Our objective is to alter metabolic states to enhance the response of human lung cancer cells to radiation at clinically relevant doses (1-5 Gy). We have produced a situation where depletion of endogenous glutathione (GSH) sensitizes A549 cells to radiation. We can reverse this sensitized state by the addition of exogenous thiols(ie., reducing agents) or increase it with tbutylhydroperoxide (tBOOH). This research will establish conditions that will maximize the radiation response of aerobic or hypoxic tumor cells in vitro. A biochemical approach will help us to rigorously define the limits for increased radiation response.
Our specific aim #1 is designed to proceed in a precise stepwise manner to understand the relative contributions of individual enzymes and substrates in the cellular compartments in the radiation response of aerobic and hypoxic cells. Our assumption is that electron transfer from glucose, via sequential coupled enzyme systems, leads to the reduction of various radicals and hydroperoxides. We will define the importance, of, in addition to GSH, thioredoxin, glutaredoxin, thioredoxin reductase, ribonucleotide reductase, GSH reductase, GSH-S-transferase, GSH peroxidase and NADPH in the radiation response of aerobic and hypoxic A549 and various small cell lung carcinomas (SCLC). We will measure the effect of radiation on pentose cycle activity. The use of various controlled states to cause additional radiosensitivity of GSH depleted cells will be investigated. We will determine the endogenous factors which contribute to the sensitivity of aerobic cells versus hypoxic cells. Monoclonal antibodies to glutathione-S-transferase will establish its presence in the nucleus in various tumor cells and lead to new strategies to inhibit its activity.
In specific aim #2 we will superimpose hormonal controls, using insulin (cell cycle alterations) or dehydroepiandrosterone (inhibition of pentose cycle), on GSH depleted A549 and SCLC lines. Some of the studies will be with non-growing nutritionally-deprived Go cultures. Mitogenic factors such as glycyl-1- histidyl-l-lysine will be used. The mechanism for misonidazole sensitization of aerobic plateau phase cultures will be elucidated.
Specific aim #3 will focus on the exogenous factors involved. Since tumor cells in vivo and in vivo are bathed with various nutrients, some of which may radioprotect external membranes or enter the cell as building blocks. Catalase, vitamin E, ergothioneine and phospholipase A2 are some of the components to be tested.
Specific aim #4 is concerned with the radiosensitizing mechanism of t-butylhydroperoxide. This chemical enhances the radiation response of GSH depleted cells. We will extend these studies to H69 cells and other SCLC lines. The importance of intracellular polyamines as chemo and radioprotectors in cells deficient in peroxidase activity will be assessed. The clinical significance of this work is the development of alternative strategies to increase radiosensitivity of human tumors in vivo.
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