293. The Epithelial Glycoprotein-2 Promoter Controls Selective and Efficient Killing of Epithelium Derived Cancer Cells

Abstract

Top of pageAbstract Replication-competent adenoviruses have oncolytic properties, but this oncolytic effect is insufficient for successful cancer therapy. In order to improve the anti-tumor activity, a replication-competent adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle was previously constructed. p53 expression from this vector significantly improved tumor cell killing and viral spread (Hum Gen Ther 2002: 13, 859). However, the full extent of this effect may be limited by the expression of inhibitory adenoviral and cellular proteins. The goal of the current work is to further enhance the anti-tumor activity of the p53-expressing replicating vector. The adenoviral E1b-55kD protein and cellular mdm2 have been shown to bind and inactivate p53. E1b-55kD is an early viral protein and p53 expression from Adp53rc has been engineered to peak late in the viral life cycle. However, the effect of p53 may still be limited by an interaction with E1b-55kD and mdm2. We hypothesized that modification of the p53 transgene to prevent binding to E1b-55kD and mdm2 would further improve the oncolytic effect of the vector. Therefore, a new vector (Adp53 Δ23) that is identical to Adp53rc except for a change of amino acid 23 (from tryptophan to serine) within the E1b-55kD/mdm2 binding region of the p53 transgene was constructed. In contrast to wild-type p53, the modified p53 protein expressed from this vector did not co-precipitate with E1b-55kD. In addition, the half-life of the modified p53 protein was prolonged. After viral infection with Adp53rc and Adp53 Δ23 for 24 hours, new protein production was inhibited with cycloheximide and p53 levels were analyzed by immunoblotting. Wild-type p53 was rapidly degraded and almost undetectable at 1 hour. In contrast, the levels of the modified p53 did not substantially change for at least 24 hours. Expression of the modified p53 protein did not impair effective virus propagation. Replication of Adp53 Δ23, measured by a modified plaque assay, was similar to replication of Adp53rc and an identical p53-negative control virus (Ad-co). Survival of three cancer cell lines (H1299, A549, H116), evaluated by WST-1 assay, was substantially decreased after infection with Adp53 Δ23 compared to Adp53rc and Ad-co. In conclusion, modification of the p53 transgene of a replication-competent adenovirus improves p53 stability and cancer cell killing without impairment of viral replication.