Epigenetic upregulation of B-cell inappropriate genes induces extinction of B-cell program in classical Hodgkin lymphoma. Homo sapiens

A unique feature of the tumour cells (Hodgkin/Reed-Sternberg (HRS)) of classical Hodgkin lymphoma (cHL) is the loss of their B-cell phenotype despite their B-cell origin. Several lines of evidence suggest that epigenomic events, especially promoter DNA-methylation, are involved in this silencing of many B-cell associated genes. Here we show that DNA-demethylation alone or in conjunction with histone-acetylation is not able to reconstitute the B-cell gene expression program in cultured HRS cells. Instead, combined DNA-demethylation and histone-acetylation of B cells induce a nearly complete extinction of their B-cell expression program and a tremendous up-regulation of numerous cHL characteristic genes including key players such as Id2 known to be involved in the suppression of the B-cell phenotype. Since the up-regulation of cHL characteristic genes and the extinction of the B-cell expression program occurred simultaneously, epigenetic changes may also be responsible for the malignant transformation of cHL. The epigenetic up-regulation of cHL characteristic genes thus play - in addition to promoter DNA-hypermethylation of B-cell associated genes – a pivotal role for the reprogramming of HRS cells and explain why DNA-demethylation alone is unable to reconstitute the B-cell expression program in HRS cells. Keywords: Epigenetic modification Overall design: 5-aza-dC treatment: The cHL derived cell lines L428, KMH2 and L1236 were treated with 5-aza-dC at a concentration of 5 µM for 5 days with drug and medium replacement after each 48 hours. Combined 5-aza-dC/TSA treatment: The Burkitt lymphoma derived cell lines (Daudi, Namalwa and Raji)were treated with 5-aza-dC at a concentration of 3 µM for 6 days. 5-aza-dC and medium was replaced at day 2 and 5. At the fifth day - in addition to 3 µM 5-aza-dC - cells were incubated for 24 hours with 625 nM TSA. The gene expression profiles of the untreated and treated cell lines were generated in duplicates.

经典霍奇金淋巴瘤（classical Hodgkin lymphoma, cHL）的肿瘤细胞——霍奇金/里-施（Hodgkin/Reed-Sternberg, HRS）细胞——的独特特征在于：尽管其起源为B细胞，却丢失了B细胞表型。多项研究证据表明，表观遗传事件——尤其是启动子区域DNA甲基化——参与了众多B细胞相关基因的沉默过程。本研究证实，单独使用DNA去甲基化试剂，或联合组蛋白乙酰化处理，均无法在体外培养的HRS细胞中重建B细胞基因表达程序。与之相反，对B细胞联合实施DNA去甲基化与组蛋白乙酰化处理，不仅会近乎完全终止其B细胞基因表达程序，还会大幅上调大量cHL特征性基因，其中包括已知可抑制B细胞表型的关键调控因子Id2等。鉴于cHL特征性基因的上调与B细胞基因表达程序的终止同步发生，表观遗传改变或许也参与了cHL的恶性转化进程。综上，除B细胞相关基因的启动子DNA高甲基化之外，cHL特征性基因的表观遗传上调，在HRS细胞的重编程过程中发挥关键作用，这也解释了为何单独使用DNA去甲基化无法在HRS细胞中重建B细胞基因表达程序。

关键词：表观遗传修饰
整体实验设计：
5-氮杂-2'-脱氧胞苷（5-aza-dC）处理：将cHL来源的细胞系L428、KMH2及L1236用浓度为5 μM的5-aza-dC处理5天，每48小时更换一次含药培养液。
5-aza-dC联合曲古抑菌素A（TSA）处理：将伯基特淋巴瘤（Burkitt lymphoma）来源的细胞系Daudi、Namalwa及Raji用浓度为3 μM的5-aza-dC处理6天，分别于第2天和第5天更换含5-aza-dC的培养液；于第5天，在维持3 μM 5-aza-dC处理的同时，加入625 nM TSA继续培养细胞24小时。
未处理组与处理组细胞的基因表达谱均设置生物学重复进行检测。