Enhancing Radiosensitivity Of Human Lung Adenocarcinoma A549 Cells By A Synthetic Radio-inducible Synchronizing Positive Feedback Gene Circuit Based On The Nitric Oxide Signaling Pathway

Background and objectiveIonizing radiation is currently used in the treatment of many tumors and its potential for eliciting the expression of exogenous therapeutic genes (radiogenetic therapy) is of considerable interest. One of the problems associated with the use of ionizing radiation to regulate targeted gene expression is that the response is transient and increased gene expression is thus only maintained for a brief period after the radiation stimulus is withdrawn, resulting in unsatisfied and disappointed therapeutic outcomes.Previous researches have shown that the positive feedback gene circuits can increase the expression level of interested genes, and the synchronization of genetic circuits can further enhance the efficacy of gene therapy. In order to obtain an enhanced and prolonged gene expression in target cells, we constructed a radiation controlled positive feedback genetic circuits via linking the c-fos promoter with the iNOS(Inducible nitric oxide synthase) cDNA, which can be synchronized by nitric oxide(NO). Ultimately, the efficacy of radio-genetic therapy for cancer will be improved.MethodsTo construct the synchronizing gene circuit, vectors pfos-iNOS/GFP, pfos-TK/GFP and pfos-iNOS/TK were generated by inserting c-fos promoter into the backbone vector pIRES2-EGFP replacing the CMV promoter, and subcloning full-length iNOS cDNA or HSV-TK cDNA into the MCS sites downstream the c-fos promoter. The constructed plasmids were then downloaded into A549 lung cancer cells using the transfect reagent lipofectamine. Outputs of green fluorescent protein(GFP), NO, iNOS and TK in the treated cells were observed and analyzed after being exposed to ionizing radiation. Expression of iNOS was detected by Western blot after adding various concentrations of NO donor SIN.The sensitivity of transfected cells to GCV was observed by MTT with or without radiation.Various doses of ionizing radiation were delivered to transfected and untransfected cells to figure out dose-survival curves.TUNEL was used to determine the ratios of apoptosis in cells in the presence of GCV with or without radiation.The clones transfected with recombinant vectors were singled out under the selection pressure of 500μg/mL G418-sulfate.The sensitivity of transfected cells to GCV and ionizing radiation in vivo was analyzed by A549 tumor xenograft models in nude mice BALB/C.Expression of TK in xenograft tumor was detected by immunohistochemistry. ResultsThe gene circuits-related plasmids were successfully constructed being verified by restriction enzyme digestion analysis. The outputs of GFP,NO and iNOS in the cells transfected with pfos-iNOS/GFP were markedly increased post irradiation with comparison of the control cells, the peak level was seen at 16-hour point post-irradiation.The variance coefficient of the expression of GFP among cells transfected with pfos-iNOS/GFP was less than that of control group post irradiation, which demonstrated that the synchronizing expression of GFP had been attained by the synthetic circuit presented here.The expression of TK in A549/pfos-iNOS/GFP was dramatically increased by the constructed gene circuit both in vitro and vivo with an apex at 16-hour post irradiation . The IC50 results indicated that A549/ pfos-iNOS/TK was much more sensitive to GCV and irradiation with comparison of A549/ pfos-TK/GFP and untransfected A549 exposed to 2Gy X-ray irradiation .TUNEL test showed that higher apoptosis ratios were found in A549/ pfos-iNOS/TK in the presence of GCV with or without radiation.Further study in nude mice demonstrate that the synthetic gene circuits transfected tumor xenografts were much more sensitive to radiation evaluated by TCD50.ConclusionIn present study, we have successfully developed a positive feedback genetic circuit composed by integrating c-fos promoter and the NO signaling pathway, which allows the synchronized expression of target gene by secreting the intercellular messenger NO. The synthetic genetic circuits presented here can markedly increase the expression level of target gene HSV-TK both in vitro and vivo test ,with significant enhancement of radiosensitivity in A549 lung cancer.Hence,this study might benefit the rdio-genetic therapy for lung cancer in further clinical investigation.