Bradley and Sharkey, 1978; Huberman and Heidelberger, 1972; Stone-Wolff and Rossman, 1981b). Reported here is a synchronization technique using a low concentration of hydroxyurea which meets the criteria of being mildly toxic, non-mutagenic and able to produce at least partially synchronized cells at high yield. The optimal conditions of this technique and the effects of DNA replication-inhibiting conditions on the UV-induced mutation frequency of V79 cells are described.
Materials and methods Cell culture
A Chinese hamster cell line, V79, strain number 743-3-6, was obtained from Dr. Dennis Yep, University of Michigan. Cells were grown in Ham's F12 medium (GIBCO) containing 5% bobby calf serum (GIBCO), 50 #g/ml gentamicin (Schering Corp.) and 2 # g / m l fungizone (Squibb), at 37°C in an atmosphere of 5% CO 2. The cells were recloned in our laboratory, and a clone which had a low spontaneous mutation frequency was isolated and stored frozen in liquid nitrogen. New batches of cells are thawed every 6-8 weeks to insure low spontaneous mutation frequencies.
496
Animal cell mutagenesis under replication-inhibiting conditions Animal cell mutagenesis experiments were performed using ouabain-resistance as a genetic marker as described by Chang et al. (1978). Survival dishes were seeded at a density of 300 cells/60-mm dish in triplicate. Mutagenesis dishes were seeded at a density of 4 × 105 cells/100-mm dish, and 10 dishes per condition were used. 4h after attachment, the cells were synchronized by incubation in 0.2 mM hydroxyurea (HU). After an incubation period of 16 h, the HU was removed and the cells were washed and immediately irradiated in 5 ml of Earle's balanced salt solution (EBSS) with a G1578 germicidal lamp at a distance of 50 cm, resulting in a fluence rate of 0.295 J/mZ/sec. Immediately following irradiation, growth medium with or without 0.2 mM HU was added to the cells for increasing durations as described in the protocols accompanying Table 1. The HU was removed and the ceils were washed with EBSS and replaced with fresh growth medium for an expression period of 48 h, at which time, the growth medium was replaced by selective medium containing 1 mM ouabain. Survival dishes were stained after 7 days, and mutagenesis dishes after 14 days, with a medium change after day 7. The mutation frequency was calculated by dividing the number of ouabain-resistant clones by the number of surviving clones, calculated from the survival dishes. Measurement of DNA synthesis Cells were seeded at a density of 105 cells/60-mm dish. After attachment, medium with or without 0.2 mM HU was added for different periods of time as described in the protocols accompanying the figures. The cells were pulse-labeled for I h in medium containing [Me-3H]thymidine (New England Nuclear, spec. act., 55.2 Ci/mmole) which was used at a final concentration of 5/iCi/ml, either in the presence of HU or after its removal. The cells were lysed by the addition of 0.2 ml of a 0.5% solution of sodium dodecyl sulfate (SDS) in EBSS, and the lysate was transferred into a tube containing 1.3 ml of EBSS. An equal volume of cold 10% TCA was added to precipitate the nucleic acids and proteins. Following a 15-rain incubation period in an ice bath, the TCA precipitates were collected on 0.45-/t Millipore filters, washed, dried, immersed in butyl PPD toluene scintillation mixture (New England Nuclear) and counted on a Packard scintillation counter. Mitotic index Cells were seeded at a density of 103 ceUs/25-mm dish. After attachment, growth media with or without hydroxyurea was added for different periods of time. After removal of the HU block, the cells were washed twice with EBSS and replaced with growth media containing colcemid (GIBCO) at a final concentration of 0.5 # g / m l for successive periods of 1 h, after which time, the cells were fixed and stained with Giemsa stain. The mitotic index was determined microscopically and expressed as the number of mitotic figures per 100 cells. Chemicals Hydroxyurea and ouabain were purchased from Sigma Chemical Company, St. Louis, MI.
