(40) INVESTIGATIONS INTO THE ROLE OF MITOCHONDRIA AND THEIR CHANNELS IN APOPTOSIS

K.W. Kinnally (1), R.C. Murphy (2), A. Moodie (1), E. Schneider (1), C.A. Mannella (1) and M.L. Campo (3)
(1) Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, NY and Department of Biomedical Sciences, University at Albany, Albany, NY.
(2) Rensselaer Polytechnic Institute, Troy, NY
(3) Dpto. de Bioquímica, Univ. de Extremadura, Spain

Several laboratories have produced evidence that mitochondria are involved in the commitment step of apoptosis and that this step is linked to the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. The released cytochrome c then forms a complex that facilitates caspase activation and progression through the apoptotic cascade. We have investigated the possible role in these processes of the protein translocation channel, MCC, of the mitochondrial inner membrane. A time line for the onset of apoptotic markers was established for human breast cells (MDA231) treated with teniposide. By patch-clamping mitochondria isolated from these cells at various times after treatment, we found the conductance through the cyclosporin A sensitive-channel MCC was opened in early apoptosis. Other early events included mitochondrial depolarization detected by JC-1 fluorescence and loss of lipid asymmetry in the plasma membrane determined by annexin-V binding. These early events were followed by caspase activation, DNA laddering and loss of plasma membrane integrity as indicated by PARP-cleavage on western blot, agarose gels, and 7AAD labeling of nuclei, respectively. Overexpression of the anti-apoptotic protein bcl-2 in untreated MDA231 cells was associated with increased mitochondrial membrane potential of mitochondria and decreased the detection of MCC activity. Further studies showed bcl-2 overexpression eliminated the calcium-activation of MCC and, hence, suggest a mechanism of action for bcl-2. These findings support a role for opening of the conductance through MCC early in the apoptotic cascade.

This work was supported by NATO CRG970210 to MLC, ES and KWK, DGICYT grant PB95-0456 and Junta de Extremadura y Fondo Social Europeo grant EIA94-11 to MLC and NSF grant MCB9513439 to KWK.