Cash Jennifer, Korchnak Amanda, Gorman Jacquelyn, Tandon Yasmeen, Fraizer Gail
Department of Biological Sciences, Kent State University, Kent, OH 44242, USA.
Oncol Rep. 2007 Jun;17(6):1413-9.
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.
为了在前列腺癌细胞中鉴定生理相关的WT1转录靶基因，我们建立了稳定转染的LNCaP细胞系，分别表达WT1(A)、其突变体DDS(R384W)或载体对照。对这些细胞进行的微阵列分析显示，血管内皮生长因子(VEGF)在构建的细胞系中差异表达。WT1对VEGF的调控可能在发育过程中促进肾脏血管生成，而WT1突变体如DDS(R384W)与以肾脏异常为特征的迪尼-德雷什综合征(DDS)相关。最近的机制研究表明，WT1(A)异构体结合VEGF启动子序列并转录调控VEGF报告构建体。然而，VEGF的调控很复杂，涉及转录和转录后过程。本研究检测了激素和放线菌素D处理对稳定转染的WT-LNCaP、DDS-LNCaP或V-LNCaP前列腺癌细胞中VEGF mRNA水平的影响。本研究的原理基于之前的一项发现，即DDS-LNCaP细胞中VEGF表达的增强仅在雄激素类似物R1881存在的情况下发生。这些结果的一种可能解释是，DDS-WT1使VEGF mRNA稳定，使其积累到更高水平。通过用放线菌素D(Act D)处理构建的LNCaP细胞，然后通过定量实时PCR测量VEGF mRNA水平来验证这一假设。在这些信息稳定性测定以及瞬时转染的LNCaP细胞的转录测定中测试了WT1或DDS(R384W)与激素的联合作用。结果表明，DDS-WT1在LNCaP前列腺癌细胞中无法调控VEGF转录或稳定VEGF mRNA。然而，我们的观察结果也与野生型WT1(A)在激素存在下对VEGF mRNA水平具有转录和转录后作用一致。这些关于WT1对VEGF的调控以及DDS(R384W)对其失调的研究表明WT1在正常和肿瘤相关血管生成中都起着重要作用。
