Mutations of the VHL tumour suppressor gene in renal carcinoma.

Multiple, bilateral renal carcinomas are a frequent occurrence in von Hippel-Lindau (VHL) disease. To elucidate the aetiological role of the VHL gene in human kidney tumorigenesis, localized and advanced tumours from 110 patients with sporadic renal carcinoma were analysed for VHL mutations and loss of heterozygosity (LOH). VHL mutations were identified in 57% of clear cell renal carcinomas analysed and LOH was observed in 98% of those samples. Moreover, VHL was mutated and lost in a renal tumour from a patient with familial renal carcinoma carrying the constitutional translocation, t(3;8)(p14;q24). The identification of VHL mutations in a majority of localized and advanced sporadic renal carcinomas and in a second form of hereditary renal carcinoma indicates that the VHL gene plays a critical part in the origin of this malignancy.

Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas.

Hereditary papillary renal carcinoma (HPRC) is a recently recognized form of inherited kidney cancer characterized by a predisposition to develop multiple, bilateral papillary renal tumours. The pattern of inheritance of HPRC is consistent with autosomal dominant transmission with reduced penetrance. HPRC is histologically and genetically distinct from two other causes of inherited renal carcinoma, von Hippel-Lindau disease (VHL) and the chromosome translocation (3;8). Malignant papillary renal carcinomas are characterized by trisomy of chromosomes 7, 16 and 17, and in men, by loss of the Y chromosome. Inherited and sporadic clear cell renal carcinomas are characterized by inactivation of both copies of the VHL gene by mutation, and/or by hypermethylation. We found that the HPRC gene was located at chromosome 7q31.1-34 in a 27-centimorgan (cM) interval between D7S496 and D7S1837. We identified missense mutations located in the tyrosine kinase domain of the MET gene in the germline of affected members of HPRC families and in a subset of sporadic papillary renal carcinomas. Three mutations in the MET gene are located in codons that are homologous to those in c-kit and RET, proto-oncogenes that are targets of naturally-occurring mutations. The results suggest that missense mutations located in the MET proto-oncogene lead to constitutive activation of the MET protein and papillary renal carcinomas.

Identification of the von Hippel-Lindau disease tumor suppressor gene.

A gene discovered by positional cloning has been identified as the von Hippel-Lindau (VHL) disease tumor suppressor gene. A restriction fragment encompassing the gene showed rearrangements in 28 of 221 VHL kindreds. Eighteen of these rearrangements were due to deletions in the candidate gene, including three large nonoverlapping deletions. Intragenic mutations were detected in cell lines derived from VHL patients and from sporadic renal cell carcinomas. The VHL gene is evolutionarily conserved and encodes two widely expressed transcripts of approximately 6 and 6.5 kilobases. The partial sequence of the inferred gene product shows no homology to other proteins, except for an acidic repeat domain found in the procyclic surface membrane glycoprotein of Trypanosoma brucei.

3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.

Suppression of growth of renal carcinoma cells by the von Hippel-Lindau tumor suppressor gene.

Clear cell renal carcinomas are most frequently characterized by loss of function of both copies of the von Hippel-Lindau (VHL) disease gene, suggesting that the VHL gene product plays an important role in regulating renal cell proliferation. To directly assess the function of the VHL gene product, we transfected the wild-type VHL gene into two renal carcinoma cell lines that lacked normal expression of the gene. Expression of the wild-type VHL gene led to a dramatic suppression of growth in two renal carcinoma cell lines, A498 and UMRC6 in vitro, as measured by colony formation and direct cell counting. Transfection of a naturally occurring mutant VHL gene (nucleotide 713 G to A, Arg to Gln) did not lead to growth suppression of these renal carcinoma cells, nor did transfection of the wild-type VHL gene into two non-renal tumor cell lines that expressed the endogenous wild-type VHL gene. Expression constructs, which included the first ATG at nucleotide 214, were sufficient to produce the strongest growth suppression. These experiments provide direct evidence that the VHL gene product functions to suppress the growth of renal carcinoma cells and also provide a model for mapping the domains of the VHL protein important in suppressing tumor growth.

One-megabase yeast artificial chromosome and 400-kilobase cosmid-phage contigs containing the von Hippel-Lindau tumor suppressor and Ca(2+)-transporting adenosine triphosphatase isoform 2 genes.

