The Research Of Aberrant CpG Island Methylation In The Promoter Of Death Associated Protein Kinase In Bladder Cancer

Bladder cancer is the most common urinary tract malignant tumor in Chinese. Most bladder cancers are transitional cell carcinomas. These tumors exhibit the entire spectrum of biologic aggressiveness, however, transitional cell carcinomas tend to occur in two principal forms: low-grade superficial and high-grade invasive cancers. Although new instruments and antineoplastic agents applied to the clinic in recent years, there still have approximately 55%～60% of cases recurrent after the operation, what's more, in roughly 16%～25%of those patients suffer from the progression of the disease . The past few years have seen a expontential accumulation of molecule information on urothelial cell carcinoma, however it is still a great challenge for us to identify the definite molecule mechanism that promote the recurrence and progression in bladder cancer.DNA methylation is an important regulator of gene transcription, and its role in carcinogenesis has been a topic of considerable interest in the last few years. Alterations in DNA methylation are common in a variety of tumors as well as in development. Of all epigenetic modifications, hypermethylation, which represses transcription of the promote r regions of tumor suppressor genes leading to gene silencing, has been most extensively studied. As methylation occurs early and can be detected in body fluids, it may be of potential use in early detection of tumors and for determining the prognosis. Bec ause DNA methylation is reversible, drugs like 5-aza-cytidine, decitabine, and histone deacetylase inhibitors are being used to treat a variety of tumors. Novel demethylating agents such as antisense DNA methyl transferase and small interference RNA are be ing developed, making the field of DNA methylation wider and more exciting.DAP-kinase gene (Death Associated Protein Kinase, DAPK) was mapped to chromosome 9q34．1 a locus that is frequently rearranged in human malignancies and loss of heterozygosity (LOH) at 9q34 is observed in the majority of bladder carcinomas. The protein product of this gene is a 160 kDa calcium/ calmodulin dependent serine/threonine kinase with a unique domain composition: an N-terminal typical serine/threonine kinase domain followed by a calmodulin binding domain, eight ankyrin repeats, two P-loop elements, several potential phosphorylation sites, a cytoskeleton binding region and a death domain. DAPK plays an important role in IFN-gamma, tumor necrosis factor (TNF)-alpha, or Fas-ligand induced apoptosis. It was found that expression of DAP kinase mRNA and protein were frequently lost in human cancer cell lines, often as a result of silencing by DNA methylation. Further analysis of DNA extracted from human tumour samples showed high incidence of hypermethylation of the DAP kinase 5'-untranslated region (UTR) in B-cell malignancies, non-small-cell lung carcinoma and head and neck cancer. The anti-tumorigenic effect of DAP kinase was demonstrated in mouse model systems where the reintroduction of DAP kinase into highly metastatic mouse lung carcinoma cells strongly reduced their metastatic capacity. Thus, it appears that loss of DAP kinase confers a selective advantage to cancer cells and may have a causative role in tumour progression.We investigated the CpG island hypermethylation of DAPK in bladder cancers cell lines and clinical UCC tissue samples in this experiment. RT-PCR and Western-blot were performed to detect the expression of DAPK in different bladder cancer lines (T24, 5637, ScaBER) respectively. Bisulfite sequencing polymerase chain reaction (BSP) were applied to study the methylation status in bladder cancers. The correlation between DNA methylation and gene expression was investigated after the treatment of DNA methyltransferases inhibitor 5-aza-deoxycytidine and antitumor drug Adriamycin in the bladder cancer cells. Flow cytometry detected apoptosis in bladder cancers to identify the relationship between DAPK expression and cell apoptosis. What's more, DAPK was transfected to T24 cells which have no expression of DAPK to authenticate its pro-apoptotic function. Methylation specific polymerase chain reaction (MSP) was performed to detect methylation of DAPK in UCC samples and normal urothelial samples. What's more, western blot and immunohistochemistry were carried out to identify the expression of DAPK in the clinical samples, with which to find out the relationship between the methylation of DAPK and the clinical characteristics of bladder cancer.Part One Analysis of aberrant methylation in CpG island of DAPK promoter inhuman bladder cancer cell linesEpigenetics can be described as a stable alteration in gene expression potential that takes place during development and cell proliferation, without any change in gene seque nce. DNA methylation is one of the most commonly occurring epigenetic events taking place in the mammalian genome. Naked CpG island DNA is unmethylated and coated by proteins which against DNA methylation establishment and/or spreading. During repeated rounds of the stem-cell mobilization and replication that accompany ageing, DNA methyltransferases are recruited to the borders of some CpG islands, depositing methyl groups and creating methylation pressure for these islands. The balance of methylation pressure and methylation protection is disrupted in the CpG island methylator phenotype (CIMP), resulting in the spread of methylation into the transcription start area and the triggering of the silencing cascade. This change, though heritable, is reversible, making it a therapeutic target. Epigenetics has evolved as a rapidly developing area of research. Recent studies have shown that epigenetics plays an important role in cancer biology, viral infections, activity of mobile elements, somatic gene therapy, cloning, transgenic technologies, genomic imprinting, developmental abnormalities, mental health, and X-inactivation.In order to investigate the expression of death-associated protein kinase (DAPK) in bladder cancer cell lines and detect the aberrant methylation status in its promoter, semiquantitative RT-PCR and Western blotting were performed to evaluate the expression of DAPK in bladder cancer cell lines (T24,5637,ScaBER). Bisulfite modification and MSP were carried out to detect the methylation status of the CpG island of DAPK in bladder cancers. The bladder cancer cells were treated with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 72hrs, 2．