| ObjectiveTumor-specific or tumor-associated antigens referrs to the molecules that are either specifically expressed or overexpressed in tumor cells when compared with normal cells.Many novel concepts for the treatment of human cancer try to use the patient's immune system, either by a nonspecific stimulus or by a specific stimulation or 'vaccination' with antigens that are specifically expressed by tumor cells. A prerequisite for the successful application of such tumor vaccines and other immunological interventions for the treatment of cancer is the recognition by the immune system of tumor-specific or tumor-associated antigens. In contrast to animal models, the existence of such antigens was not unequivocally demonstrated in human neoplasms until 1991, when Thierry Boon's group described the antigen MAGE-1 in maligant melanoma. Among tumor antigens identified to date, cancer-testis (CT) antigens have been recognized as a group of highly attractive targets for cancer vaccine. CT antigens are encoded by CT genes. More than 40 CT genes have been categorized as CT genes, including the NY-ESO-1, MAGE, and SSX gene families and so on. These CT genes are widely expressed in a variety of human cancers, such as melanoma, breast cancer, and esophageal cancer. But their expression in normal tissues is restricted to testis. Furthermore, because testis is an immune-privileged organ, cancer vaccination of CT antigens is not expected to cause damages to normal tissues due to autoimmune responses. CT antigens are of particular interest in tumor immunology since they are detectable in histologically unrelated tumors and may induce both CTL and T cell responses.Successful active specific immunotherapy of cancer might result in the immunoselection of tumor variants failing to express relevant tumor associated antigens included in vaccine preparations. Multiantigen specific immunization might help in limiting this immunoescape phenomenon. Coexpression more than 2 CT antigens expressed in high percentages of tumour tissues might be advantageously used in the treatment of defined types of tumor.Bladder carcinoma is the first common cancer in China, 95% of the bladder carcimoma are defined as transitional cell carcinoma (TCC). The main treatment modality for bladder transitional cell carcinoma is operation. But the high percentage of recurrence often distress the patients and the urologists. Renal cell carcinoma is the second common urologic neoplasm, and insentitive to the chemotherapy and the radiotherapy except for operation. The treatment is increasingly based on immunotherapeutic strategies. The first step of active immunotherapy is to identify rationalantigen targets for bladder transitional cell carcinoma and renal clear cell carcinoma. Among tumor antigens identified to date, CT antigens have been recognized as a group of highly attractive targets for cancer vaccine.The aim of our study is to evaluate the expression profile of CT gene and protein product in human urologic neoplasms tissues. The study can be divided into 3 parts:①to select the CT genes highly expressed in human urologic neoplasms tissues, ie. to evaluate the frequency of cTAGE-1, cTAGE-2, MAGE-A1, MAGE-A3, MAGE-A12 and NY-ESO-1 gene expression with the method of RT-PCR.②to evaluate the expression profile of highly expressed CT gene product (MAGE-A3 protein) in human urologic neoplasms tissues with the method of western blot.③to study the the correlation between CT gene expression pattern and pathologic characteristics of human urologic neoplasms.MethodsPatients and Tumor Samples: 42 patients with a pathological diagnosis of renal clear cell carcinoma and 35 with bladder transitional cell carcinoma were enrolled in this study including 46 men and 31 women. With the median of 61, the age is ranging from 28 to 84 years old. All patients received surgical treatment between June and December 2006 in 2nd affiliated hospital of China medical university. The clinical data included degree of histologic differentiation, and pathologic stage according to the tumor-node-metastasis system recommended by the