Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40.

Abstract

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 oflarge 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 (At,) 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 A'I is associated with a decrease in the rate ofmitochondrial 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.Mammalian cells grown in culture exhibit a finite life-span for proliferation. After a variable number of divisions they stop dividing, undergo a variety of changes, and finally die. Some oncogenes have the ability to confer an unlimited proliferative potential (immortality) to primary cells in culture. In the case of polyomavirus-induced immortalization of rodent embryo fibroblasts, it has been shown that the unlimited proliferative capacity is maintained by the large tumor (T) antigen (1)(2)(3)(4) MATERIALS AND METHODSCell Lines and Cell Culture. The REtsAF and RELPB cell lines were isolated at low cell density from a rat embryo fibroblast culture infected with SV40 (2). REtsAF was obtained by using a temperature-sensitive tsA58 mutant and is temperature sensitive for immortalization, whereas RELPB was obtained with wild-type SV40 and is immortal at both 330 and 39.50C. REtsAF-Revl was derived from REtsAF by selection for proliferation at 39.50C (8

Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death

“…It has been suggested that the loss of DC m , which may be due to the opening of PT pores, is a central and irreversible step in apoptosis Petit et al, 1995;Vayssiere et al, 1994;Zamzami et al, 1995). We observed that in Jurkat cells, changes in mitochondrial transmembrane potential occur very early, before detection of caspase activity.…”

Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation

“…20 ± 22,24,25 This is associated with collapse of the Dc m , which can be measured by a reduced mitochondrial accumulation of the fluorescent dye DiOC 6 (3). 41,42 After 2 h of exposure to okadaic acid, a significant decrease in the retention of DiOC 6 (3) was observed (P50.05), and the decline continued until 8 h Figure 3 UDCA inhibits DCA-induced release of cytochrome c in isolated mitochondria after MPT. Mitochondria were isolated and incubated (1 mg/ml) with either no addition (control), 250 mM DCA, 500 mM UDCA, DCA+UDCA, 5 mM cyclosporine A (CsA), DCA+CsA, 500 mM tauroursodeoxycholic acid (TUDCA), DCA+TUDCA, 500 mM glycoursodeoxycholic acid (GUDCA), DCA+GUDCA, 500 mM hyodeoxycholic acid (HDCA), DCA+HDCA, 500 mM taurocholic acid (TCA), or DCA+TCA in respiration buffer as described in Materials and Methods.…”

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

“…For example, strong evidences suggest that CL participates in the interaction of cytochrome c with the outer face of the inner mitochondrial membrane, thereby ensuring the presence of a pool with limited solubility close to respiratory complexes III and IV, increasing the efficiency of electron transport. 10 During the past decade, it has become unequivocally clear that most proapoptotic stimuli require a mitochondria-dependent step involving the disruption of mitochondrial bioenergetics 11,12,13,14 and outer membrane permeabilization, 15,16 leading to the release of apoptogenic factors, including cytochrome c.…”