Molecular analysis of the von Hippel-Lindau disease tumor suppressor gene in human lung cancer cell lines

The deletion of the short arm of chromosome 3 is frequently observed in lung cancer. To determine whether the von Hippel-Lindau (VHL) disease tumor suppressor gene located at 3p25 is responsible for oncogenesis in lung cancer, we searched the known open reading frame using the single-strand conformation polymorphism (SSCP) technique for mutations in the VHL gene in 72 cancer cell lines including small cell (SCLC) and non-small cell (NSCLC) lung cancers, carcinoids, and mesotheliomas. SSCP analysis showed that four cell lines have altered SSCP patterns within the coding region and one in an intron of the VHL gene. SCLC line NCI-H1672 had a somatic mutation, G to A at nucleotide (nt) 530, leading to amino acid substitution (glycine to aspartic acid) compared to normal DNA from the same patient. Mesothelioma line NCI-H28 had T to A mutation at nt 479 leading to leucine to histidine amino acid change. We found one frequent polymorphism A (0.72) or G (0.28) at nt 19 resulting in either serine or glycine at this position, changes also found in normal peripheral blood cell DNA, often in a heterozygous state. In addition, we found single rare polymorphisms which did not alter the coding region including: C to G at nt 396, G to T at nt 843, and C to T change in an intron. These results suggest that the VHL gene is only rarely mutated in thoracic malignancies.     

Putative control of angiogenesis in hemangioblastomas by the von Hippel-Lindau tumor suppressor gene

The hypoxia-inducible endothelial cell-specific mitogen vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is expressed in low amounts in adult human brain, but is highly upregulated in the perinecrotic palisading cells of glioblastomas. We observed high VEGF expression in cerebellar hemangioblastomas, which are highly vascular, nonnecrotic and presumably nonhypoxic tumors, and hypothesized that a mechanism other than hypoxia leads to VEGF upregulation. Because hemangioblastomas develop in patients with von Hippel-Lindau disease, and mutations of the von Hippel-Lindau tumor suppressor (VHL) gene have also been reported in sporadic hemangioblastomas, we investigated VHL expression in normal cerebellum and in hemangioblastomas and tested the hypothesis that mutations in the VHL gene lead to upregulation of VEGE We observed constitutive expression of VHL mRNA, but downregulation of VEGF mRNA in the postnatal cerebellum. In the adult cerebellum, VHL is predominantly expressed in neuronal cells. In hemangioblastomas, VHL expression appears to be restricted to stromal cells, suggesting that the neoplastic component is the stromal cell. VHL-deficient renal cell carcinoma cells (786-0) produced significantly higher levels of VEGF mRNA and protein compared with 786-0/ wt10 cells, which were stably transfected with the wild-type VHL gene. Our observations suggest that VHL mutations affect stromal cells in hemangioblastomas and that VEGF is upregulated in stromal cells as a consequence of mutations in the VHL gene.     

The von Hippel-Lindau tumor suppressor gene. A rare and intriguing disease opening new insight into basic mechanisms of carcinogenesis

Sindbis virus is a positive strand RNA virus that has provided a valuable model for studying virus structure and replication. It is also being developed as a vector for the expression of heterologous proteins. Many studies with this virus are carried out in cultured BHK cells where infection is usually highly cytopathic and within 1 or 2 days after infection all of the cells are dead. Weiss et al. had established a persistently infected culture of BHK cells by infecting the cells with a virus preparation highly enriched in defective interfering (DI) particles and had isolated an attenuated virus, SIN-1 virus, from the culture [Weiss et al. (1980) J. Virol. 33, 463-474]. SIN-1 virus, free of DI particles, was able to establish a persistent infection in BHK cells. We initiated studies to determine what changes in the genome of the virus were responsible for this phenotype. We describe here the cDNA cloning and sequencing of the 5' terminus and the four nonstructural protein genes from SIN-1 virus. A single coding mutation in the nsP2 gene (a predicted change of Pro-726 --> Ser) produced a virus that was able to establish persistent infection in BHK cells. Additional mutations in the other genes were required to decrease the synthesis of viral RNA to a level similar to that found in cells infected with SIN-1 virus. Incorporation of the nsP2 mutation into a Sindbis virus expression vector led to a higher level of synthesis of the reporter protein, beta-galactosidase, than that obtained with the original Sindbis virus replicon.     

von Hippel-Lindau gene mutations in N-nitrosodimethylamine-induced rat renal epithelial tumors

