COMMITMENT TO APOPTOSIS IS ASSOCIATED WITH CHANGES IN MITOCHONDRIAL BIOGENESIS AND ACTIVITY IN CELL-LINES CONDITIONALLY IMMORTALIZED WITH SIMIAN-VIRUS-40

Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potentia l that depends on temperature. At the restrictive temperature, heat-in activation of large T antigen causes p53 release, growth arrest, and c ell death. Morphological and molecular analysis indicate that the indu ced cell death corresponds to apoptosis. Flow cytometric analysis usin g a combination of Forward light scatter and side scatter allows a dis crimination of cells committed to apoptosis within the whole populatio n. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreas ed accumulation of the dye was recorded. Measures of cellular respirat ion, performed with whole cell populations, showed that the lower mito chondrial membrane potential (Delta Psi(m)) correlates, as expected, w ith an uncoupling of electron transport from ATP production and is lin ked to the induction of apoptosis. We also show that this decrease in Delta Psi(m) is associated with a decrease in the rate of mitochondria l translation. These events are detected at early stages of the apopto tic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during a poptosis.