Gene expression in thymi of Tcf1 -/-, Tcf +/- or Tcf1 -/- mice with tumor

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1-/- mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1-/- mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1-/- mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus. Using the Tcf1-/- DeltaVII/DeltaVII knockout mouse (Verbeek et al. Nature 1995), thymocytes of 17 mice (5 control Tcf+/-, 4 Tcf-/- and 8 Tcf-/- with thymic lymphoma) were homogenized for RNA isolation using Qiagen RNeasy minicolumns. The quantity and quality of total RNA was determined using spectrophotometry (Nanodrop) and an Agilent Bioanalyzer. One µg of RNA was used to generate cRNA using Affymetrix One cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA, USA), after which the samples were biotinylated using an Affymetrix IVT labeling kit (Affymetrix). The samples were hybridized overnight at 42 degrees C to GeneChip mouse genome 430 2.0 Arrays (Affymetrix). Washing and staining steps were performed on a Fluidics station 450, and the Genechips were scanned using a GeneChip scanner 3000 (Affymetrix) at the Department of Immunology, Erasmus Medical Center. Raw data were normalized and summarized using Robust Multichip Average (RMA) method. The experiment consists of 5 control Tcf+/- thymi, 4 Tcf-/- thymi and 8 Tcf-/- thymus samples with thymic lymphoma.高迁移率族蛋白盒（HMG-box）因子T细胞因子1（Tcf1）是胸腺T细胞发育过程中的必需因子，可介导细胞核对Wnt信号（Wnt signaling）的应答反应。此前已有针对Tcf1敲除（Tcf1-/-）小鼠的表征研究，该类小鼠的T细胞发育阻滞于CD4-CD8-双阴性（double negative, DN）向CD4+CD8+双阳性（double positive）的转化阶段。由于T细胞发育受阻，Tcf1-/-小鼠的胸腺通常体积显著缩小。 出乎意料的是，大量Tcf1-/-小鼠会自发形成胸腺淋巴瘤，其中50%的小鼠在16周龄时会罹患胸腺淋巴瘤/白血病。这些淋巴瘤呈克隆性且具有高转移性，矛盾的是，当与Wnt报告小鼠（Wnt reporter mice）杂交后，这些肿瘤展现出高Wnt信号活性，且Wnt靶基因Lef1与Axin2的表达水平显著升高。在野生型胸腺细胞中，Tcf1的表达水平高于Lef1，且主要以Wnt抑制型同工型的形式存在。作为Lef1的阻遏因子，Tcf1的缺失会导致Wnt信号活性异常升高，这是淋巴瘤发生的起始事件，而激活型Notch1突变会进一步加剧这一病理过程。因此，Notch1激活与Tcf1缺失在功能上属于协同致癌事件。 Tcf1缺失通过两种途径促使胸腺淋巴瘤发生：一是由于缺乏Tcf1的阻遏型同工型，导致Lef1出现异位上调；二是常伴随具有协同作用的Notch1激活突变。综上，Tcf1除了作为经典的Wnt应答转录因子外，还可作为T细胞特异性的抑癌基因发挥功能。Tcf1在胸腺T淋巴细胞发育过程中，充当增殖信号与阻遏信号之间的分子开关。 本研究使用Tcf1-/- ΔVII/ΔVII敲除小鼠（Verbeek等，《自然》，1995年），对17只小鼠的胸腺细胞进行匀浆以提取RNA：其中5只为Tcf+/-对照小鼠，4只为Tcf-/-小鼠，8只为罹患胸腺淋巴瘤的Tcf-/-小鼠。RNA提取采用Qiagen RNeasy迷你柱试剂盒。总RNA的浓度与质量通过分光光度法（Nanodrop）及Agilent生物分析仪进行检测。取1 μg RNA，使用Affymetrix单循环cDNA合成试剂盒（美国加利福尼亚州圣克拉拉市Affymetrix公司）合成cRNA，随后通过Affymetrix IVT标记试剂盒进行生物素标记。将标记后的样品与GeneChip小鼠基因组430 2.0阵列（Affymetrix）在42℃下杂交过夜。后续洗涤与染色步骤在Fluidics station 450流体工作站上完成，基因芯片扫描使用GeneChip扫描仪3000（Affymetrix），实验操作均在伊拉斯姆斯医学中心免疫学系完成。原始数据采用稳健多芯片平均（Robust Multichip Average, RMA）方法进行归一化与汇总分析。本实验共包含5份Tcf+/-对照胸腺样本、4份Tcf-/-胸腺样本及8份罹患胸腺淋巴瘤的Tcf-/-胸腺样本。