Research Project
3228166
Many polypeptide growth factors (GFs) express erythropoietic activity in culture and possibly in vivo as well. The recent availability of chemically defined media for mammalian cell culture permits evaluation of hormone effects and regulatory mechanisms at cellular and subcellular levels. We will employ a serum "free" system for the culture of human erythroid cells that we have developed in order to: 1. Determine cellular effects of defined GFs. We will determine dose-response relationships for selected peptides in single and double layer cultures under serum deprivation. We will extend our finding that one GF, L-triiodothyronine, stimulates not only protein release but also paracrine (burst promoting activity, BPA) release from cultured lymphocytes. Cumulative protein and BPA release, rates of release and subcellular localization of radiolabeled GF will be defined. Specific intracellular processing pathways will be studied. The relationship of newly synthesized BPA-like molecules to uninduced and mitogen-driven BPA will be defined biochemically and immunologically. 2. Isolate and purify undefined GFs. Soluble BPA released with and without inducting agents will be purified from liquid medium. Column chronmatograpy, lectin affinity chromatograpy and reverse-phase high-performance liquid- chromatography will be used. In addition, anti-BPA IgG will be used for purification by immunoaffinity chromatography. 3. Characterize GF target cells and examine GF binding. We will utilize hybridoma antibodies to selectively "pan" for erythroid progenitors. Together with monoclonal IgG to T- and B-cell populations, they will be used to identify marrow subpopulations that are sensitive or resistant to GF effects. Insulin and insulin-like growth factor binding to panned marrow cells will also be determined. 4. Correlate in vivo and tissue culture responses. We will accure patients with hypoproliferative anemia to determine the predictive value of in vitro androgen sensitivity testing. Studies of plasma and marrow cells from patients with hyperproliferative marrow dyscrasias will explore potential humoral abnormalities involving platelet-derived growth factor (PDGF). These investigations focus on our long term goal of evaluating the physiological relevance of tissue culture findings in health and disease.
