Jaroslav Mencl

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.

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.

Molecular analysis of hypoxanthine phosphoribosyltransferase gene deletions induced by α- and X-radiation in human lymphoblastoid cells

Mutations caused by exposure to X-radiation and to radon and its decay products were compared in the hprt gene of a human lymphoblastoid cell line. Thirty-one X-radiation-induced, 29 radon-induced, and 24 spontaneous mutants were recovered from cell cultures under identical conditions except for the exposure to radiation. Seven spontaneous point mutations were recovered and DNA sequenced. These mutations included three C:G-->T:A transitions. These spontaneous point mutations were located in the exon or splice donor regions of five of the nine hprt exons. Four X-radiation-induced and three radon-induced point mutations were also analyzed by DNA sequencing. The frequency of induced mutants at the D0 doses for radon and X-radiation respectively were 5 x 10(-6) and 4.5 x 10(-6). Deletions were the predominant mutations recovered from both radon- and X-irradiated cells. Eighty-one percent of the mutants from X-radiation-treated cultures, 86% of the radon-treated cultures, and 63% of the spontaneous mutants involved deletions. Deletions involving exon and intron DNA, as well as intron DNA alone, were found to inactivate the hprt gene and result in a selectable HPRT- phenotype. Among the deletion mutants, however, only 21% of the spontaneous mutants versus 55% of both the X-radiation- and radon-induced mutants exhibited loss of the entire hprt gene. More X-radiation-induced deletions than radon-induced deletions extended further than 800 bp in the telomeric direction from the hprt gene (six of 17 versus two of 17). The results show that at the human hprt locus of TK-6 cells the predominant kind of mutation indicative of exposure to both high LET alpha-radiation and low LET X-radiation is a large deletion, spanning the entire hemizygous hprt gene and extending into flanking sequences.

CYTOTOXIC AND MUTAGENIC EFFECTS OF THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT IN L5178Y CELLS

Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY‐R, deficient in the excision repair of UV‐induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY‐S, deficienl in the repair of DNA double‐strand breaks, was more sensitive than strain LY‐R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY‐S and LY‐R sub‐strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY‐SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 106 surviving cells. The induced frequency in strain LY‐S1 was much higher than in either tk+l‐heterozygous or ik+10 hemizygous strains of LY‐R. The rate and extent of incorporation of CAPC by the LY‐R strains was somewhat greater than for strain LY‐S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.

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.

In Vitro Exposure of Mammalian Cells to Radon: Dosimetric Considerations

We have developed a model to calculate the dose to the cell nucleus in cells exposed in suspension to radon and/or radon progeny. The model addresses the influence of (1) different radiation qualities and energies in the irradiation milieu; (2) the contribution to dose from radioactivity in the medium surrounding the cell after exposure to the radon gas as well as that from excess radon progeny associated with the cell; (3) the geometry of the cell and of the radiosensitive target, the cell nucleus; (4) the intracellular localization of the radionuclides; (5) attenuation of the alpha particles by the cytoplasm; (6) the radionuclide concentrations in the medium; and (7) the length of exposure. Investigation of the influence of these various parameters was made using an irradiation system in which cells were exposed to 212Bi, which decays to stability with the emission of an alpha particle (either 6.05 or 8.78 MeV). The information from these studies was then used to develop the system further for more complex systems in which 222Rn and its progeny are present. The model takes into account the contribution of dose from different radiation sources using scintillation counts of the medium and the cells, and it is useful for calculations of dose in situations where cells are exposed in suspension culture.

The Influence of Dose Rate on the Lethal and Mutagenic Effects of X-rays in Proliferating L5178Y Cells Differing in Radiation Sensitivity

The lethal and mutagenic effects of ionizing radiation delivered at high (53 Gy/h) and low (0.02 Gy/h) dose rates were measured in two closely related strains of mouse lymphoma L5178Y cells differing in radiation sensitivity (LY-R and LY-S). Strain LY-R was more resistant to the lethal effects of radiation than strain LY-S when exposed at either the high or low dose rate. The survival of strain LY-R was markedly enhanced by the reduction in dose rate. The dose-rate dependence of the survival of strain LY-S was less clear, because of the biphasic nature of its survival curve following low dose-rate radiation. However, if the initial slope of the low dose-rate survival curve is compared to the slope of the high dose-rate survival curve for strain LY-S, only a slight increase in survival at the low dose rate is apparent. Although more sensitive to the lethal effects of radiation, strain LY-S was less mutable at the hypoxanthine/guanine phosphoribosyl transferase locus by both low dose-rate and high dose-rate radiation than strain LY-R. Little dose-rate dependence was exhibited by either strain with regard to the mutagenic effects of radiation. Thus, for strain LY-R, which showed marked dose-rate dependence for survival but not for mutation, the ratio of mutational to lethal lesions was much greater following exposure to low dose-rate than to high dose-rate radiation.

Effects of Low Dose Rate (0·003–0·025 Gy/h) Chronic X-irradiation on Radioresistant and Radiosensitive L5178Y Mouse Lymphoma 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.

Interlaboratory comparison of different alpha-particle and radon sources : cell survival and relative biological effectiveness

Alpha radiation-induced cell killing was determined in four different laboratories in order to: 1) measure interlaboratory variability and 2) compare the effects of radon and radon daughter exposures with the effects of 238Pu (an often-used model for radon exposure). The results suggest that differences in handling from laboratory to laboratory can affect both low and high linear energy transfer responses and should be considered when comparing results from different laboratories.

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.