Energy Requirement in Programmed Cell Death

In several pathological conditions, the predominant mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Although these two modes of cell death have been regarded as conceptually and morphologically distinct, more and more evidence is accumulating that apoptosis and necrosis just represent two possible ways for a cell to die. Previous work in our laboratory has shown that the intracellular ATP concentration determines the mode of death after exposure to an, initially apoptosis-specific, stimulus, suggesting that both modes of cell death share common death-initiating signaling elements. However, high ATP levels were required to direct downstream events towards the ordered execution of apoptosis. This study shows that ATP depletion prevents the appearance of typical apoptotic features, such as chromatin condensation, proteolytic degradation of functional and structural proteins, including PARP and fodrin, and the exposure of phosphatidylserine as recognition marker for phagocytes.

Low ATP levels interfered with the apoptotic execution at two central steps: (1) the release of mitochondrial cytochrome c to the cytosol, an event directly leading to caspase activation in apoptosis, was delayed by 1 h, and (2) execution caspases were not activated. In ATP depleted cells, the extent of STS- or anti-CD95-induced cytochrome c translocation to the cytosol was similar to that found in cells with high ATP levels. Nevertheless, caspases were not activated in ATP depleted cells. This suggests that one ATP-dependent step is at the level of the caspase-activating apoptosome-complex between Apaf-1, pro-caspase-9, and cytochrome c. The absence of caspase activity in ATP depleted cells was not due to an early inactivation of the apoptosome components, because caspases in cytosols isolated from such cells could still be activated in a cell-free system. Therefore, the lack of adequate ATP concentrations seems to be directly responsible for the block of the apoptotic execution at this step. However, at least in a cell-free system, dATP promoted the apoptosome activation more efficiently than ATP.

By examining in more detail the events upstream and downstream to the mitochondrial control of apoptosis it was also demonstrated that the release of cytochrome c is due to a non-selective translocation mechanism. In Jurkat cells, this was true for apoptosis, induced by a variety of substances, and for the above necrosis model. At least one other mitochondrial intermembrane protein, namely the adenylate kinase, is released in parallel to cytochrome c, suggesting a non-selective permeabilization of the outer mitochondrial membrane during cell death. Caspases, once activated, have been shown to permeabilize the outer mitochondrial membrane. However, such a secondary proteolytic degradation was not the reason for the non-selective release in our system, since the caspase-inhibitor zVADfmk did neither alter the kinetics nor the extent of the STS-induced translocation of both proteins.

The endogenous mediator nitric oxide (NO) was able to block the execution of apoptosis, and converted death to necrosis. Like other inhibitors of mitochondrial ATP-synthesis, NO delayed the release of cytochrome c and inhibited caspase activation. This was solely due to ATP depletion by the inhibition of mitochondrial oxidative phosphorylation. Graded repletion of ATP by glucose supplementation restored activation of caspase-3, -7, and -8, and the formation of apoptotic features, including the exposure of phosphatidylserine on the cell surface. This result may be relevant for the understanding of pathologic situations with enhanced NO production and localized high rates of necrotic cell death.

The anti-apoptotic protein Bcl-2 was able to protect from STS-induced necrosis in ATP depleted cells. Overexpression of Bcl-2 blocked the release of cytochrome c in cells with high or low ATP levels. As shown before, in cells with h