Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities to the cytotoxic effects of various mutagenic agents-i.e., strain LY-S has been found to be more sensitive, less sensitive, or similarly sensitive to individual agents in comparison to strain LY-R. Nevertheless, strain LY-S has been found to be uniformly less mutable than strain LY-R at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus following treatment with x-radiation, UV radiation, or alkylating agents. In the present work we have isolated subclones of strains LY-R and LY-S that are heterozygous at the thymidine kinase (Tk) locus (chromosome 11). We have found that a heterozygous LY-S Tk+/Tk-strain shows as high or higher mutability at the Tk locus than do heterozygous LY-R strains following treatment with x-radiation, UV radiation, or ethyl methanesulfonate.Mutability of all heterozygous strains at the Tk locus is much higher than at the Hprt locus following treatment with these mutagenic agents, with the exception ofone heterozygous LY-R strain that possesses only one chromosome 11 and that is poorly mutable at the Tk locus by x-radiation. On the basis of these results, we have suggested that (i) because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; (ii) multilocus lesions lead to poor recovery of viable mutants when the target locus is closely linked to essential genes and situated on a hemizygous chromosomal region (e.g., the Hprt locus on the X chromosome or the Tk locus in strains monosomic for chromosome 11); and (iii) x-radiation is a relatively poor mutagen at loci situated on hemizygous chromosomal regions, in repair-efficient and repair-deficient cells, because of its tendency to form multilocus lesions.Mouse lymphoma strain L-5178Y-S (LY-S) was first isolated by Alexander and Mikulski (1) following a spontaneous increase in the x-radiation sensitivity ofL5178Y cells growing in vitro. The parental strain was named L5178Y-R (LY-R) to differentiate it from the newly isolated sensitive strain. In spite of the greater sensitivity of strain LY-S to the cytotoxic effects of x-radiation and alkylating agents (2-5), strain LY-S is less mutable than strain LY-R by UV radiation and x-radiation and by alkylating agents at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) and Na+,K+-ATPase loci (4-6). In the present work we have compared the mutability of the two strains at the thymidine kinase (Tk) locus situated on chromosome 11 (7, 8) using heterozygous Tk+/Tk-strains of LY-R and LY-S. We have found the mutability of the heterozygous LY-S strain (LY-Si) to be as high or higher than that of LY-R heterozygous strains (LY-R16 and LY-R83) at the Tk locus following treatment with x-radiation, UV radiation, or ethyl methanesulfonate (EtMes). Mutant frequencies are much higher at the Tk locus than at the Hprt and Na',K+-ATPase loci for LY-S and LY-R heterozygous strains treated with these age...

The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin (porfimer sodium) (PF) or aluminum phthalocyanine (AlPc) as the photosensitizer was determined at the thymidine kinase (TK) locus in the human lymphoblastic cell lines, TK6 and WTK1, and was compared to the mutagenicity of UVC and X-radiation in these cells as well as the mutagenicity of PDT in murine L5178Y lymphoblastic cell lines. Photodynamic therapy was found not to be mutagenic in TK6 cells, which possess an active p53 gene and which are relatively deficient in recombination and repair of DNA double-strand breaks. In contrast, PDT with either sensitizer was significantly mutagenic in WTK1 cells, which harbor an inactivating mutation in the p53 gene and are relatively efficient in recombination and double-strand break repair as compared to TK6 cells. The induced mutant frequency in WTK1 cells with PF as the photosensitizer was similar to that induced by UVC radiation but lower than that induced by X-radiation at equitoxic fluences/doses. The mutant frequency induced by PDT in WTK1 cells with either photosensitizer was much lower than that induced in murine lymphoblasts at equitoxic fluences. The TK6 and WTK1 cells did not differ in their sensitivity to the cytotoxic effects of PDT, but the level of PDT-induced apoptosis was greater in TK6 than in WTK1 cells. These results indicate that the mutagenicity of PDT varies in different types of cells and may be related to the repair capabilities as well as the p53 status of the cells.

