The long-term objective of this project is to understand the basic nature of the regulation of hematopoiesis which in turn should provide a base for specific approaches to marrow failure states, to ameliorating the toxicity of various cytotoxic anti-cancer therapy and to leukemias involving the bone marrow.
The specific aims of this project are to continue to define cytokine production by a specific stromal cell line termed TC-1 evaluating the relationship of a previously described hemolymphopoietic growth factor (HLGF-1) to c-kit ligand, the biology of the high molecular weight forms of CSF-1 and the nature of a possibly unique B cell cytokine. We plan to continue studies on the production of cytokines by murine Dexter stromal cells with an emphasis on the functional significance of low level or subliminal cytokine production and to define the geographic and cellular localization of marrow cytokine production in Dexter stroma and in vivo models. We plan to clone the c-kit ligand from the TC-1 murine marrow stromal line using polymerase chain reaction techniques and to continue biochemical characterization of stromal derived CSF-1 and another possibly unique B cell factor. We will assess production of cytokines by stroma evaluating mRNA by standard Northern blot or utilizing polymerase chain amplification of reverse transcribed DNA as a more sensitive approach to detecting low level mRNA. We will also assess the production of biologically active protein by stromal cells and their subpopulations. We will focus on the biologic importance of low level factor production assessing factor dependent cell lines and purified hematopoietic progenitor/stem cells for their response to stromal derived growth factors either in conditioned media or when grown directly on stroma. We will evaluate the effects of blocking low level factor production with antibodies or antisense constructs. In the latter case, we will add antisense directly to cultures, transvect cultures with retroviral constructs with antisense linked to the metalothionine promoter or create transgenic murine models with growth factor antisense linked to the metalothionine. We will first evaluate interleukin-3, GM-CSF and c-kit ligand in these various models. Lastly, we plan to assess the geographic location of both transplanted stem cells and of cytokine producing cells in in vivo models and in in vitro Dexter culture using in situ hybridization.
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