The broad, long term objectiv of this study is to determine the underlying basis for the measured difference in mitochondrial membrane potential that are exhibited by normal epithelial an carcinoma cells. The proposed study involves a comprehensive, comparative biochemical and molecular analysis of mitochondria isolated from several control and carcinoma cell lines that display differences in mitochondrial membrane potential, and investigates systematically and for the first time the biochemical and molecular alterations that likely result in increased mitochondrial membrane potential in carcinoma cells.
Specific Aim #1 is to reveal any differences in the structure of function of those molecular components directly involved in establishing and/or utilizing the mitochondria transmembrane electrical gradient in high membrane potential carcinoma vs. low membrane potential control cell types by measuring the various parameters of oxidative phosphorylation (including oxygen consumption, electron transport, ATP synthesis, etc.) in samples of mitochondria isolated from cultured carcinoma and control cells.
Specific Aim #2 is to investigate differences between cell types in the concentration and/or movement of adenine nucleotides by determining both cytosolic and mitochondrial adenine nucleotide content and by measuring the activity of mitochondrial adenine nucleotide translocase.
Specific Aim #3 is to investigate the role of calcium in establishing and/or maintaining the high mitochondrial membrane potential in carcinoma cells by comparison of cytosolic and intramitochondrial calcium concentrations, calcium uptake capacity by mitochondria, and the kinetics of calcium transport enzymes in control and carcinoma cell types.
Specific Aim #4 is to measure the expression level of BCL- 2 in control and carcinoma cell types by standard western blotting techniques. The established presence of BCL-2 in carcinoma mitochondria may suggest some causal relationship between the expression of this protein and increased membrane potential, and may further serve to link any differences between the expression of this protein and increased membrane potential, and may further serve to link any differences between high and low membrane potential cell types that had been revealed in fulfilling Specific Aims 1, 2 and 3. The results of this study will contribute to a better understanding of the phenomenon of selective uptake of lipophilic cations by carcinoma cells which occurs in response to increased mitochondrial membrane potential and which results in selective cytotoxicity of carcinoma cells. This information is relevant to the rational selection and/or design of more efficacious forms of these anti-carcinoma agents. Further, this study may reveal additional, as yet undiscovered differences in the structure and function of mitochondria from normal epithelial and carcinoma cells that could provide novel, selective and site-specific mitochondrial targets for new anti-cancer agents.
| Modica-Napolitano, Josephine S; Nalbandian, Raffi; Kidd, Marianne E et al. (2003) The selective in vitro cytotoxicity of carcinoma cells by AZT is enhanced by concurrent treatment with delocalized lipophilic cations. Cancer Lett 198:59-68 |
| Modica-Napolitano, Josephine S; Singh, Keshav K (2002) Mitochondria as targets for detection and treatment of cancer. Expert Rev Mol Med 4:1-19 |
