EVIDENCE FOR TRANSCRIPTIONAL CONTROL OF HUMAN MDR1 GENE-EXPRESSION BYVERAPAMIL IN MULTIDRUG-RESISTANT LEUKEMIC-CELLS

We investigated the mechanism of verapamil (VRP) effects on mdr1 gene expression in two leukemic multidrug-resistant (MDR) cell lines, K562/ ADR and CEM VLB(100). Exposure to VRP for 24 hr resulted in a decrease in mdr1 mRNA levels that was dose related at concentrations between 1 5 and 50 mu M. The maximal decrease of mdr1 mRNA levels was found to b e g-fold in the K562/ADR cells and 8-fold in the CEM VLB(100) cells. T he effect of VRP on mdr1 mRNA levels was, however, biphasic. At 100 mu M VRP, which strongly inhibited cell proliferation, a 2-fold increase of mdr1 mRNA levels was observed in the K562/ADR cells. To determine whether the decrease of mRNA levels resulted from post-transcriptional mechanisms, mRNA stability was studied after blocking of transcriptio n with actinomycin D in VRP-treated cells and in control cells. This s tudy revealed that mdr1 mRNA was stable in both cell lines and no incr ease in mdr1 mRNA degradation was observed in the 30 mu M VRP-treated cells versus control cells (half-lives of 23 hr versus 14 hr for the K 562/ADR cells and 15.5 hr versus 10.0 hr for the CEM VLB(100) cells). The suggestion of a transcriptional mechanism was confirmed by nuclear run-an assays. A 4-fold decrease in the mdr1 gene transcription rate was observed in the 30 mu M VRP-treated CEM VLB(100) cells. The decrea sed transcription rate could be due to the decrease in mdr1 proximal p romoter activity observed in CEM VLB(100) cells transiently transfecte d with the mdr1 promoter fused to the chloramphenicol acetyltransferas e gene. indeed, after exposure to 30 mu M VRP, chloramphenicol acetylt ransferase activity was decreased by 2-fold. This study reports for th e first time a down-regulation of mdr1 gene transcription by a pharmac ological agent. These results provide further identification of the re gulatory mechanisms involved in the overexpression of mdr1 in MDR cell s and may help in the development of new strategies for MDR reversal.