CCAAT Box, Not CArG Motifs, Identify Surviving as a Highly Potent Radiation Inducible Promoter

Abstract

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. Some previous studies concluded that CArG motifs are the elements responsible for radiation mediated transcription regulation, while other concluded that CCAAT box consensus are present in low frequencies in radioresponsive genes in comparison to normal genes. Using bioinformatics we aim to detect mechanism of radiation mediated transcription regulation in the context of glioblastoma adenoviral gene therapy. The effect of radiation was tested on 6 different in-house promoters (iNOS, FLT-1, DR5, Cox-2, VEGF, and survivin) where levels of mRNA expression of these promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by 7 different promoters; CMV, VEGF, FLT-1, DR5, iNOS, cox-2 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 (0-12Gy) at single fraction. Fluorescent Microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. A time course experiment was carried out to look for effect of time of radiation on gene expression. These promoters were screened for the presence CArG motifs and CCAAT box consensus using NCBI Blast bioinformatics software. Radiotherapy increase the expression of gene expression by1.75-fold in DR5, 2-fold in VEGF and 0 fold Flt-1 after 2 hr of radiation. Most importantly Cells treated with RT and Ad Luc driven by Survivin promoter showed 5-fold increase in expression after 2 Gy of radiation in compare to non-irradiated cells. RNA analysis was done and has shown increase copy no. of corresponding genes in level range from 2 fold for VEGF 3 fold for iNOS and as expected 30 fold more for Survivin. Using NCBI BLAST software, none of CArG motifs were found in the different 6 promoters. Survivin with the best response to radiation had the lowest number of CCAAT box (n=2) while iNOS with the lowest response to radiation had the highest number of CCAAT (n=14) with other promoters having different numbers of CCAAT ranging between these two values. Additionally, the initiator element consensus (YYANWYY) was not found in all the responsive promoters to radiation. Survivin as a radiation inducible promoter for glioblastoma viral gene therapy is independent of CArG motifs. Low frequency of CCAAT box could be responsible for this phenomena.