The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. I. Dose response and Southern blot analysis of induced mutants.

The effects of cysteamine and hypoxia on X-ray-induced mutation in human lymphoblast cells have been investigated. Irradiation of these cells under these two sets of radioprotective conditions resulted in a fourfold lower mutant fraction at the hprt locus, compared to irradiation in the absence of any protection. Using Southern blot analysis, the molecular nature of mutants induced in these cells by X rays delivered under hypoxic conditions, or in the presence of 25 mM cysteamine, has been compared with that of mutants induced by an equally mutagenic treatment of X rays alone. Of 60 mutants from cultures treated with X rays alone, 16 exhibited no change in the restriction fragment pattern and were defined as point mutations, 27 were total gene deletions, and 17 were partial deletions or rearrangements. Of 46 mutants from cultures treated with X rays in the presence of 25 mM cysteamine, 22 were point mutations, 18 were total gene deletions, and only 6 were partial deletions or rearrangements. Of 45 mutants from cultures treated with X rays delivered under hypoxic conditions, 17 were point mutations, 14 were total gene deletions, and 14 were partial deletions or rearrangements. Both radioprotective conditions reduced the level of mutation in each mutational class, when compared to the maximum expected in the absence of protection. The spectrum of mutations induced by X rays in cysteamine was significantly different from X rays alone. However, the mutational spectrum of X rays under hypoxic conditions was not different from X rays alone or from X rays in cysteamine. The extents of the deletions induced at the hprt locus were also examined by hybridizing the Southern blots to X-chromosome markers that have been mapped to within 1200 kb of hprt. There were no significant differences among the three groups; however, many mutants were missing one or more of these markers, indicating that deletions of several hundred kilobases are not uncommon.

The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. II. hprt mRNA expression and cDNA sequence analysis of induced mutants.

Mutants at the hprt locus isolated after treatment of cells of the human lymphoblastoid cell line TK6 with X rays were examined by Northern blot and cDNA sequence analysis. In previous work, Southern blot analysis showed that approximately 25% of the mutants isolated from cultures treated with X rays displayed restriction fragment patterns indistinguishable from wild type. In addition, 38 and 48% of the mutants isolated from cultures treated with X rays under two radioprotective conditions, hypoxia or 25 mM cysteamine, respectively, had normal restriction fragment patterns. In the work presented here, Northern blot and DNA sequence analyses were used to characterize these mutants further. Mutants were classified as having normal size and amount of hprt mRNA, reduced amount or undetectable mRNA, or abnormal size message. Mutants that expressed hprt mRNA were sequenced after reverse transcription and PCR amplification. Sequence analysis is reported for 7 mutants from cultures treated in the absence of protection, 11 from cultures treated under hypoxic conditions, and 11 from cultures irradiated in cysteamine. Striking differences among the three treatment groups were not apparent. All types of mutations at both AT and GC base pairs were observed, including transitions, transversions, small deletions, and insertions. Several mutations affected RNA splicing, leading to exon skipping or inclusion of intron sequences in the final message. Approximately half (14/29) of the mutants sequenced had additions or deletions of one to several nucleotides. Also, 3/29 involved tandem DNA base changes (GG-->TT, C-->AA, AA-->CG). These observations are consistent with a mechanism involving the induction of noncoding or synthesis-blocking lesions that result in polymerase slippage or error-prone bypass synthesis. In addition, potential hotspot sites for mutation by X rays were discovered. At one site in exon 3, the same complex mutation, consisting of a G-->T transversion and a nearby six-base deletion, was detected in three independent mutants. Another mutant had a G-->C transversion at the same base, but without the deletion. At another site in exon 8, three mutations occurred at a run of three consecutive cytosines; these included a -C, a -CC, and a C-->AA. Also in exon 8, two mutations (+T, T-->C) occurred at two consecutive thymines.

Mutagenicity of 2-amino-N6-hydroxyadenine to TK6 human lymphoblast cells.

TK6 human lymphoblast cells (tk +/-; hprt+) were treated with various concentrations of 2-amino-N6-hydroxyadenine (AHA) for 24 h. AHA was quite toxic to TK6 cells in the dose range 0-0.05 micrograms/ml, but additional toxicity was not observed between 0.05 and 0.10 micrograms/ml. AHA induced mutations at 2 distinct genetic loci: the autosomal thymidine kinase (tk) and the X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt). Significant levels of both tk-NG mutants (normal growth rate of 16-18 h, colonies visible after 10-11 days incubation) and tk-SG mutants (slow growth rate of greater than 24 h, colonies visible after 18 days incubation) were induced. 15 hprt- mutants were isolated and analyzed by Southern blot. 8 of these had normal restriction fragment patterns after digestion with PstI, EcoRI, and HindIII, and were defined as 'point' mutations; the remaining 7 had partial deletions of the hprt gene. 32 tk- mutants were also isolated. 3 of 22 normal growth mutants and 6 of 10 slow growth mutants had lost the active tk allele. These data suggest that both point mutations and larger-scale alterations are induced by AHA.

The relative radiosensitivity of TK6 and WI-L2-NS lymphoblastoid cells derived from a common source is primarily determined by their p53 mutational status.

The lymphoblastoid cell lines WI-L2-NS and TK6 were derived from a non-clonal pool of cells taken from a human spleen. Despite their common background they exhibit marked differences in radiosensitivities; D0 values of 93 and 67 cGy have been reported for WI-L2-NS and TK6 cells respectively. We show here that this differential radiosensitivity is due to a decreased ability of the WI-L2-NS cell line to undergo radiation-induced apoptosis. Further, the WI-L2-NS cell line overexpresses the p53 gene product as a result of a mutation in codon 237 of the p53 gene. These data indicate that WI-L2-NS cells through disruption of normal p53 function are unable to engage the radiation-induced apoptosis program and so are relatively radioresistant.