title: Effect of methylation-associated silencing of the death-associated protein kinase gene on nasopharyngeal carcinoma.
abstract: Death-associated protein kinase (DAPK) is a Ca/calmodulin-regulated serine/threonine kinase and a positive mediator of apoptosis. Loss of expression of the DAPK gene by aberrant promoter methylation may play an important role in cancer development and progression. The aim of this study was to investigate the frequency of gene promoter methylation of DAPK in nasopharyngeal carcinoma (NPC) and the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, on CNE cells, a human nasopharyngeal carcinoma cell line, and on xenografts of CNE cells. Methylation-specific PCR and RT-PCR were used to determine the promoter methylation status and mRNA expression of the DAPK gene in NPC. Furthermore, CNE cells were treated in vitro and in vivo with 5-Aza-CdR to explore the effect of demethylating agents on DAPK mRNA expression and tumor growth. Hypermethylation of the DAPK gene promoter was found in 35 (76.1%) of 46 NPC samples. There was no significant difference in the promoter hypermethylation rate among samples from patients with different TNM stages. No promoter hypermethylation of the DAPK gene was found in all six chronic inflammatory nasopharyngeal tissue specimens. DAPK mRNA expression was not detected in NPC tumor specimens with promoter hypermethylation. However, DAPK mRNA expression was observed in unmethylated NPC tumors and in the chronic inflammatory nasopharyngeal tissue specimens. Promoter hypermethylation of the DAPK gene was found and no DAPK mRNA expression was detected in CNE cells. DAPK mRNA expression in CNE cells and xenografts could be restored by treatment with 5-Aza-CdR. The CNE cell xenografts of nude mice treated with 5-Aza-CdR were obviously smaller in tumor volume than those of nude mice treated with PBS. These results demonstrate that loss of DAPK expression could be associated with promoter region methylation in NPC. 5-Aza-CdR may slow the growth of CNE cells in vitro and in vivo by reactivating the DAPK gene silenced by de novo methylation.
authors.affiliation: Department of Otolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. wjkong666@yahoo.com
authors.forename: Wei-Jia
authors.initials: WJ
authors.lastname: Kong
authors.forename: Song
authors.initials: S
authors.lastname: Zhang
authors.forename: Chang-Kai
authors.initials: CK
authors.lastname: Guo
authors.forename: Yan-Jun
authors.initials: YJ
authors.lastname: Wang
authors.forename: Xiong
authors.initials: X
authors.lastname: Chen
authors.forename: Su-Lin
authors.initials: SL
authors.lastname: Zhang
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.forename: Wen
authors.initials: W
authors.lastname: Kong
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects; D064550:Death-Associated Protein Kinases; D000077209:Decitabine; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D008969:Molecular Sequence Data; D009303:Nasopharyngeal Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D051017:Apoptosis Regulatory Proteins; D012333:RNA, Messenger; D000077209:Decitabine; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D001374:Azacitidine