Activation of human multidrug resistance-1 gene promoter in response to heat shock stress

Abstract

The multidrug resistance (MDR1) gene encodes a P-glycoprotein, which catalyzes the energy-dependent efflux of anticancer agents. Various environmental stresses including heat shock can induce the expression of endogenous MDR1 genes. In order to study the regulatory mechanisms of MDR1 gene expression, we have established human cancer KB cell lines which could stably integrate bacterial chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1 promoter. Kst-6 has an integrated plasmid, pMDRCAT1, containing the human MDR1 promoter of −2 kilobases. The MDR1 gene promoter contains a typical heat shock element (HSE) motif located −152 bp to −178 bp from the initiation site. Heat shock at 45°C for 90 min significantly induced CAT activity in Kst-6 cells. Northern blot analysis showed a 4–5 fold increase in CAT mRNA levels in Kst-6 cells. Deletion analysis of the MDR1 promoter demonstrated that the induction of CAT activity was observed in Kxh-14 cells containing a HSE-deleted MDR1 promoter construct, pMDRCAT7. However, further deletion analysis showed that heat shock could not induce CAT activity in Khp-1 cells containing −76 ∼ +121 base sequence of the promoter, suggesting that a new heat shock responsible element was located at between −136 and −76. Gel shift assay showed that the heat shock factor (HSF) could bind to the HSE motif located at −152 bp to −178 bp in the MDR1 promoter. We also found that one distinct DNA-protein complex formed specifically within the MDR1 promoter region −99 to −66 was not significantly increased, but relatively more stabilized under mild denaturing condition in the nuclear extract of heat-shocked cells. In our present assay system, activation of the MDR1 promoter in response to heat shock appears to be mediated through both a new heat shock responsive element and MDR1 specific transcription factor.