title: Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3.
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.
authors.affiliation: Department of Hematology, Oncology and Tumor Immunology, Charité-Universitätsmedizin Berlin, Campus Berlin-Buch, Berlin, Germany.
authors.forename: Philipp G
authors.initials: PG
authors.lastname: Hemmati
authors.forename: Annika
authors.initials: A
authors.lastname: Müer
authors.forename: Bernd
authors.initials: B
authors.lastname: Gillissen
authors.forename: Tim
authors.initials: T
authors.lastname: Overkamp
authors.forename: Ana
authors.initials: A
authors.lastname: Milojkovic
authors.forename: Jana
authors.initials: J
authors.lastname: Wendt
authors.forename: Bernd
authors.initials: B
authors.lastname: Dörken
authors.forename: Peter T
authors.initials: PT
authors.lastname: Daniel
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D045325:HCT116 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D020169:Caspases