| BACKGROUNDRenal Cell Carcinoma (RCC) is one of the most common malignancy in the urogenital system, accounting for 3% of adult malignancies and 85%-95% of renal cancers. The incidence of RCC has been increasing by 2 to 3% per year since 1970's, and the mortality has also increased gradually. The annually RCC incidence estimated is approximately 150,000 cases worldwide. RCC can be cured by radical nephrectomy in the early stage, though 20 to 30% of the patients will develop metastatic disease finally. Metastasises had been existed with a rate of 30% in the patients when RCC diagnosed. Metastatic RCC has an extremely poor prognosis, which had a median survival of 7-8 months. There are no effective methods for the therapy of advanced RCC. The chemo-and radiotherapy show less response to the matastatic RCC , and the immunotherapy based on IL-2 and IFN-α show an overall response rate of 10-15%. The mechanism of RCC has been unknown by now. Similar to the other malignant tumors, a series of studies suggest that activations of oncogene, mutations of tumor suppressor gene, cytokines and pathway of signal transduction are all involved in the pathogenesis of RCC. Moreover, the tumor cells must escape from the immune surveillance for their growth and development. Therefore, studies on the molecular and immunity of RCCare essential to the effective treatment of RCC.Generally, malignant cells are similar in their morphology with early embryonic cells. A possible relationship between developmental processes and oncogenesis has been suggested. There is an analogous molecular mechanism within both processes. To assure the formation of embryo in a specific way and guide the growth of cells normally, the precise spatial and temporal sequence of growth and differentiation in the developmental embryo may be achieved by the accurate control of gene expression at any given stage, only. Inappropriate control of the genes will result in abnormal proliferation , malignant transformation and cancer progression. Genes for developmental control have a pivotal role in this process. They control the expression of specific genes necessary for normal development. It is postulated that their aberrant expression and structural alteration are often the primary molecular mechanisms in tumorigenesis. Genes for developmental control encode variety of proteins. Pax genes are a gene family of developmental control, that encode nuclear transcription factors and have been implicated in the control of mammalian development. As important control genes, the nuclear transcription factors are expressed in many kinds of tumor cells. Studies confirmed that Pax genes not only control the development of important organs, but they also play a key role in development and progression of malignant tumors. Chromosome band 10q24-q25, enriched gene domain, host to a number of genes who involve in growth and development, carcinopathogenesis, abnormal neurological system, metabolism of hormone and other diseases induced by environments, etc. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other pathologyical conditions. As a member of the Pax gene family, Pax2 is localized to chromosome 10q24.3-25.1, and expressed in the developmental processs of central nervous system, eye, ear and urogenital tract. Pax2 controls multiplesteps of urogenital development, and acts as a key factor for mesenchyme-to-epithelium conversion during kidney development. Pax2 is expressed in the induced messenchyme and the early epithelial derivatives, specifically in highly mitotic and undifferentiated cells. The expression of Pax2 is rapidly down-regulated as the tubular epithelium matures, and repressed upon terminal differentiation. Consistent expression of Pax2 results in severe kidney abnormalities, and its re-activation conduces to tumorigenesis. Studies suggested that mutational activation of Pax2 is an early molecular event during malignant transformation, being considered as a proto-oncogene in RCC. Pax2 would suppress apoptosis in renal collecting duct cells distinctly, prevent the transcription of tumor suppressor gene-p53 and controls the expression of WT1 while being reactivated. Inappropriate expressions of Pax2 have been detected in some human malignant