Genistein resistance in human leukaemic CCRF-CEM cells : selection of a diploid cell line with reduced DNA topoisomerase II β isoform

Abstract Genistein, an isoflavonoid derivative initially described as an in vitro protein tyrosine kinase inhibitor, also inhibits mammalian DNA topoisomerase II both in vitro and in vivo . From a human leukaemic T cell line (CCRF-CEM), two genistein-resistant cell lines, which grow in the presence of 50 and 150 μM genistein, respectively, were selected and designated CEM/GN 50 and CEM/GN 150 . Flow cytometry and karyotype analyses revealed that more than 95% of the parental cells were tetraploid whereas both resistant sublines were essentially diploid and were likely derived from the diploid fraction in the initial population. The CEM/GN cells were 3- to 4-fold resistant to genistein, and highly crossresistant to certain metabolic inhibitors such as cytosine-arabinoside (50-fold) and 5-fluoro-2′-deoxyuridine (5000-fold). This resistance was associated with a markedly decreased uptake of thymidine and a 10-fold reduction in thymidine kinase activity. The CEM/GM cells were also 15- to 30-fold crossresistant to topoisomerase inhibitors (etoposide, m-AMSA, 2-Me-9-OH-ellipticinium). Comparison of topoisomerase II activities in the sensitive and resistant cells showed: (i) an approximatively 2-fold reduced decatenation activity in nuclear extracts from the resistant cells; (ii) an approximate 30% reduction in DNA-protein cross-links in etoposide-treated resistant cells; and (iii) a markedly reduced expression of the topoisomerase II β isoform. These data, consistent with our previous results, indicate that the cytotoxicity of genistein is at least in part related to its capacity to inhibit DNA topoisomerase II.