DOWN-REGULATION OF TOPOISOMERASE II-ALPHA IN CEM CELLS SELECTED FOR MERBARONE RESISTANCE IS ASSOCIATED WITH REDUCED EXPRESSION OF SP3

DNA topoisomerase II (topo II) is a target for many clinically useful anticancer drugs. However, a major concern in the use of these drugs i s the development of resistance, often manifested by reduced drug accu mulation or reduced topo II alpha activity, due to mutant enzyme or th e enzyme's decreased expression. To date, little is known of how the t opo II alpha is down-regulated in the resistant cells. In this study, using CEM cells selected for resistance to merbarone, we found that to po II alpha RNA levels were reduced, compared to the parental cells, a nd this corresponded to reduced protein levels, whereas there was no s ignificant difference in the RNA stability among these cell lines. Fur thermore, we detected a lower level of topo II alpha promoter activity in these resistant cells compared to the drug-sensitive parents. Thus , the down-regulation of topo II alpha appeared to occur at the transc riptional level. Nucleotide sequencing of the topo net promoter region s up to -1200 bp revealed no mutations, suggesting that some trans-act ing factors are possibly involved in this down-regulation of topo II a lpha. In this context, we found by Northern blot analysis that the tra nscription factor, sp3, was reduced in the drug-resistant cell lines c ompared to the parental cells. Furthermore, cotransfection experiments revealed that Sp3 induced topo II alpha promoter activity in a dose-d ependent manner in drug-sensitive CEM cells, but its induction of topo Hcv promoter activity was attenuated in the resistant B12 cells. Our results suggest that down-regulation of Sp3 might contribute to the re duced expression of topo II alpha in certain drug-resistant tumor cell s.