Down-Regulation of Topoisomerase ha in CEM Cells Selected for Merbarone Resistance Is Associated with Reduced Expression of 5p3

DNA topoisomeraseII (topo II) is a target for many clinically useful anticancer drugs. However, a major concern in the use of these drugs is the developmentof resistance,often manifestedby reduceddrug accumu lation or reduced topo 1kvactivIty, due to mutant enzyme or the enzyme's decreased expression. To date, little is known of how the topo ha is down-regulated in the resistant cells, In this study, using CEM cells selected for resistance to merbarone, we found that topo 1kv 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 Ha promoteractivity in these resistantcells comparedto the drug-sensitive parents. Thus, the down-regulation oftopo Ha appeared to occur at the transcriptional level. Nucleotide sequencing of the topo lla promoter regions up to â€"1200 bp revealed no mutations, suggesting that some trans-acting factors are possibly involved In this down-regulation of topo 1hz. 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 ha promoter activity In a dose-dependent manner in drug-sensitive CEM cells, but its induction of topo Ha pro moter activity was attenuated in the resistant B12 cells. Our results suggest that down-regulation of Sp3 might contribute to the reduced