American Joint Committee on Cancer. All cancer tissue and 20 adjacent cancer tissue samples were freshly obtained from surgery. Adjacent cancer tissues were taken from 5 cm away from tumor margin. All tissue samples were snap-frozen in liquid nitrogen within 10 minutes after resection and stored at-70℃.Main reagents: RT-PCR kit, mouse anti-human monoclonal antibody, trans-blot PVDF membrane,TRIZOL, agarose.Main instruments: ultrasound comminuter, electrophoresis apparatus, rocking bed, high-speed and low-temperature centrifuger, automatic electrophoresis gel imaging analysis apparatus.Isolation of total RNA was performed by TRIzol Reagent according to the manufacturer's instructions. The following primers were employed:cTAGE-1 sense 5'-GGAGACCTACCGAAAGCG-3'antisense 5'-TCAGATGAAGGCCAACCC-3'cTAGE-2 sense 5'-GGAGACCTACCGAAAGCG-3'antisense 5'-TCAGATGAAGGCCAACCC-3'MAGE-A1 sense 5'-GGAGCACCAAGGAGAAGA-3 'antisense 5'-TGATGGTAGTGGGAAAGG-3'MAGE-A3 sense 5'-AGTCCGAGTTCCAAGCAG-3'antisense 5'-GCAGGTGGCAAAGATGTA-3'MAGE-A12 sense 5'-GGAAGATGGCTGAGTTGG-3'antisense 5'-AGGCAGGTGACAAGGATG-3'NY-ESO-1 sense 5'-GAGCCGCCTGCTTGAGTT-3'antisense 5'-AGCACTGCGTGATCCACATC-3'P-actin sense 5'-GTGGGGCGCCCCAGGCACCA-3'antisense 5'-CTCCTTAATGTCACGCACGATTTC-3'Products of the reaction were subjected to electrophoresis on 2% agarose gels and monitored under UV light.A 1:500 dilution of the MAGE-A3 antibody (mouse monoclonal antibody; clone 6C1) and a 1:5,000 dilution of goat antimouse antibody were used for protein detection by western blot.Statistical Analysis: Statistical analysis was done with the SPSS program (version 11.5). Pearsonχ~2 test and Fisher exact probability test was used to compare the correlation between disease stage distribution and CT gene expression. Statistical significance was accepted at P＜0.05 (two tailed).Results1. The most frequently expressed CT genes in bladder transitional cell carcinoma was MAGE-A12 (69%), followed by MAGE-A1 (60%), cTAGE-1 (57%), MAGE-A3 (54%), cTAGE-2 (51%) and NY-ESO-1 (46%). No CT gene expression was found in the adjacent tissue. In 35 bladder transitional cell carcinoma patients, all the patients (100%) expressed at least one CT gene. We did not detect any significant correlation between CT gene expression and other clinical factors such as disease stage and grade (Fisher exact probability test, P＞0.05).2. The most frequently expressed CT genes in renal clear cell carcinoma was MAGE-A12 (71%), followed by MAGE-A3 (70%), MAGE-A1 (66%), cTAGE-1 (65%), cTAGE-2 (60%) and NY-ESO-1 (50%). No CT gene expression was found in the adjacent tissue, In 42 renal clear cell carcinoma patients, all the patients (100%) expressed at least one CT gene. We did not detect any significant correlation between CT gene expression and other clinical factors such as disease stage and grade (Pearsonχ~2 test, P＞0.05).3. In 35 bladder transitional cell carcinoma patients, 16 patients (45.7%) expressed MAGE-A3 protein. We did not detect any significant correlation between MAGE-A3 protein expression and other clinical factors such as disease stage and grade (Fisher exact probability test, P＞0.05).Conclusion1. The frequency of cTAGE-1, cTAGE-2, MAGE-A1, MAGE-A3, MAGE-A12 and NY-ESO-1 gene expression in 35 cases of bladder transitional cell carcinoma and 42 cases of renal clear cell carcinoma is relative high by using RT-PCR method. No CT gene expression was found in the adjacent tissue. No CT gene expression was found in the adjacent tissue. In all the patients, 100% expressed at least one CT gene.2. We did not detect any significant correlation between CT gene expression and other clinical factors such as disease stage and grade.3. In 35 bladder transitional cell carcinoma patients, the frequency of MAGE-A3 protein achive 45.7%. We did not detect any significant correlation between MAGE-A3 protein expression and other clinical factors such as disease stage and grade. 4. CT gene highly express in bladder transitional cell carcinoma and renal clear cell carcinoma. It may be promising target gene in human urologic neoplasm specific immunofherapy.
|
PDF Full Text Request