The Induction Of Specific Immunity Against Ovarian Cancer By MUC1/Y Gene-Modified Dendritic Cells In Vitro

According to the American statistics for 2005, ovarian cancer is the fourth leading cause of death from cancer in U.S women and is the most lethal gynecologic cancer. Although three therapeutic advances of malignant ovarian tumors in the late two decades, which are comprehensive stage laparotomy, cytoreductive surgery and omentectomy, and paclitaxel-cisplatin combined chemotherapy, have improved their five-year survival apparently, they also can not be early detected. The majority of women with ovarian cancer are diagnosed once they have disease beyond the pelvis, at which time their five-year survival is roughly 28%, and they always can not be cured completely by conventional surgery, chemotherapy and radiation therapy. About 70% women diagnosed with ovarian cancer relapse at last. For this reason, it is an obvious target to identify genes both specific and sensitive to ovarian cancer whose protein products may be candidates for early detection markers, and to further explore effective comprehensive therapeutics based on these tumor associated antigens. Recently, as the fourth tumor therapeutic pattern, biotherapy including immunotherapy and gene therapy has concentrated more and more attention, also it has become the important assistant approach to recurrence ovarian cancer.Along with the rapid development of the basic sciences such as biochemistry, molecular biology, immunology and genetics, the research for the tumor is going much further. Many kinds of tumor associated antigens are discovered, among which is MUC1, the gene frequently mentioned in the field of the tumor study. Normally MUC1 is expressed by ductal epithelial cells of many organs including airway, gastrointestinal tract, pancreas, breast, and ovaries, but in several adenocarcinomas it is overexpressed and differentially glycosylated. Previous studies proposed that MUC1 contributes to invasive and metastatic potential by interfering with cell-cell and/or cell-extracellular matrix interactions, and thereby facilitates detachment oftumor cells from the primary growth. But recent research shows that in breast carcinoma higher overall MUC1 expression is associated with a better prognosis and lower grade tumor, while another research concludes that there is no correlation between MUC1 expression and metastases with regard to breast cancer cell lines. Therefore, MUC1 is gradually becoming a controversial molecule for its function and at the same time, attracting more and more attention. MUC1/Y, as one of the MUC1 splice variants, is a new gene identified by Zrihan in 1994. MUC1/Y isoform locates on the tumor cell surface which can act as a membrane receptor and may play a pivotal role in cell transformation through ras signaling pathway. Furthermore, it is also demonstrated that MUC1/Y isoform exhibits more tumor-specificity and more powerful tumor-potentiating abilities in vivo than MUC1. Therefore, MUC1/Y may serve as an ideal target for ovarian cancer immunotherapy.T lymphocyte-dependent immunity is the major cellular immunity to tumors which relys on the effective antigen presentation. Dendritic cells (DCs) represent the most professional antigen-presenting cells (APC) of the immune system for their unique capability of capturing, processing, and presenting antigens to naive T cells. Because of their important role in initiating, modulating and keeping immune response, they are becoming the most hopeful cellular adjuvant for tumor vaccines. For these reasons, we wonder whether MUC1/Y gene-modified dendritic cells can induce specific immunity response against ovarian cancer which is MUC1/Y positive. Therefore,in the following research, we first investigate whether MUC1/Y is specifically overexpressed in ovarian cancer and then we construct a MUC1/Y gene eukaryotic expression vector and transfect it into DCs isolated from human peripheral blood. The purpose of our research is to study whether DCs transfected with MUC1/Y gene can induce the specific cytotoxic T lymphocyte which can recognize antigens presented on target ovarian tumor cells and produce strong cytotoxicity to them.PART Ⅰ THE EXPRESSION AND SIGNIFICANCE OF MUCl/Y GENE IN HUMAN OVARIAN CANCER TISSUES AND OVARIAN CNACER CELL LINESObjective To study the expression of MUCl/Y in ovarian cancer tissues and cell lines and to explore whether MUCl/Y is specifically overexpressed in ovarian cancer. The correlation between the expression of MUCl/Y and clinical and histopathologic parameters was also measured to elucidate its role in the ovarian cancer initiation and progression.Methods Real-timePCR was used to detect MUCl/Y expression in 39 cases of ovarian cancer, 11 cases of borderline ovarian tumor, 20 cases of benign ovarian tumor, 10 cases of normal ovarian tissues and six kinds of ovarian cancer cell lines were also screeened.Results The expression level of MUCl/Y in ovarian cancer tissues was significantly higher than those either in borderline or in benign ovarian tumors whereas in normal ovarian tissues, it was negative, and it was higher in epithelial ovarian cancer when compared with those derived from other tissues. There were significant differences in the MUCl/Y levels among the clinical stages, differentiations and lymph node metastases. Among the six kinds of ovarian cancer cell lines, five kinds of them, including SKOV-3, OVCAR-3, 3AO, A2780, TOV-112D, derived from ovarian adenocarcinoma were MUCl/Y positive, however, HRA which derived from ascites of ovarian serous cystadenocarcinoma was MUCl/Y negative.Conclusion MUCl/Y was mainly overexpressed in epitherial ovarian cancer tissues and closely correlated with the clinical stages, differentiations and lymph node metastases of ovarian cancer. These results indicated that MUCl/Y may play a key role in the initiation and progression of ovarian cancer and may be an ideal target for biotherapy of ovarian cancer.PART Ⅱ CONSTRUCTION OF MUC1/Y GENE EUKARYOTIC EXPRESSION VECTOR AND ITS STABLE EXPRESSION IN HRA CELLSObjective To construct MUC1/Y gene eukaryotic expression vector and to establish stable MUC1/Y-expressed HRA cell strains.Methods MUC1/Y cDNA was amplified by RT-PCR from HeLa cells and cloned into pMD18-T vector for sequence analysis. The correct cDNA was subcloned to pEGFP-Nl eukaryotic expression vector, named pEGFP-N1/MUC1/Y. The constructed vector was identified by double digestion with Hind Ⅲ and Sac Ⅱ, and then transfected into HRA cells by Lipofectamine? 