Li Q, Zhang L, Tsang B, Gardner K, Bostick-Bruton F, Reed E
Developmental Therapeutics Department, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cell Mol Life Sci. 1999 Mar;55(3):456-66. doi: 10.1007/s000180050302.
ERCC-1 is an essential gene in the nucleotide excision repair pathway, and may be essential for life. However, the mechanism of transcriptional activation and regulation of ERCC-1 gene expression is unclear. We therefore investigated the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of the ERCC-1 gene in A2780/CP70 human ovarian carcinoma cells. TPA induced a four- to sixfold increase in steady-state ERCC-1 messenger RNA (mRNA) levels that was time- and concentration-dependent. Nuclear run-on experiments demonstrated that the rate of transcription of ERCC-1 was approximately 2.8-fold higher in TPA-treated cells than in the controls. TPA stimulation of A2780/CP70 cells also resulted in a rapid but transient induction of c-jun and c-fos as determined by Northern and Western blot analyses, which peaked about 2 h before the peak in ERCC-1 expression. Electrophoretic mobility shift assays of nuclear extracts from TPA-treated cells revealed an increase in DNA-binding activity specific for the AP-1-like binding site in the 5'-flanking region of ERCC-1. c-Jun and c-Fos proteins were confirmed to be the components of the activated AP-1 complex by supershift analysis. The increase in AP-1 activity occurs immediately before the increase in ERCC-1 transcription. The increase in AP-1 DNA-binding activity and the increase in ERCC-1 mRNA expression were prevented by pretreatment with cycloheximide. These data suggest that AP-1 may contribute to the upregulation of ERCC-1 in response to TPA in human ovarian cancer cells.
ERCC-1是核苷酸切除修复途径中的一个必需基因，可能对生命至关重要。然而，ERCC-1基因转录激活和表达调控的机制尚不清楚。因此，我们研究了佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）对A2780/CP70人卵巢癌细胞中ERCC-1基因表达的影响。TPA诱导稳态ERCC-1信使核糖核酸（mRNA）水平增加4至6倍，且呈时间和浓度依赖性。细胞核连续转录实验表明，TPA处理的细胞中ERCC-1的转录速率比对照细胞高约2.8倍。通过Northern和Western印迹分析确定，TPA刺激A2780/CP70细胞还导致c-jun和c-fos快速但短暂的诱导，其峰值出现在ERCC-1表达峰值前约2小时。对TPA处理细胞的核提取物进行电泳迁移率变动分析，结果显示ERCC-1 5'-侧翼区域中AP-1样结合位点的DNA结合活性增加。通过超迁移分析证实c-Jun和c-Fos蛋白是活化的AP-1复合物的组成成分。AP-1活性的增加发生在ERCC-1转录增加之前。用环己酰亚胺预处理可阻止AP-1 DNA结合活性的增加和ERCC-1 mRNA表达的增加。这些数据表明，AP-1可能在人卵巢癌细胞中响应TPA时有助于ERCC-1的上调。
