Growth suppression by a p14ARF exon 1β adenovirus in human tumor cell lines of varying p53 and Rb status

Abstract

We have analyzed the ability of an adenoviral vector encoding the exon 1b region of the p14 ARF tumor suppressor ( ARF ) to suppress the growth and viability of an array of tumor cell lines of various origins and varying p53 and Rb status, in order to establish the clinical potential of ARF. An important activity of ARF is regulation of p53 stability and function through binding to the mdm2 protein. By sequestering mdm2, ARF may promote growth suppression through the Rb pathway as well because mdm2 can bind to Rb and attenuate its function. Whereas the high frequency of ARF gene deletion in human cancers, accounting for some 40% of cancers overall, suggests that ARF would be a strong candidate for therapeutic application, the possible dependence of ARF activity on p53 and Rb function presents a potential limitation to its application, as these functions are often impaired in cancer. We show here that a replication -defective adenovirus, Ad1b, encoding the exon 1b region of ARF is most effective in tumor cells expressing endogenous wild -type p53. Nevertheless, Ad1b suppresses tumor cell growth and viability in vitro and in vivo, inducing G1 or G2 cell cycle arrest and cell death even in tumor cells lacking both functional Rb and p53 pathways, and independently of induction of the p53 downstream targets, p21, bax, and mdm2. These results point to an activity of ARF in human tumor cells that is independent of Rb or p53, and suggest that therapeutic applications based on ARF would have a broad clinical application in cancer.

Siva1 inhibits p53 function by acting as an ARF E3 ubiquitin ligase

“…These data indicate that Siva1 regulates p53 expression in an Mdm2-dependent manner. As ARF and p53 are frequently mutated in cancer cells, generally in a mutually exclusive manner 20 , we next used a primary human fetal lung fibroblast cell line to further confirm the effect of Siva1 on the ARF-p53 pathway. As shown in Fig.…”

“…The direct binding of Siva1 to ARF appears to be required for the rapid degradation of ARF, as supported by the finding that the mutant Siva1DC, which is unable to interact with ARF, has no effect on the stability and ubiquitination of ARF. Additionally, we show that the N-terminal domain (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] of ARF is involved in its interaction with Siva1, but is not essential for this association. As this N terminus (amino acids 2-14) has been well recognized as a critical region for binding to Mdm2, it is not surprising to see that Siva1 could compete with Mdm2 to bind to ARF.…”

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.…”

DNA Damage Disrupts the p14ARF-B23(Nucleophosmin) Interaction and Triggers a Transient Subnuclear Redistribution of p14ARF

“…Both cell lines express endogenous ARF (22). The MCF-7 subclone expresses endogenous wild-type p53 and lacks endogenous ARF (22). Cell lines were maintained as described previously (23).…”

“…Cell viability assays were carried out in 96-well plates as described previously (22,23). Briefly, cells grown in medium lacking phenol red were either treated with UV-C radiation using a Stratalinker (Stratagene, La Jolla, CA) and replated in 96-well plates at 10,000 cells per well or treated with adenoviral vectors for 3 hours (22) followed 1 day later by treatment with UV-C radiation and replating.…”