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Mechanisms controlling the growth and differentiation of the bone marrow megakaryocyte are poorly understood, and no growth factor has yet made a clinically significant impact on patients with thrombocytopenia. The long- term goal of this research proposal is to gain an understanding of megakaryocytopoiesis that may enable manipulation of megakaryocyte growth in a clinical setting. During the first phase of these studies, leukemic cell lines will be used to study mechanisms controlling proliferation and differentiation of megakaryocytes. In the second phase, normal human stem cells and megakaryocytes will be used to establish the in vivo significance of the observations made in phase l. The distinct capabilities of the three investigators in this research provides a unique opportunity to address these issues. Phase 1, Aim 1. Unlike other known cell lines, c-myc expression increases upon differentiation of megakaryocyte cell lines. The role of c-myc and cyclin expression in the megakaryocyte cell cycle and endoreduplication will be characterized by determining: 1) levels of c-myc or cyclin proteins and mRNAs in megakaryocyte cell lines induced to differentiate, and 2) the effects of altering c-myc or cyclin expression on differentiation. The role of megakaryocyte cell cycle kinases and phosphatases will also be studied. Aim 2. The developmental expression of the earliest known megakaryocyte markers, GPIIb and GPIIIa, will be used as a model for megakaryocyte differentiation. Transcription factors responsible for their expression will be isolated and characterized. Phase 2. Aim 3. Preliminary studies indicate that mobilized peripheral blood stem cells (PBSC) contain a relative increase in megakaryocyte CD34+ progenitors. Highly purified bone marrow megakaryocytes and CD34+ cells from mobilized PBSC will be isolated and used to analyze the differential expression of c-myc, cyclins, megakaryocyte-specific kinases and phosphatases, and GPIIb/GPIIIa transcription factors. The ability of nuclear extracts from subsets of these progenitors to interact with regulatory regions of the GPIIb and GPIIIa genes will also be studied.
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