Molecular markers of ionizing radiation-induced gene mutations in mammalian cells.

ABSTRACT

We have isolated independent Chinese hamster ovary (CHO) cell mutants at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus from untreated, 60Co gamma-ray-exposed, and 212Bi alpha-exposed cells and identified the molecular changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Both the parental CHO-K1 cells and the X-ray-sensitive mutant xrs-5 cells were studied. The radiosensitive xrs-5 cells are defective in DNA double-strand break rejoining ability and in V(D)J recombination, which can be complemented by Ku protein. Of the 71 spontaneous CHO-K1 hprt mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of one to eight exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a high deletion spectrum with 45% of the 20 mutants analyzed showing a loss of one to eight exons and 30% showing total deletion. Exposure to an equitoxic dose of alpha radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the gamma-ray spectrum in that 75% of the 49 mutants analyzed were deletions. To alpha radiation, however, tended to produce larger intragenic deletions than gamma radiation. Of the 92 spontaneous xrs-5 mutants analyzed for deletions, 43% showed a loss of one to eight exons and 14% showed total deletion. This suggests that, in certain regions of the hprt gene, base alterations can be converted into large deletions and alteration in the Ku protein complex can influence this type of mutational process. Exposure to alpha radiation (10% survival) to xrs-5 cells resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed on change in exon number or size, 16% showed a loss of one to eight exons, and 41% showed total deletion. While the defect in xrs-5 cells has a profound effect on spontaneous mutant spectra, this defect does not appear to affect alpha-induced mutation spectra.