It has been recognized for decades that solid tumors can contain regions at very low oxygen concentrations (hypoxia) which do not occur in normal tissues under physiological conditions. During the last four years, we have used multiple technologies to identify and characterize the genomic response of untransformed and transformed mammalian cells to decreased oxygenation (hypoxia) as well as agents such as tirapazamine (TPZ) that become cytotoxic only under oxygen limiting conditions. During the last four years, we have used multiple technologies to identify and characterize the genomic response of untransformed and transformed mammalian cells to decreased oxygenation (hypoxia) as well as agents such as tirapazamine (TPZ) that become cytotoxic only under oxygen limiting conditions. A direct application of gene expression profiling to be used in this study is to identify hypoxia induced genes that code for a secreted products that could be detected in the serum of patients by ELISA assays (Project 4). Such an assay would be a non-invasive, rapid and simple means of assessing tumor hypoxia. One way to take advantage of the decreased oxygenation status of a tumor is to administer a hypoxia activated cytotoxin such as TPZ (Projects 1 & 2). While TPZ has demonstrated great promise in the clinic, the potential to enhance its toxicity could be achieved by increasing the formation of its damaging radical species (Project 1) and/or by inhibiting pathways that restitute the cellular damage induced by TPZ (Project 2). Expression of profiling of squamous cell carcinomas of the head and neck and cervix revealed that two family members, NIP3 (Nineteen K interacting protein) and NIP3L of the pro-apoptotic bcl-2 family are induced under hypoxic conditions (Project 3). These proteins may present a new approach to kill hypoxic cells using molecular cytotoxins that are regulated by low oxygen conditions (Projects 1 & 3). The overall goals of this PPG are to exploit tumor hypoxia therapeutically, and develop molecular markers to detect it. Thus, a common theme of hypoxia or TPZ induced changes in gene and protein expression run throughout the project. Project 1 will investigate the ability to use hypoxia to control the stabilization of proteins to kill cells directly by apoptosis or to metabolize TPZ into its radical damaging species. Project 2 will use yeast expression profiling and homozygous deletion strains to discover genes (and pathways) affecting TPZ sensitivity. Project 3 will use mammalian and yeast gene knockout and expression profiling to understand genes that affect the ability of NIP3 to kill tumor cells. Project 4 will use mammalian expression profiling of hypoxia induced genes that code for secreted proteins as surrogate markers for hypoxia. Few groups could be better positioned to use expression profiling of yeast and mammalian cells to address the mechanism of action of TPZ and pro- apoptotic genes, identify molecular hypoxia markers, and develop novel hypoxia based therapeutics.
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