Generation Of The Recombinant Adenovirus Expressing IκBαM Activated By A Tumor-Specific Promoter And The IκBαM Gene Expression Induces Apoptosis Of HCC Cells

The transcription factor NF-κB is an important transcript activator. It plays a pivotal role in the regulation of genes involved in a variety of immune and inflammatory responses and in the response to infection or stress in a variety of cell types. NF-κB activity affects cells survival and determines the sensitive of cancer cells to cytotoxic agents as well as ionizing radiation. In our previous study, we have successfully generated recombinat adenovirus AdIκBαM expressing the NF-κB superinhibitor IκBαM and confirmed that AdIκBαM combined with TNF-α could inhibit the proliferation of the HCC cell line HepG2 and induced cell apoptosis. But the gene transduction introduced by adenovirus doesn't target on the tumor cells so the expression of therapeutic gene will not only introduce the apoptosis of tumor cells but also the normal cells. The using of the tumor-specific promoters is a promising way to overcome this shortcoming and is becoming the focus of studies on construction of adenovirus targeting on tumors. The AFP promoter is the most commonly used promoter on the construction of tumor-specific adenovirus. But the low transcriptional activity baffled the more using of AFP promoter. Resently the study of Survivin and human telomerase transcriptase(hTERT) had becom the focus of study. hTERT is the catalysic subunit of telomerase and is only expressed in tumor cells and immortal cells with positive teloverase but not in normal cells. Survivin is a type of inhibitor of apoptosis and its expression was found only in fetal cells and tumor cells but not in full-differentiated adult tissues. The base promoter region of the two genes had become clear. A 59-bp region(-208～-150bp) is required for the maximal promoter activity of hTERT gene and the 0.4kb region upstream of the ATG of Survivin gene possesses the base promoter activity. Both of them lack the TATA box but rich in GC. The study on the regulation of hTERT and Survivin gene and the study on the use of the promoters of these genes on gene therapy have become more and more important. In this study we intent to clone the AFP, hTERT and SUR promoters, detect and compare the tumor-specific transcriptional activities of them. Then select the strongest promoter to activate the expression of IκBαM on the adenovirus vectors. On the bases of the construct of the tumor-specific recombinant adenovirus to observe the effect of the induction of apoptosis of HCC cells So that we can do some background work on the gene therapy targeting on HCC. Objective: To clone the hTERT,Survivin and AFP gene promoter and detect their transcriptional activities in various hepatocellular carcinoma cell lines and in primary human fibroblast cells in order to make groundwork for the construction of the recombinant adenovirus tumor-specifically expressing the IκBαM protein in human hepatocellular carcinoma.Methods: The fragment of hTERT, AFP and SUR promoters were acquired by PCR amplification and were cloned into the reporter plasmid pGL3-Basic in which contains a luciferase gene. The constructed eukaryotic expression plasmid in which the expression of the luciferase were drived by these tumor-specific promoter were transfected into three HCC cell lines and the primary fibroblast cells. At 24 hours post transfection (p.t.), the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System. A pGL3-CMV containing the CMV promoter controlled luciferase gene was used as positive control and a pGL3-Basic vector in which there is no promoter in front of the luciferase gene was used as negative control. Results: All the three promoters were successfully cloned into the eukaryotic reporter vector, pGL3-Basic and gained the pGL3-hTERT, pGL3-AFP and pGL3-SUR vector. All of them showed transcriptional activities in all the three HCC cell lines but no or very low transcriptional activities in the fibroblast cells. The SUR promoter showes the highest activities in the HCC cell lines, which is 52 to 98 times higher than the AFP promoter and reach the 16% to 21%...