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Photoreactivation of DNA synthesis inhibition in human cells
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breaks. The relation of the induction of chromatid aberrations to DNA replication and recombination could thereby be explained. T h i s p a p e r d e s c r i b e s w o r k c a r r i e d o u t in t h i s l a b o r a t o r y b y D. S c u d i e r o , K. K a t o a n d P. H i g g i n s a n d supported by grants from the National Institutes of Health (GM 07S16, CA 14599-03) and ERDA (E ( I i - I ) - 2 0 4 0 ,
76 B.M. Sutherland and R. Oliver, Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 (U.S.A.) Photoreactivation o f DNA synthesis inhibition in human cells
Although human cells contain photoreactivating enzyme (Sutherland, Nature, 248 (1974) 109) and can photoreactivate dimers in their DNA (Sutherland et al., Proc. Natl. Acad. Sci. U.S. 72 (1975) 103; Sutherland et al., Biochemistry, 15 (1976) 402), the biological effects of such cellular dimer photoreactivation have n o t been evaluated. We have thus examined photoreactivation of DNA synthesis in cultured human cells. Cells of the Xeroderma pigementosum line XP12BE (CRL1223, Jay Tim) grown in Dulbecco's modified Eagle's medium (SutlYerland and Oliver, submitted to Biochim. Biophys. Acta, 1976) were transferred to 35-mm culture dishes and grown for 24 h. The medium was removed, cells washed with phosphate-buffered saline (PBS) and 1 ml of PBS was added to each dish. After UV (254 nm) and/or photoreactivating light (100 W yellow bug foiler lamp) exposures, cells were treated as follows. (1) High UV exposure experiments: PBS was removed and replaced with Dulbecco's labeling medium containing 2 pCi/ ml [3H]thymidine; (2) low UV exposure experiments: some samples were labeled immediately as in procedure (1), others were treated by removing the PBS, adding 4 ml Dulbecco's medium and growing for 24, 48 or 72 h, then labeling. In all cases samples were taken after 0 . 2 , 4 or 6 h labeling by pouring off the labeling medium, washing once with PBS, and squirting on ice cold acid pyrophosphate solution. After at least 15 min incubation on ice, cells were scraped from the dish, which was washed with an aliquot of acid pyrophosphate, and filtered onto a GF/C filter in an ice cold filter block. Filters were washed with ice-cold ethanol, dried and counted in a liquid scintillation counter. In the first series of experiments, high UV exposures (54 J/m 2) and long photoreactivation times (1 h), incorporation was carried o u t immediately after light exposure. In spite of detrimental effects of the photoreactiving treatments even on unirradiated cells (5132 cpm incorporated/2 h vs 6704 for controls), some photoreactivation of DNA synthesis was observed (2263 cpm for UV only; 2828 for UV plus PR). We have also examined photoreactivation of DNA synthesis inhibition produced by low UV exposures (1.5 J/m2). Since previous data indicated that 10 min of photoreactivating light exposure was sufficient to remove most photoreactivable dimers from cellular DNA (Sutherland et al., Proc. Nat. Acad. Sci. U.S., 72 (1975) 103; Sutherland et al., Biochemistry 15 (1976) 402), even
160 short PR exposures might give significant biological photoreactivation. We thus examined DNA synthesis capacity in the following groups: no treatment, UV only, UV followed by 10 min PR, PR only, PR followed by UV. If DNA synthesis was examined immediately after UV and/or PR treatment, no differences could be discerned among the five groups. After 24-h growth, however, the UV samples and the samples exposed to PR followed by UV were able to incorporate only 2262 and 2247 cpm, respectively, into their DNA in 4 h. The samples exposed to PR only incorporated 3278 cpm, those Untreated incorporated 3568 cpm and those exposed to UV and then PR incorporated 3298 cpm. Thus, the sample given photoreactivating light after UV could synthesize DNA as well as t h a t exposed to PR only; and almost as well as those which were untreated. These results indicate that dimer photoreactivating activity of human cells can, in addition to its well-known "in vitro" effects, significantly improve the recovery of living cells from exposure to UV irradiation.
77 H. Takebe, Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Osaka (Japan)
Decreased host-cell reactivation of UV-irradiated Herpes Simplex virus and amount of excision repair in Xeroderma pigmentosum The a m o u n t of unscheduled DNA synthesis (UDS) after UV irradiation has generally been used as an indicator of the level of DNA repair in mammalian cells. Among 44 Xeroderma pigmentosum (XP) patients in Japan examined for UDS activities in their cells, there were 9 cases in which levels of UDS were between 70 and 100% of the normal level. We have tested 7 cell lines from these patients for the host-cell reactivation (HCR) activities of UV-irradiated Herpes simplex virus. All of them showed decreased activity of HCR, Do being between 13 and 18 J/m 2, while Do of normal cells was 38 J/m ~ and those of XP's with lower UDS were between 4 and 7.5 J/m 2. One of them was identified as belonging to complementation group E (by Tanaka), and two other cell lines were identified as " v a r i a n t " (by Fujiwara). Measurement of excision of pyrimidine dimers in 2 cell lines among them showed slight reduction in the excision in comparison with the normal cells in one cell line with 70% UDS level, and normal level of excision in the other cell line with normal UDS level. These results support Day's finding with adenovirus and suggest that HCR may be used as the decisive tool of diagnosis of XP. The majority of XP patients in Japan so far examined for DNA repair are, however, patients with little or no UDS activity and very low HCR activity in their cells.
