Functional Analysis of the von Hippel-Lindau Disease Tumor Suppressor Gene

我们发现，在正常人肾近端小管上皮细胞中，VHL蛋白的稳态量受到细胞密度的严格调控。细胞VHL含量在密集培养比在稀疏培养高100倍以上。缺乏完整VHL基因的肾细胞癌细胞及其表达载体为野生型或突变型VHL的衍生细胞在log-phase生长时，其生长速率无显著差异。然而，重要的是，当它们达到合流性时存在差异：缺乏野生型VHL的细胞持续生长，而表达外源性VHL蛋白的细胞生长相对有限。VHL蛋白在高细胞密度下具有生长抑制作用，这可能是VHL具有抑瘤功能的基础。我们利用啮齿动物中枢神经系统祖细胞研究了VHL基因在中枢神经系统发育中的作用。我们发现VHL蛋白的表达与神经细胞的分化有关，而与神经胶质细胞的分化无关。此外，VHL mRNA反义寡核苷酸抑制中枢神经系统祖细胞的分化并上调其细胞周期。VHL蛋白包含两个结构域，α和β。我们报道β结构域直接与非典型PKC同型，PKC zeta和PKC lambda相互作用。此外，aPKC的调控结构域足以实现这种直接的蛋白-蛋白相互作用。为了研究PTEN基因在肾肿瘤发生中的潜在作用，我们在68例原发性肾细胞癌（rcc）和17例肾癌衍生细胞系中寻找了该基因的异常。68个原发rcc中有5个（7.5%）表现出基因内突变，17个细胞系中有1个（5.9%）有插入突变。PTEN突变的5例原发肿瘤中有4例为高级别、晚期透明细胞rcc，伴有远处转移或肾静脉肿瘤侵袭，导致预后不良。PTEN突变在rcc的一个子集中被观察到，特别是在透明细胞rcc中，它发生在晚期事件，可能导致侵袭性和/或转移性肿瘤表型。少

We found that, in normal human renal proximal tubule epithelial cells, the steady-state amount of VHL protein is strictly regulated by cell density. The cellular VHL content is more than 100-fold higher in dense cultures than in sparse cultures. The growth rates of renal cell cacinoma cells lacking an intact VHL gene and their derivatives with wild-type or mutant VHL expression vector do not differ significantly when they are growing in log-phase. Importantly, however, there is a difference when they reach confluency : cells lacking wild-type VHL grew continuously, while cells expressing exogenous VHL protein showed relatively limited cell growth. VHL protein functions as a growth suppressor at high cell density, and this might be the basis of the tumor suppressor function of VHL. We investigated the role of the VHL gene in CNS development using rodent CNS progenitor cells. We show that expression of the VHL protein is correlated with neuronal differentiation but not with glial differe … More ntiation in CNS progenitor cells, and we also show that VHL gene transduction induces neuronal differentiation. In addition, a VHL mRNA antisense oligonucleotide inhibits differentiation of CNS progenitor cells and up-regulates their cell cycle. The VHL protein contains two domains, alpha and beta. We report that the beta-domain interacts directly with atypical PKC isotypes, PKC zeta and PKC lambda. Further, the regulatory domain of aPKC is sufficient for this direct protein-protein interaction. To investigate the potential role of the PTEN gene in renal tumorigenesis, we searched for abnormalities of the gene in 68 primary renal-cell carcinomas (RCCs) as well as in 17 renal carcinoma-derived cell lines. Five of 68 (7.5 %) primary RCCs exhibited intragenic mutations, and 1 of 17 (5.9 %) cell lines had an insertion mutation. Four of the 5 primary tumors with PTEN mutation were high-grade, advanced clear-cell RCCs with distant metastases or renal vein tumor invasions, resulting in poor prognostic courses. PTEN mutation is observed m a subset of RCCs and that, especially in clear-cell RCCs, it occurs as a late-stage event and may contribute to the invasive and/or metastatic tumor phenotype. Less