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A mechanism for the induction of programmed cell death (apoptosis) upon dysfunction of the mitochondrial respiratory chain has been studied. Previously, we had found that inhibition of mitochondrial cytochrome bc1, a component of the electron transport chain complex III, leads to activation of tumor suppressor p53, followed by apoptosis induction. The mitochondrial respiratory chain is coupled to the de novo pyrimidine biosynthesis pathway via the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). The p53 activation induced in response to the inhibition of the electron transport chain complex III has been shown to be triggered by the impairment of the de novo pyrimidine biosynthesis due to the suppression of DHODH. However, it remained unclear whether the suppression of the DHODH function is the main cause of the observed apoptotic cell death. Here, we show that apoptosis in human colon carcinoma cells induced by the mitochondrial respiratory chain complex III inhibition can be prevented by supplementation with uridine or orotate (products of the reaction catalyzed by DHODH) rather than with dihydroorotate (a DHODH substrate). We conclude that apoptosis is induced in response to the impairment of the de novo pyrimidine biosynthesis caused by the inhibition of DHODH. The conclusion is supported by the experiment showing that downregulation of DHODH by RNA interference leads to accumulation of the p53 tumor suppressor and to apoptotic cell death.

The opening of the cyclosporin A-sensitive permeability transition pore (MTP) in deenergized mitochondria was induced only at millimolar Ca 2÷. Pretreatment of the mitochondria with 'inducers', such as duroquinone and phenylarsine oxide, allowed observing the pore opening at 0.01-0.1 mM Ca 2+. Duroquinone caused a rapid (within 20 s) NAD(P)H oxidation which was followed by a slow (20 rain) induction of the pore sensitive to low Ca 2÷. Phenylarsine oxide capable of cross-linking of vicinal SHgroups caused pore formation without the oxidation of NAD(P)H. The pore opening by both 'inducers' was prevented by N-ethylmaleimide. We propose that oxidation or cross-linking of critical dithiol(s) in membrane proteins increase the sensitivity of a putative 'Ca2+-sensor ' that regulates the permeability transition pore opening.Key words: Mitochondria; Permeability transition pore; Ca2+; NAD(P)H oxidation; SH-groups cross-linking in the membrane resulted in a more than 100-fold increase in the sensitivity of MTP to Ca 2÷. Materials and methodsMitochondria from rat liver were isolated as described [8] in the presence of 0.25 mM EGTA. The medium for the last washing and storage contained no EGTA. The measurement medium contained: 200 mM sucrose, 20 mM Tris-MOPS, 20]zM EGTA, 2,uM rotenone, 1/zM A23187, 5 ¢tM CCCP, pH 7.4. The concentration of mitochondrial protein was 0.6 mg/ml. The total concentration of Ca was determined by atomic absorption spectroscopy and the concentration of free Ca 2÷ was calculated using the standard software. NAD(P)H was measured at 340-370 nm with an Aminco DW-2000 spectrophotometer. Lightscattering was measured at 540 nm with a Cary-219 spectrophotometer. The samples were stirred continuously and thermostated at 25°C. Results

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