Euromedim 2006 :1st European Conference on Molecular Imaging Technology

Molecular Switch of Cre/loxP for Radiation Modulated Gene Therapy on Hepatoma

11 May 2006, 14:00

1h

Palais du Pharo, Marseille

poster Molecular Imaging needs for biologists and physicians Poster session : Imaging systems, Molecular Imaging

Speaker

Ms Ya-ju Hsieh (Institute of Radiological Sciences, National Yang-Ming University, Taiwan)

Description

Alpha-fetal protein (AFP) is a specific tumor marker for hepatocellular carcinoma (HCC). The gene expression system under AFP promoter/enhancer control would be specific for AFP producing cells, but its low expression level is a problem to be overcome. For the purpose of AFP promoter enhancement for the use of radiation modulated gene therapy, we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to target cells to control the Cre/loxP system. Different gene constructs, such as pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed. HepG2, HeLa and NIH- 3T3 cell lines were then transfected by using jet PEITM reagent. Luciferase assay and MTT technique were used to analyze the luciferase gene expression and Herpes simplex virus thymidine kinase (HSV1-Tk) gene function, respectively. Cells irradiation experiments with a dose range of 1~10 Gy were performed by a 60Co gamma irradiator. HSV1-Tk gene expression of stable clone implanted on the NOD/SCID mice was assessed by in vivo 131I-FIAU imaging. The E4 enhancer showed better response to radiation after 60 hours irradiation especially at a dose range of 5~7 Gy in pE4luc HepG2 stable clone. The EIIAPA promoter provides high specificity to hepatoma but not to non-hepatoma cell lines. It also activates the Cre downstream and removes the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC, and the removal of stop cassette let the HSV1-Tk gene expresss and subsequently kill the tumor cells. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC.