The long-term objective is to identify the alterations of Ca2+ homeostasis in hepatoma cells and characterize the biochemical basis for these alterations. The approach to this problem is based on the hypothesis that the regulation of intracellular Ca2+ and (or) the metabolic sensitivity to Ca2+ in tumor cells is modified so that cell survival and proliferation are promoted even under adverse environmental conditions.
The specific aims of this study are to: 1. Accurately compare the cytosolic free Ca2+ concentration of rat hepatoma cells and rat hepatocytes under normal and metabolically-stressful conditions; 2. Compare the influence of extramitochondrial or cytosolic free Ca2+ on the activity of Ca2+- sensitive dehydrogenases and the TCA cycle in mitochondria from normal liver and hepatoma cells; 3. Test the possibility that abnormally high levels of membrane cholesterol are responsible for the abnormal Ca2+ buffering properties of hepatoma mitochondria; 4. Further characterize the difference in the response of rat liver and hepatoma microsomes to release of Ca2+ induced by inositol trisphosphate; 5. Compare the effects of transient elevation of intracellular Ca2+ and depletion of ATP on the respiratory and Ca2+-buffering characteristics of mitochondria and the Ca2+ transport activities of other Ca2+-sequestering organelles. The methods of approach will include the use of digitonin cell permeabilization, fluorescent Ca2+ indicators, O2 and Ca2+ electrode measurements, dual beam and dual wavelength spectrophotometry, steady-state measurements of 14CO2 ratios using metabolites labeled at different carbons, and TLC-flame ionization determinations of membrane phospholipids. The significance of this work is that: a. It will help explain how tumor cells are adapted for survival in harsh environments; b. It will describe the relationships between Ca2+ homeostasis and energy metabolism in tumor cells; c. It relates to recent evidence that Ca2+ homeostasis is linked to the activity of oncogene products via turnover of phosphatidylinositol; d. It may ultimately lead to improved modes of cancer chemotherapy based on manipulations of cellular Ca2+ metabolism.
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