Sustained angiopoietin-2 expression disrupts vessel formation and inhibits glioma growth.

Systematic analyses of the expression of angiogenic regulators in cancer models should yield useful information for the development of novel therapies for malignant gliomas. In this study, we analyzed tumor growth, vascularization, and angiopoietin-2 (Ang2) expression during the development of U-87 MG xenografts. We found that tumoral angiogenesis in this model follows a multistage process characterized by avascular, prolific peripheral angiogenesis, and late vascular phases. On day 4, we observed an area of central necrosis, a peripheral ring of Ang2-positive glioma cells, and reactive Ang2-positive vascular structures in the tumor/brain interface. When the tumor had developed a vascular network, Ang2 was expressed only in peripheral vascular structures. Because Ang2 expression was downmodulated in the late stages of development, probably to maintain a stable tumoral vasculature, we next studied whether sustained Ang2 expression might impair vascular development and, ultimately, tumor growth. Ang2 prevented the formation of capillary-like structures by and impaired angiogenesis in a chorioallantoic membrane chicken model. Finally, we tested the effect of sustained Ang2 expression on U-87 MG xenograft development. Ang2 significantly prolonged the survival of intracranial U-87 MG tumor-bearing animals. Examination of Ang2-treated xenografts revealed areas of tumor necrosis and vascular damage. We therefore conclude that deregulated Ang2 expression during gliomagenesis hindered successful angiogenesis and that therapies that sustain Ang2 expression might be effective against malignant gliomas.

Downmodulation of E1A protein expression as a novel strategy to design cancer-selective adenoviruses.

Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of E1A protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CR1 (Delta-39), CR2 (Delta-24) regions, or both regions (Delta-24/39) of the E1A protein. Delta-39 and Delta-24 induced a cytopathic effect with similar efficiency in glioma cells and a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, E1A protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CR1 mutant E1A proteins, and inhibition of the proteasome activity resulted in the striking rescue of E1A levels. We conclude that the level of E1A protein is a critical determinant of oncolytic phenotype and we propose a completely novel strategy for the design and construction of conditionally replicative adenoviruses.

Robust infectivity and replication of Delta-24 adenovirus induce cell death in human medulloblastoma.

The diverse advanced treatment modalities currently available to children with medulloblastoma, including surgery and radiotherapy, are associated with deleterious side effects and often with an unfavorable prognosis. A mutant adenovirus, Delta-24, which has a 24-base pair deletion in the Rb-binding region of the E1A gene, demonstrates selective replication and oncolysis in various malignant phenotypes. Here we report the ability of Delta-24 to kill medulloblastoma cells. Flow cytometric analyses of cell receptors demonstrated expression of the coxsackie adenovirus receptor and RGD-related integrins in the assessed medulloblastoma cell lines. Infectivity assays using a replication-deficient adenovirus to transduce the green fluorescence protein gene showed that the Delta-24 adenovirus infects 99% of Daoy and 46% of D283 Med medulloblastoma cells at a multiplicity of infection (MOI) of 50. Within 4 days after infecting medulloblastoma cells with Delta-24, a noticeable cytopathic effect was produced. Delta-24 induced a total cytopathic effect in Daoy and D283 Med medulloblastoma cells after 6 and 8 days of infection, respectively. In the infected population of cells, cell death correlated with the accumulation of cells in the S phase. At 5 days post-infection with 2.5 MOIs of Delta-24 adenovirus, the percentage of Daoy medulloblastoma cells in the S phase increased to 71.9+/-5.5%, compared with control values of 20.5+/-1.4%. The release of viral progeny was quantified as being increased by two orders of magnitude, indicating efficient replication of Delta-24 in medulloblastoma cells. This is the first report of the ability of oncolytic adenoviruses to infect and kill medulloblastoma cells, the findings of which suggest the potential efficacy of Delta-24 as a therapy for human medulloblastoma tumors.

A novel E1A-E1B mutant adenovirus induces glioma regression in vivo.

Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.

Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway.

BACKGROUND: Oncolytic adenoviruses are promising therapies for the treatment of gliomas. However, untargeted viral replication and the paucity of coxsackie-adenovirus receptors (CARs) on tumor cells are major stumbling blocks for adenovirus-based treatment. We studied the antiglioma activity of the tumor-selective Delta-24 adenovirus, which encompasses an early 1 A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, and of the Delta-24-RGD adenovirus. Delta-24-RGD has an RGD-4C peptide motif inserted into the adenoviral fiber, which allows the adenovirus to anchor directly to integrins. METHODS: CAR and integrin expression were examined by flow cytometry in six glioma cell lines and in normal human astrocytes (NHAs). Adenoviral vectors containing green fluorescent protein (GFP) (AdGFP and AdGFP-RGD) were used to infect glioma cell lines with high or low CAR expression. Viability of glioma cells infected with different adenoviruses was assessed by trypan blue staining. Adenovirus replication was quantified with the infection-dose replication assay. Athymic mice carrying glioma xenografts received intratumoral injections of Delta-24-RGD or Delta-24 and were followed for survival, which was analyzed by the Kaplan-Meier method and the log-rank test. All statistical tests were two-sided. RESULTS: Half the glioma cell lines expressed low levels of CAR (defined as <50% of cells expressing detectable CAR); all lines expressed integrins in more than 50% of cells. Infection of U-87 MG cells (a low-CAR-expressing line) with AdGFP-RGD resulted in approximately six times more GFP-positive cells than infection with AdGFP. Delta-24-RGD was more cytopathic to both low- and high-CAR-expressing glioma lines than Delta-24, and it replicated more efficiently in both cell lines. In the xenografted mice, intratumoral injection of Delta-24-RGD was associated with longer survival than intratumoral injection of Delta-24 (P<.001, log-rank test). Furthermore, 60% of Delta-24-RGD-treated mice but only 15% of Delta-24-treated mice survived more than 4 months (difference = 45%, 95% CI = 21% to 68%). CONCLUSIONS: The antitumor activity of Delta-24-RGD suggests that it has the potential to be an effective agent in the treatment of gliomas.