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.

Overexpression of transketolase protein TKTL1 is associated with occurrence and progression in nasopharyngeal carcinoma: A potential therapeutic target in nasopharyngeal carcinoma

“…The CNE cell line was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 18 CNE cells were maintained in RPMI 1640 with 10% FBS in a humidified 37°C incubator with 5% CO 2 .…”

Folate Status and Aberrant DNA Methylation Are Associated With HPV Infection and Cervical Pathogenesis

“…Furthermore, DAPK (but not CDH1) showed consistent results across studies, irrespective of sample type and methylation method. The three genes, CDH1, HIC1, and DAPK, resulting from our study are all plausibly associated with cervical carcinogenesis and are reported to be methylated and silenced in cancers at various other sites (49)(50)(51).…”

An Update on Epstein-Barr Virus and Nasopharyngeal Carcinogenesis

“…3p and 9p abnormalities have been identified in low-grade dysplastic lesions and normal nasopharyngeal mucosa of individuals at high risk indicating that these genetic changes are early events in the pathogenesis of NPC [10,17,20,33]. Other genes frequently inactivated by promoter methylation in NPC include TSCL1 at 11q23 and EDNRB at 13q22, E-cadherin, and death-associated protein kinase (DAPK) [34][35][36]. Gene expression profiling has shown dysregulation of the PI3K/Akt, WNT/b-catenin, TGF-b, and MAPK signaling pathways in NPC with upregulation of NF-jB2, survivin, Bcl-2 upregulation, nuclear accumulation of bcatenin, and dysregulation of integrins [37].…”