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- Title
Systematic genetic dissection of p14<sup>ARF</sup>-mediated mitochondrial cell death signaling reveals a key role for p21<sup>CDKN1</sup> and the BH3-only protein Puma/bbc3.
- Authors
Hemmati, Philipp G.; Müer, Annika; Gillissen, Bernd; Overkamp, Tim; Milojkovic, Ana; Wendt, Jana; Dörken, Bernd; Daniel, Peter T.
- Abstract
Induction of cell death by p14ARF 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 p21CDKN1. Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21CDKN1 or p53-reconstituted DU145 prostate cancer cells, we show that p14ARF-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14ARF 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, p14ARF-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14ARF-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21CDKN1 strongly enhances p14ARF-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14ARF-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14ARF-triggered apoptosis is enhanced by loss of p21CDKN1-mediated cell cycle checkpoint control.
- Subjects
CYCLIN-dependent kinases; CELL death; APOPTOSIS; MESSENGER RNA; CANCER cells
- Publication
Journal of Molecular Medicine, 2010, Vol 88, Issue 6, p609
- ISSN
0946-2716
- Publication type
Article
- DOI
10.1007/s00109-010-0606-5