The (15)(16)(17). These factors include acidic fibroblast growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor, transforming growth factors a and 13, tumor necrosis factor a, and vascular endothelial growth factor (VEGF). VEGF is secreted by a number of tumors and is normally expressed in the kidney, brain, and other tissues (18-22).VEGF is also markedly elevated in both VHL-associated and sporadic central nervous system hemangioblastomas (23, 24) and renal carcinomas (25,26). In addition to having mitogenic activity on endothelial cells, VEGF also induces vascular permeability, which may lead to extravasation of plasma proteins and deposition of fibrin, providing an extracellular support for tumor cell and endothelial cell growth.In the present study the effects of retroviral transduction of the VHL cDNA into renal carcinoma cell lines was evaluated. ITo whom reprint requests should be addressed

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel-Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (؉͞؊) mice appeared phenotypically normal, VHL ؊͞؊ mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL ؊͞؊ embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL ؊͞؊ mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL ؊͞؊ embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.Germ-line mutations in the VHL tumor suppressor gene predispose von Hippel-Lindau disease patients to develop tumors at multiple sites, including retinal angiomas, hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, and pancreatic cancers (reviewed in refs. 1 and 2). In addition, somatic VHL inactivation through deletion of one allele coupled with either mutation or hypermethylation of the remaining allele has been detected in approximately 80% of sporadic clear cell renal carcinomas examined (2), supporting a tumor suppressor function for VHL.The deduced amino acid sequence of the VHL protein gives no indication of the protein's function. Studies designed to assign VHL function through the identification of physically associating proteins found that VHL stably binds the B and C subunits of the elongin complex (3-5). Elongin, an RNA polymerase II transcription elongation factor, contains a catalytic A subunit that is stabilized and activated by the B and C regulatory subunits. In addition, the heterotrimeric VHLelongin B͞C complex (VBC) stably interacts with the human CUL-2 homolog, a member of a conserved gene family involved in cell cycle and growth control in lower eukaryotes (6). The cellular function(s) of the VBC and͞or VBC-CUL-2 complexes have yet to be elucidated.To analyze the role of VHL in normal cell growth and differentiation, we used targeted homologous recombination to develop a mouse line that is deleted for one VHL allele. Heterozygous VHL mice (ϩ͞Ϫ) have survived thus far to beyond 15 months without evidence of spontaneous disease.However, VHL-deficient mice (Ϫ͞Ϫ) develop placental lesions at 9.5 to 10.5 days of gestation (E9.5 to E10.5) and die in utero between E10.5 to E12.5 due to the absence of placental embryonal vasculogenesis and subsequent hemorrhage and necrosis. MATERIALS AND METHODSConstruction of a Murine VHL-Targeting Vector. Two genomic DNA clones corresponding to the VHL gene were isolated from a 129 mouse-derived P1 library (Genome Systems, St. Louis). Physical mapping showed that three Ba...

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