Direct correlation between DNA topoisomerase II activity and cytotoxicity in adriamycin-sensitive and -resistant P388 Leukemia cell lines

Abstract

The relationship between DNA topoisomerase II activity and drug resistance was studied in cloned cell lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia; drug resistant P388/ADR/3 (clone 3) and P388/ADR/7 (clone 7) cells are 5- and 10-fold more resistant to ADR than the sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). Topoisomerase II catalytic activity in crude nuclear extracts was reduced in drug-resistant cells as determined qualitatively by decatenation of kDNA. Using the centrifugal method of quantitative analysis, topoisomerase II catalytic activity (mean ± SE) was 81 ± 10 units/mg total nuclear protein in sensitive cells, 29 ± 2 units/mg total nuclear protein in resistant clone 3 cells, and 16 ± 2 units/mg total nuclear protein in resistant clone 7 cells; these differences were highly significant (P < 0.005). Similarly, quantitative analysis of DNA cleavage activity using 3' ³²P-end-labeled pBR322 restriction fragments showed that drug-stimulated topoisomerase II cleavage activity in nuclear extracts from sensitive cells was approximately 1.7- and 2.9-fold greater than that from resistant clone 3 and 7 cells, respectively. Western blot analysis of nuclear extracts from the three cell lines using antibody against the C-terminal half of recombinant-prepared human topoisomerase II polypeptide revealed reduced immunoreactivity of topoisomerase II protein in the drug-resistant cells. These data suggest that reduced topoisomerase II activity in resistant cells, which may represent quantitative reduction of the enzyme, may be another property contributing to multifactorial drug resistance in these cells.