Influence of mismatch repair status on the cytotoxicity and mechanism of action of 5-Fluorouracil in colon carcinoma cells

错配修复状态对5-氟尿嘧啶对结肠癌细胞的细胞毒作用及作用机制的影响

• 批准号: 53121559
• 负责人: Professor Dr. Christoph Hanski
• 金额: --
• 依托单位: Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie (CBF)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/53121559?language=en
• 关键词: Influence; mismatch; repair; status; cytotoxicity

Whether mutations in the hMLH1 gene, which is a component of the mismatch repair system, increase or decrease the sensitivity of colon carcinomas to 5-fluorouracil (5-FU) is a matter of controversy. Our preliminary work indicates that hMLH1 mutations have two opposing effects on the response to 5-FU: 1. hMLH1 loss accelerates DNA synthesis whereby precursor incorporation is increased. 2. hMLH1 loss decreases cell death by suppressing removal of incorporated 5-FU. We hypothesize that by separately analysing both influences one can better identify the processes involved and determine their contribution to the overall effect of treatment. We propose to separately analyse the contribution of hMLH1 mutations to cell proliferation and to cell death in p53wt cells, by using an adenovirus-mediated expression of hMLH1 protein and an isogenic cell system (hMLH1+ / hMLH1-). The signaling pathway dependent on hMLH1 and Chk1, which is enhanced after 5-FU treatment, will be analysed in detail. Its dependence on hMLH1 will be additionally validated through a selective knock-down of hMLH1 by and sh-hMLH1-expressing adenovirus or by siRNA. The effect of hMLH1 on the response to 5-FU treatment in vivo will be followed by monitoring the growth of hMLH1+ and hMLH1- xenografts and the analysis of cell proliferation and cell death in tumour tissue. The objective is to clarify the signaling pathways affected only by the hMLH1 protein and its effect on the response to 5-FU in vitro and in vivo.

错配修复系统中的hMLH1基因突变是否会增加或降低结肠癌对5-氟尿嘧啶(5-FU)的敏感性仍存在争议。我们的初步工作表明，hMLH1突变对5-FU的反应有两个相反的影响：1.hMLH1缺失加速了DNA合成，从而增加了前体的掺入。2.hMLH1的缺失通过抑制5-FU的去除来减少细胞死亡。我们假设，通过分别分析这两种影响，可以更好地识别所涉及的过程，并确定它们对整体治疗效果的贡献。我们建议使用腺病毒介导的hMLH1蛋白表达和同基因细胞系统(hMLH1+/hMLH1-)来分别分析hMLH1突变对p53wt细胞增殖和细胞死亡的贡献。依赖于hMLH1和Chk1的信号通路在5-FU处理后被增强，将被详细分析。它对hMLH1的依赖性将通过表达和sh-hMLH1的腺病毒或siRNA选择性地敲除hMLH1来进一步验证。通过监测hMLH1+和hMLH1-异种移植瘤的生长以及分析肿瘤组织中细胞增殖和细胞死亡来观察hMLH1在体内对5-FU治疗反应的影响。目的是阐明仅受hMLH1蛋白影响的信号通路及其在体内外对5-FU反应的影响。