三氧化二砷对淋巴瘤细胞酪氨酸磷酸酶1基因的去甲基化作用  

As2O3 induces demethylation and up-regulates transcription of SHP-I gene in human lymphoma cell line T2 cells

作　　者：杨琳[1] 罗建民[1] 李燕[1] 刘小军[1] 温树鹏[1] 杜行严[1] 姚丽[1] 杨敬慈[1] 董作仁[1] 

机构地区：[1]河北医科大学第二医院血液内科河北省血液病重点实验室,石家庄050000

出　　处：《中华肿瘤杂志》2009年第6期423-427,共5页Chinese Journal of Oncology

基　　金：基金项目：河北省自然科学基金（C2005000744）;河北省科技厅指导项目（052761615）

摘　　要：目的探讨淋巴瘤细胞系1、2和非霍奇金淋巴瘤（NHL）组织中酪氨酸磷酸酶1基因启动子区甲基化状态，三氧化二砷（As2O3）对T2细胞中SHP-1的去甲基化作用及对T2细胞生长增殖的生物学影响。方法以不同浓度As2O3处理淋巴瘤细胞株T2，采用二苯基溴化四氮唑蓝（MTT）法检测T2细胞的生长变化，采用流式细胞术检测细胞凋亡率的变化，采用实时定量聚合酶链反应（FQPCR）和Westernblot方法检测T2细胞SHP-1基因mRNA和蛋白表达及c—kit蛋白表达变化。采用甲基化特异性聚合酶链反应（MSP）检测T2细胞和32例NHL组织SHP-1基因启动子甲基化状态。结果As2O3使他细胞增殖受抑，凋亡增加，效应均具有时间和剂量依赖性。As2O3能逆转他细胞SHP-1基因启动子甲基化，SHP-1基因恢复表达，同时c-kit蛋白磷酸化水平降低。SHP-1基因在对照组淋巴组织中呈完全性非甲基化状态，而在NHL组织中启动子甲基化出现率为87．5％（28／32）。结论在淋巴瘤细胞和NHL组织中，SHP-1基因启动子区域存在高度甲基化。As2O3能逆转T2细胞DNA异常甲基化，诱导SHP-1基因表达，并可能通过抑制c-kit受体及其信号传导路径的活化，抑制肿瘤细胞增殖。Objective To investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 ceils. Methods T2 cells were treated with As2O3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 ceils. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells. Results T2 ceils contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the ceils exposed to As2O3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 phosphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apoptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1,2, 3 d treatment with As203（2.5 μmol/L）, the apoptosis rates were 6. 12% ,26.53% ,50.90% , respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% （28/32）. In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter. Conclusion Hypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effctively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating agent.

关 键 词：淋巴瘤 蛋白酪氨酸磷酸酶1 三氧化二砷 甲基化 C-KIT 

分 类 号：R686[医药卫生—骨科学]