Na Xi, Wu Guan, Ryan Charlotte K, Schoen Susan R, di'Santagnese P Anthony, Messing Edward M
Department of Urology, University of Rochester Medical Center, New York 14642, USA.
J Urol. 2003 Aug;170(2 Pt 1):588-92. doi: 10.1097/01.ju.0000074870.54671.98.
PURPOSE The von Hippel-Lindau (VHL) tumor suppressor gene is frequently inactivated in the common type of sporadic clear cell renal cell carcinoma (RCC) as well as RCCs associated with VHL disease. The VHL protein targets hypoxia-inducible factor-1 alpha (HIF-1 alpha), a transcription factor that can induce vascular endothelial growth factor (VEGF) expression, for ubiquitination and degradation. Accumulation of HIF-1 alpha caused by mutant VHL protein in tumor cells may result in VEGF over expression, which has been used to explain the increased vascularity of RCC. However, quantitative analyses of VEGF production and its correlation with VHL mutations and HIF-1 alpha expression in authentic tissues from patients with RCC are lacking.
We analyzed VHL gene mutations by direct DNA sequencing and methylation specific polymerase chain reaction in 31 paired RCC tissue samples. HIF-1 alpha protein expression detected by immunoblotting and immunohistochemical staining, and VEGF protein measured by enzyme-linked immunosorbent assay and immunohistochemical staining were performed using tumor and corresponding normal tissues.
VHL gene mutations were detected in 44% of clear cell RCCs but no differences in methylation patterns in the promoter or exon 1 were found. RCCs with VHL gene mutations or of advanced grade produced significantly higher concentrations of VEGF (p <0.0001). HIF-1 alpha protein expression was found in 40% of clear cell RCCs but 80% of them had VHL mutations (p <0.006). HIF-1 alpha expression correlated directly with higher levels of VEGF production (p <0.0001).
Our findings indicate that VHL gene alterations and HIF-1 alpha protein expression correlate with a significant increase in VEGF production by RCC. In turn it is associated with a more aggressive tumor phenotype.
目的 冯·希佩尔-林道（VHL）肿瘤抑制基因在散发性透明细胞肾细胞癌（RCC）的常见类型以及与VHL病相关的RCC中经常失活。VHL蛋白靶向缺氧诱导因子-1α（HIF-1α），一种可诱导血管内皮生长因子（VEGF）表达的转录因子，使其发生泛素化并降解。肿瘤细胞中突变的VHL蛋白导致的HIF-1α积累可能导致VEGF过度表达，这已被用于解释RCC血管增多的现象。然而，缺乏对RCC患者真实组织中VEGF产生的定量分析及其与VHL突变和HIF-1α表达的相关性研究。
我们通过直接DNA测序和甲基化特异性聚合酶链反应分析了31对RCC组织样本中的VHL基因突变情况。使用肿瘤组织和相应的正常组织，通过免疫印迹和免疫组织化学染色检测HIF-1α蛋白表达，并通过酶联免疫吸附测定和免疫组织化学染色测量VEGF蛋白。
在44%的透明细胞RCC中检测到VHL基因突变，但在启动子或外显子1的甲基化模式上未发现差异。具有VHL基因突变或高级别的RCC产生的VEGF浓度显著更高（p<0.0001）。在40%的透明细胞RCC中发现了HIF-1α蛋白表达，但其中80%存在VHL突变（p<0.006）。HIF-1α表达与更高水平的VEGF产生直接相关（p<0.0001）。
我们的研究结果表明，VHL基因改变和HIF-1α蛋白表达与RCC中VEGF产生的显著增加相关。反过来，这与更具侵袭性的肿瘤表型相关。
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