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Systematic genetic dissection of p14(ARF)-mediated mitochondrial cell death signaling reveals a key role for p21(CDKN1) and the BH3-only protein Puma/bbc3

Item Type:Article
Title:Systematic genetic dissection of p14(ARF)-mediated mitochondrial cell death signaling reveals a key role for p21(CDKN1) and the BH3-only protein Puma/bbc3
Creators Name:Hemmati, P.G. and Mueer, A. and Gillissen, B. and Overkamp, T. and Milojkovic, A. and Wendt, J. and Doerken, B. and Daniel, P.T.
Abstract: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.
Keywords:p14(ARF), Puma, p21(CDKN1), p53, Apoptosis, Mitochondria, Animals
Source:Journal of Molecular Medicine
ISSN:0946-2716
Publisher:Springer (Germany)
Volume:88
Number:6
Page Range:609-622
Date:June 2010
Official Publication:https://doi.org/10.1007/s00109-010-0606-5
PubMed:View item in PubMed

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