The long term objective of this research is to establish a biochemical basis for potentially lethal damage recovery (PLDR) in vitro. The immediate research goal is to determine the limits to which log or plateau phase human tumor models, in vitro, repair radiation damage in the presence and absence of various agents. Human tumor lines have been chosen for this research because there is evidence that these are considerably different than rodent lines, both in biochemical make-up and in their capacity to recover from radiation damage. PLDR of A549 human lung carcinoma cells can be inhibited by alkaline conditions or insulin addition. These preliminary results will be extended with the A549 cells as well as with insulin sensitive breast carcinoma (MCF-7) and human melanoma cells in vitro. The approach will be to manipulate cellular metabolism, via changes in intracellular and extracellular hydrogen ion concentration, in an attempt to make tumor cells more vulnerable to radiation damage. Acute and chronic changes in extracellular or intracellular pH will be produced in combination with changes in glucose, glutamine, phosphate and bicarbonate, or the addition of insulin, in order to determine their effect on PLDR of human tumor cells. Controlled metabolic states (i.e., lack of glucose, decreased glutathione or ATP) will be utilized to study the effects of various agents that interfere with the relationship between intracellular and extracellular pH. This work will be especially important for determining the possible dependency on pH and metabolism for the effect of dinitrophenol, amiloride and insulin on PLDR. 3-aminobenzamide, misonidazole and other agents known to inhibit (PLDR) will also be examined. Sublethal damage repair (SLDR) will be determined after a second radiation dose, under conditions giving maximal or minimal PLDR. The proposed work involves a number of techniques for measuring pH, cell cycle parameters, and the activities of enzymes and enzyme systems. The health-relatedness of this research is potentially in the field of radiation therapy. For example, if a particular tumor had an acidic pH, caused by altered circulation, or increased glucose metabolism due to hypoxia, it might also have a greater capacity for PLDR than a more alkaline tumor. Identification and subsequent alkalinization of such tumors in vivo, either pre- or post-irradiation, might improve radiocurability.
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