The overall goal of the proposed investigations is to understand important cellular and molecular mechanisms involved in the humoral regulation of normal and neoplastic hematopoiesis and apply this knowledge to develop new and effective therapies.
The specific aims are to delineate the biology and signaling events involved in colony-stimulating factor (CSF)- enhanced molecular transport in normal and neoplastic blood cells, and to elucidate the biological and structural components important in normal and neoplastic blood cells, and to CSF family of receptors. The hemopoietins to be studied include GC-CSF, IL-3 and IL-5, and the model systems involve Xenopus oocytes, monkey COS cells, human leukemia HL-60 cells, as well as normal human neutrophils and mononuclear phagocytes, and their leukemic HL-60 cells, as well as normal human neutrophils and mononuclear phagocytes and their leukemic counterparts The mechanisms involved in CSF signaling of protein interactions with the receptor alpha subunit, and by structural studies of the GM-CSF receptor. Vitamin C enters in the form of dehydroascorbic acid (DHA) through the facilitative glucose transporters and the CSF regulation of transport, oxidative generation of DHA, and its intracellular reduction will be studied in detail. Vitamin C plays a major role in defense against oxidative stress. Its function in hematopoietic cells will be defined by experiments imposing oxidative stress on hematopoietic cells containing graded concentrations of vitamin C and glutathione with peroxide, irradiation, and certain chemotherapeutic agents. The regions of the alpha subunit necessary for signaling for transport will be determined by expression of appropriate mutants in Xenopus oocytes and COS cells. The alpha subunit intracellular domain binds to free beta subunits in our yeast two-hybrid system. This interaction will be defined, the binding sites characterized, and appropriate mutants constructed and tested. The molecular links between the alpha subunit and hexose and vitamin C transport will be identified. A unique protein isolated using the yeast two-hybrid system (GRAP-1) binds to the alpha subunit and causes glycogen accumulation when disrupted in yeast. The GRAP binding site to the alpha subunit has been determined and mutagenesis of these sites on the alpha subunit and on the GRAP molecule will be used to study the functionality of this interaction. Other molecules interacting with GRAP will be identified and the molecular path in regulation of the glucose transporters dissected. We will determine the biology of alpha subunit-dependent signaling for the GM- CSF family of receptors.
Showing the most recent 10 out of 143 publications
