Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes α and θ
Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes α and θ
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DOI：
10.1046/j.1432-1327.2001.02326.x
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发表时间：
2001-08-01
期刊：
影响因子：
--
通讯作者：
Gant, TW
中科院分区：
文献类型：
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作者：
Gill, PK;Gescher, A;Gant, TW
在获得多药耐药（MDR）表型的细胞和组织中，蛋白激酶C （PKC）同工酶α和θ的水平升高与MDR1基因表达一起发生。使用PKC激活剂或针对PKC的反义策略的研究表明，PKC的激活会导致MDR1基因的转录。本研究探讨了pkc - α和PKC-theta在MDR1基因转录调控中的潜在作用。人源性MCF-7乳腺癌细胞缺乏可检测水平的pkc - α或PKC-theta的组成表达，转染由蜕皮激素启动子驱动的全长pkc - α或PKC-theta基因。通过使用合适的抗生素选择稳定的转染物。用ponasterone A处理这些细胞诱导PKC表达具有催化活性，并在暴露于12- o -十四烷醇-13-phorbol乙酸酯（TPA）时发生易位和下调。这些细胞通过进一步瞬时转染1073 bp的MDR1基因启动子或将其删除至-8 bp的片段来分析PKC介导的MDR1启动子调控，每个片段都与氯霉素乙酰转移酶（CAT）报告基因相关。在表达pkc - α的细胞中，TPA激活所有启动子片段至- 29bp。这一发现表明，TPA诱导的MDR1转录通过TPA应答因子早期生长应答1 （EGR-1）在启动子的这一区域介导，可能是由于pkc - α的激活。相比之下，PKC-theta只激活了两个MDR1片段，-982和-612 bp。PKC抑制剂GF 109203X可减弱TPA对报告基因表达的影响。这些数据表明MDR1启动子转录可由pkc - α和PKC-theta调控。这些结果支持寻找针对PKC-theta的治疗策略，以改善肿瘤对细胞毒性药物的耐药性。
Increased levels of the protein kinase C (PKC) isoenzymes alpha and theta occur in conjunction with MDR1 gene expression in cells and tissues that have acquired a multidrug resistance (MDR) phenotype. Studies using PKC activators or antisense strategies against PKC suggest that activation of PKC engenders MDR1 gene transcription. In this study the potential roles of PKC-alpha and PKC-theta in MDR1 gene transcriptional regulation were explored. Human-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-alpha or PKC-theta at detectable levels were transfected with full-length PKC-alpha or PKC-theta genes driven by the ecdysone promoter. Stable transfectants were selected by use of the appropriate antibiotics. Treatment of these cells with ponasterone A induced expression of PKC that was catalytically active and underwent translocation and downregulation on exposure to 12-O-tetradecanoyl-13-phorbol acetate (TPA), These cells were used to analyse PKC-mediated regulation of the MDR1 promoter by further transient transfection with either 1073 bp of the MDR1 gene promoter or deletion fragments thereof to -8 bp, each linked to a chloramphenicol acetyl transferase (CAT) reporter gene. In PKC-alpha expressing cells TPA caused activation of all promoter fragments to -29 bp. This finding suggests that TPA-inducible MDR1 transcription mediated through the TPA responsive factor early growth response 1 (EGR-1) in this region of the promoter may be due to activation of PKC-alpha. In contrast, PKC-theta activated only two MDR1 fragments, -982 and -612 bp. The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X. These data suggest that MDR1 promoter transcription can be regulated by PKC-alpha and PKC-theta. The results support the search for therapeutic strategies directed specifically against PKC-theta to ameliorate resistance of tumours against cytotoxic agents.