A change of mitochondrial membrane permeability is essential for apoptosis, leading to translocation of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. We recently showed that the Bcl-2 family of proteins regulate cytochrome c release and the mitochondrial membrane potential (Δψ) by directly modulating the activity of the voltage-dependent anion channel (VDAC) through binding. Here we investigated the biochemical role of the conserved N-terminal homology domain (BH4) of Bcl-x _L , which has been shown to be essential for inhibition of apoptosis, with respect to the regulation of mitochondrial membrane permeability and found that BH4 was required for Bcl-x _L to prevent cytochrome c release and Δψ loss. A study using VDAC liposomes revealed that Bcl-x _L , but not Bcl-x _L lacking the BH4 domain, inhibited VDAC activity. Furthermore, BH4 oligopeptides of Bcl-2 and Bcl-x _L , but not mutant peptides, were able to inhibit both VDAC activity on liposomes even in the presence of Bax and apoptotic Δψ loss in isolated mitochondria. It was also shown that the BH4 domain, fused to the protein transduction domain of HIV TAT protein (TAT-BH4), efficiently prevented apoptotic cell death. These results indicate that the BH4 of Bcl-2/Bcl-x _L is essential and sufficient for inhibiting VDAC activity, which in turn prevents apoptotic mitochondrial changes, and for preventing apoptotic cell death. Finally, the data suggest that the TAT-BH4 peptide is potentially useful as a therapeutic agent for diseases caused by accelerated apoptosis.

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.

TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32°C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2^F/− mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37°C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37°C was rapid and reversible, permitting induction of short periods (3–6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.