The long-term objective of these studies is to define the physiological differences between the cellular Ca?2+? homeostasis of normal and neoplastic tissues. The rationale for this project is based on observations that tumor cells exhibit abnormal growth characteristics with respect to extracellular Ca?2+? and that their maintenance and regulation of intracellular Ca?2+? may also be altered.
The specific aims of this research are: (1) to determine the cytosolic-free Ca?2+? concentration and the intracellular distribution of Ca?2+? in hepatocytes (isolated from normal and regenerating liver) as well as malignant hepatoma cells; (2) to determine the Ca?2+? buffering characteristics of normal liver and hepatoma mitochondria and endoplasmic reticulum under physiologically realistic conditions; and (3) to compare the effects of Ca?2+? on the cytoskeleton of normal and malignant tissues. Recent progress along these lines include: (1) development of a substantially improved technique for isolating mitochondria from ascites tumor cells and comparing their respiratory characteristics with those of digitonin-permeabilized cells; (2) demonstration that AS30-D hepatoma mitochondria do not release Ca?2+? as normal liver mitochondria do in response to the presence of t-butyl hydroperoxide and that this is due to a lack of net NADPH oxidation because of the presence of a NADP?+?-reducing malic enzyme in the tumor mitochondria; (3) demonstration that AS30-D hepatoma mitochondria buffer the free Ca?2+? concentration of a cytosol-like medium at a significantly higher level than normal liver mitochondria do and that this is due to an abnormally high rate of Ca?2+? efflux; and (4) evidence that microsomal vesicles from AS30-D hepatoma cells accumulate more Ca?2+? and release Ca?2+? in response to inositol trisphosphate at a faster rate than that observed with normal liver microsomes. (E)
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