Dendritic Cells Transduced With Rsf-1/HBXAP Gene Generate Specific Cytotoxic T Lymphocytes Against Ovarian Cancer In Vitro

According to the American for 2002, ovarian carcinoma is the most lethal gynecologic cancer and currently ranks as the fifth in causing cancer-related deaths among women. The major difficulty in achieving cure of ovarian carcinoma is the fact that the majority of patients are diagnosed with advanced-stage (FIGOIII-IV) disease, thereby impeding efforts directed at surgical removal of all tumor burden. 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. Gene amplification is a common mechanism underlying oncogenic activation in human cancer. Chromosome 11q13.5 amplification is one of the most frequently amplified regions in human tumors including ovarian, breast, head, and neck carcinomas. Recently, some studies have identified amplification at the 11q13.5 chromosome region and over-expression of the chromatin remodeling gene Rsf-1/HBXAP that is localized to the amplicon in high-grade ovarian carcinoma. Rsf-1/HBXAP plays a role in chromatin remodeling and transcriptional regulation may contribute to tumorigenesis in ovarian cancer. Rsf-1/HBXAP gene can disrupt the homeostatic kinetics in the chromatin remodeling machinery and fine tune gene regulation and Rsf-1 targeting RNA interference knockdown reduces cell proliferation. Rsf-1/HBXAP is upregulated in ovarian carcinoma cells in effusions and expression score is associated with more advanced (FIGO stage IV) disease at diagnosis and shows a trend for poor overall survival. Identification and characterization of new cancer-associated genes will not only elucidate the pathogenesis of neoplastic diseases but also provide new diagnostic markers and therapeutic targets for cancer patients. Therefore, Rsf-1/HBXAP may serve as an ideal target for ovarian cancer immunotherapy.T lymphocyte-dependent immunity is the major cellular immunity to tumors which relays 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. DCs express high levels of major histocompatibility complex (MHC) and adhesion and costimulatory molecules. Because of their important role in initiating, modulating and keeping immune response, they are becoming the most hopeful cellular adjuvant for tumor vaccines. It has been demonstrated that vaccination using DCs transduced with the tumor-associated antigens (TAA) gene can elicit a potent therapeutic antitumor immunity. This strategy has potential advantages as follows. First, DCs transduced with the entire TAA gene may present multiple epitopes including previously unknown epitopes associated with different MHC class I molecules. Second, TAA gene-transduced DCs may possibly present helper epitopes associated with MHC class II molecules. Third, DCs are provided with a renewable supply of the antigen for presentation by transduction with the gene, as opposed to a single pulse of peptide. For these reasons, we wonder whether Rsf-1/HBXAP-transduced dendritic cells can induce specific immunity response against ovarian cancer which is Rsf-1/HBXAP positive. Therefore, in the following research, we first investigate whether Rsf-1/HBXAP is specifically over-expressed in ovarian cancer and then we construct an Rsf-1/HBXAP gene eukaryotic expression vector and transfect it into DCs from human umbilical cord blood. The purpose of our research is to study whether DCs transduced with Rsf-1/HBXAP gene can induce the specific cytotoxic T lymphocyte which can recognize antigens presented on target ovarian tumor cells and produce strong cytotoxicity to them. We test the hypothesis that Rsf-1/HBXAP can be used a target for ovarian cancer immunotherapy using DC vaccine. PART ITHE EXPRESSION AND SIGNIFICANCE OF Rsf-1/HBXAPGENE IN HUMAN OVARIAN CANCER TISSUES ANDOVARIAN CANCER CELL LINESObjective To study the expression of Rsf-1/HBXAP in ovarian cancer tissues and cell lines and to explore whether Rsf-1/HBXAP is specifically over-expressed in ovarian cancer. The