The objective of this research is to enhance understanding of the regulation of erthropoiesis. We have developed a culture system in which the addition of the Friend polycythemia virus to murine marrow cells in vitro leads to erythroid bursts in 5 days. A variant virus (FVA) produces bursts in vitro, but these have a marked deficiency of hemoglobin and other late maturation proteins. When erythropoietin (Ep) is added to FVA bursts, it initiates synthesis of hemoglobin and the other proteins. The FVA erythroid bursts (FVA-cells) can be plucked from the cultures and provide highly enriched (90%) Ep-responsive cells with which to study the action of Ep. We now propose to enhance harvest of FVA-cells by completing application of mechanical procedures including buoyant density sedimentation, adherence to plastic, growth of FVA-cells in low calcium medium to retard leukocyte growth and """"""""panning"""""""" using lgG attachment to Fc-receptors and monoclonal antibody to granulocytes-monocytes. FVA-cells are arrested in growth after 5 days in vitro. Velocity sedimentation at unit gravity will be used to separate these cells on the basis of size which is directly related to cell cycle. The stages and length of cell cycle will be measured using 3H thymidine and flow cytometry. The effect of EP on the initiation of cell cycle, duration of each phase of the cell cycle and number of cell cycles will be determined. The precise time at which Ep initiates the onset of hemoglobin synthesis and cellular 59Fe uptake in FVA-cells and the effect of EP on the number, affinity, endocytosis, and intracellular pools of transferrin receptors will be assessed, as well as the effect on the ferritin-59Fe pool, ferritin synthesis and turnover. The effect of EP on the number and affinity of Beta-adrenergic receptors of FVA-cells also will be measured. Once the onset of these markers of Ep effect have been mapped, their appearance will be studied in relation to the different phases of cell cycle of synchronized FVA-cells. By administering short limited pulses of EP we will determine if the hormone is predominantly effective during one phase of the cell cycle. The effect of cell cycle on the time of onset of expression of these hormone induced events as well as on their magnitude will be studied. Finally, the effect of EP on protein kinase activity of FVA-cells will be measured. These studies will enhance our knowledge of the events that are controlled by EP and the relation of this control to the stages of cell cycle.
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