The Study Of The In Vitro Targeted Killing Effects Of Pcdna3-Mdr1-Promoter-CD-TK/GCV+5-FC System On Mdr Positive Leukemia Cells

Objective To construct the eukaryotic expressive vector containing CD(cytosine deaminase) and TK(thymidine kinase) double suicide genes targeted by MDR1 promoter and investigate the targeted killing effects of pcDNA3-MDR1-Promoter-CD-TK /GCV+5-FC system on MDR1 positive leukemia cells K562-A02 in vitro, to explore a more efficient targeted gene therapy for MDR leukemia.Methods The DNA fragment of MDRl promoter was amplified through PCR from genomic DNA of K562-A02 cells and was linked with Nde â…  and Hind â…¢ restriction endonuclease sites, then was inserted into the upstream of sucide gene TK in the pcDNA3-TK plasmid with the same endonuclease sites. It is tested by the methods of electrophoresis and DNA sequencing. Both pcDNA3-MDR1-Promoter-TK and pcDNA3-CD-TK were digested by BamH I, and CD was inserted into pcDNA3-MDR1-Promoter-TK. It was confirmed through electrophoresis and DNA sequencing, and transferred into K562, K562-A02 cells through lipofectin. Stable transferred tumor cell line was established after geneticin(G418) selection, RT-PCR was resorted to identify the expression of CD and TK gene. The stable lines were respectively treated at varying concentrations of GCV and 5-FC in culture for 4 days. The in vitro cytotoxic effects of pcDNA3-MDR1-Promoter-CD-Tk/GCV+5-FC system were measured by MTT assay. The apopototic ratio was observed by flow cytometry.Results The length and sequence of MDRl promoter amplified by PCR were confirmed by electrophoresis and DNA sequencing. The length ,position and orientation of inserted MDR1 promoter in pcDNA3-TK were confirmed correct by the methods of PCR and DNA sequencing. After single enzyme digestion and purification ,CD gene fragment was cloned into pcDNA3-MDR1-Promoter-TK. Thelength ,position and orientation of inserted CD gene in pcDNA3-MDRl-Promoter-CD-TK were confirmed correct through PCR and DNA sequencing. The eukaryotic expressive vector pcDNA3-MDRl-Promoter-CD-TK containing CD(cytosine deaminase) and TK(thymidine kinase) double suicide genes targeted by MDR1 promoter was successfully constructed. After transfection, Double suicide genes were expressed in K562-A02 cell while K562 cells were negative. There was a greater degree of killing effect and apoptotic ratio in K562-A02 cells treated with pcDNA3-MDRl-Promoter-CD-TK /GCV+5-FC system comparing with K562 cells. ThepcDNA3-MDRl-Promoter-CD-TK/GCV+5-FC system was more efficient in MDR positive cells.Conclusion The expressive vector containing CD-TK double sucide genes driven by MDR1 promoter was constructed successfully and it may be beneficial for the target gene therapy of the multidrug resistant leukemia cells. The pcDNA3-MDRl-Promoter-CD-TK/GCV+5-FC system mediated by lipofectin has stronger killing effect on MDR positive leukemia cells K562-A02 than on K562. The experiment reveals that the suicide genes drived by MDR1 promoter can be employed for gene therapy approaches.Objective To construct and identify the eukaryotic expressive vector containing CD(cytosine deaminase) and TK(thymidine kinase) double suicide genes targeted by MDRl promoter.Methods The genomic DNA was extracted from MDRl positive leukemia cellline K562-A02.The MDRl promoter fragment spanning from -198 to +43 was isolated by PCR from genomic DNA.and linked with Nde I and Hind III endonuclease sites. After enzyme digestion and purification, The MDRl promoter fragment was inserted into the Nde I /Hind III sites of the pcDNA3-TK plasmid. It was tested by the methods of electrophoresis and DNA sequencing. Both pcDNA3-MDRl-Promoter-TK and pcDNA3-CD-TK were digested by BamH I, and CD gene was inserted into pcDNA3-MDRl-Promoter-TK to construct pcDNA3-MDRl-Promoter-CD-TK plasmid. Electrophoresis and DNA sequencing were also used to certificate whether CD(cytosine deaminase) and TK( thymidine kinase) double suicide genes were targeted by MDRl promoter.Results The length and sequence of MDRl promoter amplified by PCR were confirmed by electrophoresis and DNA sequencing. The length, position and orientation of inserted MDRl promoter in pcDNA3-TK were confirmed correct by the methods of PCR and DNA sequencing. After single enzyme digestion and purification ,CD gene fragment was cloned into pcDNA3-MDRl-Promoter-TK. The length ,position and orientation of inserted CD gene in pcDNA3-MDRl-Promoter-CD-TK were confirmed correct through PCR and DNA sequencing. The eukaryotic expressive vector pcDNA3-MDRl-Promoter-CD-TKcontaining CD(cytosine deaminase) and TK(thymidine kinase) double suicide genes targeted by MDRl promoter was successfully constructed.Conclusion The expressive vectors containing CD-TK double suicide genes targeted by MDRl promoter was constructed successfully and it may be beneficial for the targeted gene therapy of the multidrug resistant leukemia cells.Objective To study the in vitro killing effects of CD-TK double sucide genes/GCV+5-FC system targeted by multidrug resistance gene promoter on leukemia cells.Methods Detect the P-glycoprotein and MDR1 mRNA of the MDR positive leukemia cells and the negative cells. The expressive vectors pcDNA3-MDRl-Promoter-CD-TK plasmid was transfected into different leukemia cells using lipofectin. Stably transferred tumor cell line was established after geneticin(G418) selection, PCR and RT-PCR were resorted to identify the integration and expression of CD and TK gene. The stable lines were respectively treated at varying concentrations of GCV and 5-FC in culture for 4days. The in vitro cytotoxic effects of pcDNA3-MDRl-Promoter-CD-TK/GCV+5-FC system were measured by MTT assay. The apopototic ratio was observed by flow cytometry.Results The P-glycoprotein and MDR1 mRNA were highly expressed in K562-A02 cells,while K562 cells were negative.After transfection,.By the method of PCR double suicide genes were proved to integrate into K562-A02 cell and K562 cell; and using RT-PCR CD and TK gene were proved expressed in K562-A02 cell, while K562 cells were negative., there was a greater degree of killing effect and apoptotic ratio in K562-A02 cells treated with pcDNA3-MDRl-Promoter-CD-TK/GCV+5-FC system compared with K562 cells.The pcDNA3-MDRl-Promoter-CD-TK/GCV+5-FC system was more efficient in MDR positive cells.Conclusion The pcDNA3-MDRl-Promoter-CD-TK/GCV+5-FC systemmediated by lipofectin has stronger killing effect on MDR positive leukemia cells K562-A02 than on K562. The experiment reveals that the expression of suicide genes driven by MDR1 promoter can be employed for gene therapy approaches.