We have isolated and ordered yeast artificial chromosomes (YACs) and cosmids surrounding the von Hippel-Lindau (VHL) tumor suppressor and plasma membrane Ca(2+)-transporting ATPase isoform 2 (PMCA-2) genes on chromosome 3p25-26. The YAC contig consists of six YACs and covers a region of 1 megabase. A cosmid-phage contig around VHL and PMCA-2 genes (400 kilobases) was established and integrated into the YAC map. Using these clones, we generated an EcoRI map of the 400-kilobase region. PMCA-2 and VHL complementary DNA were positioned entirely within the cosmid-phage contig as well as two polymorphic markers (D3S601 and D3S1317). This physical map of the cloned region will allow a detailed analysis of both the PMCA-2 and VHL genes. Some of the genomic clones may be useful for isolation of the full-length VHL complementary DNA.

von Hippel-Lindau syndrome: cloning and identification of the plasma membrane Ca(++)-transporting ATPase isoform 2 gene that resides in the von Hippel-Lindau gene region.

We have isolated and analyzed full-length complementary DNA clones encoded by a 200-kilobase gene encompassing the D3S601 locus that resides in the von Hippel-Lindau (VHL) gene region. The deduced amino acid sequence shows 99% identity with the published sequence of the rat plasma membrane Ca(++)-transporting ATPase isoform 2 complementary DNA, implying that we have cloned and positioned the human plasma membrane Ca(++)-transporting ATPase isoform 2 gene within the VHL critical region. The gene is expressed in VHL target tissues and should be considered a potential candidate gene for the VHL disease.

Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG.

The critical region on human chromosome 3p21.3 harboring a putative lung cancer tumor suppressor gene (TSG) was previously defined by allelotyping and recently refined by overlapping homozygous deletions. We report the construction of a 700-kb (cosmid and one P1 phage) clone contig covering the deletion overlap and its flanks. The minimal set of 23 cosmids comprises 600 kb and is extended by one P1 phage to 700 kb to cover the distal breakpoint of the overlap. The clone contig was extensively characterized by restriction and expression mapping to produce high resolution physical and transcription maps of the cloned region. Potential transcribed fragments were detected by hybridization with PCR-amplified cDNA libraries, direct cDNA selection "zoo" blotting, cDNA screening, and identification of 24 CpG islands. Thus far, 15 new genes represented by partial or full-length cDNAs were isolated, characterized, and precisely positioned on the contig. Two previously cloned genes, namely GNAI-2 and GNAT-1, were also positioned. In addition, the telomeric breakpoint of the NCI H740 deletion and centromeric breakpoint of the overlapping GLC20 deletion were discovered and mapped to define precisely the candidate TSG region. This large cosmid clone contig and high resolution maps will prove crucial in the identification of the lung cancer TSG(s).

Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies.

A 2.2 kb cDNA clone which presumably includes the complete human B-raf coding sequence was isolated from a human testes cDNA library. Sequence analysis showed the presence of all three conserved regions CR1, CR2 and CR3 previously identified in raf protein kinases. The cDNA was expressed in E. coli yielding a B-raf fusion protein which reacts with antibodies specific for B-raf raised against the C-terminal peptide as well as with monoclonal antibodies which map into the kinase domain.

Gene structure of the human MET proto-oncogene.

By direct sequencing of cosmids using primers designed from the known cDNA sequence, we identified 19 exons in the human MET proto-oncogene, and sequenced the corresponding 5' and 3' exon-intron junctions. By homology search in the database of the Washington University Genome Sequence Center (GSC), we identified one additional exon. These 20 exons, together with a previously reported exon, bring the total exon number of MET to 21. Oligonucleotide primers were designed to amplify each exon and adjacent intronic sequences to permit examination of each exon for mutations. By restriction mapping, we assembled a 110 kb genomic contig that covered almost the entire MET proto-oncogene. This information is relevant for the screening of recently reported mutations of the MET gene which cause hereditary papillary renal carcinomas and for the search for additional mutations of the same gene which may play a role in the pathogenesis of common human carcinomas including carcinomas of the breast, ovary and pancreas.

Identification of the promoter of the human von Hippel-Lindau disease tumor suppressor gene.

