Our research is focused on the role of mitochondria in apoptosis and on regulation of apoptosis-related proteins in cancer cells.

1. Mechanism of release of apoptogenic factors from mitochondria.

Apoptosis is a strictly regulated mode of cell death. Mitochondria participate in apoptosis by releasing cytochrome c and other co-factors of activation of apoptotic enzymes, caspases. The mechanism of permeabilization of mitochondrial membrane and release of apoptogenic factors is poorly understood, although it is known to be regulated by Bcl-2 family of proteins. We found that at a stage preceding release of apoptogenic factors, mitochondria transiently accumulate potassium. As a result of such potassium accumulation, mitochondria undergo cristae remodeling and ballooning that leads to localized stretching and permeabilization of a region of the outer mitochondrial membrane and release of cytochrome c. The active form of the pro-apoptotic Bcl-2 family protein Bid, tBid triggers uptake of potassium by mitochondria. The pro-apoptotic protein Bax co-operates with tBid in permeabilization of the outer mitochondrial membrane. The anti-apoptotic protein Bcl-2 prevents such potassium uptake. Once mitochondria release apoptogenic factors, they may or may not undergo the permeability transition. Interestingly, during the final stages of apoptotic program mitochondria divide and become fragmented.

2. Regulation of apoptosis and mitochondrial biogenesis in cancer cells.

They activate cell cycle and upregulate anti-apoptotic proteins such as Bcl-2, Bcl-xl and others. We are investigating the role of NF-kappaB in resistance of cancer cells to radiation-induced apoptosis. Our data indicate that NF-kappaB inhibits apoptotic response to ionizing radiation via supression of the JNK/cJun/AP1 cascade required for progression of radiation-induced apoptosis. NF-kappaB upregulates a DNA damage response protein GADD45-beta, a known inhibitor of the JNK/cJun/AP1 cascade.

We also found that NF-kappaB may regulate mitochondrial biogenesis in cancer cells. Mitochondrial DNA content and mitochondrial protein expression correlate with the level of activity of NF-kappaB. The mechanism of NF-kappaB-mediated regulation of mitochondrial biogenesis remains to be determined although preliminary results show that such regulation may be via the NRF1 transcription factor.

In our work we use various biochemical and molecular methods, such as analysis of protein expression by real-time RT-PCR and immunoblotting; gene reporter assay; regulation of protein expression via transfections or anti-sense inhibition; electron microscopy; fluorescent techniques; atomic absorption spectroscopy as well as various mitochondrial function assays.

Eliseev, R.A., Schwarz, E.M., Zuscik, M.J., O’Keefe, R. J., Drissi, H., and R.N. Rosier, “Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NFkappaB”, Exp Cell Res, in press, 2005

Eliseev, R.A., Zuscik, M.J., Schwarz, E.M., O’Keefe, R. J., Drissi, H., and R.N. Rosier, “Increased Radiation-induced Apoptosis of Saos2 Cells via Inhibition of NFkappaB: a Role for cJun N-terminal Kinase”, J Cell Biochem, in press, 2005

Eliseev, R.A., VanWinkle, B., Rosier, R.N., and T.E. Gunter, “Diazoxide-mediated Preconditioning Against Apoptosis Involves Activation of CREB and NFkB”, J Biol Chem, 279, 46748-46754, 2004

Eliseev, R.A., Alexandrov, A.A., and T.E. Gunter, “High-yield expression and purification of p18 form of Bax as an MBP-fusion protein”, Protein Expression and Purification, 35(2), 206-209, 2004

Eliseev, R.A., Gunter, K.K., and T.E. Gunter, “Bcl-2 prevents abnormal mitochondrial proliferation during etoposide-induced apoptosis”, Experimental Cell Research, 289(2), 275-281, 2003

Eliseev, R.A., Salter, J., Gunter, K.K., and T.E. Gunter, “Bcl-2 and tBid proteins counter-regulate mitochondrial potassium transport”, Biochem. Biophys. Acta, 45227, 1-5, 2003

Eliseev, R.A., Gunter, T.E., and K.K. Gunter, "Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis", Mitochondrion, 1(4), 361-370, 2002

Pateder, D.B., Eliseev, R.A., O'Keefe, R.J., Schwartz, E.M., Okunieff, P., Constine, L.S., Puzas, J.E., and R.N. Rosier, "The Role of Autocrine Growth Factors in Radiation Damage to the Epiphyseal Growth Plate", Radiation Research, 155(6), 847-858, 2001

Buntinas, L., Eliseev, R., Gunter, K.K., and T.E. Gunter, “The Rapid Mode (RaM) of Mitochondrial Calcium Transport”, Biophysical Journal, 74(2), A382, 1998

Lukianova, L.D., Eliseev, R.A., Makarenko, T., and A.I. Matiushin, "Influence of Some Steroid Hormones on Calcium Transport and Oxidative Metabolism in Isolated Mitochondria", Bulletin of Experimental Biology and Medicine, 118(12), 1307-1310, 1994

Gunter, K.K., Aschner, M., Miller, L.M., Eliseev, R., Salter, J., Anderson, K., Hammond, S.,  and T.E.Gunter, ”Determination of the oxidation states of manganese in PC12 and NGF-induced PC12 cells”, Free Rad. Biol & Med.., in press, 2004

Gunter, T.E., Yule, D.I., Gunter, K.K., Eliseev, R.A., and J.D. Salter, “Calcium and Mitochondria”, FEBS Letters, 567(1), 96-102, 2004

Gunter, T.E., Miller, L.M., Gavin, C.E., Eliseev, R., Salter, J., Buntinas, L., Alexandrov, A., Hammond, S. and K.K. Gunter, ”Determination of the oxidation states of manganese in brain, liver, and heart mitochondria”, J. Neurochem., 88, 266-280, 2004

Gunter, K.K., Miller, L.M., Aschner, M., Eliseev, R.A., Depuis, D., Gavin, C.E., and T.E. Gunter, “XANES Spectroscopy: a Promising Tool for Toxicology: a Tutorial”, Neurotoxicology, 23, 127-146, 2002

D'Souza, M.J., Zuscik, M.J., Eliseev, R.A., Gunter, T.E., Schwarz, E.M., Puzas, J.E., O'Keefe, R.J., and R.N. Rosier, “NF-kappa B over-expression protects growth plate chondrocytes from ionizing radiation-induced apoptosis”, J. Bone and Min. Res.,17 (S1): M53, 2002

Pfeiffer, D.R., Gunter, T.E., Eliseev, R.A., Broekemeyer, K.M, and K.K. Gunter, “Release of Ca2+ from Mitochondria via the Saturable Mechanisms and the Permeability Transition”, IUBMB Life, 52, 1-8, 2001

Gunter, T.E., Buntinas, L., Sparagna, G., Eliseev, R., and K.K Gunter, "Mitochondrial Calcium Transport: Mechanisms and Functions", Cell Calcium, 28(5), 285-296, 2000

Constine, L.S., Pateder, D., O'Keefe, R., Hicks, D., Eliseev, R., Williams, J., Puzas, E., and R. Rosier, “Radiation injury to growth plate/epiphyseal chondrocytes: molecular mechanisms”, International Journal of Radiation Oncology, Biology, Physics, 48(3 S1), 284, 200