Experimental Study On Treatment Of Colon Cancer With E.Coli. Cytosine Deaminase Suicide Gene

1 The Construction of Targeted Recombinant Adenovirus Vector Containing E. Coli. Cytosine Deaminase Gene and Its Application. Objective To kill carcino-embryonic antigen (CEA) positive colorectal carcinoma cells specifically, a new replication-deficient recombinant adenoviral vector was constructed in which the E. Coll cytosine deaminase (EC-CD) suicide gene was controlled under CEA promoter and its in vitro and in vivo cytotoxic effects were evaluated.Methods Shuttle plasmid containing EC-CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay, in vitro cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC50) of 5-FC was calculated using a curve-fitting parameter. The expression of EC-CD mRNA of infected cells was determined with RT-PCR The human colorectal carcinoma cell lines, Lovo and HCT1116BG cell, which were CEA-producing, and the CEA-nonproducing cell lines, Hela and CT26 cell, were applied in in vitro cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the EC-CD gene was controlled by cytomegalovirus(CMV) promoter, was used as virus control. Lovo cells and CT26 cells were inoculated to Balb/c nu/nu nude mice subcutaneously(s.c.) followed by intratumoral injection of AdCEACD and AdCMVCD for EC-CD gene therapy respectively, 5-fluorocytosine(5-FC) was administered intraperitoneally(i.p.) for the next 15 days. The tumor volume among differentgroups was compared. The glutamic-pyruvic 1rarisarninase(ALT) and glutamic-oxaloacetic transarninase(AST) were measured and the proliferation of bone marrow was examined at the end of treatment. Quantitative results were expressed as the mean + SD of the mean. Statistical analysis was performed using one-way ANOVA test by SPSS 11.0 software.Results The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and EC-CD gene. Virus liter was about 5.0 x 1014pfu.L-1 after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD ( The IC50 values of 5-FC in parent Lovo cells, Lovo cells infected with 100 m.o.i AdCEACD and Lovo cells infected with 10 m.o.i AdCMVCD were 62498.11 +4426.06, 133.97 + 12.21 and 129.77 + 15.46 u mol.L-1, respectively, P=0.002). The cytotoxicity of 5-FC increased accordingly when the rao.i of adenoviruses were enhanced (The value of IC50 of 5-FC was reduced to 16.76 + 4.63 u mol.L-1 in 500 m.o.i AdCEACD infected Lovo cells and 19.95 +3.94 umol.L-1 in 100 mo.i AdCMVCD infected Lovo cells,p=0.001,p=0.008, respectively). No effect was found in CEA-nonproducing Hela cells infected with AdCEACD, but Hela cells infected with AdCMVCD had the cytotoxic sensitivity to 5-FC ( The IC50 of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 mo.i was 842.29+149.31 and 25.37 + 6.22 umol.L-1, respectively,p =0.01). which were similar to that of HCT1116BG cells and CT26 cells. AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. RT-PCR showed that EC-CD mRNA was expressed in Lovo cells infected with both AdCMVCD and AdCEACD, and in Hela cells infected with AdCMVCD, however, EC-CD mRNA was not detected in Hela cells infected with AdCEACD. Both AdCMVCD/5-FC and AdCEACD/5-FC systems retarded the development of Lovo tumor in nude mice(p<0.01), and CT26 tumor in mice was inhibited by AdCMVCD/5-FC system(p<0.01), but AdCEACD/5-FC system did not affect the growth of CT26tumor. Both AdCMVCD/5-FC and AdCEACD/5-FC treatments increased the values of ALT and AST in mice. The proliferation of bone marrow wa...