title: Methylation-associated silencing of death-associated protein kinase gene in laryngeal squamous cell cancer.
abstract: OBJECTIVE
  Death-associated protein kinase (DAPK) is a Ca/calmodulin-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It has been found that DAPK gene is frequently inactivated by its promoter hypermethylation in some cancers and tumor cell lines. However, it is not clear whether promoter hypermethylation of DAPK gene exists in laryngeal squamous cell cancer (LSCC). The aim of this study was to investigate the promoter methylation status of the DAPK gene in LSCC and the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, on Hep-2 cells, a human laryngeal cancer cell line, and on xenografts of Hep-2.
  
  
  METHODS
  Methylation-specific polymerase chain reaction (PCR) and reverse-transcription PCR techniques were used to determine the promoter methylation status and mRNA expression of DAPK gene in LSCC. Furthermore, Hep-2 cells in vitro and in vivo were treated by 5-Aza-CdR to explore the effect of demethylating agents on DAPK mRNA expression and tumor growth.
  
  
  RESULTS
  Hypermethylation of DAPK gene promoter was found in 39 (67.2%) of 58 LSCC samples. There was no significant difference in the promoter hypermethylation rate among the samples of different histologic grades or samples from patients with different T stages. However, there was significant difference in methylation status of DAPK gene between the samples from patients in N0 stages and those from patients in N1 stages. No promoter hypermethylation of DAPK gene was found in any of the five normal laryngeal tissue samples. DAPK mRNA expression was not detected in tumor specimens with promoter hypermethylation. On the contrary, DAPK mRNA expression was observed in the unmethylated tumor specimens, specimens from tissues adjacent to the tumor, and normal laryngeal tissues samples. Promoter hypermethylation of DAPK gene was found, and no DAPK mRNA expression was detected in Hep-2 cells. DAPK mRNA expression in Hep-2 cells and xenografts could be restored by treating cells and xenografts with 5-Aza-CdR. The tumors' xenografts, induced by way of Hep-2 cell injection in nude mice treated with 5-Aza-CdR, were obviously smaller than those in nude mice treated with phosphate-buffered saline.
  
  
  CONCLUSIONS
  Abnormal loss of DAPK expression could be associated with aberrant promoter region methylation in the LSCC. 5-Aza-CdR may slow the growth of Hep-2 cells in vitro and in vivo by reactivating tumor suppressor gene DAPK silenced by de novo methylation.
authors.affiliation: Department of Otolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Dadao Avenue, Wuhan, Hubei 430-022, China. wjkong888@yahoo.com
authors.forename: Wei-Jia
authors.initials: WJ
authors.lastname: Kong
authors.forename: Song
authors.initials: S
authors.lastname: Zhang
authors.forename: Changkai
authors.initials: C
authors.lastname: Guo
authors.forename: Sulin
authors.initials: S
authors.lastname: Zhang
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Wang
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor / Q000166:cytology / Q000187:drug effects*; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012016:Reference Values; D012494:Sampling Studies; D012680:Sensitivity and Specificity; D014183:Transplantation, Heterologous
chemical_list: D012333:RNA, Messenger; D011494:Protein Kinases; D001374:Azacitidine