Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3

Induction of cell death by p14 ARF is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21 CDKN1 . Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21 CDKN1 or p53-reconstituted DU145 prostate cancer cells, we show that p14 ARF -induced apoptosis is attenuated in the absence of Puma. Upon expression of p14 ARF in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14 ARF -induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma . The sensitivity of Puma-deficient cells to p14 ARF -induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21 CDKN1 strongly enhances p14 ARF -induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14 ARF -induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14 ARF -triggered apoptosis is enhanced by loss of p21 CDKN1 -mediated cell cycle checkpoint control.