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Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs.

ABSTRACT: BACKGROUND:Radiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation. AIM:To select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation. METHODS:To select initial candidates for promoter inducible elements, the levels of mRNA expression of six different promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by five different promoters; CMV, VEGF, FLT-1, DR5 and survivin were constructed. Glioma cell lines were infected with different multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0-12 Gy) at single fraction. Fluorescent microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. Different glioma cell lines and normal astrocytes were infected with Ad survivin and exposed to radiation. The promoters were analyzed for presence of CArG radio-responsive motifs and CCAAT box consensus using NCBI blast bioinformatics software. RESULTS:Radiotherapy increases the expression of gene expression by 1.25-2.5 fold in different promoters other than survivin after 2 h of radiation. RNA analysis was done and has shown an increase in copy number of tenfold for survivin. Most importantly cells treated with RT and Ad Luc driven by survivin promoter showed a fivefold increase in expression after 2 Gy of radiation in comparison to non-irradiated cells. Presence or absence of CArG motifs did not correlate with promoter response to radiation. Survivin with the best response to radiation had the lowest number of CCAAT box. CONCLUSION:Survivin is a selective potent radiation inducible promoter for glioblastoma viral gene therapy and this response to radiation could be independent of CArG motifs.

SUBMITTER: Naoum GE

PROVIDER: S-EPMC5332320 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs.

Naoum George E GE Zhu Zeng B ZB Buchsbaum Donald J DJ Curiel David T DT Arafat Waleed O WO

Clinical and translational medicine 20170301 1

<h4>Background</h4>Radiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation.<h4>Aim</h4>To select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation.<h4>Methods</h4>To select initial candidates for promoter indu ...[more]

PMID: 28251571

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Project description:The aim of this study was to investigate the selective killing effect of the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro. Recombinant plasmid vectors driven by the survivin promoter were constructed. HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression was detected using RT-PCR and western blot analysis. The selective killing effects after GCV application were evaluated by tetrazolium assay, FCM and western blot analysis. Statistical analysis was performed by ANOVA. After transfection with GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in the HepG2 cells, but not in the L02 cells and TK gene expression was evidently detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were successfully established. Compared with the HepG2/TK/pSURV group, a significant 'bystander effect' was observed in the HepG2/TAT-TK/pSURV group with the incorporation of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly inhibited. This finding was further corroborated by FCM and immunoblot analysis revealed the repressed expression of proliferating cell nuclear antigen (PCNA). Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion protein gene driven by the survivin promoter were successfully constructed and their specific expression in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Dataset Information

Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs.

Publications

Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs.

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