Experimental Models for Understanding the Role of PUMA During Hypoxia

Abstract

This is Emily Backues presenting her poster at URCAS 2005 to Dr. Robert Strandburg, Dean of Academic Affairs at Rhodes College. Gerard Zambetti and John Jeffers also worked on the project. Hypoxia often occurs in the centers of solid tumors due to a lack of sufficient oxygenated blood transported to the tissue. In response to hypoxia, cells express a multitude of genes, some of which eventually destroy the irreversibly damaged cells. p53, a well known tumor suppressor, is accumulated in vivo during hypoxic conditions, and triggers p53 dependent apoptosis. Little is known about p53 targets that are induced by p53 or that mediate p53-dependent apoptosis during hypoxia. Puma (p53 upregulated modulator of apoptosis) is a downstream target of p53 that mediates cell death through both p53-dependent and independent pathways. We sought to investigate the necessity of Puma for an apoptotic response during hypoxic conditions. Initially, untransformed MEF cells (mouse embryonic fibroblasts) were utilized, and exposed to a level of 0.1% oxygen for 6-48 hrs, after which a series of cell viability assays, RT-PCR, and western blots were performed. The resulting ambiguous data, as well as further experimentation on both thymocytes and bone marrow cells, led to a conclusion that oncogenetically transformed MEF cells would be the most suitable model for the hypoxia experimentation. This will facilitate in a more complete understanding of the mechanism by which cell death occurs during hypoxia.