The von Hippel-Lindau (VHL) disease gene is a novel multiple tumor suppressor gene which plays a causal role in the origin of some common cancers including clear cell renal carcinomas and hemangioblastomas of the central nervous system. Here we report the identification of transcription start sites and the promoter of the human VHL gene. The promoter sequence does not contain TATA and CCAAT boxes. Transcription is initiated around a putative SP1 binding site about 60 bp upstream from the first AUG codon in the VHL mRNA. Several putative transcription factor binding sites, notably for nuclear respiratory factor 1 and PAX, were found upstream of the transcription start sites. Promoter-luciferase expression constructs demonstrate, that the promoter is functional when transfected into 293 cells (transformed primary human embryonal kidney cells) and UMRC 6 renal carcinoma cells. Activity is dependent on correct orientation of the promoter. A minimal promoter region of 106 bp was delineated. A set of VHL minigenes, containing the 5' flanking VHL genomic region, was constructed and transfected into UMRC 6 cells. In these cells the level of transcription from the minigenes driven by VHL promoter was comparable with endogenous VHL expression.

Analysis of aberrant methylation of the VHL gene by transgenes, monochromosome transfer, and cell fusion.

Several tumor suppressor genes were shown to be inactivated by a process involving aberrant de novo methylation of their GC-rich promoters which is usually associated with transcriptional repression. The mechanisms underlying this process are poorly understood. In particular this abnormal methylation may be caused and/or maintained by either deficiency of some trans-acting factor(s) or by various malfunctions acting in cis. Here we studied the nature of aberrant methylation of the von Hippel-Lindau (VHL) disease tumor suppressor gene in a human clear cell renal carcinoma cell line, UOK 121, that contains a silent hypermethylated endogenous VHL allele. First, we transfected unmethylated VHL transgenes, driven by the VHL promoter, into UOK 121 cells. Next, to exclude possible position effects that may influence methylation of the introduced VHL genes, we transferred a single chromosome 3, carrying an apparently normal hypomethylated VHL allele into the UOK 121 cells. Finally, we created somatic cell hybrids between UOK 121 and UMRC 6 cells containing a mutant VHL-expressing hypomethylated allele. In these three experiments both the methylation of the VHL promoter and the transcriptional status of the introduced and endogenous VHL alleles remained unchanged. Our results demonstrate that the putative trans-acting factors present in the UOK 121 and UMRC 6 cells are unable to induce changes in methylation pattern of the VHL alleles in all cell lines and hybrids studied. Taken together, the results indicate that cis-specific local features are pivotal in maintaining and perpetuating aberrant methylation of the VHL CpG island. Contribution of some putative trans-acting factors cannot be excluded during a period when the aberrant VHL methylation pattern was first generated.

Cloning of a breast cancer homozygous deletion junction narrows the region of search for a 3p21.3 tumor suppressor gene.

Chromosome 3p abnormalities and allele loss are frequent in lung and breast cancers, and several lung cancer cell lines exhibit homozygous deletions of 3p indicating potential sites of tumor suppressor genes at regions 3p21.3, 3p14.2 and 3p12. We have identified and characterized a new 3p21.3 homozygous deletion in a breast cancer cell line and the primary tumor that overlaps those previously described in small cell lung cancer (SCLC). This homozygous deletion is approximately 220 kb in length and represents a somatically acquired change in the primary breast cancer. Cloning and sequencing of the breakpoint demonstrated that this resulted from an interstitial deletion and precisely pinpoints this deletion within the three SCLC homozygous deletions previously reported. This deletion significantly narrows the minimum common deleted region to 120 kb and is distinct from the previously reported region that suppresses tumor formation of the murine A9 fibrosarcoma cells. These findings suggest that a common homozygous deletion region on 3p21.3 is important in both lung and breast cancers. It is likely that this very well characterized region either contains one tumor suppressor gene common to both tumor types or two closely linked tumor suppressor genes specific for each tumor.

cDNA cloning and expression of the human homolog of the sea urchin fascin and Drosophila singed genes which encodes an actin-bundling protein.

cDNA clones having extensive sequence identity with the sea urchin fascin and the Drosophila singed gene products were isolated from a human teratocarcinoma cDNA library. The human homolog, termed hsn, is a single-copy gene that was localized to human chromosome 7p22 by fluorescence in situ hybridization and is predicted to encode a 493-amino-acid product with a molecular mass of approximately 55,000. This protein would be similar in size to the fascin and singed proteins, as well as a previously described 55-kD actin-bundling protein that was purified from HeLa cells. Monoclonal antibodies directed against the 55-kD HeLa protein were reactive against a bacterially expressed hsn fusion protein, indicating that the hsn gene probably encodes the 55-kD protein. The hsn mRNA was variably expressed in all human tissues analyzed and was highly expressed in actively growing renal carcinoma cell lines and in activated, but not in resting, lymphocytes, suggesting a functional role for hsn in proliferation. The fascin family lacks homology with other characterized actin-binding proteins, and the high degree of evolutionary conservation of these proteins indicates a functional importance of their actin-bundling properties.

