Tumor development and progression are stepwise processes involving the accumulation of multiple genotypic and phenotypic alterations. These alterations include all types of genome rearrangements and point mutations that activate dominant oncogenes or eliminate tumor suppressors. While considerable progress has been made in the identification of genes altered in tumors, little is known of the mutation mechanisms affecting them. It has been argued that the rate of point mutation in tumor cells is too low to account for all mutations involved in tumor development. The goal of the work proposed here is to elucidate mechanisms of hypermutation in human tumor cells and to determine how these contribute to the genetic instability of tumor cells. In particular, mechanisms underlying the production of multiple mutations (in which a single low frequency initiating event apparently results in the production of multiple alterations in a target sequence) and microsatellite instability in human colorectal carcinoma cell lines will be investigated. Dr. Meuth proposes that these forms of hypermutation are the result of error prone DNA synthesis or altered mismatch repair and could significantly contribute to the accumulation of point mutations in tumor cells.
Specific aims are i) to examine the cell and locus specificity of these mutations. if these events contribute to genome instability, then they should occur in a wide range of tumor cells and throughout the genome. The nature and rate of mutation in lines showing multiple mutations will be a particular focus. ii) to determine the structure and origin of multiple mutations. The frequency of multiple mutations relative to single point mutations and the complexity of these events (the distance over which the mutations fall and the frequency of mutations in this tract) will be determined. Such data provide a putative """"""""patch"""""""" size for the error prone synthesis and an in vivo measurement of the fidelity of replication in this patch. The ability of DNA damaging agents to trigger these mutations will be examined. iii) to examine the role of tumor suppressors in these forms of genetic instability. Mutant forms of the tumor suppressor gene p53 will be transfected into colorectal carcinoma cell lines to determine whether they stimulate the production of multiple mutations. and iv) to investigate biochemical mechanisms underlying multiple mutations. Assays to examine the role of replicational fidelity and mismatch repair in the formation of multiple mutations at selectable genetic loci in colorectal carcinoma cell lines will be undertaken. Single strand conformation polymorphism analysis and multiplex DNA sequencing will be used to characterize spontaneous and induced mutations. In vitro assays will be used to examine the integrity and fidelity of DNA replication and mismatch repair.
