MOLECULAR MECHANISM OF CHEMOPREVENTION BY APIGENIN

描述：这是一个竞争性续订申请，用于调查 芹菜素参与皮肤癌化学预防的分子机制 抑制紫外线诱导皮肤的无毒和非诱变的生物素 致癌作用。前一 在资助期间，申请人研究了芹菜素治疗对 p53肿瘤抑制基因在小鼠角质形成细胞中的表达。在 申请人证明，具有野生型p53状态的角质形成细胞系 芹菜素在提高野生型p53的水平方面非常有效， 角质形成细胞中的蛋白质（27倍）。这种p53诱导水平是 显著高于UVB照射在角质形成细胞中诱导的， 示例（5倍）。申请人进一步证明， p53蛋白表达的增加是由于蛋白质稳定，同时伴随着p53蛋白表达的增加。 p53在Ser15的磷酸化和随后的转录激活， p53应答基因包括p21WAF1。有趣的是，申请人没有 观察到MDM2蛋白在芹菜素处理的细胞中的任何增加的积累， 这是出乎意料的，因为MDM2基因是 p53转录激活和一般负责反馈 通过泛素化促进p53降解来抑制p53。的 申请人的结果表明，芹菜素处理角质形成细胞诱导 p53通路中的许多相同事件通常在 细胞UV DNA损伤反应，除了阴性 反馈MDM2控制回路似乎不存在。缺乏反馈 MDM2的抑制可能延长p53蛋白的有益作用 稳定芹菜素处理的角质形成细胞。待检验的假设 这种更新的应用是， 从其增强正常细胞p53通路对 紫外线诱导的角质形成细胞损伤。申请人提出了四个具体目标 为了验证这一假设：（1）研究芹菜素 治疗诱导p53的翻译后修饰，通过鉴定p53 磷酸化位点，参与磷酸化的激酶，以及是否 芹菜素诱导角质形成细胞中的p53乙酰化;（2）表征p53 由芹菜素单独处理诱导的蛋白质翻译后修饰， 研究芹菜素处理加UVB照射对 p53蛋白水平、稳定性和翻译后修饰;（3） 研究芹菜素处理对MDM2基因表达的影响， p53和MDM2蛋白的相互作用，以及芹菜素处理是否导致 抑制MDM2介导的p53泛素化和降解;（4）研究 芹菜素处理与UVB照射联合作用对MDM2基因的影响 表达、MDM2/p53蛋白相互作用和MDM2/p53蛋白相互作用，以及 MDM2介导的p53泛素化。

DESCRIPTION: This is a competitive renewal application to investigate the molecular mechanisms involved in skin cancer chemoprevention by apigenin, a nontoxic and nonmutagenic bioflavonoid which inhibits UV-induced skin carcinogenesis when topically applied to mouse skin. During the previous funding period, the applicant investigated the effect of apigenin treatment on expression of the p53 tumor suppressor gene in mouse keratinocytes. In keratinocyte cell lines with wildtype p53 status, the applicant demonstrated that apigenin is extremely potent in elevating the level of wildtype p53 protein in keratinocytes (27-fold). This level of p53 induction is substantially higher than that induced in keratinocytes by UVB irradiation, for example (5-fold). The applicant further demonstrated that the increased level of p53 protein was due to protein stabilization, accompanied by increased phosphorylation of p53 at Ser15 and subsequent transcriptional activation of p53-responsive genes including p21WAF1. Interestingly, the applicant did not observe any increased accumulation of MDM2 protein in apigenin-treated cells, which is unexpected considering that the MDM2 gene is a downstream target of p53 transcriptional activation and generally responsible for feedback inhibition of p53 by promoting its degradation through ubiquitination. The applicant's results indicate that apigenin treatment of keratinocytes induces many of the same events in the p53 pathway that are normally triggered during the cellular UV DNA damage response, with the exception that the negative feedback MDM2 control loop appears to be absent. The lack of feedback inhibition by MDM2 may prolong the beneficial effects of p53 protein stabilization in apigenin-treated keratinocytes. The hypothesis to be tested in this renewal application is that apigenin's chemopreventive activity is derived from its ability to enhance the response of the normal cellular p53 pathway to UV-induced damage in keratinocytes. The applicant proposes four specific aims to test this hypothesis: (1) Investigate the mechanism(s) by which apigenin treatment induces posttranslational modification of p53, by identifying the p53 phosphorylation sites, the kinases involved in phosphorylation, and whether apigenin induces p53 acetylation in keratinocytes; (2) Having characterized p53 protein post-translational modification induced by apigenin treatment alone, investigate the combined effects of apigenin treatment plus UVB irradiation on p53 protein levels, stabilization, and post-translational modification; (3) Investigate the impact of apigenin treatment on MDM2 gene expression, on interaction of p53 and MDM2 protein, and whether apigenin treatment results in inhibition of MDM2-mediated p53 ubiquitination and degradation; (4) Investigate the combined effects of apigenin treatment plus UVB irradiation on MDM2 gene expression, MDM2/p53 protein interaction, and MDM2/p53 protein interaction, and MDM2-mediated p53 ubiquitination.