Isolation and characterization of the full-length 3' untranslated region of the human von Hippel-Lindau tumor suppressor gene.

We have isolated the 3' untranslated region (3'UTR) of the human von Hippel-Lindau (VHL) tumor suppressor gene from a P1 phage containing the entire VHL genomic sequence. Several putative noncanonical (ATTAAA) poly(A) signals were identified, and the functional significance of these signals was examined by preparing VHL mammalian expression constructs with this DNA fragment and the previously isolated partial cDNA. Northern blot analysis from transfected renal carcinoma cells showed that both the endogenous and transgene VHL transcripts were the same length. Use of VHL transgene deletion mutants indicated that an ATTAAA sequence located between nucleotide (nt) +4237 and nt +4379 most likely serves as an active poly(A) signal in renal carcinoma cells, yielding a 3.6-kb 3'UTR. This work indicates that, together with the 5'UTR and the coding region, these sequences comprise the full-length human VHL cDNA. Sequence analysis revealed a 300- to 600-bp region conserved in human, murine, and rat VHL UTRs. In addition, the human 3'UTR was extremely rich in Alu repetitive elements.

Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation.

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for approximately 25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

The human homolog of the rodent immediate early response genes, PC4 and TIS7, resides in the lung cancer tumor suppressor gene region on chromosome 3p21.

Recently, human chromosome band 3p21.3 was shown to undergo overlapping homozygous deletions in several small cell lung cancer lines further defining a putative tumor suppressor gene(s) region. We report the cloning and mutational analysis of a novel human gene, SKMc15, from the commonly homozygously deleted region in three small cell lung cancer lines (NCI-H1450, NCI-H740, GLC20). It has 11 exons ranging in size from 50 to 541 bp with an open reading frame of 442 amino acids. The gene covers 7 to 10 kb of genomic DNA; the message of 1.8 to 2 kb is expressed in all analyzed fetal and adult human and mouse tissues including heart, brain, placenta, lung liver, skeletal muscle, kidney, testis and pancreas and in small cell and non-small cell cancer lines. The intron/exon boundaries were used to analyze the gene for mutations by exon PCR-SSCP sequencing in 60 small cell lung cancer cell lines. No loss-of-function mutations were detected. The cDNA sequence has high homology, 75% at the protein level, to the rat early response gene PC4 and its murine homolog TIS7. In addition, the known partial sequence of the putative mouse interferon beta2 (64 amino acids) gene is highly conserved in PC4/TIS7 (94%) and in SKMc15 (83%) at the amino acid level. The sequence TAAAT, which is thought to be involved in mRNA degradation, is present in the 3' UTR of SKMc15 and in the 3' UTR of PC4 and TIS7 genes.

von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis.

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19-63'. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.

Germline mutations in the von Hippel-Lindau disease tumor suppressor gene: correlations with phenotype.

von Hippel-Lindau disease (VHL) is an inherited neoplastic disease characterized by a predisposition to develop retinal angiomas, central nervous system hemangioblastomas, renal cell carcinomas, pancreatic cysts, and pheochromocytomas. The VHL gene was recently isolated by positional cloning. The cDNA encodes 852 nucleotides in 3 exons. The VHL gene is unrelated to any known gene families. We identified germline mutations in 85/114 (75%) of VHL families. Clinical heterogeneity is a well-known feature of VHL. VHL families were classified into 2 types based on the presence or absence of pheochromocytoma. The types of mutations responsible for VHL without pheochromocytoma (VHL type 1) differed from those responsible for VHL with pheochromocytoma (VHL type 2). Fifty-six % of the mutations responsible for VHL type 1 were microdeletions/insertions, nonsense mutations, or deletions; 96% of the mutations responsible for VHL type 2 were missense mutations. Specific mutations in codon 238 accounted for 43% of the mutations responsible for VHL type 2. The mutations identified in these families will be useful in presymptomatic diagnosis. The identification of mutations associated with phenotypes contributes to the understanding of fundamental genetic mechanisms of VHL disease.