497 Results
Synchronization of V79 cells with a low concentration of hydroxyurea (HU) Fig. 1 is a dose-response curve representing the effects of 16-h exposure to various concentrations of hydroxyurea on the clonal survival of V79 cells. A 16-h treatment with 2 mM H U was very toxic, yielding a cloning efficiency of only 20% of control cells. This was surprising in light of the fact that many cell synchronization protocols use 2 mM H U as a minimum concentration. A 16-h treatment with 0.2 m M H U was only mildly toxic, yielding a cloning efficiency of 80% of control cells. It was of interest to determine whether this concentration of hydroxyurea could be substituted for the more toxic one in the synchronization technique. Fig. 2 demonstrates that 0.2 m M H U can inhibit up to 95% of D N A synthesis of control cells within the first 4 h. A complete reversal of this inhibition occurs immediately upon removal of the HU. Fig. 3a shows the rate of incorporation of [ 3H]thymidine into cold-acid-precipitable material at 1-h intervals following release from a single 16-h block with 0.2 mM HU. The rate of D N A synthesis increases immediately and reaches a maximum level between 1 and 2 h after removal of the H U block. The length of this S phase is approx. 3.25 h which was estimated by measuring the time interval between the end of the H U block and the point at which the labeling index had fallen to half the maximum (see Fig. 3a). This parasynchronous wave of D N A synthesis is followed by a wave of mitosis which peaks 4-5 h following the removal of the H U block. A second wave of DNA synthesis begins 6 - 7 h after the removal of the H U block. It has been demonstrated that the use of a double block improves the synchrony caused by excess thymidine (Peterson and Anderson, 1964). The rationale is that a single thymidine treatment results in some cells being blocked within S phase (Puck, 1964). If the block is lifted and the cells are resuspended in growth medium for a period of 5 - 6 h (a length of time longer than S phase), the blocked cells will progress through S phase but will be caught at the G 1 / S boundary in the next cycle by a second block of thymidine. It was of interest to see whether a similar double block protocol with H U would improve the synchrony of V79 cells. Cells were treated for 16 h with 0.2 mM hydroxyurea, the H U was removed and the cells were washed and incubated in fresh growth medium for 6 h. A second 16 h treatment with 0.2 mM H U was given and the rate of [3H]thymidine incorporation at 1-h intervals was determined following removal of the second block. The effects of a double H U block on the rates of [3H]thymidine incorporation and mitotic index are shown in Fig. 3b. The double hydroxyurea block yields similar results with respect to synchrony as those obtained following a single H U block. The rate of DNA synthesis increases immediately and reaches a peak between 1 and 2 h after removal of the second H U block, as was seen after removal of the first H U block. The length of S phase is also the same as after a single H U treatment. A wave of mitosis occurs following S which also reaches a maximum 4-5 h after the removal of the HU. However, the maximum mitotic index was increased from 27% after a single block to 32% after a double block. The second wave of DNA synthesis starts 8-9 h after removal of the second H U block. This is approx. 2 h later than the start of the second cycle after a single
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Fig. 1. Effects of hydroxyurea on clonal survival of V79 cells. Cells were incubated with different concentrations of hydroxyureafor 16 h. The hydroxyureawas then removedand the cells were incubated with growth medium for a period of 1 week or until colonies became macroscopic. Fig. 2. The inhibition and recoveryof DNA synthesis in V79 cell treated with 0.2 mM hydroxyurea.V79 cells (10~ cells/dish) were given 1-h pulses of [3H]thymidinein the presence of 0.2 mM HU (added at time zero and after its removal).
