Regulation of p21 induction by butyrate in colonic cells

点击翻译按钮获取中文摘要

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.