This five-year project will continue to focus on the molecular and cell biology of the human acidic fibroblast growth factor (aFGF). Acidic FGF, also known as heparin-binding growth factor 1, is the prototype member of the FGF family, and is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells. Remarkably, it is one of the best characterized mitogen for endothelial cells and acts as an angiogenic factor in vivo. Thus, aFGF may play a role in tumorigenesis, blood vessel homeostasis and embryonic development. Using RNase protection analyses with riboprobes derived from four different aFGF cDNA clones isolated in our laboratory, we demonstrate the presence of at least four upstream, untranslated exons which are alternatively spliced to the first protein- coding exon. Expression of these transcripts is regulated in a tissue- and tumor-specific manner. The transcriptional start site of one of the transcripts (aFGF1.A) has been defined by RNase mapping and primer extension analyses. It will be confirmed by 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 luciferase in glioblastoma or kidney carcinoma cells expressing aFGF1.A. The cis-acting sequences will be further defined by gel-retardation and DNase I footprinting analyses. A better understanding of the gene organization including its promoter/enhancer regions will provide insight into how this gene is regulated during cellular differentiation and/or malignant transformation. Additionally, the interaction of the full-length and truncated forms of recombinant aFGF with their receptor(s) in fibroblasts will be studied using cross-linking and Scatchard analysis. Our recombinant cell lines, which are able to discern the mitogenic activity and the induced anchorage-independent growth activity of different forms of aFGF, are vital tools for these studies. The aFGF proteins and their receptors will be localized using immunofluorescence analyses such that the site(s) of action for aFGF can be identified. We will also examine the effects of constitutively-expressed aFGF in pheochromocytoma cells and endothelial cells using retroviral expression constructs. The study of uncontrolled cell growth in mammalian cells expressing high levels of aFGF will bring us closer to the control of abnormal cell growth in cancer.
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