tumors, and the level of expression in renal cancer tissues is high.Researches on tumor immunity found that lacking the essential elements to activate body immune response is the main mechanism of tumor escaping from immune surveillance. Disability of tumor antigen presenting plays a key role in this mechanism. Antigen presenting cells (APCs) are responsible for presenting the tumor antigen. Having been identified as the most potent professional-APCs, Dendritic cells (DCs) take up variety of antigen, and express plentiful MHC molecules, costimulatory molecules and adhesion molecules. DCs are powerful to activate the T cells. It is confirmed that DCs can induce specific cytotoxic T lymphocytes (CTLs) both in vitro and in vivo, and act as pivotal role in body anti-tumor immune response. DCs are extremely rare in tumor tissue and peripheral blood and possess a low immune activity. While the methods to generate DCs having been developed, DCs can be cultured in large scale from bone marrow, cord and peripheral blood. They can exert powerful effects on specific anti-tumor immune response after cultured, expanded, pulsed with antigen and matured in vitro.However, DCs must be pulsed with tumor antigen in order to activate the most effectively and specific anti-tumor immune response. Pulsed with polypeptide, lysate of tumor cells, apoptotic body, purified protein and nucleic acid, as vaccines, DCs have been applied to RCC patients for clinical trials. Considering the affectivity and safety, these vaccines are restricted to further clinical application. People focus on new strategies to prepare more effective and safe DCs vaccines for RCC patients.OBJECTIVESDevelopmental control gene-Pax2 , encoding nuclear transcription factor, plays a pivotal role not only in kidney embryonic development, but also in oncogenesis of kidney. Pax2 is one of the earliest molecular events during cells malignant transformation, and acts as a proto-ongcogene. RCC is one of the most common malignancy in the urogenital system. This study focus on:1. Investigate the expression of Pax2 in normal kidney and RCC tissues, determine whether it is a frequent and highly specific event in renal cell carcinoma, and confirm the importance of Pax2 gene expression in RCC.2. Clone Pax2 open reading frame from human RCC tissues, construct an efficient prokaryotic expression vector of hPax2, preparation and purification recombinant human Pax2 protein(rhPax2).3. Generate DCs from peripheral blood PBMCs of RCC patient, evaluate the possibility of obtaining substantial DCs with high immune activity.4. Construct efficient eukaryotic expression vector of hPax2, transfect DCs with the recombinant plasmid, prepare the hPax2/DCs vaccine, stimulate Pax2-specific CTLs response in vitro, evaluate the applicability of this vaccine stragety.METHODS1. Experimental specimen: 30 cases of RCC tissues and 10 cases of normal tissues surrounding the tumors obtained from radical nephrectomy patients,10ml peripheral blood rich of PBMCs obtained from one of the RCC patients.2. By the PV6000PicTure?current type immunohistochemical (IHC) two-step method, analyzed the expression of Pax2 in different cell type, grade and stage RCC tissues were analyzed using the mouse-anti-human monoclone antibody, normal tissues were examined as a control.3. Extracted RCC Total RNA by QIAamp RNA Blood Mini Kit, analyzed the integrality of RNA by 0.8% agarose gel electrophoresis, and examined the concentration and purity of RNA by OD260/28O.4. hPax2 primers were derived in GeneBank (M89470), the expression of Pax2 mRNA were analyzed in RCC and normal tissues by RT-PCR using the QIAGEN OneStep RT-PCR Kit.5. Construction of recombinant plasmid, pcDNA4/HisMax-hPax2, by PCR cloning: RT-PCR product and plasmid vector pcDNA4/HisMax were digested by restriction endonuclease Kpn I and Xba I to create the sticky ends. Vectors and PCR products were ligted by T4 DNA ligase in a condition according to user's manual. Ligation products were transformed in E.coli strain, JM109, then. Recombinant plasmids, pcDNA4/HisMax-hPax2 were extracted by alkaline lysis, further restriction enzyme analysis was carried out by endonuclease BamH I . Data of DNA sequencing was obtained through ABI PRISM?310 