Gene-specific damage produced by topoisomerase inhibitors in human lung cancer cells and peripheral mononuclear cells as assayed by polymerase chain reaction stop assay

We have peviously reported that the polymerase chain reaction (PCR)-stop as say is an useful technique for investigating gene-specific damage induced b y cisplatin, and that the degree of gene-specific damage sustained by perip heral blood mononuclear cells (MNC) when exposed to cisplatin in vitro pred icts the response to cisplatin-based chemotherapy. In the current study, we investigated whether PCR-stop assay was suitable for investigating gene- s pecific damage induced by the topoisomerase I inhibitor CPT-11 or the topoi somerase II inhibitor VP-16, in the human lung cancer cell lines PC-7 and H 69, respectively. The cells were incubated with CPT-11 or VP16 for 24 hours in vitro and the hypoxanthine-phosphoribosyl transferase gene was amplifie d by PCR. Amplification of a 2.7kb fragment of this gene was clearly inhibi ted by each drug in a dose dependent manner. The concentration which reduce d amplification by 63% (D63, the dose that produces an average of one lesio n per single strand of the 2.7kb fragment), was 318 mu g/ml for PC-7 treate d with CPT-11 and 35 mu g/ml for H69 treated with VP-16. We also used PCR-s top assay to investigate gene-specific damage induced by CPT-11 or VP-16 in adenocarcinoma cells from pleural effusions and MNC from freshly isolated blood obtained from 4 patients with lung cancer. Between-patient variations in the extent of gene-specific damage were observed in both types of cells . These results suggest that PCR-stop assay is suitable for the analysis of interindividual variations in the extent of gene-specific damage induced b y topoisomerase inhibitors in human cells.