The PI's immediate interest will primarily focus on the molecular, cellular and developmental biology studies of class 1 heparin-binding growth factor (HBGF-1) HBGF-1 is a mitogen for a variety of mesoderm- and neuroectoderm- derived cells in vitro and can induce neovascularization in vivo. It may play a role in tumorigenesis, development and blood vessel homeostasis. Genomic DNA and cDNA clones encoding human HBGF-1 have been isolated in the PI's laboratory. The 5' end(s) of the gene will be characterized by RNase mapping, primer extension and in vitro transcription studies. The promoter/enhancer sequences will be identified by constructing a series of recombinant molecules which will direct the expression of the gene coding for the easily assayed chloramphenicol acetyl transferase in mammalian cells expressing HBGF-1. Better understanding of the gene organization including its enhancer/promoter region will help us understand how this gene is regulated during cellular differentiation and/or malignant transformation. A chromosome deletion was observed in patients of refractory anemia or acute non-lymphocytic leukemia; the distal breakpoint resides at the region where HBGF-1 gene is located. We proposed to identify and to characterize the breakpoint in the abnormal chromosomes of these patients at the molecular level by both pulse-filed gel electrophoresis and long-range DNA cloning. We also propose to study the transforming potential and the functional domains of HBGF-1 in fibroblasts, chromaffin cells and endothelial cells with retroviral expression constructs. Lastly, we propose to study the roles of HBGF-1 during limb regeneration in newts (salamander). Limb regeneration involves dedifferentiation and proliferation of mesenchymal cells at the severed end of the limb, then blastema formation and growth and finally redifferentiation. HBGF-1 is involved in the stimulation of growth during limb regeneration. Thus, we propose to isolate and to characterize the HBGF-1 cDNA and genomic DNA clones from the newt, Notophthalamus viridescens. We'll also isolate the HBGF-1 protein thereof and characterize its mitogenic, chemotactic and receptor-binding activities. The temporal and spatial expression of the HBGF-1 gene in the regenerating newt limb blastema will be studied by in situ hybridization and immunohistochemistry methods. It is envisioned that studying uncontrolled cell growth i mammalian cells expression high levels of HBGF-1 as well as studying controlled cell growth and differentiation in limb regeneration involving HBGF-1 will bring us one step closer toward the control of abnormal cell growth in cancer.
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