The long-term goal of this project is to understand the molecular mechanisms underlying leukemia relapse. There are more than 21,000 deaths to relapsed leukemia each year in the United States, but the molecular basis of this deadly disease is still unknown. Since leukemia relapse is always associated with chemotherapy, and since cells exposed to therapeutic agents usually acquire a mismatch repair (MMR) deficient phenotype, we hypothesize that leukemia relapse in many cases is caused by a small fraction of leukemic cells that have adopted an MMR deficient phenotype during the course of chemotherapy. Our preliminary studies have showed that defects in MMR are indeed associated with cell lines derived from relapsed leukemia and patients with relapsed leukemia. To further test our hypothesis, this application proposes both genetic and biochemical approaches to analyze the relationship between relapsed leukemia and MMR proficiency. First, real-time PCR will be used to analyze the status of microsatellite sequences in adult acute leukemia patient samples at presentation, remission, and relapse to dynamically and quantitatively determine how leukemic cells transform from a microsatellite stable form to a microsatellite instable form during the development of relapse. Second, leukemic cells from relapsed patients will be analyzed for genetic alterations and epigenetic modifications in key MMR genes such as hMSH2 and hMLH1 using combination technologies of PCR-based single strand conformation polymorphism, DNA sequencing, and methylation-specific PCR. Third, individual alterations identified in leukemia patients will be introduced into MMR genes to express recombinant mutant proteins. To determine the actual impact of these alterations on MMR function, individual recombinant mutant proteins will be examined for their ability to restore MMR to the corresponding MMR mutant extracts using an in vitro functional MMR assay. This study will provide significant insight into the mechanism underlying leukemia relapse as well as vital information for designing therapeutic regimens for the disease.
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