Hormone treatment enhances WT1 activation of Renilla luciferase constructs in LNCaP cells.
The zinc finger transcription factor, WT1, regulates many growth control genes, repressing or activating transcription depending on the gene and cell type. Based on earlier analyses of the effect of WT1 on androgen responsive genes, we hypothesized that there may be an interaction between the androgen signaling pathway and WT1, such that the commonly used Renilla luciferase control vectors were activated in LNCaP prostate cancer cells. Using cotransfection assays we tested the effects of WT1 and/or the androgen analog, R1881, on two Renilla luciferase vectors, pRL-SV40 and the promoter-less pRL-null. To determine whether the zinc finger DNA binding domain was required, the zinc finger mutant DDS-WT1 (R394W) was tested; but it had no significant effect on the Renilla luciferase vectors. To determine whether the androgen signaling pathway was required, WT1 was co-transfected with Renilla vectors in cells with varied hormone responsiveness. The WT1 effect on pRL-null varied from no significant effect in 293 and PC3 cells to very strong enhancement in LNCaP cells treated with 5 nM R1881. Overall, these results suggest that hormone enhanced WT1 mediated activation of Renilla luciferase and that these interactions require an intact WT1 zinc finger DNA binding domain.
Hanson
Julie
J
Department of Biological Sciences, Kent State University, Kent, OH 44242, USA.
Reese
Jennifer
J
Gorman
Jacquelyn
J
Cash
Jennifer
J
Fraizer
Gail
G
D045744:Cell Line, Tumor; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000032:analysis* / Q000235:genetics; D015741:Metribolone / Q000494:pharmacology; D045165:Testosterone Congeners / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D025721:WT1 Proteins / Q000378:metabolism*
D045165:Testosterone Congeners; D025721:WT1 Proteins; D015741:Metribolone; D049410:Luciferases, Renilla