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

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 microM genistein, respectively, were selected and designated CEM/GN50 and CEM/GN150. 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 cross-resistant 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 cross-resistant 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 approximately 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 beta 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.