5μmol/l, then DAPK expression were detected. Flow cytometry detected the apoptosis rate in bladder cancer cells including agent treated or not to identify the pro-apoptosis function of DAPK.Our results showed that DAPK was not detected in T24 cells either at mRNA level or protein level, and low expression of DAPK existed in 5637 and ScaBER cells. Its expression could be up-regulated by in vitro treatment with the inhibitor 5-aza-2'-deoxycytidine 2．5μmol/l for 72hrs. Aberrant methylation of DAPK was detected in 5637 and ScaBER cells, methylation specific PCR demonstrated the relationship between the CpG island methylation and the gene expression. Flow cytometry indicates that cells with high expression of DAPK have an apoptosis trend comparing to the cells with no or less expression of DAPK.In summery, these data strongly suggested that important correlation between the abnormal expression of DAPK and the development of UCC, and the aberrant methlytion of CpG island plays an important role in the expression of DAPK. The up-regulation of DAPK can significantly promote the apoptosis in UCC cells. Our results also indicate that there may exist other mechanisms in the regulation of DAPK expression in bladder cancer cells besides the CpG island methylation.Part Two The regulation of DAPK expression in bladder cancer cells and the study of it's pro-apoptotic mechanisms.In the previous study, we have demonstrated that the aberrant meth ylation of DAPK in bladder cancer cell lines, moreover, the hypermethylation of CpG island in the promoter of DAPK plays a important role in the expression of this gene.Methylation can also occur in almost any site in the CpG island, such as the far upstream region of the transcript start site, where methylation occurs without causing gene silencing. So to identify genes that are silenced by methylation, studies should be focused on methylation of the core region within a promoter CGI. This will help rese archers to avoid obtaining 'false positives', genes that look like they should be silenced because of methylation, but in reality are not. Methylation of non-core regions within a promoter CGI does not block gene transcription, and is frequently observed in cancer and even in normal cells. Ageing and chronic inflammation are associated with increased DNA methylation, which is usually observed at non-core regions.In this study, we will investigate the core region of the CpG island in the promoter of DAPK. Bisulfite sequencing PCR was performed to identify the methylation status of different regions in the promoter of DAPK in two groups of cells which have different expression of DAPK. In order to further study the pro-apoptosis function of DAPK, Full length of DAPK plasmid was transfected to T24 cells which have no DAPK expression, then flow cytometry was carried out to evaluate the apoptosis rate. What is more, wound-healing assays of migration was applied. In this study, anti-tumor agent adriamycin treated cells with 0．01μg/ml, 0．1μg/ml and 1μg/ml for 24hrs respectively, then DAPK expression and CpG island methylation were evaluated as well.As a result, we clarified that the CGI2 region which is around the transcription start site is the core region of the promoter in DAP K from the BSP study, which means that the DNA methylation of CGI2 can effectively block the gene transcription of DAPK in bladder cancer. The DAPK transfected T24 displayed a relatively high apoptosis rate (24．15%) comparing to the non-transfected cells (0．09%). What is more, T24 cells transfected with DAPK revealed a slow migration in the wound-healing assay. More interested is that adriamycin can significantly up-regulate the protein expression of DAPK in 5637 cells, and this regulation have a dose dependent.Over all, we demonstrated that CGI2 was the core region in the DAPK promoter, which played a important role in the regulation of gene expression. Further more, the plasmid transfection verified the pro-apoptosis function of DAPK. Also, we found that the extensive applied chemotherapeutic agent adriamycin can transform the CpG island methylation of DAPK in bladder cancer cells which may indicate a new mechanism in tumor therapy.Part ThreeAnalysis of CpG island methylation of DAPK in tissuesof human bladder cancerBased on the previous studies, we select two groups of clinic samples to clarify the CpG island methylation of DAPK in bladder cancer.In this study, we collect 24 fresh bladder cancer samples and 22 cases of normal urothelia] as control. Methylation specific PCR detected the CpG island methylation of DAPK in two groups of samples. Western bloting were performed to identify the protein expression of DAPK and DNMT1 in bladder samples, what's more, immunohistochemistry were carried out to verify the expression of DAPK in bladder tissues.Aberrant methylation was observed in 14 (58．3%) of 24 cases of tumor tissues, and no methylation was founded in normal tissues. There is no significant relationship between the hypermethylation of DAPK and the tumor grade, however it seems to have relationship with muscle invasive (P=0．0446) and tumor recurrence(P=0．0337) . Western blotting confirmed the protein expression of DAPK in bladder cancer samples is lower than the normal tissues. Loss expression is observed in tumor samples with hypermethylation of DAPK. Immunohistochemistry staining provided the similar results as the western blotting. Moreover, western blotting also demonstrated that 18 (75%) of 24 bladder cancer samples express DNMT1 protein, and all of the tumor tissues with hypermethylation of DAPK can detect the expression of DNMT1 in western blotting. No expression was detected in normal tissues.Taken together, all of these results demonstrated that there is a close relationship between the hypermethylation of DAPK and the development of bladder cancer. The most important is that the aberrant methylation of DAPK tend to happen in recurrent tumors, which indicate that the methylation DAPK may present us a useful molecule marker to prognose the recurrence of bladder cancer. Also the present study further imply that DNMT1 plays an important roll in the hypermethylation of DAPK.