Colon carcinogenesis is a multi-step process dependent upon the accumulation of mutations in tumor suppressors and proto-oncogenes. Recent work indicates that an inherited form of colon cancer (HNPCC) the accumulation of mutations may be driven by the loss of mismatch repair. Cells losing this repair pathway have a distinctive mutator phenotype including microsatellite instability, a high rate of spontaneous mutation, and resistance to DNA alkylating agents. Our understanding of the biological consequences of mismatch repair deficiency stems largely from studies of colon cancer cell lines. However, these lines have many other alterations that could contribute to the mutation phenotype.
In Specific Aims 1 and 2 the hypothesis that mismatch repair deficiency is sufficient for induction of the mutation phenotype seen in these colorectal carcinoma cell lines will be tested.
In Aim 1 the ability of inducible cDNA expression constructs of the mismatch repair genes to correct the mutator phenotype in repair deficient colon cancer cell lines will be investigated. Dominant negative alleles of the mismatch repair genes that can induce a mutator phenotype in repair proficient cell lines will be sought in Aim 2. Expression constructs of mutant forms of these mismatch repair cDNAs will be introduced into repair proficient cell lines (in particular non-tumorigenic cell lines of epithelioid origin) to determine the effects on spontaneous mutation rate, cell cycle checkpoint controls, and apoptosis. The strength of this approach is the use of isogenic repair proficient and deficient cell lines to determine effects on the determinants of genome stability and cell growth.
In Specific Aim 3 the lethal effects of over-expression of the mismatch repair cDNAs will be examined by microinjection of wild type and mutant constructs. This will test the hypothesis that the mismatch repair genes are involved in cell cycle regulation.
In Specific Aim 4 the role of the mismatch repair homologs in the repair process will be determined by the manipulation of these genes in cultured cells. Long term objectives are reflected in Specific Aim 5 where we will determine whether mutations of these genes in cultured cells. Long term objectives are reflected in Specific Aim 5 where we will determine whether mutations of other repair genes contribute to inherited forms of colon cancer. These studies should significantly improve our understanding of the role of mismatch repair deficiency in early events of colon carcinogenesis.
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