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Article Dans Une Revue Cell Death and Differentiation Année : 2004

p53 can promote mitochondria- and caspase-independent apoptosis

Résumé

The tumour suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis. Cell cycle arrest is mediated by transcriptional induction of genes whose products inhibit cell cycle progression. Conversely, the molecular events that lead to p53-dependent apoptosis are less clear. Transcriptional activation is commonly implicated but growing evidences show that transrepression and transcription-independent functions can also play a central role in p53-dependent apoptosis. 1 At the cellular level, all studies converge to the crucial role of both the mitochondrial pathway (cytochrome c release, 2 ROS production or/and DCm drop) 3 and caspase activation in p53-induced apopto-sis. 4 In this way, the Bcl-2 antiapoptotic protein as well as caspase inhibition were shown to protect cells from p53-induced apoptosis. We previously showed in rat embryo fibroblasts (e.g. the REtsAF cell line) expressing a temperature-sensitive mutant (tsA58) of the simian virus 40 large tumour antigen (LT) that LT inactivation leads to p53-mediated apoptosis. Moreover, we reported that while bcl-2 overexpression inhibits apopto-sis, caspase inhibition surprisingly accelerates apoptosis and moreover abolishes the protective effect of Bcl-2. 5 These data led us to postulate that caspase inhibition would unmask an alternative route for p53-induced cell death signal, which would lead to a caspase-independent and Bcl-2-insensitive cell death process. In order to ascertain that this new cell death process observed in the presence of ZVAD is dependent on p53 activity, REtsAF cells were transiently transfected with genes encoding temperature-sensitive dominant-negative mutants of p53 (p53 val135 and p53 ala143). 6,7 At restrictive temperature, these mutants are defective in their DNA binding domain and consequently loss their sequence-specific transactivation and transrepression properties. We observed that overexpression of either p53 val135 or p53 ala143 led to a decreased rate of apoptosis in the absence or in the presence of ZVAD (Figure 1a), demonstrating that active p53 is required for these two cell death pathways. Next, we controlled that this unexpected effect of ZVAD on p53-induced cell death could be reproduced in a more physiological model. For this purpose, we induced a p53-dependent apoptosis in primary rat embryo fibroblasts (RE) by addition of 100 mM etoposide, in the absence or in the presence of ZVAD. As observed in REtsAF cells, ZVAD treatment accelerated commitment to death of RE cells. As control, ZVAD reduced staurosporine-induced apoptosis of RE cells, showing its usual protector effect against this p53-independent cell death (Figure 1b). These results argue for a physiological relevance of the proapoptotic effect of ZVAD on p53-mediated apoptosis. In order to characterise the pattern of the p53-induced death of REtsAF cells in the presence of ZVAD, we examined the associated nuclear and cytological alterations. In the absence of ZVAD, the earliest morphological changes observed are the rounding up, the brightening phase, the shrinkage of the cells as well as the blebbing of the plasma membrane (Figure 1c). These events are associated with typical apoptotic chromatin compaction and fragmentation in globular, crescent-shaped figures (stage II chromatin condensation), and they preceded the breaking up of the cells into fragments as well as the fragmentation of DNA, as judged by the flow cytometric analysis of TUNEL-stained cells. In the presence of ZVAD, light microscopy observation showed that some typical morphological features of apoptosis such as loss of adherence and condensed cytoplasm were evident in dying REtsAF cells. Further fluorescence microscopy examination revealed chromatin condensed in lumps (stage I chromatin condensation), and rounded cells without microvilli or protu-sions on the surface (Figure 1c). However, specific events of the final degradation phase such as the nucleus and cell fragmentation do not occur whereas cells completely detached from the substratum. These observations indicate that p53 can promote two cell death pathways showing apoptotic features, one that is caspase-dependent and another that is independent of caspases. Recent data suggest that noncaspase proteases including calpains, cathepsins or serine proteases can also mediate cell death and bring about many of the morphological changes characteristic of apoptosis in a caspase-independent manner. 8 We investigated the possible involvement of these proteases in the novel route by which p53 signal cell death through the use of specific inhibitors: serine protease inhibitors (TPCK, TLCK), calpain protease inhibitors (ALLN, MDL), cysteine protease inhibitors (Lactacystine, Z-FA-fmk). Microscopic studies of cell morphology did not show notable differences in the amount of Z-VAD-dependent cellular death whether the cells were cultured in the presence or in the absence of the drugs. These results suggest that none of the tested proteases are involved in p53-induced apoptosis of REtsAF cells in the presence of ZVAD. Inasmuch as the mitochondrial pathway plays a central role in many models of p53-dependent apoptosis, we investigated
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Dates et versions

hal-03002800 , version 1 (21-12-2020)

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N Godefroy, C Lemaire, F Renaud, V Rincheval, S Perez, et al.. p53 can promote mitochondria- and caspase-independent apoptosis. Cell Death and Differentiation, 2004, 11, pp.785 - 787. ⟨10.1038/sj.cdd.4401398⟩. ⟨hal-03002800⟩

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