COMPARISONS OF THE FREQUENCIES AND MOLECULAR-SPECTRA OF HPRT MUTANTS WHEN HUMAN CANCER-CELLS WERE X-IRRADIATED DURING G(1)-PHASE OR S-PHASE

In an attempt to elucidate mechanisms underlying the variation in radi osensitivity during the cell cycle, mutations in the HPRT gene were se lected with 6-thioguanine, quantified and characterized in synchronous human bladder carcinoma cells (EJ30-15) that were irradiated in G(1) or S phase with 3 or 6 Gy. Synchronous cells were obtained by mitotic selection, with similar to 98% of the cells in G(1) phase when they we re irradiated after 3 h of incubation, and 75% in S phase when they we re irradiated after 14 h of incubation. The mutant frequencies were si milar to 4-fold higher (P < 0.01) when cells were irradiated in G(1) p hase compared with S phase, and the lowest frequency (1.5 X 10(-5) for 3 Gy during S phase) was similar to 10-fold higher than the spontaneo us frequency. Exon analysis by multiplex polymerase chain reaction was performed on DNA isolated from each independent mutant. The different types of mutants were categorized as class 1, which consisted of base -pair changes or small deletions less than 20 bp; class 2, which consi sted of deletions greater than 20 bp but with one or more HPRT exons p resent; and class 3, which consisted of deletions encompassing the ent ire HPRT gene and usually genomic markers located 350-750 kbp from the 5' end of the gene and/or 300-1400 kbp from the 3' end. A ''hotspot'' for class 2 deletions was observed between exons 6 and 9 (P < 0.01). For cells irradiated during G(1) phase, the percentages for the differ ent classes (total of 78 mutants) were similar for 3 and 6 Gy, with a selective induction of class 3 mutants (34-38%) compared with spontane ous mutants (3%, total 20). When S-phase cells were irradiated with 3 Gy, there were fewer class 1 mutants (21%, total 37) than when cells w ere irradiated in G(1) phase with 3 Gy (45%, total 42) (P < 0.01). The greatest change was observed when the dose was increased in S phase f rom 3 Gy to 6 Gy (total of 43 mutants), with the frequency of class 2 mutants decreasing dramatically from 30% to 1% (P < 0.005). A similar decrease in class 2 mutants with an increase in dose has been observed by others in asynchronous cultures of normal human fibroblasts. We hy pothesize that these differences occur because: (a) there is more erro r-free repair of double-strand breaks (DSBs) during S than G(1) phase; (b) a single DSB within the HPRT gene causes a class 2 mutation or a certain percentage of class 1 mutations, while two DSBs, with one in e ach similar to 1-Mbp region 5' and 3' of the gene, cause a class 3 mut ation; and (c) a repair process that is induced when the dose during S phase is increased from 3 to 6 Gy results in a preferential decrease in class 2 mutations. (C) 1997 by Radiation Research Society.