The experiments described in this proposal are designed to define the role of transcriptional response to hypoxia during tumorigenesis. Our focus is on a principal effector of this response: the hypoxia-activated transcription factor HIF-1, a dimeric transcription factor made up of two basic helix-loop-helix molecules, HIF-1a and ARNT. We have created mutations in mice at the locus encoding the oxygen responsive component of this factor, HIF-1a; we have also derived differentiated cell lines from these mice and transformed them via stable transfection with the activated H-ras oncogene. These reagents and others derived from them will be used in genetic and biochemical studies of how HIF-1 regulates vascular function and energy metabolism, the two most essential and important responses to hypoxia. We will also continue to pursue novel targets of HIF-1 regulation, in order to fully map the response pathway in terms of the effector genes it controls.
Specific aim l: Role of HIF-1 in the control of solid tumor expansion. The response to hypoxia includes growth arrest, apoptosis, and increased expression of angiogenic factors. The two sub-aims of this aim will determine the role of HIF-1a in regulation of p53-induced apoptosis and HIF-1 induced angiogenesis during solid tumor formation.
Specific aim 2 : Role of HIF-1 in regulation of cellular metabolism in transformed cells. Experiments in this aim will dissect the role played by HIF-1 in regulating cellular metabolism in primary, immortalized, and H-ras transformed cells. In sub-aim 2.1 we will analyze the effects of loss of HIF-1a on cell survival in various metabolically challenged states, and determine to what extent these effects are influenced by loss of p53 and transformation by H-ras. In sub-aim 2.2 we will dissect the pathway of insulin/IGF-1 activation of HIF-1, in order to determine if there are common effectors and inhibitors, as well as differential responses acting through HIF-1.
Specific aim 3 : Gene control by HIF-1: discovery and analyses of target genes.
This aim focusses on the identification and characterization of novel targets in the HIF-1-induced and HIF-1-suppressed expression program, using large array EST blots in combination with HIF-1a mutant cell lines.
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