Adenovirus-Mediated p14^ARFGene Transfer Cooperates with Ad5CMV-p53 to Induce Apoptosis in Human Cancer Cells

Abstract

p14(ARF), a product of the INK4A/ARF locus, induces p53 upregulation by neutralizing the effects of MDM2, a transcriptional target of p53 that antagonizes its function. Here we report that adenovirus-mediated p14(ARF) gene transfer leads to the accumulation of ectopically transduced p53 and to apoptosis in human cancer cells. We constructed an adenoviral vector expressing p14(ARF) (Ad-ARF) and examined its synergistic effect with p53-expressing adenovirus (Ad5CMV-p53 or Ad-p53) in human lung and esophageal cancer cells. Simultaneous Ad-ARF and Ad-p53 infection increased p53 protein levels not only in a wild-type p53-expressing cell line, but also in cell lines with deleted p53. This resulted in a significant in vitro cytotoxicity compared with Ad-p53 infection alone. Coinfection of Ad-ARF and Ad-p53 also resulted in an increase in expression of p53-inducible genes, including p21(WAF-1/Cip1), p53R2, and Noxa. In addition, the growth of human lung cancer tumors subcutaneously implanted into nu/nu mice was inhibited significantly by intratumoral injection with Ad-ARF and Ad-p53. Our data demonstrate that overexpression of ectopic p14(ARF) may render cells more sensitive to p53-mediated apoptosis, an outcome that has important implications for the treatment of human cancers.

Strategies for reversing drug resistance

“…Importantly, we lack data on whether these various proteins are coregulated, and how they might interact. Despite our limited insight, numerous investigators have shown that levels of p53 above a certain 'threshold' almost invariably lead to apoptosis or to enhanced drug sensitivity (Blagosklonny and el-Deiry, 1998;Waugh et al, 1999;Boulay et al, 2000;Tango et al, 2002). While this is not conclusive proof that p53 has a central role in mediating drug sensitivity, it suggests at least an ancillary role.…”

DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

“…Various approaches to restore the pathway include gene transfer of p53 (Edelman et al 2003;Peng 2005), gene transfer of p53 plus ARF (Lu et al 2002;Tango et al 2002;Huang et al 2003), small molecules to restore the function of mutant p53 (Wang et al 2003), small molecule inhibitors of the p53/mdm2 interaction Vassilev et al 2004), antisense oligonucleotide inhibitors of mdm2 expression , and small molecule inhibitors of the ubiquitin ligase activity of mdm2 (Yang et al 2005). Subnuclear compartmentalization of ARF may now provide an additional target for therapies to activate endogenous p53 and optimize other p53-based strategies.…”

Gene Therapy Progress and Prospects: cancer gene therapy using tumour suppressor genes

“…41 In the past 2 years, more interest has focused on p14 Arf . Several reports have demonstrated that adenoviral delivery of the p14 Arf gene is capable of inducing cell cycle arrest and apoptosis in a wide variety of tumour models [42][43][44][45][46][47] and can sensitize cells to chemotherapy. 48 Initial reports suggested that intact p53 pathways were required for p14 Arf -mediated cytotoxicity [47][48][49] and that cotransfection with wild-type p53 could enhance the p14 Arf effect.…”

“…48 Initial reports suggested that intact p53 pathways were required for p14 Arf -mediated cytotoxicity [47][48][49] and that cotransfection with wild-type p53 could enhance the p14 Arf effect. 46 It now appears that p14 Arf is capable of affecting proteins other than p53, such as E2F-1, 50 HIF1a 51 and topoisomerase I. 52 Cluster analysis of gene expression patterns in mouse embryo fibroblasts indicates that p19 ARF induces expression of both p53-dependent and -independent genes, the latter including members of the B-cell translocation family (Btg/Tob) that can inhibit proliferation in cells regardless of p53 status.…”