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 y-ray-exposed, and 212Bi a-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-Kl 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-Kl 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-Kl cells to 6 Gy of y 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 cx radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the tray spectrum in that 75% of the 49 mutants analyzed were deletions. The cx radiation, however, tended to produce larger intragenic deletions than y 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 ax radiation (10% survival) to xrs-5 cells resulted in a deletion spectrum similar to that seen in CHO-Kl cells. Of the 49 mutants analyzed, 43% showed no 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 a-induced mutation spectra. EnvironHealth Perspect 104(Suppl 3): 675-678 (1996)

Characterization of gpt deletion mutations in transgenic Chinese hamster cell lines

“…This differs signifi-G12 cells (Fig. Whereas and in vivo exposures of mammalian cells to these agents [Morgan et al, 1990;Nicklas et al, 1991; Aghamoham-the presence of a secondary selection marker can serve to reduce spontaneous deletion loss of gpt or other trans-madi et al, 1992;Fuscoe et al, 1992;Skandalis et al, 1992;An and Hsie, 1993;Simpson et al, 1993; Kro-genes in some mammalian cell systems [Debenham et al, 1988;Romac et al, 1989;Sockett et al, 1991], the dele-nenberg et al, 1995;Hsie et al, 1996;Suzuki and Hei, 1996;Turker et al, 1997]. The high frequency of deletions induced by these clastogens was not unexpected, since fre-cantly from other transgenic gpt / cells, such as the CHOderived AS52 cells, which are reported to spontaneously quent chromosomal aberrations, rearrangements, and deletions have been noted as a result of numerous in vitro lose the integrated gpt sequences in 55-79% of isolated mutants Stankowski, 1987, 1989].…”