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

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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.

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Acknowledgments

This work was supported by the Deutsche Krebshilfe Grant 10-2088-Da3 to PTD and PGH. HCT116 cells and mutants were generously provided by Dr. Bert Vogelstein, Johns Hopkins Cancer Center, Baltimore, USA.

Disclosure statement

All authors have read and approved the final version of the manuscript. None of the authors has any type of financial interest to disclose.

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Authors and Affiliations

  1. Department of Hematology, Oncology and Tumor Immunology, Charité—Universitätsmedizin Berlin, Campus Berlin-Buch, Berlin, Germany

    Philipp G. Hemmati, Annika Müer, Bernd Gillissen, Tim Overkamp, Ana Milojkovic, Jana Wendt, Bernd Dörken & Peter T. Daniel

  2. Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany

    Philipp G. Hemmati, Bernd Gillissen, Bernd Dörken & Peter T. Daniel

  3. Clinical and Molecular Oncology, Charité—Universitätsmedizin Berlin, Campus Virchow Klinikum, Augustenburger Platz 1, 13353, Berlin, Germany

    Peter T. Daniel

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  1. Philipp G. Hemmati
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Correspondence to Peter T. Daniel.

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Philipp G. Hemmati and Annika Müer contributed equally to this paper.

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Hemmati, P.G., Müer, A., Gillissen, B. et al. Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3. J Mol Med 88, 609–622 (2010). https://doi.org/10.1007/s00109-010-0606-5

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