The production and repair of radiation-induced DNA damage were measured by filter elution in strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. The induction of radiation-induced damage, as measured by filter elution at pH 12.1, 9.6, and 7.2, was similar in the resistant-strain LY-R and the sensitive strain LY-S. The repair of single-strand breaks and alkali-labile sites, as measured by filter elution at pH 12.1 at various times after irradiation, was somewhat slower in strain LY-S than in strain LY-R, although after a 20-min repair period the extent of repair was equal in the two strains. However, when filter elution was performed at either pH 9.6 or pH 7.2, the repair of x-radiation-induced damage was much less extensive in strain LY-S than in strain LY-R. We have assumed that the extent of filter elution at pH 9.6 is a measure of the occurrence of frank double-strand breaks as well as closely opposing single-strand breaks and pH 9.6-labile sites (and combinations thereof), and that the extent of elution at pH 7.2 is a measure of the occurrence of frank double-strand breaks alone. If these assumptions are correct, the results suggest that the sensitivity of strain LY-S to the cytotoxic effects of ionizing radiation is caused by a deficiency in the ability of this strain to repair frank double-strand breaks and pH 9.6-labile lesions. The repair of pH 9.6-labile lesions was temperature sensitive in strain LY-S, as previously found for cellular recovery processes in this strain. Two independent radiation-resistant variants of strain LY-S, isolated after protracted exposure of LY-S cells to low-dose-rate radiation, showed a deficiency in the repair of pH 9.6-labile lesions similar to that observed in strain LY-S. However, the repair of frank double-strand breaks was more extensive in the radiation-resistant variants than in strain LY-S and was similar in extent to that occurring in strain LY-R after a 60-min postirradiation incubation. The results suggest that there is a difference In the nature of DNA damage measured by filter elution at pH 9.6 vs. pH 7.2 and that a deficiency in the repair of pH 9.6-labile lesions does not contribute to cell lethality in the case of the radiation-resistant variants. The radiation resistance of these variants in comparison to strain LY-S may be due at least in part to recovery of the ability to rejoin frank DNA double-strand breaks.Mouse lymphoma strain L5178Y-S (LY-S) was isolated in 1961 by Alexander and Mikulski (1) following a spontaneous increase in sensitivity of a culture of LY5178Y cells to the cytotoxic effects of ionizing radiation. The parental culture was designated LY-R to differentiate it from the newly isolated sensitive strain. LY-S cells have been shown to be deficient in cellular recovery processes following exposure to ionizing radiation [reviewed by Beer et al. (2)], particularly when postirradiation incubation was carried out at 370C (3-5). Conflicting reports have appeared concerning the difference in the abi...

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.

TK6, WI-L2, SB and three other B-lymphoblast lines were deficient in the rejoining of DNA double-strand breaks (DSBs) induced by ionizing radiation. Cells of these cell lines rejoin less than 50% of the breaks in 60 min after exposure, as assayed by filter elution at pH 9.6. The deficiency in TK6 cells was confirmed using the comet assay. IN TK6 cells the percentage of DSB rejoining did not vary markedly with dose and was similar for G1, S, and G2 + M-phase cells. Two B-lymphocyte lines (Raji and GM0606), three T-lymphoblast lines (MOLT-4, Jurkat, and CCRF-HSB-2), HL-60 promyelocytes, and GM3440 human skin fibroblasts rejoined more than 50% of the DSBs in this period after exposure. Radiation sensitivity in terms of cell survival was measured in those cells forming colonies. Of the cell lines tested, those that were deficient in DSB rejoining were radiation-sensitive (TK6 and WI-L2: D0 = 0.64 Gy). However, not all lines that were proficient in DSB rejoining were radiation-resistant, since Jurkat and GM0606 cells were relatively radiation-sensitive (D0 = 0.63-0.73 Gy). TK6 and WI-L2 cells were more sensitive to bleomycin (D0 = 8-9 micrograms/ml) than were HL-60 and Raji cells (D0 = 40-54 micrograms/ml). No relationship of DSB rejoining to V(D)J recombinase activity was observed, since no mRNA hybridizing to the cDNA probes for RAG-1 or RAG-2 was detected in any of the cell lines tested.