Development of specific prevention against DNA damage caused by environment

GADD45基因在紫外线照射下通过P53依赖或非P53依赖的途径被转录激活。为了研究紫外线照射在P53非依赖途径中诱导GADD45的序列，我们在P53失活的细胞系中对融合了荧光素酶报告基因的人GADD45启动子进行了突变分析。我们发现紫外光响应元件参与了OCT-1结合位点相对于转录起始点的-99个碱基。凝胶迁移率改变分析表明，转录因子Oct-1将该元件结合到GADD45基因上，但紫外光照射并未改变延迟条带的强度和迁移率模式。这些结果表明，OCT-1调节元件可能是紫外线辐射激活GADD45启动子的关键元件之一，其途径不依赖于P53。组蛋白脱乙酰酶(HDAC)抑制剂可导致多种肿瘤细胞生长停滞于G1和/或G2/M期、分化和/或凋亡。我们证明，GADD45可导致细胞周期停滞于G2/M期，并参与遗传毒性应激诱导的细胞凋亡，也可由典型的HDAC抑制剂曲古抑素A(TSA)通过其启动子以不依赖于p53的方式诱导。为了确定GADD45启动子的激活机制，我们进行了荧光素酶报告分析和电泳性迁移率改变分析。对GADD45启动子的分析表明，OCT-1和CCAAT位点都是TSA完全激活所需的。我们还发现，转录因子OCT-1和NF-Y特异性地结合到每个位点。我们的结果表明，HDAC抑制剂可以通过其启动子诱导Gadd4S，而不是功能性的P53，OCT-1和NF-Y共同参与了TSA诱导的GADD45启动子的激活。

The gadd45 gene is transcriptionally activated by UV irradiation through p53-dependent or p53-independent pathways. To investigate the sequences involved in induction of gadd45 by UV irradiation in a p53-independent pathway, we performed mutation analyses of the human gadd45 promoter fused to the luciferase reporter gene in cell lines in which p53 was inactivated. We found that the UV-responsive element was involved in the Oct-1 binding site at -99 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that Oct-1, a transcription factor, bound this element on the gadd45 gene, although the intensity and mobility pattern of the retarded bands were not altered by UV irradiation. These results suggest that the Oct-1 regulatory element might be one of the essential elements involved in the activation of the gadd45 promoter by UV irradiation in a p53-indepeadent pathway.Histone deacetylase (HDAC) inhibitors have been revealed to cause growth arrest at G1 and /or G2/M phases, differentiation, and/or apoptosis in a wide variety of tumor cells. We demonstrated that gadd45, which has been shown to cause cell cycle arrest at G2/M phase and take part in genotoxic stress-induced apoptosis, is also induced by a typical HDAC inhibitor, trichostatin A (TSA), through its promoter in a p53-independent manner. To identify the mechanism of activation of the gadd45 promoter, we performed luciferase reporter analyses and elecrophoretic mobility shift assays. Analysis of the gadd45 promoter revealed that both the Oct-1 and CCAAT sites are needed for the full activation by TSA. We also found that transcription factors, Oct-1 and NF-Y, specifically bind to each site. Our results suggest that HDAC inhibitors can induce gadd4S through its promoter without functional p53 and both the Oct-1 and NF-Y concertedly participate in TSA-induced activation of the gadd45 promoter.