Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferas e (hprt) gene and the DNA changes underlying induced mutations were analyse d in Chinese hamster ovary cells. Camptothecin treatments increased hprt mu tations up to 50-fold over the spontaneous levels at highly cytotoxic doses . Genomic DNA was isolated from 6-thioguanine resistant clones and subjecte d to multiplex PCR to screen for gross alterations in the gene structure. T he molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon d eletions. Furthermore, a fraction of the analysed clones showed deletions o f more than one exon that were characterised by the absence of non-contiguo us exons. These data show that single locus mutations induced by camptothec in are characterised by large deletions or complex rearrangements rather th an single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level. (C) 2001 Elsevier Science B.V. All rights reserved.