| Traditional non-invasive therapy for tumor (such as radio-therapy andchemo-therapy) can kill tumor cells as well as normal somatic cells at thesame time (2:1 to 6:1) due to lack of tumor-specific effect, and often leads tosevere side effects. Gene therapy for tumors is a new treatment for tumorsfollowing traditional therapy such as chemo-therapy, radio-therapy andsurgical treatment, etc, which functions to tumors through transferring therapygene to tumor cells, or modifying tumor cells or immune cells in vitro, thentransfuse these cells back to patients. Now there is increasing interests in thisfield. Many tumors including salivary tumors have been tested extensivelywith various gene therapy, among which resuming tumor cell apoptoticprocedure is a promising strategy for tumor gene therapy. This method mainlyuse pro-apoptosis gene to induce tumor cell apoptosis directly. But it is stilldifficult to apply gene therapy clinically for many reasons, especially forlacking of tumor-specific effect, that is gene transfer system and manyapoptosis-inducing genes, such as TNF, FasL, Bax, etc affect normal cellsnon-specifically, they are all lack of favorable tumor-specific effect (ortargeting effect), and have side effects to some degree. It is non-selectedtransfection that hinders application of gene therapy to malignant tumors.The salivary glands of mammalian (parotid and submandibular gland) areexcellent target of gene therapy due to their anatomy characteristic: ①throughorifice of salivary duct in mouth, we can inject all sorts of gene to salivaryglands directly without trauma; ②acinic cell and duct cell are monolayer,which greatly increases the opportunity to touch gene transfer vector directlyand promote transgene expression efficiency; ③there is intact envelope forhuman salivary glands, which can restrict vector diffusion so as to avoid sideeffects to other tissues; ④salivary glands are secretive and produce abundantproteins, which function as exocrine and endocrine. Nevertheless, finding genetransfer vector with targeting effect but without side effects is still animportant goal for salivary gene therapy. The common method currently usedis to control transgene expression via tumor-specific promoters in specifictissue, cell or organ. Several promoters have been identified that are moreactive in particular tumor types than in the tissues from which they arise, andthese promoters have been exploited to target transgene expression in tumors.These promoters include the AFP promoter, CEA promoter and Tyr enzymepromoter. But these promoters are limited to specific tumor histologies andcannot be used universally in tumors of various origins. Second, most of thesepromoters are much weaker than commonly used viral promoters such as theCMV promoter. Consequently, their uses are hampered by the problem of lowexpression. Recent years, telomerase was found to be responsible for cellimmortalized and tumor-genesis. It is a specialized DNA polymeraseresponsible for the replication of chromosomal ends, or telomeres. Telomeraseis highly active in immortalized cell lines and >85%of human cancers but isinactive in most somatic cells, therefore telomerase is thought to be verypromising not only as a tumor-specific marker but also as a target foranticancer therapy. The enzyme is a ribonucleoprotein complex composed ofan essential RNA template and several associated proteins, among which is theessential catalytic subunit named human telomerase reverse transcriptase(hTERT), which controls telomerase expression. Activation of telomerase istightly regulated at the transcriptional level of hTERT. It has shown thathTERT promoter has much higher transcriptional activity in telomerasepositive cells (such as cancer cells) than that in telomerase negative cells, andit can effectively induce anti-tumor gene expression in tumor cells with highefficiency and targeting effect, while there is no trans-gene expression innormal tissue (in additional to stem cell and embryo cell), also hTERT activityin cancer cells is similar to CMV promoter. Therefore hTERT promoter as apowerful and universal tumor-selective promoter gives gene transfer vectorshighly tumor-specific characteristic, and will have potential application intargeted salivary cancer gene therapy. Adenoid cystic carcinoma (ACC) is a common salivary malignant tumor,which infiltrates facial nerve and metastasis earlier and often leads to obviousmaxillo-facial malformation after operation. It also reoccurs easily and is notsensitive to radio-therapy or chemo-therapy, so has a deep effect to patient'slife. In this study, we took ACC as a representative to test the feasibility oftargeted gene therapy for salivary tumors. The field of this research isfollowing: 1 We first assessed hTERT expression in ACC tissue and SACC-83cell using polyclonal antibody H231 by immunohistochemistry. The resultsshowed that hTERT activity is high and the signal is mainly located in thecytoplasm of tumor cells, which indicates the level of hTERT activity may beassociated with malignant degree of ACC and its prognosis, and alsoestablished basis for next targeted gene transfer. 2 We transfected SACC-83using EGFP-including plasmids pACCMV-EGFP and pACTERT-EGFPinduced by lipofectin, and showed that the transfection efficiency can reach to>30% in suitable proportion of plasmid to lipofectin (plasmid 2μg, lipofectin8μl), which reached the requirement of transient gene transfer vector. Thisprovided experiment basis for us to construct vectors with TRAIL gene andtransfect SACC-83 with it. 3 pACCMV-TRAIL and pACTERT-TRAIL weretransfected to SACC-83 and normal human fibroblast -----HEL respectively,and demonstrated that hTERT promoter can be used to drive tumor-specificTRAIL gene expression, apoptosis induction and growth inhibitation ofSACC-83 cell while negating the toxicity of TRAIL gene to normal cells. Incomparison, CMV promoter drove TRAIL expression and induced cytotoxic inboth SACC-83 and HEL due to lack of targeting effect, this will inevitablyhave some side effects. Also our results showed that hTERT promoter activityin SACC-83 was strong enough to be used in targeting gene therapy forsalivary tumors compared with CMV promoter and with greater safety. 4 Wefirst constructed replication-defective adeno-virus vector with hTERTpromoter------AdTERT-TRAIL, as well as positive control vector------AdCMV-TRAIL by homologus recombination. After transfecting SACC-83 and HEL ,the results also showed that AdTERT-TRAIL can be used favorably intargeting transgene expression to SACC-83 and induces much higherapoptosis rate than plasmid vector. The innovation of this study is that hTERTpromoter was first used to drive pro-apoptotic gene expression in salivarytumor gene therapy, and testify that hTERT promoter is a favorable promoterin targeting transgene expression to SACC-83. Our research achievements... |
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