The long-term objective of this research is to characterize the molecular mechanisms involved in the regulation of cellular proliferation and differentiation in erythropoiesis. An established hematopoietic cell line, B6SUt, will be employed to test the hypothesis that the activation of phospholipases A2 and C through a pertussis-sensitive G protein is essential for the signal transduction events in erythropoiesis.
Specific Aim #1 will address the question of whether or not diacylglycerol accumulation in response to erythropoietin occurs in a timedependent manner as well as the source(s) of diacylglycerol in these cells. Since it is expected that phospholipase activation will accompany growth factor stimulation, the goal of Specific Aim #2 is to determine the involvement of guanyl nucleotide regulatory proteins (G proteins) in the mechanism of action of erythropoietin. If, as anticipated, G protein function is necessary for subsequent signal transduction events in erythropoiesis, specific G proteins involved will be determined by means of Western blotting experiments with specific antibodies to G proteins and by means of Northern and slot blotting (messenger RNA).
Specific Aim #3 will test the hypothesis that internalization of receptors for erythropoietin and the growth factor/hormone itself is essential for the proliferation of B6SUT cells in response to erythropoietin. Transcription of the protooncogenes,c-fos, c-jun, and c-myc which is associated with the proliferative response will be determined in nuclei exposed to erythropoietin. Erythropoietin, its receptor and specific isoforms of protein kinase C will be localized by means of immunocytochemical methods and electron microscopy. Alterations of the signal sequence for erythropoietin gene will be carried out to determine whether or not erythropoietin must be in the nucleus in order for proliferation to occur. In addition to their inherent biological relevance the molecular mechanisms elucidated will have long-range implications for our understanding of alterations in signal transduction events which result in the loss of differentiation and unregulated proliferation of cancer cells.
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