Induction of oxidants distinguishes susceptibility of prostate carcinoma cell lines to p53 gene transfer mediated by an improved adenoviral vector: Beyond the abstract
P53 is an obvious choice as the therapeutic gene since it is one of the most important tumor suppressors and has long been considered the “guardian of the genome”.2 Except for Gendicine, progress in the field of p53 gene therapy has been limited. As applied to prostate carcinoma (PCa), p53 gene transfer has not been tested clinically even though its application in PCa cell lines was first reported more than 20 years ago 3, 4. Though many factors may have contributed to this situation, one central issue is the reliability of the gene transfer system at both the transductional and transcriptional levels.
Our group has developed adenoviral vectors that feature improvements that should facilitate virus entry as well as promote high levels of expression of the gene of interest. We developed a p53-responsive promoter (called PG), which directs the transgene expression in the presence of p53, and have shown the utility of the PG promoter in several vector platforms.5, 6, 7 When the PG promoter is employed to direct the expression of p53, a positive feed-back loop is established that induces high levels of p53.8 We introduced this expression cassette into an adenoviral vector and observed high levels of p53 expression in prostate carcinoma cell lines whereas a vector similar to Gendicine was quite limited in its ability to express p53.9 Our improved vector with autoregulated expression of p53 (AdPG-p53) was also superior in killing PC3 prostate carcinoma cells in vitro and in vivo as compared to the traditional rAd-p53 vector. However, PC3 cells were quite difficult to transduce. With this study we achieved high-level expression of p53, but were frustrated by the limited tropism of this adenoviral vector.
In order to surpass this limitation we have employed a fiber modified adenoviral vector (AdRGD), with the insertion of an RGD motif, directing the viral particle to the ubiquitous integrin receptor. In our recent study, we showed increased transduction efficiency and high levels of transgene expression in prostate carcinoma cell lines when using the AdRGD platform.10 We then showed that the adenoviral vector with improved transduction efficiency and autoregulated expression of p53 (AdRGD-PGp53) conferred even higher levels of p53 protein as compared to our AdPG-p53 vector. The new AdRGD-PGp53 vector was also shown to be superior for the induction of cell death as compared to the AdPG-p53 vector in PC3 cells.
We then explored the possible mechanisms responsible for the cell killing associated with the exceptionally high levels of p53 expression. Since many cell death mechanisms converge on the generation of oxidants, especially radical oxygen species (ROS) that damage DNA, we looked for these indicators of the cellular response upon treatment with the new AdRGD-PGp53 vector. Indeed, we observed accumulation of superoxide and peroxide only when PC3 cells were treated with the new vector, yet treatment with catalase or an inhibitor of NOX1 reduced cell killing, revealing the importance of ROS in the response to our gene therapy approach. Upon treatment with AdRGD-PGp53, the induction of oxidants correlated with reduced mitochondrial membrane potential and accumulation of phosphorylated H2AX.
We then explored the impact of gene therapy on the expression of key cellular genes. Strikingly, expression of the NOX-1 gene, an important factor in the production of ROS, was markedly increased only in the presence of AdRGD-PGp53 in PC3 cells. Note that NOX1 is not a known p53 target, a point that may be further explored in future studies. We also showed that the new, improved vector was especially effective for the induction of known p53 target genes (p21, Sestrin2, NOXA and PIG3).
When applied in a xenograft mouse model of in situ gene therapy, our vector retarded tumor progression and increased overall survival significantly. Upon treatment with AdRGD-PGp53, cell death was induced and was correlated with signs of DNA damage (phosphorylated H2AX) induced, presumably, by oxidative stress. These assays indicate that our new vector has a superior capacity to kill prostate cancer cells in vitro and in vivo by a mechanism that involves the production of oxidants.
While AdRGD-PGp53 maximizes transductional and transcriptional mechanisms, overcoming limitations associated with other p53-expressing adenoviral vectors, it did not halt tumor progression. Thus, further refinements, such as alteration of the treatment regime and association with chemotherapeutics, may offer even better control over tumor progression. Clearly, additional work is required before proposing pre-clinical evaluation of our approach. However, we have made a considerable advance in the design and study of virus-mediated gene therapy in a model of prostate carcinoma.
Written by: Rodrigo E. Tamura and Bryan E. Strauss
References:
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