BACKGROUND: Mutations in the von Hippel-Lindau (VHL) gene are common in human clear cell kidney cancers. Carcinogens in cigarette smoke, especially nitrosamines, are known to induce kidney tumors of a variety of histologic types in rodents--but with no evidence of VHL mutations; however, none of these tumors resembled human clear cell carcinomas. We examined N-nitrosodimethylamine-induced kidney tumors of the clear or mixed clear/granular cell type in Wistar rats to assess the presence of VHL mutations. METHODS: Sections of eight clear or mixed clear/granular cell kidney tumors that had been formalin fixed and paraffin embedded were microdissected. DNA was extracted from the microdissected tissue, and exons 1-3 of the rat VHL gene were examined by use of polymerase chain reaction and cycle sequencing techniques. RESULTS: Four VHL gene mutations (three G:C to A:T and one A:T to G:C) were detected in three of the tumors in contrast to no mutations in 40 previously reported rat kidney tumors of other histologic types (three of eight tumors versus none of 40; two-sided Fisher's exact test; P=.003). Only tumors showing prominent swollen clear cell cytology with a signet-ring appearance had VHL mutations. CONCLUSIONS: To our knowledge, this is the first report of VHL mutations in kidney tumors after direct chemical exposure and provides a possible molecular pathway linking tobacco smoking to kidney cancer.     

von Hippel-Lindau gene deletion detected in the stromal cell component of a cerebellar hemangioblastoma associated with von Hippel-Lindau disease

In adults, lead toxicity is most commonly caused by occupation in a lead industry. Whereas lead toxicity has been described in workers who are involved in bridge rehabilitation, as of this date there has been no systematic evaluation published regarding the conditions responsible for lead toxicity in ironworkers. This is a report of a study designed to identify risk factors for elevated blood-lead levels in ironworkers. One hundred fifty members of a 2,400-member local ironworkers union volunteered to have their blood drawn for lead and zinc protoporphyrin analysis and to complete a questionnaire regarding demographics, health, and occupation. The relationships between these variables and blood-lead level were analyzed using student's t-test, chi-square, and logistic regression. Current work on a lead job, rivet busting as the predominant job task, and cigarette smoking were all found to be significantly associated with elevated blood-lead level. Whereas cigarette smoking and current work with lead have been previously identified as risk factors for toxicity, interventions to prevent lead toxicity in ironworkers should also focus on work practices during rivet busting.     

Efficacy of gene testing for von Hippel-Lindau disease

OBJECTIVE: To determine the efficacy of genetic testing of individuals presenting with features possibly indicative of von Hippel-Lindau (VHL) disease, regardless of other relevant family and clinical details. SETTING AND PARTICIPANTS: Between September 1994 and December 1997, 16 unrelated individuals were referred to Genetic Services of Western Australia by local clinicians and by similar genetic services in other States, for VHL gene mutation analysis because of clinical manifestations suggestive of the diagnosis. METHODS: The subjects were investigated by screening for mutations in the polymerase chain reaction products of the three VHL gene exons using single-stranded conformational polymorphism analysis (SSCP). If no mutations were detected the exons were sequenced, and if no variations were found DNA was examined by Southern analysis for germinal rearrangements. RESULTS: Mutations in the VHL gene were detected in eight of 16 individuals (50%), including 3 individuals with no family history suggestive of VHL disease. Five mutations were detected by SSCP, two by gene sequencing and one by Southern analysis. Each mutation occurred only in a single family and three had not been previously reported. CONCLUSION: Genetic screening of individuals presenting with clinical features suggestive of VHL facilitates confirmation of the diagnosis, accurate genetic counselling and surveillance of at-risk family members. The necessity for costly and time-consuming screening programs can be reduced and screening directed at those carrying the mutation. Our low stringency criteria are justified for screening for VHL mutations.     

Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel-Lindau tumor suppressor gene loss of function

Hemangioblastomas are highly vascular tumors of the central nervous system that overexpress the hypoxia-inducible gene, vascular endothelial growth factor (VEGF), as a consequence of mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL). Previous reports showed that hemangioblastomas can also express erythropoietin (Epo), which is also hypoxia-inducible. However, Epo expression in hemangioblastomas was observed only in individual cases, and the analyses were mainly based on indirect determination of erythropoiesis-stimulating activity. Therefore, we analyzed a series of 11 hemangioblastomas for Epo, VEGF, and VHL expression by Northern blot analysis and compared the results with normal brain and glioblastomas. Surprisingly, we observed Epo mRNA expression in all hemangioblastoma specimens analyzed, but in none of four glioblastomas. In contrast, VEGF mRNA was expressed in all hemangioblastomas and all glioblastomas. In situ hybridization revealed neoplastic stromal cells as Epo- and VEGF-producing cells in hemangioblastomas. These results suggest that in the nonhypoxic microenvironment of hemangioblastoma, Epo, similar to VEGF, might be negatively regulated by the VHL gene product.     

Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.     

Identification of novel mutations in the human EXT1 tumor suppressor gene

Hereditary multiple exostoses (EXT) is a genetically heterogeneous bone disorder caused by genes segregating on human chromosomes 8, 11, and 19 and designated EXT1, EXT2 and EXT3, respectively. Recently, the EXT1 gene has been isolated and partially characterized and appears to encode a tumor suppressor gene. We have identified six mutations in the human EXT1 gene from six unrelated multiple exostoses families segregating for the EXT gene on chromosome 8. One of the mutations we detected is the same 1-bp deletion in exon 6 that was previously reported in two independent EXT families. The other five mutations, in exons 1, 6, 9, and the splice junction at the 3' end of exon 2, are novel. In each case, the mutation is likely to result in a truncated or nonfunctional EXT1 protein. These results corroborate and extend the previous report of mutations in this gene in two EXT families, and provide additional support for the EXT1 gene as the cause of hereditary multiple exostoses in families showing linkage to chromosome 8.     

