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.
Evans, H. H., Evans, T. E. and Horng, M. F. Antimutagenicity of WR-1065 in L5178Y Cells Exposed to Accelerated 56Fe Ions. Radiat. Res. 158, 110–114 (2002).The ability of the aminothiol WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] to protect L5178Y (LY) cells against the cytotoxic and mutagenic effects of exposure to accelerated 56Fe ions (1.08 GeV/nucleon) was determined. It was found that while WR-1065 reduced the mutagenicity in both cell lines when it was present during the irradiation, the addition of WR-1065 after the exposure had no effect on the mutagenicity of the radiation in either cell line. No marked protection against the cytotoxic effects of exposure to 56Fe ions was provided by WR-1065 when added either during or after irradiation in either cell line. We reported previously that WR-1065 protected the LY-S1 and LY-SR1 cell lines against both the cytotoxicity and mutagenicity of X radiation when present during exposure, but that its protection when administered after exposure was limited to the mutagenic effects in the radiation-hypersensitive cell line, LY-S1. The results indicate that the mechanisms involved differ in the protection against cytotoxic compared to mutagenic effects and in the protection against damage caused by accelerated 56Fe ions compared to X radiation.
Abstract— Two closely related strains of mouse lymphoma L5178Y cells, LY‐R and LY‐S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY‐R is more sensitive to photodynamic cell killing than strain LY‐S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single‐strand breaks (which includes frank single‐strand breaks and alkali‐labile lesions), but substantially more DNA‐protein cross‐links were formed in strain LY‐R by CAPC and light. Repair of single‐strand breaks proceeded with similar kinetics in both strains for the first 30 min post‐irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY‐R. DNA degradation, as measured by the conversion of prelabled [ 14 C] DNA to acid‐soluble radioactivity, was more rapid and extensive in strain LY‐R.
Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (porfimer sodium) (PF) or aluminum phthalocyanine (AIPc) 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‐radia‐tion 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 faiences/ 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.
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.
Cultures of radioresistant (LY-R) and radiosensitive (LY-S) strains of L5178Y mouse lymphoma cells were exposed continuously to X-rays delivered at dose rates ranging from 0.003 to 0.025 Gy/h for up to 35 days. Populations of both strains proliferated actively during the exposure, but the growth rates were reduced in a dose rate-dependent manner. The reduction of growth rate occurred for strain LY-S earlier during the exposure and at lower dose rates than for strain LY-R. The survival (as measured by colony forming ability) of strain LY-R was affected only slightly at all dose rates applied. For strain LY-S, a decrease in the surviving fraction was observed in the initial part of the exposure. This decrease was followed by a plateau and eventually by an increase, in some cases to values close to the control level. The increase in the surviving fraction indicated that the radioresistance of the exposed LY-S cells had increased. This pattern was particularly clear for dose rates greater than 0.014 Gy/h. The pre-irradiated cells exhibited radioresistance when exposed to acute X-radiation after termination of the chronic exposure. The increase in radiation resistance was stable for at least 70 days after termination of the protracted exposure. These results show that mutagenic and/or selective phenomena leading to an increase in radiation resistance of mammalian cells can be caused by protracted exposures to X-rays at dose rates permitting active proliferation.
Evans, H. H., Horng, M. F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Diverse Delayed Effects in Human Lymphoblastoid Cells Surviving Exposure to High-LET 56Fe Particles or Low-LET 137Cs Gamma Radiation. Radiat. Res. 156, 259–271 (2001).To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to 56Fe particles or 137Cs γ 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 56Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived γ irradiation. The phenotypes of the unstable clones that survived exposure to 56Fe particles were also qualitatively different from those of the clones that survived γ irradiation. A greater percentage (20%) of the unstable clones that survived γ irradiation than those that survived exposure to 56Fe 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 56Fe particles than in those exposed to γ 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.
