Diverse delayed effects in human lymphoblastoid cells surviving exposure to high-LET (56)Fe particles or low-LET (137)Cs gamma radiation.

To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to (56)Fe particles or (137)Cs gamma radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na(+)/K(+) ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to (56)Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived gamma irradiation. The phenotypes of the unstable clones that survived exposure to (56)Fe particles were also qualitatively different from those of the clones that survived gamma irradiation. A greater percentage (20%) of the unstable clones that survived gamma irradiation than those that survived exposure to (56)Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to (56)Fe particles than in those exposed to gamma rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.

Differential antimutagenicity of WR-1065 added after irradiation in L5178Y cell lines.

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.

Mutagenicity of photodynamic therapy as compared to UVC and ionizing radiation in human and murine lymphoblast cell lines.

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.

Induction of multilocus mutations at the Tk1 locus after X irradiation of L5178Y cells at different times in the mitotic cycle.

TK1+/- L5178Y-R16 cells were separated into G1, S and G2/M-phase populations by centrifugal elutriation and were treated with 1.5 Gy X radiation. Cells irradiated in the G1 and G2/M phases were most sensitive to the cytotoxic effects of radiation, while cells irradiated in the G2/M phase showed the highest mutant frequency at the thymidine kinase (Tk1) locus. DNA isolated from independent TK1-/- mutants was analyzed for loss of heterozygosity (LOH) at the Tk1 locus and two microsatellites, D11Mit48 and D11Nds7. Homogenates of each mutant were assayed for activity of galactokinase (GLK), the product of the galactokinase (Glk) gene neighboring the Tk1 gene on chromosome 11. Irradiated G1-phase cells had the highest percentage of mutants showing no LOH. The frequency of mutants with LOH at both Tk1 and D11Nds7 with no loss of GLK activity was high in all cell populations: There was no significant difference in the observed frequency of these mutants between the populations. The frequency of mutants losing GLK activity was low, particularly in cells irradiated in the S or G2/M phases. The possibility that the loss of GLK activity is not indicative of LOH at the Glk gene under the conditions of the present experiments is discussed.

Characterization of multilocus lesions in human cells exposed to X radiation and radon.

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK-/-) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone or extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome (Bao et al., Mutat. Res. 326, 1-13, 1995). In the present study, we observed that the percentage of slowly growing and very slowly growing TK-/- mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells.

Cytotoxic and mutagenic effects of radon and radon daughters on murine L5178Y lines differing in DNA repair.

The effects of 222Rn were measured in mouse L5178Y (LY) lymphoblasts that differ in repair capabilities. Line LY-S1 is deficient in the repair of X-radiation-induced DNA doublestrand breaks, while lines LY-R16 and LY-R83 are presumed to be deficient in the excision of UV-radiation-induced pyrimidine dimers. Line LY-R83 is hemizygous while the other two lines are heterozygous at the thymidine kinase (tk) locus. After exposure to radon the D0's were found to be very similar for the three lines (0.30-0.31 Gy), whereas for X radiation the D0 for line LY-S1 is lower (0.7 Gy) than that for the two LY-R lines (1.3 Gy). Mutant frequencies at the tk locus were higher per gray after treatment with radon than X radiation, but at equitoxic doses the mutant frequencies were similar for X and alpha-particle radiation. A low radon-induced mutant frequency was observed for the hemizygous line, in agreement with the hypothesis that multilocus lesions were induced by the alpha-particle radiation and that mutants bearing intergenic lesions were not recovered in the TK+/- line. The entire active tk allele was lost by 81% of the TK-/- mutants of line LY-R16. In lines LY-S1 and LY-R16, 39-43% of the TK-/- mutants exhibited loss of galactokinase activity, indicating that the mutational lesion inactivating the tk gene frequently extended to the neighboring galactokinase gene.

The sensitivity to DNA topoisomerase inhibitors in L5178Y lymphoma strains is not related to a primary defect of DNA topoisomerases.

DNA topoisomerase-targeting antitumor drugs are potent inducers of protein-concealed strand breaks in mammalian cells and act by trapping DNA topoisomerases on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes. It has been proposed that the cleavable complex is an unusual form of DNA damage that elicits cellular responses analogous to those caused by DNA damaging agents. The relationship between topoisomerase-targeting drug-induced damage and radiation-induced damage has been investigated by analyzing the properties of DNA topoisomerases in mouse L5178Y lymphoma strains that are cross-sensitive to topoisomerase I-II inhibitors and to UV light or X-ray irradiation. The strains are LY-R, isolated from L5178Y cells on the basis of increased resistance to ionizing radiation, and strain LY-S, isolated from LY-R cells following a spontaneous increase in the sensitivity to ionizing radiation. LY-S cells, deficient in the rejoining of DNA double-strand breaks, show enhanced sensitivity to topoisomerase II-targeting inhibitors, whereas LY-R cells have an increased sensitivity to UV radiation and to the topoisomerase I inhibitor, camptothecin. The cellular availability of DNA topoisomerase I and II and the sensitivity of the enzymes to their specific inhibitors have been measured in the two related strains. In the LY-R strain, we found a 30% decrease in topoisomerase I content but no difference in camptothecin sensitivity, while no quantitative or qualitative differences were observed for the topoisomerase II. The results indicate that variations in sensitivity of the L5178Y strains to topoisomerase inhibitors are unlikely to be related to primary defects of the target enzymes, and thus it is possible that common pathways exist for processing of topoisomerase- and radiation-induced damage.

DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines.

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.

Induction of multilocus lesions by UVC-radiation in mouse L5178Y lymphoblasts.

The survival, the mutant frequency and the nature of the DNA alteration responsible for the inactivation of the thymidine kinase (tk) locus were investigated in 5 strains of mouse L5178Y lymphoblasts exposed to UVC radiation. The nature of the DNA alteration was investigated in independent TK-/- mutants using Southern blot analysis. The concomitant loss of galactokinase (GK) activity in homogenates of individual TK-/- mutants was taken as an indication that the lesion inactivating the tk allele extended to the neighboring galactokinase (gk) allele. The survival of strains LY-R16 and LY-R83 was decreased to a greater extent than that of strains LY-S1, LY-SR1, and LY-3.7.2C, reflecting a deficiency in excision repair in strains derived from LY-R cells. The TK-/- mutant frequency of strain LY-R83, which is monosomic for chromosome 11 and thus hemizygous for the tk and gk genes, was only 50% of the mutant frequency of strain LY-R16 which is heterozygous for the tk gene. Moreover, a greatly reduced percentage of individual spontaneous and UVC-induced TK-/- mutants of strain LY-R83 showed loss of GK activity in comparison to the other strains. This result indicates that UVC irradiation induces intergenic mutations and that such mutants are poorly recovered in the hemizygous strain. Strain LY-3.7.2C appears to have only one active galactokinase (gk) allele, and very few TK-/- mutants of this strain showed loss of GK activity, possibly because this strain, although heterozyogous for the tk gene, is hemizygous in the region of the gk gene. Strains LY-R16 and LY-S1 are deficient in the repair of UVC- and X-radiation-induced damage, respectively, and the percentage of TK-/- mutants with intergenic mutations was higher for strain LY-R16 after UVC-radiation and for strain LY-S1 after X-radiation. These results indicate that unrepaired DNA lesions lead to an increase in intergenic mutations.

Mutagenicity of 2-amino-N6-hydroxyadenine at the tk locus in L5178Y strains differing in repair capabilities and karyotype.

The cytotoxicity and mutagenicity of 2-amino-N6-hydroxyadenine (AHA) were measured in strains of L5178Y differing in repair capabilities and karyotype. Strain LY-R83 is monosomic for chromosome 11 and is therefore hemizygous for the tk gene, while strains LY-R16 and LY-S1 are TK+/- heterozygotes. Both strain LY-R83 and LY-R16 are sensitive to UV light and are presumed to be deficient in the excision of pyrimidine dimers as shown for the parental strain, LY-R (Hagen et al., 1988; Szumiel et al., 1988). Strain LY-S1 is sensitive to the cytotoxic effects of ionizing radiation and is presumed to be defective in the repair of radiation-induced DNA double-strand breaks, as shown for the parental strain, LY-S (Evans et al., 1987a; Wlodek and Hittelman, 1987). The sensitivities of the three strains to the cytotoxic effects of AHA were similar. After a 4-hour treatment with AHA at 37 degrees C, the D37 for all three strains was approximately 35 ng/ml. The AHA-induced mutant frequency was similar for the hemizygous TK+ strain LY-R83 and the heterozygous TK +/- strain LY-R16, but was slightly higher for strain LY-S1 than for either LY-R strain at an AHA concentration of 100 ng/ml. The proportion of AHA-induced LY-S1 TK -/- mutants forming colonies with diameters less than 0.3 mm was much lower than following treatment with X radiation (24% vs. 61% for AHA and X radiation, respectively). These results indicate that the vast majority of AHA-induced TK -/- mutants harbor single gene mutations. AHA did not result in cyanide-insensitive oxygen uptake, and treatment with this compound did not induce a significant number of DNA single-strand breaks, DNA alkali labile lesions, or DNA degradation in either strain. However, two hours after AHA removal, DNA single-strand breaks and/or alkali-labile lesions, possibly due to the occurrence of DNA repair, were apparent in the DNA of both strain LY-R16 and strain LY-S1.

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells.

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.

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains.

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.

Deficiency in DNA repair in mouse lymphoma strain L5178Y-S.

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.

Locus specificity in the mutability of L5178Y mouse lymphoma cells: the role of multilocus lesions.

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 of one 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 because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; 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 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.