| Epigenetic inactivation of certain tumor suppressor genes by aberrant promoter methylation seems to play an important role in the pathogenesis of tumor. Epigenetic gene silencing refers to nonmutational gene inactivation that can be faithfully propagated from parental cells to clones of daughter cells, this modifications could be restored which is different to gene mutations. Two key changes in chromatin are associated with epigenetic transcriptional regulation: (1) DNA methylation (2) histone modifications. In human cancer cells, DNA methylation mainly refers to the CpG island hypermethylation of tumor suppressor gene. The aberrant promoter hypermethylation and its association with loss of gene function in cancer cells suggest that CpG island methylation is an important mechanism in inactivating tumor suppressor genes.DAP-kinasel (death-assosiated protein kinasel) is a tumor suppressor gene and also a proapoptotic gene, which is mapped at 9q34. 1 of the chromosome 9 and encodes a 160-kD protein. DAP kinase is a new type of calcium /calmodul in-dependent enzyme that phosphorylates serine/threonine residues on proteins. It is inactivated in many tumors, such in B-cell lymphoma cell,thyroid lymphoma, and gastric neoplastic, et al. Hyperniethylation of death-associated protein kinasel CpG island is the main cause of its loss.Methylation specific PCR (MSP) is a rapid and very sensitive technique to screen DNA squence for methylation, in which, the sodium bisulfite treatment converting unmethylated, but not methylated, cytosines to uracil modifies DNA. Following removal of bisulfite and completion of the chemical conversion, this modified DNA is used as a template for PCR. Primers are designed to amplify either the methylated strand or unmethylated strand, and a simple gel electrophoresis will reveal the methylation status of target DNA squence. MSP is the most sensitive technique available, and can detect methylation accurately.In this research, we investigated the methylation status of DAP-kinaselgene, followed by examination of its mRNA and protein expression in cellsafter treated with 5-azacytidine, 5-azacytidine and interferon-y synergisticinduction on the apoptosis of uterine cervical cell line.1. Detection the methylation of DAP-kinasel in uterine cervical cancer. The CpG island methylation of DAP-kinasel gene was detected by MSP and the expression of DAP-kinasel protein was examined with immunohistochemistry in 32 cases of uterine cervical carcinoma including 18 cases of squamous cell carcinoma and 14 cases of adenocarcinoma. We show that 11 of 18 cases of squamous cell carcinoma have the CpG island hypermethylation of DAP-kinasel gene and 15 samples have negative protein staining. Among 14 cases of adenocarcinoma, 7 samples have the CpG island hypermethylation of DAP-kinasel gene and 12 samples have negative protein staining. In normal cervical cells, the DAP-kinasel has no CpG island hypermethylation and the corresponding protein was detected. It is suggest that DAP-kinasel protein was decreased in uterine cervical carcinoma, and the hypermethylation of CpG island may play an important role ininactivating the DAP-kinasel expression, which is dependent upon the form of uterine cervical cancer.2.Effect of 5-azacytidine on the restoration of methylation of DAP-kinase 1 expression in cervical carcinoma cell lines.DNA methyltransferase inhibitor, 5-azacytidine could restore many hypermethylated gene. In this part we analyzed DAP-kinasel methylation in cervical cell line SiHa and HeLa by using MSP, the expressions of mRNA and protein of DAP-kinasel were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry after treated with 5-azacytidine. We showed that DAP-kinasel gene were methylated and inactivated in SiHa cell, which could be restored by 5-azacytidine. But no hypermethylation of DAP-kinasel gene was detected in HeLa cell and 5-azacytidine has no effect on its expression. It suggested that DAP-kinasel gene methylation might take contribute to the...
|
PDF Full Text Request