Hypermethylation of the death-associated protein kinase CpG island in canine B-cell lymphoid tumors
Hypermethylation of the death-associated protein kinase CpG island in canine B-cell lymphoid tumors
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DOI：
10.1016/j.vetimm.2014.08.011
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发表时间：
2014-10-15
影响因子：
1.8
通讯作者：
Tsujimoto, Hajime
中科院分区：
文献类型：
--
作者：
Sato, Masahiko;Mochizuki, Hiroyuki;Tsujimoto, Hajime
死亡相关蛋白激酶（DAPK）是一种160 kD的丝氨酸/苏氨酸激酶，是干扰素-γ（IFN-γ）诱导的细胞凋亡和肿瘤抑制的关键分子。在多种人类恶性肿瘤中，DAPK中CpG岛的超甲基化使该基因失活。本研究旨在检测DAPK在犬淋巴瘤细胞中的失活情况。从正常犬外周血单个核细胞中提取DAPK基因，经RT-PCR扩增得到犬DAPK cDNA序列。通过快速扩增5 ′-cDNA末端，确定了DAPK基因的转录起始位点。通过使用搜索算法鉴定位于翻译起始位点上游的CpG岛。使用亚硫酸氢盐序列分析和甲基化特异性PCR（MSP）检查CpG岛的甲基化状态。在3种犬淋巴肿瘤细胞系GL-1（B细胞白血病）、CLBL-1（B细胞淋巴瘤）和CL-1（T细胞淋巴瘤）中检测DAPK基因的失活。实时定量RT-PCR检测DAPK mRNA表达。末端脱氧核苷酸转移酶缺口末端标记法（TUNEL）检测IFN-γ诱导的细胞凋亡。用5-氮杂-2 '-脱氧胞苷（5-氮杂-dC）检查了去甲基化的影响。用MSP法检测了14例犬淋巴瘤的甲基化状态。在犬DAPK的翻译起始位点上游发现了一个1926 bp的CpG岛，包含280个CpG重复序列。硫酸氢盐序列分析和MSP显示GL-1细胞中CpG岛的高甲基化，但在CLBL-1或CL-1细胞中没有。GL-1细胞中DAPK mRNA的表达量明显低于CLBL-1和CL-1细胞。IFN-γ诱导的细胞凋亡在CLBL-1和CL-1细胞中被检测到，但在GL-1细胞中未检测到。用5-aza-dC处理显著增加GL-1细胞中DAPK mRNA和IFN-γ诱导的凋亡的量。这些结果揭示了DAPK在GL-1细胞中通过其CpG岛的甲基化而失活。MSP在8个原发性B细胞淋巴瘤样品中的5个中显示出DAPK CpG岛的高甲基化，但在从犬患者获得的6个原发性T细胞淋巴样肿瘤样品中的任何一个中都没有。在犬B细胞淋巴肿瘤细胞中，DAPK通过其CpG岛的超甲基化而失活。本研究将导致使用犬B细胞淋巴瘤作为动物模型，以评估去甲基化剂的疗效。(C)2014爱思唯尔有限公司版权所有。
Death-associated protein kinase (DAPK) is a 160-kD serine/threonine kinase known as a key molecule in interferon-gamma (IFN-gamma)-induced apoptosis and tumor suppression. Hypermethylation of the CpG island in DAPK inactivates the gene in a variety of human malignancies. This study aimed to detect the inactivation of DAPK in canine lymphoid tumor cells. The sequence of canine DAPK cDNA was obtained from normal dog peripheral blood mononuclear cells after reverse transcription polymerase chain reaction (RT-PCR). By rapid amplification of 5'-cDNA ends, the transcription initiation site of the DAPK gene was identified. The CpG island located upstream of the translation initiation site was identified by using a search algorithm. The methylation status of the CpG island was examined using bisulfite sequence analysis and methylation-specific PCR (MSP). The inactivation of DAPK gene was examined in 3 canine lymphoid tumor cell lines, GL-1 (B-cell leukemia), CLBL-1 (B-cell lymphoma), and CL-1 (T-cell lymphoma). DAPK mRNA expression was measured by real-time RT-PCR. IFN-gamma-induced apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. The influence of demethylation was examined with 5-aza-2'-deoxycytidine (5-aza-dC). The methylation status in 14 dogs with various lymphoid tumors was screened by MSP. A 1926-bp CpG island containing 280 CpG repeats was identified upstream of the translation start site of canine DAPK. Bisulfate sequence analysis and MSP revealed hypermethylation of the CpG island in GL-1 cells, but not in CLBL-1 or CL-1 cells. The amount of DAPK mRNA was significantly smaller in GL-1 cells than CLBL-1 and CL-1 cells. IFN-gamma-induced apoptosis was detected in CLBL-1 and CL-1 cells but not in GL-1 cells. Treatment with 5-aza-dC significantly increased the amount of DAPK mRNA and IFN-gamma-induced apoptosis in GL-1 cells. These results revealed the inactivation of DAPK through methylation of its CpG island in GL-1 cells. MSP showed hypermethylation of the DAPK CpG island in 5 of 8 primary B-cell lymphoma samples, but not in any of the 6 primary T-cell lymphoid tumor samples obtained from canine patients. DAPK was inactivated through hypermethylation of its CpG island in canine B-cell lymphoid tumor cells. This study will lead to the use of canine B-cell lymphoid tumors as an animal model to evaluate the efficacy of demethylating agents. (C) 2014 Elsevier B.V. All rights reserved.