title: Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis.
abstract: The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.
authors.affiliation: Department of Hematology, Oncology and Tumor Immunology, Charité-Campus Berlin-Buch, Humboldt University, Lindenberger Weg 80, 13125 Berlin-Buch, Germany.
authors.forename: Philipp G
authors.initials: PG
authors.lastname: Hemmati
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gillissen
authors.forename: Clarissa
authors.initials: C
authors.lastname: von Haefen
authors.forename: Jana
authors.initials: J
authors.lastname: Wendt
authors.forename: Lilian
authors.initials: L
authors.lastname: Stärck
authors.forename: Dilek
authors.initials: D
authors.lastname: Güner
authors.forename: Bernd
authors.initials: B
authors.lastname: Dörken
authors.forename: Peter T
authors.initials: PT
authors.lastname: Daniel
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D001859:Bone Neoplasms / Q000473:pathology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D008928:Mitochondria / Q000502:physiology; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000502:physiology; D012516:Osteosarcoma / Q000473:pathology; D011471:Prostatic Neoplasms / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014407:Tumor Cells, Cultured; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000502:physiology*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
chemical_list: C494826:BAX protein, human; C494811:BCL2L1 protein, human; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein