Thäte C, Englert C, Gessler M
Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg, Physiologische Chemie I, Germany.
Oncogene. 1998 Sep 10;17(10):1287-94. doi: 10.1038/sj.onc.1202055.
The Wilms' tumour suppressor gene WT1 encodes a zinc finger protein that is mutated in a subset of Wilms' tumours. Mutation screening and animal studies revealed essential roles during development and later function of the kidneys and the entire genitourinary system. Sequence similarity suggested a possible role for WT1 as a transcription factor. Indeed, sequence specific DNA binding and transcriptional activation or repression potential could be demonstrated in transient transfection assays with various reporter constructs. To identify endogenous WT1 target genes we established HEK293 cell lines expressing the different WT1 isoforms in a tetracycline dependent manner. Differential display PCR (ddPCR) was performed on RNA from stable WT1 transfected HEK293 cell lines and two other WT1 transfected lines (G401 and Saos-2). In an extended survey of several thousand ddPCR bands only few differences in intensity were seen and none of these could unambiguously be verified as being WT1 regulated by subsequent Northern blot analysis. In addition, almost none of the WT1 target genes identified to date in transient co-transfection assays could be confirmed by either ddPCR or Northern hybridization in the three stable transfected cell lines. Among the nine genes expressed, the only exceptions were CSF1 and to a lesser extent IGF1R being induced in Saos-2/G401 and HEK293 cells, respectively. At least two of the cell lines tested had previously shown clear biological effects though -- either WT1 dependent apoptosis (Saos-2) or greatly reduced tumorigenicity (G401). This suggests that WT1 may regulate only a very small set of genes that escape the detection methods used or it may not act as a transcription factor that influences steady state levels of mRNA.
威尔姆斯瘤抑癌基因WT1编码一种锌指蛋白，在一部分威尔姆斯瘤中发生突变。突变筛查和动物研究揭示了其在肾脏及整个泌尿生殖系统发育和后期功能中的重要作用。序列相似性表明WT1可能作为转录因子发挥作用。确实，在使用各种报告基因构建体的瞬时转染实验中可证明其序列特异性DNA结合以及转录激活或抑制潜能。为了鉴定内源性WT1靶基因，我们建立了以四环素依赖方式表达不同WT1异构体的HEK293细胞系。对稳定转染WT1的HEK293细胞系以及另外两个转染WT1的细胞系（G401和Saos-2）的RNA进行差异显示PCR（ddPCR）。在对数千条ddPCR条带的扩展研究中，仅观察到强度上的少数差异，且后续的Northern印迹分析均无法明确证实这些差异是由WT1调控的。此外，在这三个稳定转染的细胞系中，几乎没有一个在瞬时共转染实验中鉴定出的WT1靶基因能够通过ddPCR或Northern杂交得到证实。在所表达的九个基因中，唯一的例外是CSF1，以及在较小程度上的IGF1R，它们分别在Saos-2/G401和HEK293细胞中被诱导表达。不过，至少有两个测试的细胞系之前已显示出明确的生物学效应——要么是WT1依赖的细胞凋亡（Saos-2），要么是致瘤性大幅降低（G401）。这表明WT1可能仅调控一小部分逃避所用检测方法的基因，或者它可能并非作为影响mRNA稳态水平的转录因子发挥作用。
