Previous work on X-ray-mutagenesis in human cells was done with TK6 lymphoblasts. WTK1 cells, derived from the same male donor, are more resistant to the toxic effects of X-rays, more sensitive to their mutagenic effect, and are better than TK6 at catalyzing intermolecular DNA recombination. Thus a recombinational repair system apparently functions better in WTK1 than in TK6. These cell lines afford an opportunity to evaluate the genetic consequences of recombinational repair. The molecular natures of spontaneous and X-ray-induced mutants from these two lines will be compared at the X-linked hprt locus. Excess mutants in WTK1 may be deletions arising by intrachromosomal recombination, or multiple point mutations resulting from gene conversions from a pseudogene. These possibilities will be distinguished by comparing mutational spectra. Each spectrum will consist of 60 mutants, characterized as to (i) proportion of deletions and their extent within a 2 MB region around hprt, (ii) size and amount of hprt mRNA, and (iii) DNA sequence of point mutants and partial deletion breakpoints. Alternatively, non-homologous recombination between the X-chromosome and autosomal sites could produce hprt mutants. Chromosome painting- fluorescence in situ hybridization (FISH) will be used to determine if more X-ray-induced translocations are induced in WTK1 than in TK6. FISH also will indicate if any individual mutants resulted from a translocation of the X chromosome. TK6 and WTK1 exhibit an 'adaptive response': i.e., a low X-ray dose reduces the mutagenicity of a later one. There is greater protection in TK6, and so adaptation may protect largely against the mutagenic process not mediated by recombinational repair. To investigate this further, the spectra of mutants induced after adaptation will be determined. Dimethylsulfoxide (DMSO) protects TK6 cells from the toxicity of X-rays, but has not effect on the induced mutant frequency. Surprisingly, there is a considerable shift in the mutational spectrum, and it is hypothesized that two competing processes account for this. In WTK1, there is a protective effect against induced mutant frequency,and it is hypothesized that DMSO eliminates mutants arising via recombination. To investigate this further, mutational spectra will be determined after irradiation in DMSO. The final two aims will explore possible reasons for the differential mutagenic response in WTK1 and TK6. The status of p53 will be determined because it is involved in response to DNA damage, and there are differences between WTK1 and TK6 on chromosome 17p. Both lines will be sequenced and if there are different genotypes, a dominant-negative p53 will be transferred into the wild-type cell, and a wild-type p53 into the deficient cell. Resulting effects on radiation toxicity and mutagenicity will be determined. If quantitative differences are seen at the hprt focus in the transfectants, the X-ray-induced mutational spectra will be characterized. Finally, to investigate whether apoptosis is involved in differential mutability in the cell lines. TK6 and WTK1 will be transfected with the bcl-2 gene. If in this way radiation-induced apoptosis can be modulated, then survival and mutational dose-response studies will be conducted and mutational spectra characterized.
