Reversion in Chinese hamster lines amplified at the AMPD2 locus: spontaneous and benzamide-stimulated gradual loss of amplified alleles of marker genes

Abstract

The HC47 and HC474 cell lines of Chinese hamster fibroblasts resist coformycin through the intrachromosomal amplification of the AMP deaminase 2 (AMPD2) gene. Due to the coamplification of a μ glutathione S-transferase (GST) gene, these mutant lines are more sensitive than GMA32 wild-type parental cells to buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis. This property was exploited to select revertants of amplification from HC474 cells. Reversion in that line is frequently a gradual process that does not involve extrachromosomal intermediates. The terminal products of this process are commonly cells with a complete deletion of the amplified allele of marker genes and are therefore haploid for these loci on the homologous chromosome. Exposing HC474 cells to benzamide (BA), an inhibitor of polyADP-ribosylation, increased the recovery of revertants to an extent allowing the detection of reverting cells without BSO selection. This effect of BA was used to isolate revertant cells from the HC47 line that is extremely stable and to demonstrate that the mechanism of gradual reversion also occurs in this line. The gradual deletion of amplified copies within the chromosomes suggests that breakage-fusion-bridge (BFB) cycles drive this process.