ANALYSIS OF WT1 TARGET GENE-EXPRESSION IN STABLY TRANSFECTED CELL-LINES

The Wilms' tumour suppressor gene WT1 encodes a zinc finger protein th at is mutated in a subset of Wilms' tumours. Mutation screening and an imal studies revealed essential roles during development and later fun ction of the kidneys and the entire genitourinary system. Sequence sim ilarity suggested a possible role for WT1 as a transcription factor. I ndeed, 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 ta rget genes we established HEK293 cell lines expressing the different W T1 isoforms in a tetracycline dependent manner. Differential display P CR (ddPCR) was performed on RNA from stable WT1 transfected HEK293 cel l lines and two other WT1 transfected lines (G401 and Saos-2), In an e xtended 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 addit ion, almost none of the WT1 target genes identified to date in transie nt co-transfection assays could be confirmed by either ddPCR or Northe rn hybridization in the three stable transfected cell lines. Among the nine genes expressed, the only exceptions were CSF1 and to a lesser e xtent IGF1R being induced in Saos-2/G401 and HEK293 cells, respectivel y. At least two of the cell lines tested had previously shown clear bi ological effects though - either WT1 dependent apoptosis (Saos-2) or g reatly reduced tumorigenicity (G401). This suggests that WT1 may regul ate only a very small set of genes that escape the detection methods u sed or it may not act as a transcription factor that influences steady state levels of mRNA.