breaks. The relation of the induction of chromatid aberrations to DNA replication and recombination could thereby be explained. T h i s p a p e r d e s c r i b e s w o r k c a r r i e d o u t in t h i s l a b o r a t o r y b y D. S c u d i e r o , K. K a t o a n d P. H i g g i n s a n d supported by grants from the National Institutes of Health (GM 07S16, CA 14599-03) and ERDA (E ( I i - I ) - 2 0 4 0 ,
76 B.M. Sutherland and R. Oliver, Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717 (U.S.A.) Photoreactivation o f DNA synthesis inhibition in human cells
Although human cells contain photoreactivating enzyme (Sutherland, Nature, 248 (1974) 109) and can photoreactivate dimers in their DNA (Sutherland et al., Proc. Natl. Acad. Sci. U.S. 72 (1975) 103; Sutherland et al., Biochemistry, 15 (1976) 402), the biological effects of such cellular dimer photoreactivation have n o t been evaluated. We have thus examined photoreactivation of DNA synthesis in cultured human cells. Cells of the Xeroderma pigementosum line XP12BE (CRL1223, Jay Tim) grown in Dulbecco's modified Eagle's medium (SutlYerland and Oliver, submitted to Biochim. Biophys. Acta, 1976) were transferred to 35-mm culture dishes and grown for 24 h. The medium was removed, cells washed with phosphate-buffered saline (PBS) and 1 ml of PBS was added to each dish. After UV (254 nm) and/or photoreactivating light (100 W yellow bug foiler lamp) exposures, cells were treated as follows. (1) High UV exposure experiments: PBS was removed and replaced with Dulbecco's labeling medium containing 2 pCi/ ml [3H]thymidine; (2) low UV exposure experiments: some samples were labeled immediately as in procedure (1), others were treated by removing the PBS, adding 4 ml Dulbecco's medium and growing for 24, 48 or 72 h, then labeling. In all cases samples were taken after 0 . 2 , 4 or 6 h labeling by pouring off the labeling medium, washing once with PBS, and squirting on ice cold acid pyrophosphate solution. After at least 15 min incubation on ice, cells were scraped from the dish, which was washed with an aliquot of acid pyrophosphate, and filtered onto a GF/C filter in an ice cold filter block. Filters were washed with ice-cold ethanol, dried and counted in a liquid scintillation counter. In the first series of experiments, high UV exposures (54 J/m 2) and long photoreactivation times (1 h), incorporation was carried o u t immediately after light exposure. In spite of detrimental effects of the photoreactiving treatments even on unirradiated cells (5132 cpm incorporated/2 h vs 6704 for controls), some photoreactivation of DNA synthesis was observed (2263 cpm for UV only; 2828 for UV plus PR). We have also examined photoreactivation of DNA synthesis inhibition produced by low UV exposures (1.5 J/m2). Since previous data indicated that 10 min of photoreactivating light exposure was sufficient to remove most photoreactivable dimers from cellular DNA (Sutherland et al., Proc. Nat. Acad. Sci. U.S., 72 (1975) 103; Sutherland et al., Biochemistry 15 (1976) 402), even
160 short PR exposures might give significant biological photoreactivation. We thus examined DNA synthesis capacity in the following groups: no treatment, UV only, UV followed by 10 min PR, PR only, PR followed by UV. If DNA synthesis was examined immediately after UV and/or PR treatment, no differences could be discerned among the five groups. After 24-h growth, however, the UV samples and the samples exposed to PR followed by UV were able to incorporate only 2262 and 2247 cpm, respectively, into their DNA in 4 h. The samples exposed to PR only incorporated 3278 cpm, those Untreated incorporated 3568 cpm and those exposed to UV and then PR incorporated 3298 cpm. Thus, the sample given photoreactivating light after UV could synthesize DNA as well as t h a t exposed to PR only; and almost as well as those which were untreated. These results indicate that dimer photoreactivating activity of human cells can, in addition to its well-known "in vitro" effects, significantly improve the recovery of living cells from exposure to UV irradiation.
77 H. Takebe, Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Osaka (Japan)
Decreased host-cell reactivation of UV-irradiated Herpes Simplex virus and amount of excision repair in Xeroderma pigmentosum The a m o u n t of unscheduled DNA synthesis (UDS) after UV irradiation has generally been used as an indicator of the level of DNA repair in mammalian cells. Among 44 Xeroderma pigmentosum (XP) patients in Japan examined for UDS activities in their cells, there were 9 cases in which levels of UDS were between 70 and 100% of the normal level. We have tested 7 cell lines from these patients for the host-cell reactivation (HCR) activities of UV-irradiated Herpes simplex virus. All of them showed decreased activity of HCR, Do being between 13 and 18 J/m 2, while Do of normal cells was 38 J/m ~ and those of XP's with lower UDS were between 4 and 7.5 J/m 2. One of them was identified as belonging to complementation group E (by Tanaka), and two other cell lines were identified as " v a r i a n t " (by Fujiwara). Measurement of excision of pyrimidine dimers in 2 cell lines among them showed slight reduction in the excision in comparison with the normal cells in one cell line with 70% UDS level, and normal level of excision in the other cell line with normal UDS level. These results support Day's finding with adenovirus and suggest that HCR may be used as the decisive tool of diagnosis of XP. The majority of XP patients in Japan so far examined for DNA repair are, however, patients with little or no UDS activity and very low HCR activity in their cells.