The Experimental Study On Targeted Treatment Of Double Suicide Genes Controlled By MDR1 Promoter In Drug-Resistant Glioma Cells

Objective:Multidrug resistance(MDR)presented in some glioma cells is the major cause of chemotherapy-based treatment failure.The suicide gene therapy is presently considered to be available and applicable method.But how to improve the targeting ability and specificity is a key to kill tumor cells of suicide gene.MDR1 promotor of multidrug resistance gene has been chosen as the targeting promotor in suicide gene therapy due to its highly active feature.In this study,we will investigate the targeted killing effects of CD-TK double suicide gene controlled by MDR1 promoter with GCV and 5-FC in MDR glioma cell.Methods:The eukaryotic expression vector of CD-TK double suicide gene controlled by MDR1 promoter(pcDNA3.MDR1P.CD.TK)was transferred into C6/ADR cells through lipofectin and 500μg/ml genetiein(G418)was used to establish stable transferred tumor cell line.Transferred C6/CDTK cell and untransferred C6/ADR were chosen as control groups.After bolting,PCR method was used to test the integration of CD and TK suicide genes and RT-PCR was resorted to identify their expression in C6 and C6/ADR cells.After the addition of prodrugs,morphologic change of C6/ADR/CDTK cells were observed with light microscope,cell proliferation was determined by MTT assays,apoptosis was detected by TUNNEL assay,the cell cycle was examined by flow cytometry and cloning efficiency was detected by clone formation assay.Results:PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells.RT-PCR reveled that CD and TK genes expressed in C6/ADR/CDTK cells,whereas not in C6/CD-TK cells.After the addition of prodrugs,the light microscope demonstrated that cell endoparticle of C6/ADR/CDTK increased gradually,cell morphous were shrinking,the ability of adherence became weak,the growth condition went bad and the cell number decreased.MTT assay indicated that the inhibition of cell proliferation in C6/CDTK was slightly obvious compared with C6 cell;following increasing drug concentration,the inhibition of cell proliferation in C6/ADR/CDTK was more predominance than C6 and C6/ADR cells with no marked change;TUNNEL assays replayed that the result of C6/ADR/CDTK was positive and others were negative;flow cytometry manifested that the cell cycle of C6 and C6/ADR cells was mostly in S-stage;the number of C6/CDTK cell was a little more in G1-stage,and least in S-stage;the cell cycle of C6/ADR/CDTK stayed in G1-stage without DNA synthesis.Clone formation assay expressed that cloning efficiency of C6,C6/ADR and C6/CDTK cells went up to ninety percent above,but the cloning efficiency degraded significantly in C6/ADR/CDTK cell with increasing prodrug concentration. Conclusion:The PcDNA3.MDR1P.CDTK/GCV+5-FC system could kill the MDR cell(C6/ADR)much more than non-drug-resistant C6 cell and it was proved that it is targeted expression in MDR1 positive cells.We can believe that this study will provide a new consideration and method for treatment in MDR glioma.