Study of differentiation and proliferation of normal and leukemic myeloid hematopoietic cells is my major research. In this arena the proposal focuses on three areas: 1) control of expression of myelopecoxidase (MPO): 2) effects of tumor necrosis factor (TNF) on normal and leukemic hematopoiesis: 3) role of p53 in normal and abnormal hematopoiesis. 1) MPO is an enzyme important in killing microorganisms and is expressed abundantly at only one stage of myeloid differentiation Using a variety of myeloid leukemic cell lines, we shall: A.) examine chromatin structure, methylation pattern, and association with the nuclear matrices of the MPO gene region during myeloid differentiation and correlate the results with MPO expression; B.) identify by CAT and electrophoretic mobility shift assays those DNA sequences that regulate MPO expression; C.) purify and study the biological activities of proteins regulating MPO expression; D.) utilize recombinant MPO/oncogenes in transgenic mice to define the activity of 5' sequences of MPO in different tissues; E.) define the cause of MPO deficiency in patients with hereditary MPO deficiency and in those with myeloid leukemia. 2.) TNF is a monokine which we recently discovered can cause production of granulocyte (G-) and granulocyte-macrophage (GM-) colony stimulating factor (CSF) is mesenchymal cells. This may constitute a major mechanism to control production of granulocytes and macrophages. We will: A) characterize at the cellular and molecular level how TNF stimulates mesenchymal cells to synthesize CSF; B) define the effects of TNF on normal and leukemic hematopoiesis in vivo using both mouse and man. 3.) p53 is nuclear protein associated with cell division and capable of causing cell transformation. We found that p53 is phosphorylated in transformed T-lymphocytes but not in their normal countpart. We shall determine the site of p53 phosphorylation and attempt to characterize its importance. Also, we will define the role of p53 in mediating transcription. In another arena, we discovered that p53 is frequently rearranged in human osteosarcomas; this finding may help explain how osteosarcomas develop. Using human osteosarcomas we shall: A.) Determine the frequency of p53 rearrangement; B.) Define the structure of the p53 rearrangement and look for alterations in gene expression as a consequence of this rearrangement; C.) Develop an in vivo murine model of osteosarcoma to study the rearranged p53.
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