The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix

Fibronectin coimmunoprecipitated with wild-type von Hippel-Lindau protein (pVHL) but not tumor-derived pVHL mutants. Immunofluorescence and biochemical fractionation experiments showed that fibronectin colocalized with a fraction of pVHL associated with the endoplasmic reticulum, and cold competition experiments suggested that complexes between fibronectin and pVHL exist in intact cells. Assembly of an extracellular fibronectin matrix by VHL-/- renal carcinoma cells, as determined by immunofluorescence and ELISA assays, was grossly defective compared with VHL+/+ renal carcinoma cells. Reintroduction of wildtype, but not mutant, pVHL into VHL-/- renal carcinoma cells partially corrected this defect. Finally, extracellular fibronectin matrix assembly by VHL-/- mouse embryos and mouse embryo fibroblasts (MEFs), unlike their VHL+/+ counterparts, was grossly impaired. These data support a direct role of pVHL in fibronectin matrix assembly.     

Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes

OBJECTIVE: von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia type 2 (MEN2) are autosomal dominant cancer syndromes. In both conditions, phaeochromocytoma is a prominent feature. It has recently been suggested that phaeochromocytoma can be the presenting and sole clinical manifestation of these multi-organ syndromes. The aim of this study was to ascertain the incidence of VHL and MEN2 among patients with sporadic phaeochromocytoma by mutational analysis. PATIENTS: Twenty-seven unrelated patients with biochemically and/or anatomically proven sporadic phaeochromocytoma were evaluated. DESIGN AND MEASUREMENTS: Constitutional DNA obtained from the patients was analysed by single stranded conformational analysis (SSCP) for mutations within the VHL gene coding sequence and by denaturing gradient gel electrophoresis (DGGE) for predominant mutations in exons 10, 11 and 16 of the RET proto-oncogene. The incidence of patients positive for either VHL or RET germline mutations was assessed. RESULTS: Twenty-six of 27 patients had normal SSCP patterns in all three VHL gene exon segments and only one patient, with an atypical clinical presentation, had an aberrant pattern in exon 3 which upon DNA sequencing was shown to harbor a G to A transversion mutation at nucleotide 695. All patients had normal RET exon 10, 11 and 16 DGGE migration patterns. CONCLUSION: Most, if not all, patients with typical unilateral sporadic phaeochromocytoma do not have von Hippel-Lindau disease or MEN2. Thus, clinical and/or molecular investigation for von Hippel-Lindau disease and MEN2 in this patient population does not appear to be indicated.     

Treatment of hemangioblastomas in von Hippel-Lindau disease with linear accelerator-based radiosurgery

OBJECTIVE: Stereotactic radiosurgery is increasingly being used to treat hemangioblastomas, particularly those that are in surgically inaccessible locations or that are multiple, as is common in von Hippel-Lindau disease. The purpose of this study was to retrospectively evaluate the effectiveness of radiosurgery in the treatment of hemangioblastomas. METHODS: From 1989 to 1996, 29 hemangioblastomas in 13 patients with von Hippel-Lindau disease were treated with linear accelerator-based radiosurgery. The mean patient age was 40 years (range, 31-57 yr). The radiation dose to the tumor periphery averaged 23.2 Gy (range, 18-40 Gy). The mean tumor volume was 1.6 cm3 (range, 0.07-65.4 cm3). Tumor response was evaluated in serial, contrast-enhanced, computed tomographic and magnetic resonance imaging scans. The mean follow-up period was 43 months (range, 11-84 mo). RESULTS: Only one (3%) of the treated hemangioblastomas progressed. Five tumors (17%) disappeared, 16 (55%) regressed, and 7 (24%) remained unchanged in size. Five of nine patients with symptoms referable to treated hemangioblastomas experienced symptomatic improvement. During the follow-up period, one patient died as a result of progression of untreated hemangioblastomas in the cervical spine. Three patients developed radiation necrosis, two of whom were symptomatic. CONCLUSION: Although follow-up monitoring is limited, stereotactic radiosurgery provides a high likelihood of local control of hemangioblastomas and is an attractive alternative to multiple surgical procedures for patients with von Hippel-Lindau disease.     

Tumor suppressor genes with special emphasis on the APC tumor suppressor gene

Tumor suppressor genes play a central role in the genesis and progression of human cancers. Genetic alterations of tumor suppressor genes have been found in a variety of hereditary and nonhereditary cancers. Persons that carry a hereditary mutation in tumor suppressor genes are strongly predisposed to one or more kinds of cancer. This review brings current developments in the field of tumor suppressor genes. Special emphasis is dedicated to recently discovered tumor suppressor gene APC (adenomatous polyposis coli) whose mutations are responsible for familial adenomatous polyposis (FAP). The known mutations of the APC gene are described. The role of the APC gene in tumor development, as well as the possibility for presymptomatic genetic testing is also discussed in the paper.