The Selective Antitumor Effect Of Adenovirus Mediated Double Suicide Gene System In Vitro

ObjectiveTo evaluate the selective killing effect of adenovirus(Ad) mediated double suicide gene with KDR promoter on human colorectal cancer LOVO cells. Two recombinant Adenoviral plasmid pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed in a "Two-step transformation protocol", The two plasmids were transfected into 293 packaging cells for amplification of the infectious Ad, and generated in the cells. The infectious viruses were used to infect KDR-expressing cells(ECV304 and LOVO) and the KDR-non-expressing cells(LS174T) respectively . Then The three cells were treated with 5-FC and /or GCV . Its killing effects on various kinds of cells and at different doses were evaluated with the aid of GFP expression. Moreover, the distribution of cell cycle was detected by flow cytometric assay. Methods 1. Recombinant adenovirus constructionKDR promoter sequence, CD gene sequence and TK gene sequence were generated by PCR protocol to construct pKDR-CDglyTK. ThenpAdKDR-CDglyTK were generated with AdEasy-1 system in a "two step transformation protocol". And pAdCMV-CDglyTK was constructed in the same way. The two recombinant adenoviral plasmids were then transfected to 293 packaging cells to grow Adenovirus, which were further multiplied and purified. The resultant recombinant adenoviruses were identified by PCR protocols. 2. Experiment of antitumor effect in vitroECV304 cells> LOVO cells and LS174T cells were infected with the recombinant adenoviruses at different MOI, and infection rate were measured with the aid of GFP expression visualized by fluorescence microscopy. Transgeneic cells were treated with different concentration of GCV and/or 5-FC, cell survival rate were measured 3d latter. Moreover the distribution of cell cycle(LOVO cells) was detected by flow cytometric assay. Results1. we constructed KDR promoter CD gene and TK gene successfully, and the resultant segment were sequenced and identified by Sangon Biotechnology (Shanghai) Co. Ltd.2. pAdKDR-CDglyTK and pAdCMV-CDglyTK were then transfected to 293 packaging cells to grow Adenovirus, which were further multiplied and purified. The resultant recombinant adenoviruses were identified by PCR protocols.3. ECV304> LOVO and LS174T cells were infected with the two recombinant adenoviruses: the infection rate were of no differernce, and it increasing with the increasing of the MOI of these viruses.4. The LOVO cells and ECV304 cells infected with pAdKDR-CDglyTK(MOI=100) were sensitive highly and similar to the prodrugs ( P>0.7ompared with the former two, the infected LS174T cells appeared to be unsensitive (P<0.001) .5. LOVO cells infected with pAdKDR-CDglyTK or pAdCMV-CDglyTK at MOI on 100 were maintained in culture medium of different concentration of GCV and 5-FC for 3d: All of them were sensitive highly and similar.6. The killing effect of CD/TK fusion gene (AdKDR-CDglyTK) on the target cells was much powerfully than that of either suicide gene(P< 0.001).7. The cell cycle of LOVO cells,which was infected with pAdKDR-CDglyTK and then treated with 5-FC and GCV ,was arrested at S phase.Conclusions1. The two-step transformation protocol is a method generating recombinant adenovirus more efficient and convenient than the traditional ones.2. Prodrug/KDR-CDglyTK system is effective in killing ECV304 and LOVO cells; its killing effect correlates to the concentration of the prodrugs and the recombinant Adenovirus's MOI.3. The killing effect of CD/TK fusion gene (AdKDR-CDglyTK) on the target cells was much powerfully than that of either suicide gene.4. The cell cycle of LOVO cells,which was infected with pAdKDR-CDglyTK and then treated with 5-FC and GCV ,was impacted markedly.5. The CD/TK fusion gene system driven by KDR promoter hasselectively killing effect on the KDR-expressing human LOVOcells and ECV304 cells .The result was as same as CMV promoter .And this would be provided an optional way in targeting gene therapyof colorectal neoplasms and its blood vessel.Note: the study was supported by biological and modern agriculture technology research foundation of "863 project" of China (2001AA217171) and natural science foundation of Guangdong province (013072) respectively.