Methylation-associated silencing of death-associated protein kinase gene in laryngeal squamous cell cancer
Methylation-associated silencing of death-associated protein kinase gene in laryngeal squamous cell cancer
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DOI：
10.1097/01.mlg.0000166708.23673.3a
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发表时间：
2005-08-01
期刊：
影响因子：
2.6
通讯作者：
Zhang, D
中科院分区：
文献类型：
--
作者：
Kong, WJ;Zhang, S;Zhang, D
目标/假设。死亡相关蛋白激酶(DAPK)是一种受钙离子/钙调蛋白调节的丝氨酸/苏氨酸蛋白激酶，是细胞程序性死亡的正向调节因子。研究发现，在某些肿瘤和肿瘤细胞系中，DAPK基因经常被其启动子高甲基化失活。然而，目前尚不清楚喉鳞状细胞癌是否存在DAPK基因启动子高甲基化。本研究旨在探讨喉鳞状细胞癌中DAPK基因启动子甲基化状态及去甲基化药物5-氮-2‘-脱氧胞苷(5-aza-2’-deoxcytidine，5-aza-CDR)对喉癌Hep-2细胞及Hep-2移植瘤的影响。用5-aza-CDR处理体内外Hep-2细胞，探讨去甲基化药物对DAPK基因表达和肿瘤生长的影响。58例喉癌组织中39例(67.2%)存在DAPK基因启动子区高甲基化。不同组织学分级和不同T分期患者的标本之间启动子甲基化发生率差异无统计学意义。然而，N-0期患者和N-1期患者的DAPK基因甲基化状态存在显著差异。在5例正常喉组织中均未发现DAPK基因启动子区高甲基化。在启动子高甲基化的肿瘤标本中未检测到DAPK基因的表达。相反，DAPK mRNA在未甲基化的肿瘤组织、癌旁组织和正常喉组织中均有表达。Hep-2细胞DAPK基因启动子高甲基化，未检测到DAPK基因的表达。5-aza-CDR可恢复Hep-2细胞和异种移植瘤DAPK基因的表达。Hep-2细胞注射5-aza-CDR诱导的裸鼠移植瘤明显小于磷酸盐缓冲液处理的裸鼠移植瘤。结论：喉鳞状细胞癌中DAPK表达异常缺失可能与启动子区异常甲基化有关。5-aza-cdr可能通过重新激活甲基化沉默的抑癌基因DAPK，在体内外延缓Hep-2细胞的生长。
Objectives/Hypothesis. Death-associated protein kinase (DAPK) is a Ca2+/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 N-0 stages and those from patients in N-1 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.