Regulation of p21 induction by butyrate in colonic cells

描述（由申请人提供）： 丁酸盐通常在结肠腔中通过微生物发酵产生 膳食纤维和未消化的淀粉。丁酸盐减小了尺寸， 在动物模型中结肠肿瘤的发病率，并影响培养的结肠 腺癌细胞通过抑制生长和刺激凋亡， 分化更好地理解丁酸盐 达到其效果可能会导致更有效地利用膳食 预防做法、治疗干预措施和治疗战略。在 这个建议，我们追求我们最近的观察，丁酸盐引起快速 p21 mRNA持续升高。已知p21影响细胞周期， 通过抑制激酶生长。我们假设细胞生长抑制 丁酸盐的作用是由于p21的诱导，我们假设这是由于p21的诱导。 通过组蛋白乙酰化诱导的染色质变化介导诱导 结构，并通过磷酸化。为了证实初步调查结果， 将结肠细胞系（p53野生型和突变型）暴露于丁酸盐。 将测量细胞生长和p21 mRNA水平。利用瞬态 转染后，我们将确定丁酸盐是否通过快速转染增加p21 mRNA， 刺激p21启动子。我们还将确定是否增加 p21 mRNA也导致p21蛋白的快速增加。以确定是否 丁酸改变了p21周围染色质的构型 启动子“开放”构型，DNaseI超敏位点将被 沿着包括p21并位于p21侧翼的11 kb基因组DNA片段定位沿着。我们 我希望发现丁酸盐增加了这些位点对DNaseI的敏感性 乳沟在我们的最终目标中，我们将确定磷酸化是否参与 丁酸盐诱导的结肠腺癌细胞基因转录。 磷酸酶和激酶抑制剂的初步研究将在 培养和p21瞬时转染的细胞。这些研究将提供新的 知识他们还将提供数据，用于支持 后续R01应用。

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.