2000 and G418 was used to select the stable MUC1/Y-expressed monoclonal cell strains.The transfection effect was identified on both protein and mRNA levels through observing the green fluorescence of the cells under the adversed fluorescence microscope and detecting MUC1/Y mRNA by RT-PCR, respectively.Results The sequence of our cloned full-length MUC1/Y cDNA was identical with that of the Genebank and we successfully subcloned it into the N-terminal of EGFP gene of pEGFP-Nl eukaryotic expression vector to make these two genes locate in the same open reading frame (ORF) and express fusion protein. 18 hours after transfecting into HRA cells, green fluorescence located on the cell membranes could be observed in the pEGFP-N1 /MUC1/Y transfected cells, whereas in pEGFP-Nl transfected cells, it located in the cytoplasts. A 256bp specific fragment was amplified by RT-PCR in the stable transfected HRA strains with resistance to G418.Conclusion We constructed pEGFP-N1/MUC1/Y eukaryotic expression vector successfully. The fusion of MUC1/Y and EGFP gene did not influence the character of MUC1/Y protein being located on the cell membranes.We also established the stable MUC1/Y-expressed HRA cell strains.This experiment made it possible to further investigate the function of MUC1/Y gene and develop the nucleic acid vaccines of ovarian cancer based on this gene.PART Ⅲ THE IMMUNE EFFECTS INDUCED BY MUC1/Y GENE-MODIFIED DENDRITIC CELLS IN VITROObjective To investigate the influence of MUC1/Y gene transfection to the biological function of dendritic cells derived from human peripheral blood monocyte and explore whether MUC1/Y gene-modified DC can elicit antigen-specific cytotoxic T lymphocyte responses.to MUC1/Y+ ovarian cancer cells.Methods Dendritic cells were generated from human peripheral blood monocyte using combined cytokines and transfected with DNA plasmid pEGFP-N1/MUC1/Y or pEGFP-N1 (as control group) using LipofectamineTM 2000 or Human Dendritic Cell Nucleofector? Kit. After transfection, DCs were observed under adversed fluorescence microscope analysed MUC1/Y mRNA expression by RT-PCR. Then the DC phenotypes, T-cell stimulatory capacity, endocytic activity and migration capacity were explored by flow cytometry analysis, allogeneic mixed lymphocyte reaction assay, endocytosis assay and transwell chemotaxis assay, respectively. After stimulating autogeneic T-cells generated from the peripheral blood monocytes with MUC1/Y gene-modified DCs through four sequential rounds for 4 weeks, the effector cells were harvested as MUC1/Y specific CTLs and were tested for the capacity to recognize MUC1/Y antigen and specific cytotoxic to HRA-MUC1/Y cells by ELISPOT assay and 51Cr-release assay.Results Dendritic cells were successfully induced from peripheral blood monocytes in combined cytokines including GM-CSF, IL-4 and TNF-α. Most of induced cells expressed DC surface markers, such as DC-SIGN /CD209, CD1a, CD86 and HLA-DR, but few cells expressed CD 14. The transduction efficiency using primary cell nucleofector may achieve 50% which could meet the need of function research. 8 hours after transfecting with primary cell nucleofector, green fluorescence could be observed on the cell membranes in DC-MUC1/Y group, but in control group, green fluorescence mainly located on the cytoplasts. After 48 hours, MUC1/Y expression was detected on the mRNA level in the pEGFP -N1/MUC1/Y transfected cells, but in control group, it was negative. Allogeneic T cell proliferation induced by DC-MUC1/Y was obviously higher than nontransfected DC group (P<0.05), but therewere no significant differences either between the DC-MUC1/Y group and the DC-pEGFP-N1 group or between the DC-pEGFP-N1 group and nontransfected DCs (P>0.05). The expression of DC surface markers (CD83 and CD86) and the chemotaxis index were similar among three groups. After incubated with FITC-OVA, the fluorescence intensity detected by FACS was also similar among three groups. When restimulated with same tumor antigen, the DC-MUC1/Y induced CTL in vitro can secret IFN-γ, also when conculture with target cells, they can kill the HRA-MUC1/Y cells in an antigen specific manner. However, no lysis of HRA-pEGFP-N1 or HRA in cytotoxicity assay was observed.Conclusion The transgene efficiency to DC could be enhanced by primary cell nucleofector method. MUC1/Y gene transfection could promote allogeneic stimulatory capacity to some extent. However, DC surface markers (CD83 and CD86), endocytosis capability and migratory ability were not influenced by transfection. MUC1/Y gene-modified DCs could induce antigen-specific CTL to produce IFN-γ and generate most potent cytotoxicity to ovarian cancer cells that are MUC1/Y positive.In conclusion, we comfirmed that MUC1/Y was specifically expressed in ovarian cancer tissues and the majority of common cell lines derived from ovarian adenocarcinom.Therefore it may be an ideal taget of immutherapy to ovarian cancer. The eukaryotic expression vector containing cDNA sequence of MUC1/Y was successfully constructed and transfeced into human dendritic cells.We concluded that MUC1/Y gene transfection could improve allogeneic stimulatory capacity and had no obvious influence to the most of the biological function of dendritic cells derived from human peripheral blood monocyte. MUC1/Y gene-modified DC can induce antigen-specific CTL to produce IFN-γ and generate most potent cytotoxicity to ovarian cancer cells that are MUC1/Y positive. This study will lay the experimental foundation both for the future research of MUC1/Y-based ovarian cancer vaccine and further clinical application.