title: Radiation enhances the anti-tumor effects of vaccinia-p53 gene therapy in glioma.
abstract: The overall goal of this study was to analyze the effect and mechanism of radiation in combination with vaccinia viruses (VV) carrying the p53 gene against glioma. Comparison of two alternative treatments of cultured C6 (p53(+)) and 9L (p53(-)) rat glioma cells showed significantly reduced survival for both cell lines, especially 9L, when radiation was applied prior to virus versus radiation alone. High p53 protein expression mediated by VV-TK-p53 was measured in infected cells. Single modality treatment of C6 cells with psoralen and UV (PUV)-inactivated VV-TK-p53 (PUV-VV-TK-53) or radiation significantly decreased survival compared with PUV-inactivated L-15 (PUV-L-15) control virus. However, no difference was observed between radiation and combination treatments of C6 cells. In contrast, radiation followed by PUV-VV-TK-53 resulted in dramatic reduction of 9L cell viability, compared to single modality treatment. Flow cytometry analysis of Annexin-V-stained 9L cells showed that radiation and PUV-VV-TK-53 caused a significant decrease in live cells (17.2%) as compared to other treatments and control (61.6-98.3%). Apoptosis was observed in 37.2% of cells, while the range was 0.7-7.8% in other treatment groups; maximal p53 level was measured on day 7 post-infection. In athymic mice bearing C6 tumors, VV-TK-53 plus radiation in both single and multiple therapies resulted in significantly smaller tumors by day 30 compared to the agents given only once. Immunohistochemical analysis of tumor sections demonstrated p53 protein expression over 20 days after VV-TK-53 treatment. Analysis of blood and spleen cells of mice given multiple combination treatments showed significant splenomegaly, leukocytosis, and increased DNA synthesis and response to mitogen. Multiple combination treatments were also associated with significantly elevated natural killer and B cells in the spleen. There were no overt toxicities, although depression in red blood cell and thrombocyte parameters was noted. Collectively, the data demonstrate that radiation significantly improves the efficacy of VV-mediated tumor suppressor p53 therapy and may be a promising strategy for glioma treatment. Furthermore, the results support the conclusion that the mechanisms underlying the enhanced anti-tumor effect of combination treatment include apoptosis/necrosis and upregulation of innate immune defenses.
authors.affiliation: Center for Molecular Biology & Gene Therapy, Loma Linda University & Medical Center, School of Medicine, 11085 Campus Street, Loma Linda, CA 92350, USA.
authors.forename: Tatyana M
authors.initials: TM
authors.lastname: Timiryasova
authors.forename: Daila S
authors.initials: DS
authors.lastname: Gridley
authors.forename: Bing
authors.initials: B
authors.lastname: Chen
authors.forename: Melba L
authors.initials: ML
authors.lastname: Andres
authors.forename: Radha
authors.initials: R
authors.lastname: Dutta-Roy
authors.forename: Glen
authors.initials: G
authors.lastname: Miller
authors.forename: Erben J M
authors.initials: EJ
authors.lastname: Bayeta
authors.forename: Istvan
authors.initials: I
authors.lastname: Fodor
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D016923:Cell Death; D003131:Combined Modality Therapy; D016158:Genes, p53; D015316:Genetic Therapy / Q000379:methods*; D005910:Glioma / Q000473:pathology / Q000532:radiotherapy / Q000628:therapy* / Q000821:virology; D007694:Killer Cells, Natural / Q000276:immunology; D051379:Mice; D008819:Mice, Nude; D011878:Radiotherapy / Q000379:methods*; D051381:Rats; D013154:Spleen / Q000276:immunology; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay / Q000379:methods; D014616:Vaccinia virus / Q000276:immunology*