p53 enhances the Δ-24 conditionally replicative adenovirus anti-glioma effect

Abstract

Abstract. Previous studies have demonstrated that the conditionally replicative adenovirus Ad5¢24 is a powerful cytolytic agent against glioma selectively affecting cells with a defective p16/Rb/E2F pathway. The p53 protein is also known to be an apoptotic factor for glioma cells. In this study, we examined the simultaneous delivery of the combination of exogenous p53 and Ad5¢24 adenovirus in glioma cells. Infecting cells with low doses of adenovirus p53 and Ad5¢24 resulted in an additive effect on cell death. The cell death induced by both agents was independent of the p53 status of cells. Flow cytometry revealed that the potent anti-tumor effect induced by the mixture of Ad5CMV-p53 and Ad5¢24 adenoviruses was due to a combination of apoptosis and cell lysis. Our results indicate that Ad5CMV-p53 enhances the oncolytic effect of the Ad5¢24 adenovirus, and the combination of adenovirus Ad5¢24 and Ad5CMV-p53 may thus be a potential therapeutic tool for gliomas. IntroductionMalignant gliomas are the most common primary brain tumors in humans, and highly resistant to all current therapies, including widely investigated approaches to gene therapy. At the molecular level, glioblastomas are highly heterogeneous tumors, as several populations of cells with different gene abnormalities co-exist within a given tumor (1). Secondary glioblastomas that develop from pre-existing low-grade astrocytomas frequently contain p53 mutations (1), and abnormalities of the p16/Rb/E2F pathway are present in most gliomas (2,3).Previous studies have demonstrated that the transfer of exogenous wild-type p53 cDNA causes apoptosis in glioma cells expressing endogenous mutant p53, but not in cells expressing endogenous wild-type p53 (4,5). However, only 50% of gliomas exhibit mutant p53, and the mutant cell population usually constitutes <30% of tumor mass (6).More than 90% of malignant gliomas exhibit abnormalities in the p16/Rb/E2F pathway (7). The adenovirus Ad5¢24 carries a 24-bp deletion in the E1 region corresponding to amino acids 122-129 specific for binding the Rb protein. This virus produces an anti-glioma effect in vitro and in vivo in tumors with altered p16/Rb/E2F pathway (8). The conditionally replicative adenoviruses (e.g. Ad5¢24 and ONYX-15) kill tumor cells selectively, and their replication leads to amplification of their oncolytic potential. Considerable preclinical data support this notion (8)(9)(10)(11). In this study, we tested if the combination of an apoptotic molecule (p53) with an oncolytic adenovirus (Ad5¢24) could have an additive effect on cell death in malignant glioma cells. Materials and methodsCell lines, adenoviral vectors, and infection conditions. The human glioma cell line U-251 MG was obtained from Dr W.K. Alfred Yung (Department of Neuro-Oncology, M.D. Anderson Cancer Center, Houston, TX). U-87 MG was obtained from ATCC (Manassas, VA) and HFK (human kidney fibroblasts) from Dr T. Fotsis (Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina, Greece). The conditionally replic...

Natural and genetically engineered viral agents for oncolysis and gene therapy of human cancers

“…In human brain tumor cells (glioblastoma, medulloblastoma) the E2F-1 promoter and in human melanoma cells the tyrosinase enhancer/promoter (hTyrE/P) drive the replication of the Ad-Delta24 viruses [415]. Ad5CMV--p53 and Ad5Delta24 doubly infected glioma cells were killed more efficiently than with either one of the viruses applied singly [255]. In killing glioblastoma cells, the topoisomerase antagonist irinotecan (CPT-11), or temozolomide, and an Ad-Delta24 derivative acted additively/synergistically [127].…”

Potential adenovirus-mediated gene therapy of glioma cancer

“…Replacement of the p53 tumor suppressor gene is another rational approach to the management of malignant gliomas, since it is frequently mutated or inactivated in this cancer (Ito et al 2005;Nashimoto et al 2005;Gil-Perotin et al 2006;Mitlianga et al 2006;Senatus et al 2006). However, the transfer of exogenous wild-type p53 cDNA could cause apoptosis in glioma cells expressing endogenous mutant p53, but not in cells expressing endogenous wild-type p53 (Gomez-Manzano et al 1997).…”

“…However, the transfer of exogenous wild-type p53 cDNA could cause apoptosis in glioma cells expressing endogenous mutant p53, but not in cells expressing endogenous wild-type p53 (Gomez-Manzano et al 1997). In vitro experiments showed that the combination of a replication-incompetent adenoviral vector serotype 5 (Adv5) carrying the apoptosis-inducing p53 gene and a cytolytic Adv (Adv5D24) specific to cells with a defective p16/Rb/ E2F pathway could enhance the oncolytic effect on gliomas (Mitlianga et al 2006). Recently, p53 upregulated modulator of apoptosis (PUMA) was identified as a p53-inducible pro-apoptotic molecule.…”

Viruses, gene therapy and stem cells for the treatment of human glioma

“…These results indicated that Ad5CMV-p53 enhanced the oncolytic effect of the Delta-24 adenovirus. 43 E1 transcomplementation of a replication-competent adenovirus-encoding p53 resulted in dramatic augmentation of cell killing and circumvented resistance to apoptosis obviating the potential utility of therapeutic transgene expression by a replicating adenovirus. 44 Evaluation of AdDelta24-p53 in comparison with its parent Delta-24, showed better efficiency and caused more frequent regression of glioma xenografts.…”