Expression data of cHL cell lines after FOXO1 activation

FOXO1 is highly expressed in normal B cells and in most types of non-Hodgkinl lymphoma. In Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma(cHL) expression of FOXO1 is low or absent. We overexpressed constitutively active mutant of FOXO1 fused in frame with estrogen receptor ligand-binding domain (FOXO1(3A)ER), which can be activated by 4-Hydroxytamoxifen (4-OHT), in cHL cell lines KM-H2 and L428. Activation of the FOXO1 with 4-OHT resulted in inhibition of proliferation and apoptosis. Using gene-expression array we found that FOXO1 activates transcription of known and potential tumor suppressor genes: CDKN1B, PMAIP1, BCL2L11, TNFSF10, FBXO32, CBLB). Of note, FOXO1 repressed transcription of several cytokines and cytokine receptors, which are known tobe involved in pathogenesis of cHL (e.g. CCL5, CXCR5, TNFRSF8). Taken togather our data indicate important role of FOXO1 repression in pathogenesis of cHL. KM-H2, L428, L1236, UH0-1, and SUP-HD1 cells expressing constitutively active mutant of human FOXO1 fused in frame with estrogen receptor ligand-binding domain were incubated with 200 µcle (ethanol). After 24 h, total RNA was isolated with RNeasy mini kit (QIAGEN). Microarray analyses were performed using 200 ng of total RNA as starting material and 5.5 ug ssDNA per hybridization (GeneChip Fluidics Station 450; Affymetrix, Santa Clara, CA). The total RNAs were amplified and labeled following the Whole Transcript (WT) Sense Target Labeling Assay (http://www.affymetrix.com). Labeled ssDNA was hybridized to Human Gene 1.0 ST Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA). The chips were scanned with a Affymetrix GeneChip Scanner 3000 and subsequent images analyzed using Affymetrix Expression Console Software (Affymetrix). Probe level data were obtained using the robust multichip average (RMA) normalization algorithm.

叉头框蛋白O1（FOXO1，forkhead box O1）在正常B细胞及多数非霍奇金淋巴瘤（non-Hodgkin lymphoma）中呈高表达。而在经典霍奇金淋巴瘤（classical Hodgkin lymphoma, cHL）的霍奇金细胞与里德-施特恩贝格细胞（Reed-Sternberg cells）中，FOXO1的表达水平较低或检测不到。我们将与雌激素受体配体结合域（estrogen receptor ligand-binding domain）框内融合的组成型激活突变体FOXO1（FOXO1(3A)ER）在cHL细胞系KM-H2和L428中过表达，该融合蛋白可被4-羟基他莫昔芬（4-Hydroxytamoxifen, 4-OHT）激活。使用4-OHT激活FOXO1后，可显著抑制细胞增殖并诱导细胞凋亡。通过基因表达芯片（gene-expression array）分析，我们发现FOXO1能够激活多种已知及潜在抑癌基因的转录，包括CDKN1B、PMAIP1、BCL2L11、TNFSF10、FBXO32及CBLB。值得注意的是，FOXO1还抑制了多种已知参与cHL发病机制的细胞因子及细胞因子受体的转录，例如CCL5、CXCR5与TNFRSF8。综上，本研究数据表明FOXO1的表达抑制在cHL的发病机制中发挥关键作用。将表达人源组成型激活突变FOXO1（与雌激素受体配体结合域框内融合）的KM-H2、L428、L1236、UH0-1及SUP-HD1细胞，以200 µcle乙醇进行孵育。孵育24小时后，采用RNeasy迷你试剂盒（QIAGEN）提取总RNA。以200 ng总RNA为起始材料，每次杂交使用5.5 μg单链DNA（single-stranded DNA, ssDNA），通过GeneChip Fluidics Station 450工作站（Affymetrix，美国加利福尼亚州圣克拉拉）完成芯片分析。总RNA的扩增与标记按照全转录组（Whole Transcript, WT）正义靶标标记实验方案（http://www.affymetrix.com）进行。将标记后的ssDNA与人类基因1.0 ST Affymetrix基因芯片（Affymetrix，美国加利福尼亚州圣克拉拉）进行杂交。使用Affymetrix GeneChip Scanner 3000扫描芯片，随后通过Affymetrix Expression Console软件（Affymetrix）分析扫描得到的图像。采用稳健多芯片平均（robust multichip average, RMA）归一化算法获取探针水平的表达数据。