Yan Qing-Wu, Reed Eddie, Zhong Xiao-Song, Thornton Keith, Guo Yi, Yu Jing Jie
Department of Biochemistry and Molecular Pharmacology, and Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, 1610-C Health Sciences South, Morgantown, WV 26506-9300, USA.
Biochem Pharmacol. 2006 Mar 14;71(6):761-71. doi: 10.1016/j.bcp.2005.12.015. Epub 2006 Jan 19.
ERCC1 is a critical gene within the nucleotide excision repair pathway. Overexpression of ERCC1 through promoter-mediating transcriptional regulation is associated with repair of cisplatin-induced DNA damage and clinical resistance to platinum-chemotherapy. Several transcriptional repressors and activators within the 5'-flanking region of the ERCC1 gene may be involved in the up-regulation of this gene. Minimal sequence within the promoter region required for ERCC1 transcription was analyzed by CAT assay and demonstrated that the region of -220 to -110 is essential to constitutive expression of ERCC1 gene in ovarian cancer cell line A2780/CP70. A more forward upstream region seems to be responsible for cisplatin-induced expression. Study of the functional cis-element in this region by electrophoretic mobility shift assay indicates that a MZF1-like site as well as an AP1-like site responded in a time-dependent manner to cisplatin stimulation with altered binding activities. EMSA with MZF1 ZN1-4 consensus oligonucleotides suggests that the MZF1 N-terminal domain of zinc finger cluster may bind to the MZF1-like site of the ERCC1 promoter region. MZF1 mRNA in A2780/CP70 cells decreased upon cisplatin exposure as analyzed by quantitative PCR, suggesting that MZF1 may mediate cisplatin-invoked gene expression in these cells. Overexpression of MZF1 repressed the ERCC1 promoter activity as determined in co-transfection assay, suggesting that MZF1 might be a repressor of ERCC1 transcription upon cisplatin exposure. In summary, our studies revealed a core promoter region and adjacent drug-responsible region within the ERCC1 promoter. The drug-responsible region contains cis-elements of activator, AP1 and repressor, MZF1. In response to cisplatin treatment, decreased MZF1 and increased AP1 binding activities appear to be the leading mechanism of up-regulation of ERCC1 expression. Our findings imply potential therapeutic strategies to antagonize drug resistant mechanisms in treatment of human ovarian cancer.
ERCC1是核苷酸切除修复途径中的一个关键基因。通过启动子介导的转录调控使ERCC1过表达与顺铂诱导的DNA损伤修复及对铂类化疗的临床耐药性相关。ERCC1基因5'侧翼区域内的几种转录抑制因子和激活因子可能参与了该基因的上调。通过CAT分析对ERCC1转录所需启动子区域内的最小序列进行了分析，结果表明 -220至 -110区域对于卵巢癌细胞系A2780/CP70中ERCC1基因的组成型表达至关重要。更靠前的上游区域似乎负责顺铂诱导的表达。通过电泳迁移率变动分析对该区域的功能性顺式元件进行研究表明，一个类MZF1位点以及一个类AP1位点对顺铂刺激呈时间依赖性反应，其结合活性发生改变。用MZF1 ZN1 - 4共有寡核苷酸进行的电泳迁移率变动分析表明，锌指簇的MZF1 N端结构域可能与ERCC1启动子区域的类MZF1位点结合。通过定量PCR分析发现，顺铂处理后A2780/CP70细胞中的MZF1 mRNA减少，这表明MZF1可能介导这些细胞中顺铂引发的基因表达。共转染实验确定，MZF1过表达会抑制ERCC1启动子活性，这表明顺铂处理后MZF1可能是ERCC1转录抑制因子。总之，我们的研究揭示了ERCC1启动子内的一个核心启动子区域和相邻的药物反应区域。药物反应区域包含激活因子、AP1和抑制因子MZF1的顺式元件。顺铂处理后，MZF1减少和AP1结合活性增加似乎是ERCC1表达上调的主要机制。我们的研究结果暗示了在治疗人类卵巢癌中对抗耐药机制的潜在治疗策略。
