MECHANISMS OF ABERRANT IG EXPRESSION IN CHRONIC LEUKEMIA

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Chronic Lymphocytic leukemia (CLL) is the most prevalent form of leukemia in Western countries, and is characterized by a monoclonal proliferation of primarily mature CD5+B lymphocytes. Surface immuno-globulin (Ig) expression, which is often decreased in CLL, normally requires the protein product of the B29 gene for translocation of the B cell antigen receptor complex (BCR) to the cell surface and for signal transduction. Because B29 is essential for intracellular assembly and transport of the B cell antigen receptor complex to the cell surface, we postulate that a perturbationin the B29 gene could result in diminished expression and function of surface ig in leukemic Cll cells. Our studies have now revealed aberrations in the expression of the B29 gene in CLL. Analysis of 18 unselected cases of CLL demonstrate that over 80% had low to absent B29 expression which correlated directly to their level of surface ig expression. Half of these surface B29low/-cases had either no or barely detectable levels of B29 mRNA by RNAse protection assay. To date, all of the CLL samples with normal B29 mRNA levels have had point mutations or truncations identified by RT-PCR and sequencing which would significantly effect the structure and/or function of B29 protein. The primary objectives of this research proposal ae to define the spectrum of specific aberrations of the B29 gene in CLL, to determine their consequences and then finally to relate these abnormalities to disease pathogenesis. PCR/SSCP analysis of genomic DNA from a larger panel of CLL cases is expected to identify B29 mutations which will be confirmed by direct sequencing. RNAse protection assays and RT-PCR will be used to identify alternative spliced and other B29 mRNA variants. Cotransfection of mutant B29 constructs with othe rBCR component expression vectors should reproduce the defects seen in CLL. Primary CLL cells maintained on a CD40L expressing geeder layer will be infected with a normal B29 vaccinia viral expression vector and assayed for BCR surface expression. BCR signal transduction, cell cycle progression and sensitivity to apoptosis of "corrected" CLL cells will also be determined. From the studies proposed, it will be clearly demonstrated that mutations of directed at correctign these B29 mutations are expected to induce increased ig surface expression in CLL and may improve the sensitivity of CLL to cytotoxic chemotherapy.