Chronic myelogenous leukemia (CML) begins with a more benign chronic phase and then transforms into a fatal accelerated phase or blast crisis. The long range goal of these investigations is to understand the pathogenesis of the disease, hopefully providing new insights into diagnosis and treatment. CML is associated with the Philadelphia (Ph) chromosome, which result from a reciprocal translocation between chromosomes 9 and 22. This translocation generates a fusion gene which includes sequence from the break point cluster region (bcr) on chromosome 22, and sequence from the abl oncogene chromosome 9. This grant will focus on three areas: 1. The structural alterations of the abl gene, and their functional consequences, especially: a. the identification of patients' translocation breakpoints, their cloning and sequencing, and a correlation between the breakpoint location and the expression of the bcr/abl gene; and b. the effect of different recombinations on the transforming ability of the abl gene. 2. The specific DNA sequences which effect the recombination. 3. The structure of the RNA transcripts, and the sequences and proteins which regulate bcr/abl expression, more specifically: a. the structure of the bcr/abl transcript; b. the relationship between cellular proliferation, differentiation, and abl-abc/abl expression; c. the characterization and isolation of DNA binding proteins related to the expression of the bcr/abl gene; and d. the use of anti-sense RNA transcripts to block the bcr/abl expression. The project will analyze samples of peripheral blood from CML patients, and cultured cells. Southern and Northern blotting will be used to examine the DNA and RNA of the abl and bcr/abl genes. Portions of the DNA will be cloned and sequenced to look for important unique sequences or homologies. New combinations of abl, bcr, and non-abl DNA will be made in the laboratory and transfected into tissue culture cells. This will make it possible to observe the effect of new sequence combinations upon bcr/abl expression and transforming ability, and upon the bcr/abl recombination. The SP6 polymerase will be used to generate RNA probes for use in RNAase protection assays of bcr/abl transcription. Potential changes in bcr/abl structure or expression which correlate with the clinical course, particularly progression into blast crisis, will be examined. The investigation of abl and bcr/abl structure and function will hopefully provide some understanding of the leukemic process, and suggest ways to alter its course at a molecular level.
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