| Cancer is one of the most deadly diseases in the world. There are 7600000 people died of cancers, taking 13% in the whole number of people's death in 2005. The routine methods such as surgery, radiotherapy and chemotherapy, are not competent enough for the treatment. Therefore the gene therapy is a very good substitution method to cure the cancer.Transcriptional targeting of gene therapy is a novel and prospect method for cancer's treatment. It is a strategy for enhancing the specificity of gene expression to target tumor cells of interest. This targeting approach is based on the use of tissue- or tumor-specific promoters (TSP) in a heterologous context to direct the expression of therapeutic genes specifically to the tumor. An efficient gene therapy regimen requires transgene expression in the tumor and absence of expression in relevant normal tissues, especially in the liver. This combination of"tumor on"and"liver off"profile can result in increasing the therapeutic index and limiting the toxicity of vectors and transgenes in vivo. Many promoters have already been evaluated for transcriptional targeting in cancer gene therapy, such as AFP, hTERT, CXCR4 as well as Survivin and COX-2 promoter. Among all the promoters above, hTERT promoter, survivin promoter and cox-2 promoter were validated to be more predominance than the others. They are demonstrated to be more sufficient specific to avoid gene expression in normal tissues, while shows noticeable expression levels in tumor cells. Telomeres are the distal ends of human chromosomes composed of tandem repeats of the sequence TTAGGG.Numerous studies have demonstrated that telomerase is activated in more than 90% of malignant tumors but is stringently repressed in normal somatic cells. hTERT is a catalytic subunit homologue protein of human telomerase, and the expression of hTERT is observed at high levels in malignant tumors and cancer cell lines but not in normal tissues or telomerase-negative cell lines. The hTERT promoter was considered to be one of the typical example of tumor specific promoter and researched in many tumor cell lines to determined the activity in them. It shows good prospect for the utilization in the cancer gene therapy.Survivin is a novel member of the inhibitor of apoptosis (IAP) protein family, which plays an important role in the survival of cancer cells and the progression of malignancies. Survivin expression is found during embryonic and fetal development, but is undetectable in terminally differentiated adult tissue. Recently, various investigations have evaluated the activity of the survivin promoter in breast cancer, pancreatic cancer, esophageal carcinoma, primary glioblastoma, melanoma, and ovarian cancer. The survivin promoter shows noticeable activity in them and appears to be negligible in the majority of adult tissues.Cyclooxygenase (Cox), a rate-limiting enzyme in the synthesis of prostanoids, is encoded by two genes, Cox-1 and Cox-2, which are differentially expressed and regulated. The expression of Cox-1 is maximal in quiescent cells; Cox-2 expression is induced by growth factors and cytokines. Cox-2 is virtually undetectable in most tissues under physiological conditions. In contrast, it is expressed in several cancer tissues and may have an important role in carcinogenesis. The up-regulation of Cox-2 has been well established in breast, colon, and lung cancers, bladder cancer as well as in melanoma. And the Cox-2 promoter was demonstrated to have favorable utility in many cancers'gene therapy with a'tumor-on/ normal tissue-off'status such as in melanoma.In this study, we construct a series of recombinant plasmid each with a candidate TSP (the Cox-2, Survivin, or hTERT), a reporter Luciferase gene, and a poly-A signal (with an SV40 Enhancer or not), all of which were inserted into the pGL-3 vector. A pGL-3 recombinant plasmid containing the CMV promoter and Luciferase gene was used as a positive control to normalize the Luciferase activity. Luciferase activity was measured in multiple tumor cell lines after infection with the plasmids. Human embryonic lung cells, 2BS, were used as normal control. Moreover, flow cytometry was used to evaluate the activation of the TSP in the corresponding cell lines. The result shows that all the three TSPs tested in this study shows distinct induction activities in most cancer cell lines. COX-2 promoter exhibits the highest transcriptional activity while hTERT promoter shows the lowest activity in them. Compared to the CMV promoter, the three promoters especially hTERT promoter and Survivin promoter are also negative in the normal cells, exhibiting a'normal tissue-off'profile. It was also showed that SV40Enhancer can up-regulate the expression of reporter Luciferase gene remarkably in varies cell lines. It was demonstrated that SV40Enhancer plays an important role to improve the promoters'transcriptional activity.In this study, we abstracted Survivin promoter and COX-2 promoter successfully from human genome. And then we compared the transcriptional activation of the hTERT promoter, Survivin promoter and COX-2 promoter in cancer cell lines derived from tumors of the breast, head&neck, lung and ovary by the transfection and flow cytometry experiments, with CMV promoter as control. The results of this study will give some important information for the following tumor targeting therapy.
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