| Backgroud and Objective:Gastric cancer is one of the most common cancers.It is the fourth most common malignancy worldwide and the second most common cause of death from cancer.Most of gastric cancer patients are diagnosed in advanced stages,when surgery can only be palliative.Meanwhile, gastric cancers are largely resistant to chemotherapy and radiotherapy.So there is a pressing need for novel therapeutic strategies and gene therapy may be an option.Because of its slight side-effects and strong bystander effects,suicide gene therapy is of high clinical interest.In suicide gene therapy,the gene encoding an enzyme is delivered to tumor cells,followed by administration of a prodrug,which is converted locally to a cytotoxin by the enzyme.The producer cells as well as surrounding bystanders are subsequently killed.Cytosine deaminase: uracil phosphoribosyltransferase/5-fluorocytosine(CD:UPRT/5-FC)is developed on the basis of CD/5-FC.CD is a bacterial or yeast enzyme that can convert the antifungal agent 5-FC into the chemotherapeutic agent 5-FU(5-fluorouracil).However,certain cancers demonstrate relative resistance to 5-FU,possibly attributable to defects in downstream cellular pathways that are responsible for the metabolism of this enzyme. UPRT is a pyrimidine salvage enzyme that catalyzes the conversion of 5-FU directly into 5-FUMP.Co-expression of CD and UPRT has been reported to increase the sensitivity to 5-FC dramatically.Efficient gene therapy regiment requires transgene expression especially in tumors.One of strategies to achieve this goal is transcriptional targeting.Gene regulatory elements that drive transcription of some special proteins in tumors have the potential capacity to control gene expression in a tumor-specific manner. Transcriptional targeting is based on the use of tissue or tumor-specific promoters.Telomerase is the most extensive tumor molecular marker up to date and accounts for the ability of cancer cells to proliferate in a manner that is out of control.Telomerase is highly active in more than 85%of human cancers but inactive in most somatic cells.hTERT(human telomerase reverse transcriptase)is an essential catalytic subunit of telomerase and has been found to be expressed at high levels in primary tumors and cancer cell lines but repressed in most somatic cells.Recent data suggest that hTERT is a key determinant of the telomerase activity and highly correlated with telomerase activity.Because the expression of hTERT gene is regulated at the transcription level,we hypothesized that the hTERT promoter may be used for tumor-specific expression of transgene.So we constructed the expression vector carrying CD:UPRT genes under control of the hTERT promoter to study its targeted killing effects on human gastric cancer cells to find a new method for gastric cancer gene therapy.Methods:1.The hTERT core promoter was PCR amplified from the total genomic DNA of HeLa cells and cloned into pGL3-Basic vector.The recombinant was named as hTERT-pGL3Basic,hTERT-pGL3Basic was transfected into SGC7901 cells with high telomerase activity and HLF cells without telomerase activity by using cationic liposome to detect the transcriptional activities of the hTERT promoter.2.The expression vector containing CD:UPRT genes under control of the hTERT promoter named as hTERT-CD:UPRT was constructed. This vector and the vector containing CD:UPRT genes under control of cytomegalovirus(CMV)promoter named as pcDNA3.1-CD:UPRT were transfected into SGC7901 cells and HLF cells,respectively.The transfected cells were selected by G418.The expression of the CD gene was detected by RT-PCR and Western Blot.MTT analysis was used to determine the cytotoxic effect of the CD:UPRT/5-FC system and its bystander effect.Apoptosis was determined by flow cytometry.3.Mice models bearing subcutaneous tumors of SGC7901 cells were established,hTERT-CD:UPRT was transfered into tumors and tumor growth was observed.After treatment,all mice were sacrificed and tumors were excised for routine histological examination and immunohistochemical staining of CD. Results:1.The hTERT promoter was PCR amplified successfully.Luciferase assay showed the relative luciferase activity of the hTERT promoter was (21.5±2.2)%in SGC7901 cells and only(0.4±0.1)%in HLF cells.2.The expression vector hTERT-CD:UPRT was successfully constructed.SGC7901 cells and HLF cells transfected with pcDNA3.1-CD:UPRT both expressed CD gene and were sensitive to 5-FC.Flow cytometry showed apoptosis was induced significantly. Similar results were found in HLF cells transfected with pcDNA3.1-CD:UPRT,but not in HLF cells transfected with hTERT-CD:UPRT.The bystander effect of hTERT-CDUPRT/5-FC existed.When SGC7901 cells transfected with hTERT-CDUPRT reached 10%,the survival rate was 48.4%.3.The tumor growth in 5-FC treatment group was significantly inhibited as compared with PBS treatment group and growth inhibition ratio(GIR)was 59.6%.Conclusion:1.The hTERT core promoter is transcriptional active specifically in human gastric cancer cells SGC7901 but not in normal human fibroblast cells HLF.2.CD:UPRT/5-FC system under control of hTERT promoter can specially kill SGC7901 cells in vitro and in vivo and is a promising method for gastric cancer gene therapy. |
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