Kong Wei-Jia, Zhang Song, Guo Changkai, Zhang Sulin, Wang Yanjun, Zhang Dan
Department of Otolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Dadao Avenue, Wuhan, Hubei 430-022, China.
Laryngoscope. 2005 Aug;115(8):1395-401. doi: 10.1097/01.MLG.0000166708.23673.3A.
OBJECTIVES/HYPOTHESIS: 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.
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.
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.
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.
目的/假设：死亡相关蛋白激酶（DAPK）是一种受钙/钙调蛋白调节的丝氨酸/苏氨酸激酶，作为程序性细胞死亡的正向介质发挥作用。已发现在某些癌症和肿瘤细胞系中，DAPK基因常因启动子高甲基化而失活。然而，尚不清楚DAPK基因启动子高甲基化是否存在于喉鳞状细胞癌（LSCC）中。本研究的目的是调查LSCC中DAPK基因的启动子甲基化状态，以及去甲基化剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对人喉癌细胞系Hep-2细胞及其异种移植物的影响。
采用甲基化特异性聚合酶链反应（PCR）和逆转录PCR技术，检测LSCC中DAPK基因的启动子甲基化状态和mRNA表达。此外，对体外和体内的Hep-2细胞用5-Aza-CdR进行处理，以探讨去甲基化剂对DAPK mRNA表达和肿瘤生长的影响。
58例LSCC样本中有39例（67.2%）发现DAPK基因启动子高甲基化。不同组织学分级的样本或不同T分期患者的样本之间，启动子高甲基化率无显著差异。然而，N0期患者的样本与N1期患者的样本之间，DAPK基因的甲基化状态存在显著差异。5例正常喉组织样本中均未发现DAPK基因启动子高甲基化。在启动子高甲基化的肿瘤标本中未检测到DAPK mRNA表达。相反，在未甲基化的肿瘤标本、肿瘤旁组织标本和正常喉组织样本中观察到DAPK mRNA表达。在Hep-2细胞中发现了DAPK基因启动子高甲基化，且未检测到DAPK mRNA表达。用5-Aza-CdR处理细胞和异种移植物后，Hep-2细胞和异种移植物中的DAPK mRNA表达得以恢复。在用5-Aza-CdR处理的裸鼠中，通过注射Hep-2细胞诱导的肿瘤异种移植物明显小于用磷酸盐缓冲盐水处理的裸鼠中的肿瘤异种移植物。
DAPK表达的异常缺失可能与LSCC中启动子区域的异常甲基化有关。5-Aza-CdR可能通过重新激活因从头甲基化而沉默的肿瘤抑制基因DAPK，在体外和体内减缓Hep-2细胞的生长。
