Construction and I-125 induced-expression of lentiviral vector carrying radiation-responsive promoter and CD gene in EJ cells

1214 Objectives Radionuclide therapy is widely used in tumor therapy. The radiotherapy efficacy may be enhanced and the side effects may be reduced due to less radioactive dose used by inducing radiation sensitive gene to tumor cells. In this study, the construction and I-125 induced-expression of lentiviral vector carrying radiation-responsive promoter and Cytosine Deaminase gene (CD gene) in bladder cancer EJ cells was investigated. Methods To package recombinant lentivirus, recombinant lentiviral vector carrying CD gene and Green Fluorescent Protein (GFP) reporter gene and using synthetic radiation-responsive promoter which contains eight CArG elements(E8) as their upstream regulation sequence was constructed and transfected into 293T cells with pHelper 1.0 vector and pHelper 2.0 vector. The titer of recombinant lentivirus was measured by Realtime PCR. EJ cells was infected by recombinant lentivirus and the lentivirus-infected EJ cells were exposed to different doses of I-125. The expression of GFP gene was observed and the ability to converting 5-Fluorocytosine (5-FC) to 5-Fluorourial (5-FU) was assayed by HPLC. Results The recombinant lentiviral vector was established successfully and the titer of recombinant lentivirus was 2×108TU/ml. Exposure of lentivirus-infected EJ cells to 125I, green fluorescence can be observed and 5-FU which was converted from 5-FC in cell supernatant can be detected. The green fluorescence was obviously observed at the dose of greater than 55.5kBq, and the ultraviolet peak of 5-FU was the sharpest at the dose of 148kBq. Conclusions The results indicated that synthetic promoter can induce the expression of downstream CD gene and reporter gene after exposure of 125I and provided a valuable contribution to the continued experiments on radionuclide 125I therapy combination with CD gene/5-FC suicide gene therapy of tumor