No abstract

The mechanism by which caspase-2 executes apoptosis remains obscure. Recent findings indicate that caspase-2 is activated early in response to DNA-damaging antineoplastic agents and may be important for the engagement of the mitochondrial apoptotic pathway. We demonstrate here that fully processed caspase-2 stimulates mitochondrial release of cytochrome c and Smac/ DIABLO, but not apoptosis-inducing factor (AIF). This event occurs independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2, and inactivation experiments reveal that the proteolytic activity of caspase-2 is not required for the effect. Further, functional studies of mitochondria indicate that processed caspase-2 stimulates state 4 respiration and decreases the respiratory control ratio as a result of, in large part, an uncoupling effect. Combined, our data suggest that caspase-2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity.

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cell's ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3r deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53-and Chk2-related proteins.

Recent findings have established caspase-2 as an important apical regulator in apoptotic pathways leading from DNA damage to release of mitochondrial cytochrome c and subsequent activation of effector caspases. Yet, the molecular map connecting the embarking stimuli of genotoxic stress with caspase-2 activation remains to be elucidated. Here, we address the question of potential caspase-2 regulators by examining 5-fluorouracil (5-FU)-induced apoptosis in wild-type and p53-deficient human colon carcinoma cells. Apoptosis was observed only in p53 þ / þ cells and was preceded by caspase-2 activation. Hence, although no direct interaction between p53 and caspase-2 was observed in the cell system used, our data clearly demonstrate that a functional connection between these two proteins is essential for initiation of the 5-FUinduced apoptotic process. Proposed mediators of caspase-2 activation include PIDDosome complex proteins PIDD and RAIDD. Surprisingly, the presence of a complex encompassing at least RAIDD, PIDD and caspase-2 was verified in both p53 þ / þ and p53 À/À cells, also in the absence of 5-FU treatment. Thus, our results confirm the participation of PIDD and RAIDD in PIDDosome complex formation but question their role as sole mediators of caspase-2 activation. This assumption was further supported by siRNA transfections targeting PIDD or RAIDD. In conclusion, our findings support the hypothesis of p53 as an upstream regulator of caspase activity and provide data concerning caspase-2 processing mechanisms. As suppression of caspase-2 expression in 5-FU-treated cells also affects the level of the p53 protein, possibilities of a reciprocal interaction between these proteins are discussed.

The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signaling cascade regulated by sequential activation of caspases. Mechanisms that lead from p53 to activation of individual initiator caspases are still unclear. The present model for caspase-2 activation includes PIDDosome complex formation. However, in certain experimental models, elimination of complex constituents PIDD or RAIDD did not significantly influence caspase-2 activation, suggesting the existence of an alternative activation platform for caspase-2. Here we have investigated the link between p53 and caspase-2 in further detail and report that the latter is able to utilize the CD95 DISC as an activation platform. The recruitment of caspase-8 to this complex is required for activation of caspase-2. In the experimental system used, the DISC is formed through a distinct, p53-dependent upregulation of CD95. Moreover, we show that caspase-2 and -8 cleave Bid, and that both act simultaneously upstream of mitochondrial cytochrome c release. Finally, a direct interaction between the two caspases and the ability of caspase-8 to cleave caspase-2 are demonstrated. Thus, the observed functional link between caspase-8 and -2 within the DISC represents an alternative mechanism to the PIDDosome for caspase-2 activation in response to DNA damage.