In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations
In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations
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DOI：
10.1158/0008-5472.can-04-1430
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发表时间：
2004-12-01
期刊：
影响因子：
11.2
通讯作者：
Simon, MC
中科院分区：
文献类型：
--
作者：
Rathmell, WK;Hickey, MM;Simon, MC
von Hippel-Lindau （VHL）肿瘤抑制基因的突变导致组织特异性肿瘤，具有显著的基因型-表型相关性。VHL表达缺失易导致血管母细胞瘤和透明细胞肾细胞癌，而特异性点突变易导致嗜铬细胞瘤、红细胞增多症，或血管母细胞瘤、肾细胞癌和/或嗜铬细胞瘤的组合。VHL蛋白（pVHL）参与许多细胞活动，包括缺氧反应、细胞周期阻滞、细胞凋亡和细胞外基质重塑。我们在Vhl（-/-）小鼠胚胎干细胞中表达错义pVHL突变，以测试整倍体细胞中基因型-表型的相关性。我们首先检测了突变型pVHL直接降解缺氧诱导因子（HIF）亚基hif1 α和hif2 α的能力。所有突变的pVHL蛋白都恢复了对hif2 α的适当缺氧调节，尽管一个VHL突变（VHLR167Q）显示与长链蛋白c的结合受损，该突变也未能恢复hif2 α的调节。在单独的实验中，这些胚胎干细胞被用来在免疫功能低下的小鼠中产生畸胎瘤，从而可以独立评估特定VHL突变对肿瘤生长的影响。令人惊讶的是，尽管恢复HIFalpha调节，表达VHLY112H突变蛋白的畸胎瘤表现出生长劣势。最后，我们观察到来自Vhl（-/-）以及VHLY112H、VHLR167Q和VHLR200W胚胎干细胞的畸胎瘤微血管密度增加。总之，这些观察结果支持pVHL在细胞中发挥多种作用的假设，并且这些活动可以通过离散的VHL点突变分离。在整倍体模型中解剖带有错义突变的特定VHL功能的能力为阐明VHL作为肿瘤抑制因子的活性提供了新的机会。
Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause tissue-specific tumors, with a striking genotype-phenotype correlation. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to pheochromocytoma, polycythemia, or combinations of hemangioblastoma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein (pVHL) has been implicated in many cellular activities including the hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix remodeling. We have expressed missense pVHL mutations in Vhl(-/-) murine embryonic stem cells to test genotype-phenotype correlations in euploid cells. We first examined the ability of mutant pVHL to direct degradation of the hypoxia inducible factor (HIF) subunits HIF1alpha and HIF2alpha. All mutant pVHL proteins restored proper hypoxic regulation of HIF1alpha, although one VHL mutation (VHLR167Q) displayed impaired binding to Elongin C. This mutation also failed to restore HIF2alpha regulation. In separate assays, these embryonic stem cells were used to generate teratomas in immunocompromised mice, allowing independent assessment of the effects of specific VHL mutations on tumor growth. Surprisingly, teratomas expressing the VHLY112H mutant protein displayed a growth disadvantage, despite restoring HIFalpha regulation. Finally, we observed increased microvessel density in teratomas derived from Vhl(-/-) as well as VHLY112H, VHLR167Q, and VHLR200W embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the cell, and that these activities can be separated via discrete VHL point mutations. The ability to dissect specific VHL functions with missense mutations in a euploid model offers a novel opportunity to elucidate the activities of VHL as a tumor suppressor.