Regulation of p21 induction by butyrate in colonic cells

描述(由申请人提供)： 丁酸通常是通过微生物发酵在结肠腔中产生的。 膳食纤维和未消化的淀粉。丁酸盐减小了尺寸和 动物模型中结肠肿瘤的发生率及其对培养结肠的影响 腺癌细胞通过抑制生长和刺激细胞凋亡 差异化。更好地理解丁酸酯的作用机理 达到它的效果可能会导致更有效地利用饮食 预防做法、治疗干预措施和治疗战略。在……里面 这一提议，我们坚持我们最近的观察结果，丁酸盐导致快速 P21基因表达持续增加。已知p21影响细胞周期，并且 通过抑制酪氨酸酶来生长。我们假设细胞生长抑制 丁酸的作用是由于p21的诱导，我们假设这是 诱导是通过组蛋白乙酰化引起染色质的变化来实现的 结构，并通过磷酸化。为了确认初步发现，有六个人 结肠细胞系(野生型和突变的P53)将暴露于丁酸盐。 将测量细胞生长和p21mRNA水平。使用瞬变 ，我们将确定丁酸盐是否迅速增加p21mrna 刺激p21启动子。我们亦会决定是否会增加 P21基因的表达也导致p21蛋白的快速增加。以确定是否 丁酸改变p21周围染色质的构型 启动子到“开放”的配置，DNaseI超敏部位将 沿着基因组DNA的11kb片段定位，该片段包括p21及其侧翼。我们 预计丁酸盐会增加这些站点对DNaseI的敏感度 乳沟。在我们的最终目标中，我们将确定是否涉及磷酸化 在丁酸诱导的结肠腺癌细胞基因转录中。 将于#年进行磷酸酶和激酶抑制剂的初步研究。 培养p21瞬时转染细胞。这些研究将提供新的 知识。他们还将提供数据，用于支持 跟进R01申请。

DESCRIPTION (provided by applicant): Butyrate is normally produced in the colonic lumen via microbial fermentation of dietary fiber and undigested starch. Butyrate reduces the size and incidence of colonic tumors in animal models, and influences cultured colonic adenocarcinoma cells by inhibiting growth and stimulating apoptosis and differentiation. A better understanding of the mechanism by which butyrate achieves its effects may lead to more effective exploitation of dietary prevention practices, therapeutic interventions and treatment strategies. In this proposal, we pursue our recent observation that butyrate causes a rapid and sustained increase in p21 mRNA. p21 is known to affect the cell cycle and growth by inhibiting kinases. We hypothesize that the cell growth inhibition effects of butyrate are due to p21 induction and we hypothesize that this induction is mediated via histone acetylation induced changes in the chromatin structure, and via phosphorylation. To confirm preliminary findings, six colonic cell lines (p53 wild-type and mutated) will be exposed to butyrate. Cell growth and p21 mRNA levels will be measured. Using transient transfection, we will determine whether butyrate increases p21 mRNA by rapidly stimulating the p21 promoter. We will also determine whether the increase in p21 mRNA results also in rapid increases in p21 protein. To determine whether butyrate changes the configuration of the chromatin surrounding the p21 promoter to an "open" configuration, DNaseI hypersensitivity sites will be mapped along an 11 kb segment of genomic DNA that includes and flanks p21. We expect to find that butyrate increases sensitivity of these sites to DNaseI cleavage. In our final aim, we will determine if phosphorylation is involved in butyrate-induced gene transcription in colonic adenocarcinoma cells. Initial studies with phosphatase and kinase inhibitors will be conducted in cultured and p21 transiently transfected cells. These studies will provide new knowledge. They will also provide data that will be used to support a follow-up R01 application.