Tetraploidy can result in cancer-associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax-dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl-2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax-proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53-inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and c-irradiation. These data indicate the existence of p53-dependent alterations in apoptosis regulation in tetraploid cells.

Apaf-1 is an essential factor for cytochrome c-driven caspase activation during mitochondrial apoptosis but has also an apoptosis-unrelated function. Knockdown of Apaf-1 in human cells, knockout of apaf-1 in mice, and loss-of-function mutations in the Caenorhabditis elegans apaf-1 homolog ced-4 reveal the implication of Apaf-1/CED-4 in DNA damage-induced cell-cycle arrest. Apaf-1 loss compromised the DNA damage checkpoints elicited by ionizing irradiation or chemotherapy. Apaf-1 depletion reduced the activation of the checkpoint kinase Chk1 provoked by DNA damage, and knockdown of Chk1 abrogated the Apaf-1-mediated cell-cycle arrest. Nuclear translocation of Apaf-1, induced in vitro by exogenous DNA-damaging agents, correlated in non-small cell lung cancer (NSCLC) with the endogenous activation of Chk-1, suggesting that this pathway is clinically relevant. Hence, Apaf-1 exerts two distinct, phylogenetically conserved roles in response to mitochondrial membrane permeabilization and DNA damage. These data point to a role for Apaf-1 as a bona fide tumor suppressor.

On binding to its receptor, transforming growth factor ␤ (TGF␤) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGF␤-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGF␤-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGF␤-treated cells. TGF␤ induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGF␤ induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGF␤-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGF␤-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGF␤-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGF␤ induced an apoptotic pathway via FADD-independent activation of caspase-8. INTRODUCTIONApoptosis is a highly regulated process involving various intracellular signaling pathways and a large number of molecules. Among these molecules, the proteases of the caspase family play a crucial role in triggering and controlling the execution of apoptosis (Cohen, 1997). These caspases are cysteine-related proteases that are synthesized as inactive proenzymes and are activated by most apoptotic stimuli. The proenzymes are activated by proteolysis at specific aspartate sites. The cleavage products form dimers, which are the active enzymes (Alnemri, 1997). There are 14 known caspases, of which caspase-8 and caspase-3 play key roles in control of the various steps of apoptosis. In recent years, an increasing number of investigations has contributed to elucidate the mechanisms underlying the activation of these two caspases (Kumar, 1999). Thus, caspase-3 may be activated via mitochondria-dependent or -independent pathways (Porter and Janicke, 1999). One of these pathways is dependent on the release by mitochondria of cytochrome c, which, in the presence of ATP, associates with the cytoplasmic Apaf1 and inactive proforms of caspase-9 to form a complex called apoptosome (Li et al., 1997;Qin et al., 1999). Autocleavage and activation of caspase-9 occur in this complex. In turn, caspase-9 then directly cleaves and activates caspase-3 proforms.An alternative pathway, observed in type I Jurkat T cells in response to Fas ligation, is independent of mitochondrial activation and requires the direc...

Aneuploidy and chromosomal instability, which are frequent in cancer, can result from the asymmetric division of tetraploid precursors. Genomic instability may favor the generation of more aggressive tumor cells with a reduced propensity for undergoing apoptosis. To assess the impact of tetraploidization on apoptosis regulation, we generated a series of stable tetraploid HCT116 and RKO colon carcinoma cell lines. When comparing diploid parental cells with tetraploid clones, we found that such cells were equally sensitive to a series of cytotoxic agents (staurosporine [STS], hydroxyurea, etoposide), as well as to the lysis by natural killer cells. In strict contrast, tetraploid cells were found to be relatively resistant against a series of DNA-damaging agents, namely cisplatin, oxaliplatin, camptothecin, and gamma- and UVC-irradiation. This increased resistance correlated with a reduced manifestation of apoptotic parameters (such as the dissipation of the mitochondrial transmembrane potential and the degradation of nuclear DNA) in tetraploid as compared to diploid cells subjected to DNA damage. Moreover, tetraploid cells manifested an enhanced baseline level of p53 activation. Inhibition of p53 abolished the difference in the susceptibility of diploid and tetraploid cancer cells to DNA damage-induced apoptosis. These data point to an intrinsic resistance of tetraploid cells against radiotherapy and DNA-targeted chemotherapy that may be linked to the status of the p53 system.

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EBV infects a large proportion of the human population worldwide and is one of the major viruses with human B lymphocyte tropism. It can immortalize human B lymphocytes and controls their resistance to apoptosis. EBV infection is associated with several lymphomas, including Burkitt’s lymphoma. In this report we show that EBV infection leads to the post-transcriptional down-regulation of expression of the proapoptotic protein Bim. This process involves the phosphorylation of BimEL by the constitutive EBV-activated kinase ERK1/2, followed by its degradation through the proteasome pathway. We also show that ectopic expression of BimEL in EBV-positive Burkitt’s lymphoma cells can enhance the sensitivity of these cells to serum deprivation-dependent apoptosis. Thus, EBV-mediated resistance to growth factor deprivation in human B lymphocytes is dependent on BimEL expression. Our data suggest that this regulatory pathway is an important contributor to the oncogenic potential of EBV.

Studies in Bim-deficient mice have shown that the proapoptotic molecule Bim plays a key role in the control of B cell homeostasis and activation. However, the role of Bim in human B lymphocyte apoptosis is unknown. We show in this study that, depending on the degree of cross-linking, B cell receptors can mediate both Bim-dependent and apparent Bim-independent apoptotic pathways. Cross-linked anti-μ Ab-mediated activation induces an original pathway governing the expression of the various Bim isoforms. This new pathway involves the following three sequential steps: 1) extracellular signal-regulated kinase-dependent phosphorylation of the BimEL isoform, which is produced in large amounts in healthy B cells; 2) proteasome-mediated degradation of phosphorylated BimEL; and 3) increased expression of the shorter apoptotic isoforms BimL and BimS.

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.