Analysis of WT1 target gene expression in stably transfected cell lines

Abstract

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.