The objective of this research proposal is to increase the sensitivity of cells to radiation by altering pentose cycle (PC) activity. We will test the hypothesis that changes in electron flow from the PC are involved in a radiation-sensitive signaling pathways that ultimately lead to apoptosis. We will test this hypothesis using as a model, glucose-6-phosphate dehydrogenase deficient (G6PD) cell lines and the appropriate wild type controls (G6PD). We propose to use G6PD cells to study the role of NADPH, generated by the PC, in the radiation and apoptotic responses. The cells with wild type G6PD activity will be used to study the potential use of inhibitors of G6PD, and related enzymes (i.e., glutaredoxin) as radiation sensitizers.
In specific aim 1 the cDNA for wild type G6PD will be transfected into G6PD cells. G6PD activity in the selected clones will be correlated with apoptosis and clonogenic cell kill. The hypothesis tested in specific aim 2 is that the PC plays a direct role in regulating protein tyrosine phosphorylation in response to ionizing radiation. Tyrosine phosphorylation of cellular proteins is elevated in the G6PD cells, when compared to the wild type cells after radiation exposure. We propose that the changes in phosphotyrosyl proteins observed in response to radiation are necessary for the onset of radiation-induced apoptosis. Immunological and physical chemical techniques will be used to identify component(s) of this signaling pathway.
Specific aim 3 will test the hypothesis that the PC protects against radiation-induced apoptosis and clonogenic cell death by maintaining thiols, e.g., glutathione, thioredoxin, glutaredoxin and ultimately protein thiols, in the reduced state (electron transfer). The experiments outlined in Specific aim 4 will examine the ability of inhibitors of G6PD, i.e., dehydroepiandrosterone (DHEA) and its halogenated analogues, to influence radiation-induced apoptosis and clonogenic cell death. We will also examine if blocking other cellular sources of NADPH, i.e., malic enzyme and isocitrate dehydrogenase, will result in further enhancement of the radiation response. Some studies will be done in A549 cells (human lung tumor with no detectable apoptosis) and H69 cells (human tumor containing myc with a high incidence of apoptosis). The results of specific aim 4 will determine if inhibiting G6PD activity in vivo can be used clinically to sensitize tumors to radiation or chemotherapy. In addition, proteins identified as components of the radiation- sensitive signaling pathway that leads to apoptosis, could be potential targets for new drug development.
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