Mechanisms of epithelial development and neoplasia in the metanephric kidney.
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Recent studies on the mechanisms of normal epithelial development in the kidney, and on the aetiology of renal neoplasms, are converging to reveal remarkably close relationships between the phenotypes and behaviours of normally-developing and neoplastic cells. Normal renal epithelia arise from two sources; those of the collecting duct system develop by arborisation of an initially-unbranched ureteric bud, in a manner similar to the development of other glandular organs, while epithelial nephrons develop via an unusual mesenchyme-to-epithelial transition. Both types of development require controlled proliferation, cell-cell and cell-matrix interactions, protease activity etc., but of the two tissues, the development of the nephrons is arguably the more complex. It includes many defined stages, signals and checkpoints that ensure that events happen at the right time, and that processes such as proliferation, apoptosis and differentiation are properly balanced. Detailed investigation of renal neoplasms has revealed some to be caused by mutations in molecules with known roles in normal nephrogenesis (e.g. Wilms' tumour and the WT-1 gene, renal cell carcinoma and the c-met receptor tyrosine kinase gene), some to be caused by mutations in genes expressed during normal development (e.g. renal cell carcinoma and the TSC-2 gene, renal cell carcinoma of the clear cell variety and the VHL gene). Furthermore, these and other tumours of unknown aetiology re-express genes such as Pax-2 that are expressed during the normal mesenchyme-to-epithelium transition but are shut off during terminal differentiation. Their re-appearance in tumours suggests that the cells have 'regressed' in an ontogenic sense, and their biology may therefore be understood most clearly by reference to the properties of normal developing cells rather than cells of a mature kidney. (+info)
Constitutional von Hippel-Lindau (VHL) gene deletions detected in VHL families by fluorescence in situ hybridization.
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von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer syndrome predisposing to a variety of tumor types that include retinal hemangioblastomas, hemangioblastomas of the central nervous system, renal cell carcinomas, pancreatic cysts and tumors, pheochromocytomas, endolymphatic sac tumors, and epididymal cystadenomas [W. M. Linehan et al., J. Am. Med. Assoc., 273: 564-570, 1995; E. A. Maher and W. G. Kaelin, Jr., Medicine (Baltimore), 76: 381-391, 1997; W. M. Linehan and R. D. Klausner, In: B. Vogelstein and K. Kinzler (eds.), The Genetic Basis of Human Cancer, pp. 455-473, McGraw-Hill, 1998]. The VHL gene was localized to chromosome 3p25-26 and cloned [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. Germline mutations in the VHL gene have been detected in the majority of VHL kindreds. The reported frequency of detection of VHL germline mutations has varied from 39 to 80% (J. M. Whaley et al., Am. J. Hum. Genet., 55: 1092-1102, 1994; Clinical Research Group for Japan, Hum. Mol. Genet., 4: 2233-2237, 1995; F. Chen et al., Hum. Mutat., 5: 66-75, 1995; E. R. Maher et al., J. Med. Genet., 33: 328-332, 1996; B. Zbar, Cancer Surv., 25: 219-232, 1995). Recently a quantitative Southern blotting procedure was found to improve this frequency (C. Stolle et al., Hum. Mutat., 12: 417-423, 1998). In the present study, we report the use of fluorescence in situ hybridization (FISH) as a method to detect and characterize VHL germline deletions. We reexamined a group of VHL patients shown previously by single-strand conformation and sequencing analysis not to harbor point mutations in the VHL locus. We found constitutional deletions in 29 of 30 VHL patients in this group using cosmid and P1 probes that cover the VHL locus. We then tested six phenotypically normal offspring from four of these VHL families: two were found to carry the deletion and the other four were deletion-free. In addition, germline mosaicism of the VHL gene was identified in one family. In sum, FISH was found to be a simple and reliable method to detect VHL germline deletions and practically useful in cases where other methods of screening have failed to detect a VHL gene abnormality. (+info)
The impact of molecular genetic analysis of the VHL gene in patients with haemangioblastomas of the central nervous system.
