MOLECULAR MECHANISM OF CHEMOPREVENTION BY APIGENIN

描述：这是一份竞争续订申请，旨在调查 芹菜素化学预防皮肤癌的分子机制 无毒、无诱变的抑制紫外线皮肤的生物类黄酮 当局部应用于小鼠皮肤时会致癌。在上一次 在资助期内，申请人调查了芹菜素治疗对 P53抑癌基因在小鼠角质形成细胞中的表达。在……里面 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水平方面是极其有效的 角质形成细胞中的蛋白质(27倍)。这种对P53的诱导水平是 大大高于UVB辐射对角质形成细胞的诱导， 示例(5折)。申请人进一步证明，增加的水平 P53蛋白的增加是由于蛋白的稳定，并伴有增加 P53在Ser15位的磷酸化及其随后的转录激活 P53反应基因包括p21WAF1。有趣的是，申请者并没有 观察在芹菜素处理的细胞中MDM2蛋白的任何增加， 这是意想不到的，因为mdm2基因是 P53转录激活，一般负责反馈 通过泛素化促进P53的降解，从而抑制P53。这个 申请人的结果表明，芹菜素治疗角质形成细胞诱导 P53通路中许多相同的事件，通常在 细胞紫外线DNA损伤反应，但阴性的例外 反馈MDM2控制回路似乎不存在。反馈的缺乏 MDM2的抑制可能延长P53蛋白的有益作用 芹菜素对角质形成细胞的稳定化作用。待检验的假说 这一新的应用是芹菜素的化学预防活性派生出来的 从其增强正常细胞P53途径对 紫外线对角质形成细胞的损伤。申请者提出了四个具体目标 为了验证这一假设：(1)研究芹菜素的作用机制(S) 通过识别P53，治疗诱导P53的翻译后修饰 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P53乙酰化；(2)具有P53特征 芹菜素单独处理引起的蛋白质翻译后修饰， 观察芹菜素治疗加中波紫外线照射的联合作用 P53蛋白水平、稳定性和翻译后修饰； 研究芹菜素治疗对MDM2基因表达的影响 P53和MDM2蛋白的相互作用以及芹菜素治疗是否会导致 抑制MDM2介导的p53泛素化和降解；(4)调查 芹菜素联合UVB照射对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 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.