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 potential that depends on temperature. At the restrictive temperature, heat-inactivation of large T antigen causes p53 release, growth arrest, and cell death. Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of Forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. 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 decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole cell populations, showed that the lower mitochondrial membrane potential (Delta Psi(m)) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in Delta Psi(m) is associated with a decrease in the rate of mitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.