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OBJECTIVES: Haemangioblastoma of the CNS occurs as a sporadic entity and as a manifestation of the autosomal dominant von Hippel-Lindau disease with the major additional components retinal angioma, renal cancer, and pheochromocytoma. Genetic testing for germline mutations predisposing to von Hippel-Lindau disease has been available since identification of the VHL tumour suppressor gene. The impact of this testing was evaluated in patients with haemangioblastomas seen in this centre. METHODS: A register and database of patients with symptomatic haemangioblastomas for the last 15 years was evaluated. The VHL gene was analysed by the SSCP method for all exons and Southern blotting for mutations and deletions of the gene. RESULTS: 141 patients with haemangioblastoma of the CNS were registered. In 81 patients (57%) there was a disease predisposing germline mutation including eight novel mutations. Population related calculation of patients from the administrative district of Freiburg disclosed VHL germline mutations in 22% of the patients with haemangioblastoma. Analysis of mutation carriers for clinical information suggestive of the syndrome showed (1) a positive family history of a brain tumour in 50%, (2) a history for the patient of extracranial manifestations in 36% (retinal angioma 30%, pheochromocytoma 6%), and (3) 19% presenting with multiple brain tumours when first admitted. By genetic testing of haemangioblastoma patients without any indications of von Hippel-Lindau disease mutation carriers were identified in 14%. Sensitivity of VHL germline testing was 86%. CONCLUSIONS: DNA analysis for VHL germline mutations is clearly superior to clinical information in the diagnosis of von Hippel-Lindau disease. Although the percentage of von Hippel-Lindau disease associated haemangioblastoma decreases after the fourth decade of life and is infrequent in patients without other symptomatic lesions and a negative family history, it is recommended that every patient with CNS haemangioblastoma should be screened for von Hippel-Lindau disease germline mutations. This provides the key information and enables screening for extraneurological tumours of the patients and investigations of the patient's family to ameliorate management of von Hippel-Lindau disease. (+info)
The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2.
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We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2. (+info)
Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein.
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The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease. (+info)
The antiangiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice.
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The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2-3-mm fragments for "prevention" experiments; and (b) 2-3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment. (+info)
Mosaicism in von Hippel-Lindau disease: lessons from kindreds with germline mutations identified in offspring with mosaic parents.
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von Hippel-Lindau disease (VHL [MIM 193300]) is a heritable autosomal dominant multiple-neoplastic disorder with high penetrance. It is characterized by brain and spinal-cord hemangioblastomas, retinal angiomas, clear-cell renal carcinoma, neuroendocrine tumors and cysts of the pancreas, pheochromocytomas, endolymphatic-sac tumors, and papillary cystadenomas of the epididymis and broad ligament. Although most index cases have a positive family history of VHL, some do not and may represent de novo cases. Cases without a family history of VHL may or may not have a germline mutation in their VHL tumor-suppressor gene. We present two cases of VHL mosaicism. In each of two families, standard testing methods (Southern blot analysis and direct sequencing) identified the germline mutation in the VHL gene of the offspring, but not in their clinically affected parent. Additional methods of analysis of the affected parents' blood detected the VHL-gene mutation in a portion of their peripheral blood lymphocytes. In one case, detection of the deleted allele was by FISH, and, in the second case, the 3-bp deletion was detected by conformational sensitive gel electrophoresis and DNA sequencing of cloned genomic DNA. Mosaicism in VHL is important to search for and recognize when an individual without a family history of VHL has VHL. Patients diagnosed without family histories of the disease have been reported in as many as 23% of kindreds with VHL. Identification of individuals potentially mosaic for VHL will affect counseling of families, and these individuals should themselves be included in clinical screening programs for occult disease. (+info)
Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC.
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von Hippel-Lindau (VHL) disease is caused by loss of function of the VHL tumor suppressor protein. Here, we demonstrate that the folding and assembly of VHL into a complex with its partner proteins, elongin B and elongin C (herein, elongin BC), is directly mediated by the chaperonin TRiC/CCT. Association of VHL with TRiC is required for formation of the VHL-elongin BC complex. A 55-amino acid domain of VHL is both necessary and sufficient for binding to TRiC. Importantly, mutation or deletion of this domain is associated with VHL disease. We identified two mutations that disrupt the normal interaction with TRiC and impair VHL folding. Our results define a novel role for TRiC in mediating oligomerization and suggest that inactivating mutations can impair polypeptide function by interfering with chaperone-mediated folding. (+info)