Activation of DNA Methyltransferase 1 by EBV LMP1 Involves c-Jun NH2-Terminal Kinase Signaling

Abstract

EBV latent membrane protein 1 (LMP1) activates cellular DNA methyltransferases, resulting in hypermethylation and silencing of E-cadherin. However, the underlying mechanism remains to be elucidated. In this study, we show that LMP1 directly induces the dnmt1 promoter activity through its COOH-terminal activation region-2 YYD domain. Using (i) LMP1 mutants, (ii) dominant negative mutants c-jun NH 2 -terminal kinase (JNK)-DN, p38-DN, and constitutive active mutant IKB, as well as (iii) dsRNAs targeting c-Jun, JNK, and tumor necrosis factor receptor-associated death domain protein, and (iv) signal transduction inhibitors, we show that LMP1-mediated DNA methyltransferase-1 (DNMT1) activation involves JNK but not nuclear factor KB and p38/mitogenactivated protein kinase signaling. In addition, LMP1 is unable to activate dnmt1-P1 promoter with activator protein-1 (AP-1) site mutation. Chromatin immunoprecipitation assay results also confirm that LMP1 activates P1 promoter via the JNK-AP-1 pathway. Furthermore, chromatin immunoprecipitation assay data in LMP1-inducible cells disclose that LMP1 induces formation of a transcriptional repression complex, composed of DNMT1 and histone deacetylase, which locates on E-cadherin gene promoter. Treatment with JNK inhibitor, SP600125, prevents the formation of this repression complex. Statistical analyses of the immunohistochemical staining of 32 nasopharyngeal carcinoma (NPC) biopsies show LMP1 expression (18 of 32, 56.25%), DNMT1 expression (31 of 32, 97%), and phospho-c-Jun (27 of 32, 84.38%), suggesting that overexpression of these proteins is observed in NPC tumor. Overall, these results support a mechanistic link between JNK-AP-1 signaling and DNA methylation induced by the EBV oncogene product LMP1. (Cancer Res 2006; 66(24): 11668-76)

DNMT1 as a Molecular Target in a Multimodality-Resistant Phenotype in Tumor Cells

“…The DNMT genes clearly meet several of these criteria. Alterations in methylation patterns and activity have been observed in countless tumors and tumor cell lines, and agents such as 5-aza-CdR, which inhibits DNMT activity, have been shown to inhibit tumor cell proliferation, induce cell death (32,42), and in the present study, to reverse MMRP seen in several tumor cells. Thus, based on the results from this work, combined with previous findings, it seems logical to assume that the DNMT genes might be effective molecular targets in cancer therapy.…”

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

“…Reverse transcription of RNA (1 mg) was performed using ImProm-II (Promega, Madison, WI, USA) and Oligo(dT) 15 primers (Promega). The primers used to amplify the TP cDNA were 5 0 -GCAAGGTGCCAATGAT and 5 0 -CTCCACGAGTTTCTT ACTG, and those used to amplify the internal control cDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were as previously described (Tsai et al, 2006). Quantitative RT-PCR was performed on a Light-Cycler (Roche Diagnostics), according to the manufacturer's instructions, using the FastStart DNA Master SYBR Green I reagent (Roche Diagnostics, Mannheim, Germany).…”

Viral epigenomes in human tumorigenesis

“…Paradoxically, LMP1 might promote the methylation of its own encoding EBV genome by enhancing the expression of DNA methyltransferase 1 (DNMT1) (Tsai et al, 2006). In the same manner, tumor-suppressor genes in nasopharyngeal carcinoma have been found to be hypermethylated through LMP1-induced DNMT1 activation (Niemhom et al, 2008).…”