DNA.6. Subcloning: Deleting terminator codon of hPax2 ORF by PCR downstream primer 5'-modification, meanwhile, a new recognizing site of endonuclease was constructed by upperstream primer 5'-modification. PCR product and plasmid vector pTrcHis2B were digested by restriction endonuclease Kpn I and Xba I, and the recombinant pTrcHis2B-hPax2 was cloned by T4 DNA ligase. Transformation, preparation, enzyme analysis and inserts identification were carried out. rhPax2 proteins were expressed by IPTG induction in JM109. rhPax2 was purified by Ni-NTA system, and analyzed by SDS-PAGE.7. Generated immature DCs and mature DCs from peripheral blood PBMCs with rhGM-CSF, rhIL-4 and rhTNF-a, analyzed their morphology and surface phenotypes by invert phase-contrast microscope and FCM, tranfected autologous immature DCs with RCC total RNA and eukaryotic expression vector pcDNA4/HisMax-hPax2 by Effectene Transfection Reagent, stimulated CTLs in vitro , and analyzed CTLs response by 51Cr release experiment.8. Statistic analysis: The SPSS (Verson 11.0) Chi-Square test and One-way ANOVA program were used for statistic anylysis. Significance was considered as P<0.05.RESULTS1. Expression of Pax2: the negative control (normal kidney tissues) showed no expression of Pax2 both in IHC and RT-PCR products. 23 of 30 specimens (76.67%) of tumor tissues expressed Pax2 examined by IHC, 22 of 30 specimens (73.33%) of tumor tissues expressed Pax2 mRNA detected by RT-PCR. All of histologic subtypes of RCC expressed Pax2 protein and mRNA, though, difference among grades and stages of RCC shown no statistically significance(P>0.05).2. Identification RCC total RNA: full length 28S and 18S rRNA observed clearly by 0.8% agarose gel electrophoresis, the concentration and purity of RNA were assessed by UV spectrophotometer (OD26o=0.0892, OD26o/OD28o=1.83, total RNA=0.18ug/ul).3. Analyzed by agarose gel electrophoresis, the product of RT-PCR was about 1.2kb, coincides with hPax2 ORF mRNA sequence in GenBank.4. Analyzed by restriction endonuclease and agarose gel electrophoresis, hPax2 was confirmed to have been subcloned into the prokaryotic expression plasmid. DNA sequencing showed that the cloning product has been identified as human Pax2 in GeneBank.5. By the induction of IPTG and purification of Ni-NTA, rhPax2 proteinwas obtained from E.coli JM109, and was identifyied by SDS-PAGE analysis.6. Identification of DCs: After 7 days cultured with rhGM-CSF and rhIL-2, PBMCs displayed morphological changes, such as, the large body, plentiful cytoplasm, typical cytomembrane processes, and irregular shape. The level of CDla, CD80, CD83, CD86 and HLA-DR expression was low. These proved to be typical characteristics of immature DCs. In addition to display more typical in shape, and the level of CDla, CD80, CD86, HLA-DR expression was increased significantly (P<0.05). After 10 days cultured with rhTNF-a, these characteristics represented to be mature DCs.7. By RT-PCR analysis, the recombinant eukaryotic expression vector pcDNA4/HisMax-hPax2 and RCC total RNA are both successfully transfected into immature DCs.8. By measuring the released 51Cr, in vitro cytotoxicity assay demonstrated that hPax2/DCs were capable of stimulating hPax2-specific CTLs that recognized and lysed both autologous hPax2/DCs and RNA/DCs with similar efficacy (P>0.05), but the lytic activity against non-transfected DCs targets was weak and significantly less than those against hPax2/DCs and RNA/DCs targets (P<0.05). Data also showed that DCs transfected with RCC total RNA stimulated tumor-specific CTLs that recognized and both lysed RNA/DCs and hPax2/DCs. The tumor-specific CTLs were consistently superior to CTLs stimulated with hPax2 RNA-transfected DCs in recognizing and lysing tumor targets, suggesting that tumor-specific CTLs represent a polyclonal response providing more effective antitumor activity than T cell responses directed against a single antigen in the form of Pax2.CONCLUSIONS1. Developmental control gene-Pax2 has an important role not only in kidney embryonic development, but also in the tumorigenesis and progress of RCC. Mutational re-activation of Pax2 is essential to oncogenic transformation... |
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