Induction of Oxidants Distinguishes Susceptibility of Prostate Carcinoma Cell Lines to p53 Gene Transfer Mediated by an Improved Adenoviral Vector.

Abstract

Previously, the authors developed an adenoviral vector, Ad-PG, where transgene expression is regulated by a p53-responsive promoter. When used to transfer the p53 cDNA, a positive feedback mechanism is established. In the present study, a critical comparison is performed between Ad-PGp53 and AdRGD-PGp53, where the RGD motif was incorporated in the adenoviral fiber protein. AdRGD-PGp53 provided superior transgene expression levels and resulted in the killing of prostate carcinoma cell lines DU145 and PC3. In vitro, this effect was associated with increased production of cytoplasmic and mitochondrial oxidants, DNA damage as revealed by detection of phosphorylated H2AX, as well as cell death consistent with apoptosis. Differential gene expression of key mediators of reactive oxygen species pathways was also observed. Specifically, it was noted that induction of known p53-target genes Sestrin2 and PIG3, as well as a novel target, NOX1, occurred in PC3 cells only when transduced with the improved vector, AdRGD-PGp53. The participation of NOX1 was confirmed upon its inhibition using a specific peptide, resulting in reduced cell death. In situ gene therapy also resulted in significantly improved inhibition of tumor progression consistent with oxidant-induced DNA damage only when treated with the novel AdRGD-PGp53 vector. The study shows that the improved adenovirus overcomes limitations associated with other p53-expressing vectors and induces oxidant-mediating killing, thus supporting its further development for cancer gene therapy.