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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA032946-05
Application #
3170830
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1983-07-01
Project End
1991-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
George Washington University
Department
Type
Schools of Medicine
DUNS #
City
Washington
State
DC
Country
United States
Zip Code
20052
Holleran, A L; Briscoe, D A; Fiskum, G et al. (1995) Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation. Mol Cell Biochem 152:95-101
Briscoe, D A; Fiskum, G; Holleran, A L et al. (1994) Acetoacetate metabolism in AS-30D hepatoma cells. Mol Cell Biochem 136:131-7
Clawson, G A; Norbeck, L L; Hatem, C L et al. (1992) Ca(2+)-regulated serine protease associated with the nuclear scaffold. Cell Growth Differ 3:827-38
Baumgold, J; Paek, R; Fiskum, G (1992) Calcium independence of phosphoinositide hydrolysis-induced increase in cyclic AMP accumulation in SK-N-SH human neuroblastoma cells. J Neurochem 58:1754-9
Kobryn, C E; Fiskum, G (1992) Differential sensitivity of AS-30D rat hepatoma cells and normal hepatocytes to anoxic cell damage. Am J Physiol 262:C1384-7
Lyons, H T; Kharroubi, A; Wolins, N et al. (1991) Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver. Arch Biochem Biophys 285:238-45
Madsen, K R; Fiskum, G; Clawson, G A (1990) Regulation of nuclear scaffold protease activity by calcium. Exp Cell Res 187:343-5
Murphy, A N; Kelleher, J K; Fiskum, G (1990) Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms. J Biol Chem 265:10527-34
Staley, J; Fiskum, G; Moody, T W (1989) Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells. Biochem Biophys Res Commun 163:605-10
Mahmoud, S; Palaszynski, E; Fiskum, G et al. (1989) Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues. Life Sci 44:367-73

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