This resubmission of a competing renewal application builds on accomplishments made during prior support and progress made in the past year, dealing with the characterization of two novel polypeptides in acute lymphoblastic leukemia (ALL) that were detected using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). One polypeptide, designated OP18 was identified as a highly conserved novel proliferation regulated cytosolic phosphoprotein that appears to be involved in signal transduction. Unlike other proliferation regulated proteins we have identified, OP18 is overexpressed at the protein and RNA levels in All relative to nonleukemic proliferating lymphoid cells. It is also phosphorylated to a much lesser extent in acute leukemia cells. In this application, we propose to determine the role of OP18 in lymphoid proliferation and to define more precisely the basis for differences in OP18 phosphorylation observed between normal lymphoid cells and acute leukemia, using well defined normal lymphoid cell populations obtained from thymus and bone marrow. Having completed genomic sequencing of Op18, we also propose to study the regulation of Op18 gene expression by identifying cis-acting elements using functional assays; by identifying transcription factors involved in activation of the Op18 gene; and determining which if any may be responsible for its overexpression in leukemia. Three polypeptides (L2,L3,L4) expressed in pre-B ALL, a maturation defined subtype which accounts for most cases of ALL, were identified as the phosphorylated and unphosphorylated forms of a heat shock protein hsp27, not previously known to be constitutively expressed in lymphoid cells outside of the heat shock response. In pre-B ALL, phosphorylation of hsp27 occurs to a variable extent among patients. We have ascertained through follow up that diminished phosphorylation of hsp27 associated with poor prognosis. We propose to study the mechanism of phosphorylation of hsp27; to identify phosphorylation sites; and to generate monoclonal antibodies to hsp27 phosphopeptides to allow studies of hsp27 in bone marrow B lymphoid precursors and to more precisely determine the extent of variability of hsp27 phosphorylation in pre-B ALL. Clinical as well as laboratory features of ALL, which are a part of a leukemia database we have developed, will be investigated to identify which features may correlate with Op18 and hsp27 phosphorylation. The studies proposed are aimed at providing a better understanding of the deregulated cell proliferation in ALL and of the biochemical basis of heterogeneity observed between patients with pre-B ALL.
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