p53 can promote mitochondria- and caspase-independent apoptosis

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 t