correlation between the expression of Rsf-1/HBXAP and clinical and histopathologic parameters was also measured to elucidate its role in the ovarian cancer initiation and progression.Methods Real-time PCR was used to detect Rsf-1/HBXAP mRNA expression in 40 cases of ovarian cancer, 9 cases of borderline ovarian tumor, 24 cases of benign ovarian tumor, 12 cases of normal ovarian tissues and 8 kinds of ovarian cancer cell lines, control cell line human embryonic kidney (HEK) 293 cells were also screened. Immunohistochemical assay was performed to detect Rsf-1/HBXAP protein expression in 30 cases of ovarian cancer, 10 cases of borderline ovarian tumor, 20 cases of benign ovarian tumor and 8 cases of normal ovarian tissues.Results The expression level of Rsf-1/HBXAP 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 serous carcinoma when compared with those derived from other tissues. There were significant differences in the Rsf-1/HBXAP levels among the clinical stages, differentiations and lymph node metastases. The eight kinds of ovarian cancer cell lines, SKOV-3, OVCAR-3, 3AO, A2780, A549, TOV-112D, HRA, and Caov-3 were Rsf-1/HBXAP positive, and however, the control cell line human embryonic kidney (HEK) 293 cells was Rsf-1/HBXAP negative.Conclusion Rsf-1/HBXAP was mainly over-expressed in epitherial ovarian cancer tissues, especially in ovarian serous carcinoma, and closely correlated with the clinical stages, differentiations and lymph node metastases of ovarian cancer. These results indicated that Rsf-1/HBXAP 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 IICONSTRUCTION OF Rsf-1/HBXAP GENE EUKARYOTICEXPRESSION VECTOR AND ITS STABLEEXPRESSION IN HEK293 CELLSObjective To construct Rsf-1/HBXAP gene eukaryotic expression vector and to establish stable Rsf-1/HBXAP-expressed HEK293 cell strains.Methods Rsf-1/HBXAP gene cDNA was obtained from the plasmid pcDNA.6/V5-His B encoding the Rsf-1/HBXAP gene by restriction endonuclease Hind IIIand Sac II, and cloned into pMD18-T vector for sequence analysis. The correct cDNA was subcloned to pEGFP-C1 eukaryotic expression vector, named pEGFP-C1 /Rsf-1/HBXAP.The constructed vector was identified by double digestion Hind Illand Sac II, and then transfected into HEK293 cell by by using Lipofectamine^TM 2000 and G418 was used to select the stable Rsf-1/HBXAP expressed monoclonal cell strains. The transfection effect was identified through observing the green fluorescence of the cells under the adversed fluorescence microscope and detecting Rsf-1/HBXAP mRNA level by RT-PCR and protein level by Western blot respectively.Results The sequence of Rsf-1/HBXAP cDNA was identical with that of the Genebank and was successfully subcloned it into the N-terminal of EGFP gene of pEGFP-Cl eukaryotic expression vector to make these two genes locate in the same open reading frame (ORF) and express fusion protein. The vector was named pEGFP-C1/Rsf-1/HBXAP. 18 hours after transfecting into HEK293 cells, green fluorescence located on the cell nucleus, whereas in pEGFP-Cl transfected cells, it located in the cytoplasts. A 478bp specific fragment was amplified by RT-PCR and a single protein band at the molecular weight of approximately 215 kDa in the stable transfected HEK293 strains with resistance to G418.Conclusion The eukaryotic expression vector pEGFP-C1/Rsf-1/HBXAP was constructed successfully. The fusion of Rsf-1/HBXAP and EGFP gene did not influence the character of Rsf-1/HBXAP protein being located on the cell nucleus. The stable Rsf-1/HBXAP-expressed HEK293 cell strains were established. This experiment made it possible to further investigate the function of Rsf-1/HBXAP gene and develop the DC vaccines of ovarian cancer based on this gene. PART IIITHE IMMUNE EFFECTS INDUCED BY Rsf-1/HBXAP GENE TRANSDUCED DENDRITIC CELLS IN VITROObjective To investigate the influence of Rsf-1/HBXAP gene transfection to the biological function of dendritic cells from human umbilical cord blood monocyte and explore whether Rsf-1/HBXAP gene-transduced DC can elicit antigen-specific cytotoxic T lymphocyte responses to Rsf-1/HBXAP⁺ ovarian cancer cells.Methods Dendritic cells were generated from human umbilical cord blood monocyte using combined cytokines and transfected with DNA plasmid pEGFP-C1/Rsf-1/HBXAP or pEGFP-C1 (as control group) using Lipofectamine^TM 2000 or Human Dendritic Cell Nucleofector ? Kit. After transfection, DCs were observed under adversed fluorescence microscope analysed Rsf-1/HBXAP mRNA expression by RT-PCR and protein expression by Western blot. 