Biological Characteristics Of Esophageal Carcinoma Cells Transduced By Recombinant Adenovirus Expressing HIFN-β Gene In Vitro

Background and Objective Type I IFNs including the IFN-αfamily and IFN-βare multifunctional cytokines with antiviral, antiproliferative, antiangiogenic activity and immune cell stimulation. Many investigators have demonstrated that IFN-βcould inhibit significantly the growth of many tumors. However, the problem in use of IFN-βprotein in clinical treatment is its short half-life. Moreover, clinical studies have shown that the serum concentrations of IFN-βafter systemic administration of the maximally tolerated dose is far below that required to achieve the anti-proliferative effect observed in vitro . Gene therapy may overcome this limit because of continuous intracellular secretion of cytokine with its gene transduction. Preclinical studies have confirmed that hIFN-βgene mediated by recombinant adenovirus can express at high levels on the tumor cells and tissues, resulting in significant growth inhibition and a profound apoptotic response in the tumor cells or tissues. So, gene therapy provides a promising approach for patients. The vector can be delivered directly into the target organ relative easily and potentially provide transgene expression. Recombinant adenovirus vectors have gained increasing usage in cancer gene therapy strategies. Potential advantages of adenovirus vectors include the safety of transient expression, ease of producing high titers of virus, and relatively high transduction efficiency when compared with other gene therapy vectors.Esophageal carcinoma is one of the most common malignant tumors in human disease. 300 thousands people died from it in the world every year and half of that is in China, which is a quarter of all who died from malignant tumors in China. It has done great harm to people's health. We supposed that the IFN-βgene had possibilities for gene therapy against esophageal carcinoma. We then constructed recombinant adenovirus vector containing human IFN-βgene and to evaluated the anti-proliferation effect of AdhIFN-βagainst esophageal carcinoma cells in vitro and offered basic experiment evidences.Materials and Methods1. The HEK293 cells were infected with recombinant adenovirus AdhIFN-βand proceed with a large-scale amplification of viral recombinant particles in HEK293 cells. Subsequently the virus was purification and concentrated. The titer of AdhIFN-βwas determined by plaque assay in HEK293 cells.2. KYSE150 cells were infected with AdGFP and AdhIFN-βat various MOIs, respectively. After 48h, we observed the expressing of the GFP under the fluorescent microscopy and assessed the infection rate.3. Total RNA was isolated from cells which treated with AdGFP and AdhIFN-βaccording to the protocol. RT-PCR was performed according to the manufacturer's instruction to evaluated the expression of hIFN-βmRNA. rAd-untreated cells were subjected to the same course as a negative control. Human genome DNA was amplified in parallel with the cDNA samples as a positive control for the PCR reactions of hIFN-β. PCR products were analyzed by 2% agarose gel electrophoresis.4. The control cells and the cells infected with AdGFP and AdhIFN-βwere collected. The expressing of hIFN-βprotein in KYSE150 cells was observed by Immunocytochemistry.5. rAd-treated and control groups of cells were plated into 6-well plates respectively. Cloning efficiency was calculated about two weeks after incubation.6. KYSE150 cells were plated into 96-well plates and then added 30MOI AdGFP and AdhIFN-βrespectively. The control group cells added nothing. MTT method were used to detected the proliferation activity of the KYSE150 cells.7. AdGFP and AdhIFN-βinfect the KYSE150 cells. After 3d of incubation, Cells were harvested, fixed in 70% ice-cold ethanol with an overnight incubation at 4°C, then were digested with 50ug/ml of RNase A for 1h at 37°C and were stained with 100ug/ml of PI for 30min at ordinary temperature. Cell cycle was determined by flow cytometer.8. DNA was isolated from infected cells and control cells according to the protocol. Products were analyzed by 1.5% agarose gel electrophoresis and the DNA bands was observed under the ultraviolet transilluminator. 9. statistical treatment: we took SPSS 10.0 as the statistics tool. The data are marked as x|-±sand use the t test to analyze the data. test criterion:α=0.05.Results AdhIFN-βwas propagated in HEK293 cells and purified using Adeno-X^TMvirus purification kits. The titer of AdhIFN-βreached 2×10¹¹ pfu/ml and more than 95% KYSE150 cells could be infected by 30 MOIs rAd. The results of RT-PCR and immunocytochemistry showed that the hIFN-βmRNA and protein expressing of KYSE150 cells were obvious after infection. The proliferation of KYSE150 cells was obviously inhibited after infected with AdhIFN-βthrough the cloning efficiency test and growth curve of KYSE150 cells compared with that of the control cells. The results of flow cytometry test showed that AdhIFN-βcould slow progression of cells through the S phase. And the DNA fragmentation observed the DNA ladder of apoptotic cells.Conclusion the cells infected with AdhIFN-βcan express hIFN-β. The active hIFN-βcan inhibit obviously the growth of cells and induces S phase lengthening and apoptosis in KYSE150 cells. These findings indicate that hIFN-βgene mediated by recombinant adenovirus may have an antitumor activity against human esophageal carcinoma cell, suggesting its potential clinical application and provides an elementary basis for gene therapy against esophageal carcinoma.