Apoptosis induced by adenovirus-mediated p14 ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression q Min Kim, a,b, * Magda Sgagias, b Xiyun Deng, c Yun-Jin Jung, d Toshiki Rikiyama, d Kwangmoon Lee, b Michel Ouellette, b and Kenneth Cowan b a Children’s National Medical Center, Washington, DC 20010, USA b Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA c Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, PR China d Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Received 10 May 2004 Available online 9 June 2004 Abstract The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14 ARF ). Adp14 ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24 h. In addition, Adp14 ARF -mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14 ARF infection demonstrated that the growth of U2OS cells was in- hibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14 ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14 ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14 ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14 ARF - mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14 ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression. Ó 2004 Elsevier Inc. All rights reserved. Keywords: p14ARF; Apoptosis; Fas; p53; Adenovirus The INK4a/ARF gene locus produces two transcripts which use alternative first exons, E1a and E1b, each of which is joined through the identical splice acceptor site to sequences in exon 2 [1,2]. The exon 1a–exon 2–exon 3 transcript encodes the CDK4/6-specific inhibitor p16 INK4a , while the exon 1b–exon 2–exon 3 transcript uses an alternative reading frame and encodes ARF. p16 INK4a and ARF have no homology at the amino acid level [3]. Translation from the mouse b cDNA yields a polypeptide of 169 amino acids with a molecular mass of 19 kDa (p19 ARF ) while human ARF protein is predicted to be 132 amino acids long with a molecular mass of 14 kDa. Mouse and human ARF polypeptides are 45% identical in exon 1b segments and 50% identical overall. Homozygous deletion of the INK4a/ARF locus, which causes a loss of both the INK4a gene and the ARF gene, occurs frequently in a variety of human cancers. Thus, determining the individual contributions of each of the two genes to tumor suppression has been difficult [4,5]. However, mice lacking p19 ARF but retaining an q Abbreviations: ARF, alternative reading frame; MDM2, mouse double minute 2; CMV, cytomegalovirus; pfu, plaque-forming unit; FACS, fluorescence-activated cell sorting; TUNEL, terminal deoxy- nucleotidyl transferase-mediated dUTP nick end labeling; FITC, fluorescein isothiocyanate. * Corresponding author. Fax: 1-202-884-5800. E-mail address: minkim@cnmc.org (M. Kim). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.135 Biochemical and Biophysical Research Communications 320 (2004) 138–144 BBRC www.elsevier.com/locate/ybbrc