H U block and is in better agreement with published values for the V79 cell cycle (Robbins and Scharff, 1967). Therefore, these differences imply that the double block showed a small improvement in the level of synchrony in V79 cells over the single block. Although the number of cells, concentration of [3H]thymidine, and labeling conditions were the same in both synchronization treatments, the amount of [3 H]thymidine incorporation after the double block equalled approximately one half of the rate of [3H]thymidine incorporation observed following the single block. One possible explanation for this difference is that the d o u b l e block caused increased toxicity in V79 cells. A comparison of the effects of a double verses a single H U block on clonal survival of V79 cells showed that a single 16-h treatment with 0.2 m M H U resulted in a survival of 88% of untreated cells, while a double treatment resulted in 54.4% of control survival. Since the double block showed increased toxicity without significantly improving the level of synchrony in the first S phase, the single 16-h treatment was employed in the following experiments.
Effects of replication-inhibiting conditions on UV-mutagenesis in synchronized V79 cells Following a 16-h treatment with 0.2 mM HU, V79 cells were washed with buffer and UV-irradiated as described in the Methods section. Post-irradiation D N A synthesis was blocked with 0.2 mM H U for different durations from 0 to 10h. Table 1 shows the effects of these 'hquid-holding' conditions on UV-mutagenesis.
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Fig. 3a. Cell synchronization after a single treatment with 0.2 mM hydroxyurea. The effects of a single 16-h block with 0.2 mM HU on DNA synthesis (0) and mitotic index (dk) are demonstrated here. V79 cells were given I-h pulses of [3H]thymidine at various times after the removal of the block. Mitotic indices were calculated each hour after the removal of the HU block as described in the Methods section. Fig. 3b. Cell synchronization after a double treatment with 0.2 mM hydroxyurea. The effects of a double HU block on DNA synthesis (Q) and mitotic index (&) are demonstrated here. V79 cells were given 1-h pulses of [3H]thymidine at various times after the removal of the second 16-h HU block. Mitotic indices were calculated each hour after the removal of the block as described in the Methods section.
Treatments in which cells were allowed to enter S phase immediately after UV (Table 1, 0 h) demonstrated the highest UV-mutation frequencies. Cells whose replication was blocked following UV-irradiation demonstrated gradually decreasing mutation frequencies with respect to the amount of time that DNA synthesis remained inhibited. For example, in Expt. 1, the UV-induced mutation frequency was reduced by almost 50% when the cells were held for 6 h. Therefore, V79 cells are able to demonstrate replication-inhibited recovery from potentially mutagenic UV lesions.
Discussion
Chinese hamster cells were synchronized by a new technique using a low concentration of hydroxyurea which provides a mildly-toxic, non-mutagenic method of producing large quantities of synchronized cells needed for studying mutagenesis. Cells treated with 0.2 mM HU for 16 h demonstrate a parasynchronous wave of DNA synthesis which reaches a maximum between 1 and 2 h after removal of the
500 TABLE ! EFFECTS OF REPLICATION-INHIBITING CONDITIONS ON THE U V - I N D U C E D MUTATION F R E Q U E N C Y IN V79 CELLS a Duration of postirradiation inhibition of DNA synthesis by 0.2 mM H U
Survival (% control)
Total number of Oua r mutants
Mutation frequency (mutants/106 survivors)
Expt. 1 (a) Unirradiated control b (b) Oh (c) 3 h (d) 6 h
100.00 88.00 70.80 67.10
3 183 133 72
1.84 127.97 115.70 66.10
Expt. 2 (a) Unirradiated control b (b) Oh (c) 2 h (d) 4 h (e) 6 h (f) 8 h (g) 10 h
100.00 103.10 107.10 111.40 81.70 72.00 62.90
5 186 153 133 90 70 60
2.80 100.54 79.69 73.89 68.18 60.34 58.82
a All UV-irradiations were performed at 3.5 J/m 2, b Unirradiated controls were blocked for 16 h with 0.2 mM HU, washed with buffer, given a mock UV-irradiation treatment.
block, followed by a wave of mitosis which peaks 4 - 5 h following removal of the H U block. The S period which immediately follows the release from the single H U block in these experiments is shorter than the S period reported by Robbins and Scharff (1967) for the same cell line. This could be the result of incomplete inhibition of D N A synthesis by hydroxyurea. Fig. 3 shows that a small amount of [3H]thymidine incorporation still occurs in the presence of 0.2 mM H U even after a 16-h treatment. Since many of the cells have already synthesized some D N A during the block, completion of D N A synthesis after the block requires less time than a normal S period. Bostock et al. (1971) observed this same phenomenon in Chinese hamster ovary cells synchronized with 2 mM thymidine. A double block with 0.2 mM H U had no effect on this foreshortened S period. The double H U block delayed the onset of the second wave of D N A synthesis by 2 h as compared to the single block. This may be an indication that the double block was more inhibitory with respect to allowing cells to progress through S phase. However, a double block with H U was too toxic for use in these experiments. Although, the synchronization after a 16-h treatment with 0.2 mM H U is only partial, its low toxicity and lack of mutagenicity, makes it a useful technique in cases where large quantities of synchronized cells are needed for mutagenesis analysis. . We previously demonstrated that post-irradiation treatment of V79 cells with the translation inhibitors puromycin or cycloheximide decreased the UV-mutation
501 frequency, and proposed that this decrease was due to their inhibitory effects on DNA synthesis rather than their inhibition of de novo protein synthesis (Stone-Wolff and Rossman, t981a). Inhibition of post-irradiation DNA synthesis may have allowed more time for the limited excision repair processes in these cells to remove dimers or other photoproducts before the misreplication of unexcised photoproducts could lead to increased mutagenesis. These results bear some similarities to the decrease in UV-mutagenesis observed in density-inhibited human fibroblasts, a phenomenon similar to 'liquid-holding recovery' in bacteria. In order to see whether this interpretation of the cycloheximide and puromycin effects was correct, the effects of replication-inhibiting conditions on UV-mutagenesis in Chinese hamster cells were examined. The protocol developed for this study involved UV-irradiation of synchronized V79 cells which were blocked at the G 1 / S boundary. Immediately following UV-irradiation, the cells were either allowed to enter S phase or remained blocked for increasing periods of time by the addition of hydroxyurea. Cells which were allowed to enter S phase immediately following UV-irradiation contained the highest frequencies of ouabain-resistant mutants, while cells whose replication was blocked following UV-irradiation showed decreasing mutation frequencies with respect to time. Therefore, a delay in post-irradiation DNA synthesis by cycloheximide, puromycin or hydroxyurea results in a decrease in the UV-induced mutation frequency. These results imply that the reduction in UV-mutagenesis following replication-inhibiting conditions results from the increased time available for the limited excision-repair processes in these cells to remove UV lesions before the misreplication of unexcised lesions can lead to mutagenesis. It has been demonstrated that complete excision repair of pyrimidine dimers caused by UV light requires at least 24h for human cells (Rauth, 1970; Cleaver, 1974; Regan and Setlow, 1974) and much longer for Chinese hamster cells (Meyn et al., 1974). According to Yarosh and Setlow (1981), V79 cells remove less than 15% of the dimers induced by 10 J / m 2 of UV within 9 h. Our results demonstrating that inhibition of post-irradiation DNA synthesis for a period of 6 h in V79 cells causes a 50% reduction in the mutation frequency induced by 3.5 J / m 2 of UV light, might be interpreted in 2 ways. First, the rate of excision repair in these cells may be faster at non-lethal UV fluences, and may even approach human excision-repair rates. This would imply that the differences between human and rodent excision-repair capacities may be due to different saturation levels rather than to any specific deficiency in the rodent cell excision-repair system. A second interpretation of our data may be that mutagenesis induced by UV light in V79 cells is dependent upon a combination of dimer and non-dimer photoproducts whose composite rates of excision repair approaches a half-time of 6 h.
Acknowledgements This study was funded by Grant No. CA19421 (National Cancer Institute) and is part of Center programs supported by Grant ES-00260 from the National Institute of Environmental Health Sciences and by Grant CA 13343 from the National Cancer Institute.
502
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