title: VEGF transcription and mRNA stability are altered by WT1 not DDS(R384W) expression in LNCaP cells.
abstract: To identify physiologically relevant WT1 transcriptional target genes in prostate cancer cells, we have established stably transfected LNCaP cell lines expressing either WT1(A), its mutant counterpart DDS(R384W), or vector control. Microarray analyses of these cells revealed that vascular endothelial growth factor (VEGF) was differentially expressed in the engineered lines. Regulation of VEGF by WT1 likely contributes to kidney angiogenesis during development and WT1 mutants such as DDS(R384W) are associated with the Denys-Drash syndrome (DDS), characterized by renal abnormalities. Recent mechanistic studies have demonstrated that the WT1(A) isoform binds VEGF promoter sequences and transcriptionally regulates VEGF reporter constructs. However, regulation of VEGF is complex, involving both transcriptional and post-transcriptional processes. This study examined the ability of hormone and Actinomycin D treatment to alter VEGF mRNA levels in stably transfected WT-LNCaP, DDS-LNCaP, or V-LNCaP prostate cancer cells. The rationale of this study was based on a previous finding that enhancement of VEGF expression in DDS-LNCaP cells occurred only in the presence of the androgen analog, R1881. One possible explanation for these results was that DDS-WT1 stabilized VEGF mRNA so that it accumulated to higher levels. This hypothesis was tested by treating engineered LNCaP cells with Actinomycin D (Act D) and then measuring VEGF mRNA levels by quantitative real-time PCR. The combined effects of WT1 or DDS(R384W) and hormone were tested in these message stability assays and also in transcription assays of transiently transfected LNCaP cells. The results indicated that DDS-WT1 is unable to regulate VEGF transcription or stabilize VEGF mRNA in LNCaP prostate cancer cells. However our observations are also consistent with wild-type WT1(A) having both transcriptional and post-transcriptional effects on VEGF mRNA levels in the presence of hormone. These studies of VEGF regulation by WT1 and dysregulation by DDS(R384W) suggest an important role for WT1 in both normal and tumor-related angiogenesis.
authors.affiliation: Department of Biological Sciences, Kent State University, Kent, OH 44242, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Cash
authors.forename: Amanda
authors.initials: A
authors.lastname: Korchnak
authors.forename: Jacquelyn
authors.initials: J
authors.lastname: Gorman
authors.forename: Yasmeen
authors.initials: Y
authors.lastname: Tandon
authors.forename: Gail
authors.initials: G
authors.lastname: Fraizer
mesh_terms: D045744:Cell Line, Tumor; D003609:Dactinomycin / Q000494:pharmacology; D030321:Denys-Drash Syndrome / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D008297:Male; D009154:Mutation; D009389:Neovascularization, Pathologic / Q000235:genetics*; D018919:Neovascularization, Physiologic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D014158:Transcription, Genetic / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism; D025721:WT1 Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D006728:Hormones; D042461:Vascular Endothelial Growth Factor A; D025721:WT1 Proteins; D003609:Dactinomycin