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

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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.