Hideyuki Tanimura

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

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 degrees 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 approximately +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.

Involvement of protein kinase in environmental stress-induced activation of human multidrug resistance 1 (MDR1) gene promoter

The human MDR1 gene can be induced in response to various environmental stimuli. To examine whether such stress‐induced activation of the MDR1 gene can be modulated by protein kinase, we employed a stable human cancer KB cell line which contained the bacterial chloramphenicol acetyltransferase (CAT) gene directed by the MDR1 gene promoter. H‐7, a protein kinase C inhibitor, at more than 40 μM inhibited activation of the MDR1 promoter that was induced by ethylmethane sulfonate, 5‐fluorouracil or UV irradiation. DNA binding activity of nuclear factors recognizing the MDR1 promoter was augmented in KB cells treated with UV, but decreased in cells treated concomitantly with H‐7. Okadaic acid alone was able to induce the promoter activation, and this induction was dependent on specific promoter sequences. Okadaic acid also enhanced the DNA binding activity of nuclear factors recognizing the MDR1 promoter. The phosphorylation of transacting factors may modulate the MDR1 gene promoter activity.

The human multidrug resistance 1 promoter has an element that responds to serum starvation

We have previously demonstrated in transient expression assay systems that a human multidrug resistance 1 (MDR1) promoter can be directly activated by cytotoxic anticancer agents. In this study, we examined whether the MDR1 promoter could be regulated in response to growth arrest induced by serum starvation. We have established human and rodent cell lines which stably expressed the chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1, the viral thymidine kinase (TK) and the simian virus 40 (SV40) promoters. Serum starvation caused enhanced expression of CAT gene with MDR1 promoter, but not with two viral gene promoters in human cancer KB cells. Hydroxyurea activated the MDR1 promoter, but not TK and SV40 promoters. By contrast, the DNA topoisomerase II inhibitor, etoposide, equally activated the MDR1, TK and SV 40 promoters. Increased CAT gene expression by serum starvation was also specifically observed in stable transfectants of human adrenal SW-13 cell lines, but not in stable transfectants of mouse fibroblast NIH3T3 and adrenal Y-1 cell lines when the human MDR1 promoter-CAT was introduced. Etoposide, however, effectively induced CAT activity in both human and rodent cells. Assays with deletion constructs of the MDR1 promoter showed that serum starvation activated the MDR1 promoter carrying -258 approximately +121 base sequence of the promoter, but not -198 approximately +121 of the promoter. These results suggest that the expression of the MDR1 gene induced by serum starvation is regulated at the transcriptional level in a promoter sequence-specific manner in human cells.

Reversal by a Dihydropyridine Derivative of Non-P-Glycoprotein-Mediated Multidrug Resistance in Etoposide-Resistant Human Prostatic Cancer Cell Line

PURPOSE We have isolated etoposide-resistant prostatic cancer cell lines, P/VP10 and P/VP20, to investigate the multidrug resistance (MDR) mechanism and to find MDR reversal agents. MATERIALS AND METHODS We examined expression of MDR-related genes and screened reversal agents of MDR in P/VP20 cells. RESULTS These cells demonstrated a non-P-glycoprotein (P-gp)-mediated MDR phenotype with overexpression of MDR-associated protein (MRP) mRNA due to MRP DNA amplification. A 1,4-dihydropyridine derivative, bis(4-pyridylmethyl)4-[2-(3-methyl-5,6- dihydro-1,4-dithiinyl)]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicar boxylate (NIK250), was found to overcome MDR in P/VP20 cells. CONCLUSIONS NIK250 might be useful in reversing MDR, which often develops during chemotherapy of advanced or hormone-resistant prostatic cancer.

Enhanced Expression of the Multidrug Resistance Gene in Vindesine-Resistant Human Esophageal Cancer Cells

We developed and characterized a new series of low- and high-grade multi-drug-resistant (MDR) cell lines of human esophageal carcinoma. Eight vindesine-resistant clones, SH-1-V1, SH-1-V2, SH-1-V3, SH-1-V4, SH-1-V5, SH-1-V6, SH-1-V7, and SH-1-V8 were isolated from the human esophageal cancer cell line, SH-1, by stepwise selection on exposure to increasing doses of vindesine. SH-1-V1 to SH-1-V8 acquired resistance to vindesine, in a stepwise manner, from 3- to 115-fold over findings in the parental SH-1 cells. The most resistant clone, SH-1-V8, was cross-resistant to other anticancer agents such as vincristine, actinomycin D, and daunomycin, thereby suggesting acquisition of the MDR phenotype. In SH-1-V8 cells, cellular accumulation of vincristine decreased and an MDR reversal agent, cepharanthine, potentiated the cytocidal action of vindesine. The expression of MDR1 mRNA was enhanced and amplification of the MDR1 gene was observed in clones SH-1-V4, SH-1-V5, SH-1-V6, SH-1-V7 and SH-1-V8; expression of MDR1 mRNA was detectable without gene amplification in the remaining 3 clones. The enhanced expression of the MDR1 gene may be involved in the acquisition of vindesine resistance in human esophageal cancer cells.