The objective of this proposal is to determine how the transcript for vascular endothelial growth factor (VEGF) is transiently stabilized by hypoxia in normal cells and becomes constitutively stabilized in a subset of tumor cells that highly overexpress VEGF. VEGF is a potent angiogenic peptide produced by solid tumors by at least two different mechanisms. First, concentrated zones of VEGF expression appear in hypoxic regions of solid tumors and are implicated in mediating hypoxia-induced angiogenesis. and growth. We find that upregulation of VEGF by hypoxia, TPA, and cyclohexamide is accompanied by a 3- to 6-fold stabilization of the VEGF transcript (from t 1/2 of 26-32 minutes to t 1/2 of 103-158 min), and that VEGF transcripts in human tumor cell lines that overexpress VEGF are constitutively stabilized 3- to 6-fold (1 2 of 113 to 181 min.) and exhibit little or no additional stabilization by hypoxia, TPA, or cycloheximide. This suggests that a normal signal transduction pathway involving an oxygen sensor regulates the stability of VEGF mRNA, and that mutations in this regulatory system cause persistent stabilization in some tumors.
Our specific aims are designed to identify the primary sequences in VEGF mRNA that mediate basal instability and hypoxia-inducible stabilization, and to determine the molecular basis for constitutive stabilization of VEGF transcripts in breast carcinomas and other VEGF overexpressing tumors.
Aim 1 : Clone full-length, normal, 4.2 kb human and chicken VEGF transcripts from hypoxia-induced HBL 100 breast epithelial cells and chicken embryo fibroblasts. Evolutionarily conserved sequences represent potential elements that regulate message stability, and provide a framework for initiating mutagenic studies (Aim 3).
Aim 2 : Determine whether tumor cell lines exhibiting constitutive VEGF mRNA stabilization have mutations in their message stabilization pathways or in their VEGF transcripts. A slightly altered version of the normal human VEGF transcript, which can be distinguished from the endogenous transcript using RNase protection, will be expressed in tumor cell lines exhibiting constitutive VEGF message stabilization Basal and hypoxia- or TPA-induced stability will be measured for both the endogenous and exogenous VEGF mRNA's, using RNAse protection. Lines that confer the stabilized, stimulus-unresponsive half-life on the exogenous transcript would contain stabilization pathway mutations. Those that process the exogenous VEGF mRNA mRNA with a normal half-life would contain VEGF transcript mutations.
Aim 3 : Identify VEGF RNA sequence elements responsible for basal instability and for hypoxia- or TPA-induced message-stabilization. Determine whether these elements mediate constitutive stabilization in tumor cells. Regulatory RNA elements will be mapped using a panel of NIH3T3 clones expressing deleted forms the normal human VEGF mRNA. Inherent stability and hypoxia- or TPA-induced stability will be measured. Elements will be tested for transferable function in rabbit beta-globin fusion transcripts. Altered function of individual elements in tumor cell lines will be examined in the context of chimeric transcripts.
Aim 4 : Begin to identify, purify, clone, sequence, and characterize an RNA-binding protein(s) that mediates basal instability and hypoxia-induced upregulation of VEGF in normal cells, and constitutive upregulation in some solid-tumors. These proteins represent potential targets for anti-angiogenic therapy. Their identification and purification will be based on their binding to 32P-labeled RNA probes in nondenaturing gels and their lack of binding to mutant versions of specific RNA elements that were demonstrated to abrogate function in vivo (in Aim 3).
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