Down-regulation of topoisomerase IIalpha 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 is the development of resistance, often manifested by reduced drug accumulation or reduced topo IIalpha activity, due to mutant enzyme or the enzyme's decreased expression. To date, little is known of how the topo IIalpha is down-regulated in the resistant cells. In this study, using CEM cells selected for resistance to merbarone, we found that topo IIalpha RNA levels were reduced, compared to the parental cells, and this corresponded to reduced protein levels, whereas there was no significant difference in the RNA stability among these cell lines. Furthermore, we detected a lower level of topo IIalpha promoter activity in these resistant cells compared to the drug-sensitive parents. Thus, the down-regulation of topo IIalpha appeared to occur at the transcriptional level. Nucleotide sequencing of the topo IIalpha promoter regions up to -1200 bp revealed no mutations, suggesting that some trans-acting factors are possibly involved in this down-regulation of topo Ilalpha. In this context, we found by Northern blot analysis that the transcription factor, sp3, was reduced in the drug-resistant cell lines compared to the parental cells. Furthermore, cotransfection experiments revealed that Sp3 induced topo IIalpha promoter activity in a dose-dependent manner in drug-sensitive CEM cells, but its induction of topo IIalpha promoter activity was attenuated in the resistant B12 cells. Our results suggest that down-regulation of Sp3 might contribute to the reduced expression of topo IIalpha in certain drug-resistant tumor cells.