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. Secretion of IL-12 by DC was detected by ELISA. Transfected, matured dendritic cells were used to stimulate autologous T cells. After stimulating autogeneic T-cells generated from four sequential rounds for 4 weeks, the effector cells were harvested as Rsf-1/HBXAP specific CTLs. Specific antigen presentation and specific effector T-cell generation were analyzed by an IFN-γrelease Elispot assay. Cytotoxicity of DC against tumor cell lines OVCAR3 and activity of cytotoxic T lymphocytes (CTLs) were determined by ⁵¹Cr-release assay.Results Dendritic cells were successfully induced from human umbilical cord blood monocytes in combined cytokines including GM-CSF, IL-4. Immatured DCs cultured in the presence of GM-CSF and IL-4 were transduced with Rsf-1/HBXAP gene by nucleofection and mature CD83⁺DC generated by additional stimulation with TNF-a and lipopolysaccharide(LPS). Most of induced cells expressed DC surface markers, such as CD1a⁺, CD86⁺, CD83⁺ and HLA-DR et al, but few cells expressed CD14. The transduction efficiency using primary cell nucleofector may achieve 50% which could meet the need of function research. 18 hours after transfecting with primary cell nucleofector, green fluorescence could be observed on the cell nucleus in DC-Rsf-1/HBXAP group, but in control group, green fluorescence mainly located on the cytoplasts. After transfection 24 hours, Rsf-1/HBXAP expression was detected on the mRNA level and protein level in the transfected DCs, but in control group, it was negative. Allogeneic T cell proliferation induced by transfected DC was obviously higher and produced higher levels of IL-12 than nontransfected DC group (P<0.05), but the endocytosis capacity and migratory ability were not influenced by transfection. Rsf-1/HBXAP gene-transduced DCs could induce antigen-specific CTL to produce IFN-γand generate most potent cytotoxicity to OVCAR3 and Rsf-1/HBXAP-HEK293. However, no lysis of pEGFP-C1-HEK293 or HEK293 in cytotoxicity assay was observed.Conclusion The transgene efficiency to DC could be enhanced by primary cell nucleofector method. Rsf-1/HBXAP gene transfection could promote allogeneic stimulatory capacity to some extent. However, DC surface markers (CD1a and CD86) endocytosis capability and migratory ability were not influenced by transfection. Rsf-1/HBXAP gene transduced DCs could induce antigen-specific CTL against ovarian cancer in vitro. These data suggest that Rsf-1/HBXAP gene-transduced DCs may have potential as an adjuvant immunotherapy for ovarian cancer, and may be useful for clinical application.In conclusion, it was comfirmed that Rsf-1/HBXAP was specifically expressed in ovarian cancer tissues and the majority of common ovarian carcinoma cell lines. Therefore it may be an ideal taget of immutherapy to ovarian cancer. The eukaryotic expression vector containing cDNA sequence of Rsf-1/HBXAP was successfully constructed and transduced into dendritic cells. It was concluded that Rsf-1/HBXAP 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 cord blood monocyte. Rsf-1/HBXAP gene transduced DC can induce antigen specific CTL to produce IFN-y and generate most potent cytotoxicity to ovarian cancer cells that are Rsf-1/HBXAP gene positive. This study will lay the experimental foundation both for the future research of Rsf-1/HBXAP-based ovarian cancer vaccine and further clinical application.