In order to identify the biochemical events occurring in immature normal lympho-hematopoietic cells upon exposure to mitogen, it is necessary to analyze subpopulations enriched for lymphohematopoietic blast cells. Several colony-stimulating factors (CSFs) providing mitogenic stimulation to marrow precursor cells have recently been cloned and produced in pure form. In this research project, we will determine the involvement of certain second messenger pathways linking the exposure of marrow blast cells to recombinant CSFs with subsequent initiation of DNA synthesis. The purification of bone marrow blast cells is facilitated by the anti-My-10 monoclonal antibody, supplemented by other antibodies which subdivide the My10+ cell population, and immune-mediated separation. Recently-developed fluorescence imaging technology is used to detect rapid changes in intracellular free calcium concentration at the single cell level. First, the kinetics (lag, time-course) of S phase entry and the CSF dose and exposure-time requirements will be analyzed. Second, the interactions between different CSFs, the specificity of CSFs for subsets of precursor cells defined by the lineage of their progeny, and the early requirement for c-myc expression will be determined. Third, quantitative characterization of changes in intracellular free calcium concentration upon stimulation by rhCSFs, and the relation of such changes to subsequent entry into DNA synthesis, will be studied, again in lineage-defined subsets of the my10+ cell population. Fourth, the involvement of certain phospholipase-dependent pathways in physiologic stimulation of normal marrow blast cells will be defined, specifically, the necessity for metabolism of arachidonate via the lipoxygenase pathway to leukotrienes and for the activation of protein kinase C. At each stage, the relationships between immediate (Ca+2, c- myc), intermediate (DNA synthesis), and ultimate (clonal proliferation) responses of antigenically-defined blast cell subsets to recombinant CSF will be examined. With improved understanding of the mechnmisms governing proliferation in normal immature cells, the disordered proliferation in malignant cells may be clarified.
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| Kastan, M B; Strauss, L C; Civin, C I (1989) The role of hematopoietic growth factors and oncogenes in leukemogenesis. Am J Pediatr Hematol Oncol 11:249-67 |
