Tumor necrosis factor (TNFalpha) receptor signaling can simultaneously activate caspase 8, the transcription factor, NF-kappaB and the kinase, JNK. While activation of caspase 8 is required for TNFalpha-induced apoptosis, and induction of NF-kappaB inhibits cell death, the precise function of JNK activation in TNFalpha signaling is not clearly understood. Here, we report that TNFalpha-mediated caspase 8 cleavage and apoptosis require a sequential pathway involving JNK, Bid, and Smac/DIABLO. Activation of JNK induces caspase 8-independent cleavage of Bid at a distinct site to generate the Bid cleavage product jBid. Translocation of jBid to mitochondria leads to preferential release of Smac/DIABLO, but not cytochrome c. The released Smac/DIABLO then disrupts the TRAF2-cIAP1 complex. We propose that the JNK pathway described here is required to relieve the inhibition imposed by TRAF2-cIAP1 on caspase 8 activation and induction of apoptosis. Further, our findings define a mechanism for crosstalk between intrinsic and extrinsic cell death pathways.

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. In the present study, we report that mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. We show that the Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. Our results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis.

The POT1 (protection of telomeres) protein binds the single-stranded G-rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53-dependent growth arrest and an ATM-dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR-dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATMdependent DDR. Our results uncover a novel ATR-dependent DDR at telomeres that is normally shielded by POT1 binding to the single-stranded G-overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo.

Induction of wild-type p53 in mouse fibroblasts causes cell cycle arrest at the G1 phase, whereas coexpression of p53 and the protooncogene c-myc induces apoptosis. Although p53 transcriptional activity generally is required for both pathways, the molecular components mediating p53-dependent apoptosis are not well understood. To identify factors that could mediate p53-induced cell death, we used a comparative RNA differential display procedure. We have identified Pw1͞Peg3 as a gene product induced during p53͞c-myc-mediated apoptosis. Pw1͞Peg3 is not induced during p53-mediated G 1 growth arrest nor by c-myc alone. Although it is not clear whether the induction of Pw1͞Peg3 depends on p53 activity, we show that Pw1͞Peg3 interacts with a p53-inducible gene product Siah1a. We demonstrate that coexpression of Pw1͞ Peg3 with Siah1a induces apoptosis independently of p53 whereas expression of Pw1͞Peg3 or Siah1a separately has no effect on cell death. These data suggest that Siah1a and Pw1͞Peg3 cooperate in the p53-mediated cell death pathway. Furthermore, we show that inhibiting Pw1͞Peg3 activity blocks p53-induced apoptosis. The observation that Pw1͞Peg3 is necessary for the p53 apoptotic response suggests a pivotal role for this gene in determining cell death versus survival.