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 inhibited 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 p 14 expression. (C) 2004 Elsevier Inc. All rights reserved.