Search PubMed⌕ Search

Biomedical subjects

M I Lerman

Publications and source records attributed to M I Lerman.

At least 55 records · Page 3Linked to original sources
PubMed1996

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

Amino Acid Sequence↗
PubMed1996

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).

Chromosome Mapping↗
PubMed1996

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.

Animals↗
PubMed1996

Localization of the human vascular endothelial growth factor gene, VEGF, at chromosome 6p12.

Using overlapping cosmids representing the vascular endothelial growth factor (VEGF) locus, the VEGF gene was mapped by fluorescence in situ hybridization to chromosome 6p12. This localization permits linkage analysis and the identification of gene interaction in the region, as well as alterations of the VEGF structure or expression in cancer cells with chromosome abnormalities.

Base Sequence↗
PubMed1995

Cellular proteins that bind the von Hippel-Lindau disease gene product: mapping of binding domains and the effect of missense mutations.

The von Hippel-Lindau disease (VHL) gene is a novel tumor suppressor gene that plays a role in the pathogenesis of renal cell carcinomas and hemangioblastomas of the central nervous system. To begin an evaluation of the biological functions of the VHL gene product (pVHL), we prepared bacterial fusion protein between glutathione S-transferase and wild-type or mutant pVHLs. The fusion proteins were used to identify cellular proteins that bind to pVHL in vitro. Monkey kidney cells transfected with wild-type or mutant VHL cDNAs were used to identify cellular proteins that bind to pVHL in vivo. Wild-type pVHL consistently bound two cellular proteins with apparent molecular masses of 10 and 14 kilodaltons that were designated p10 and p14, respectively. Mapping studies with a panel of VHL deletion mutant proteins demonstrated that p10 and p14 bound to a 32-amino acid peptide located in the carboxy terminal portion of pVHL. Missense mutation located within this 32-amino acid peptide abrogated the ability of the VHL protein to bind p10 and p14. Of 67 VHL families with identified germline mutations, 42 families had mutations predicted to affect the p10/p14-binding region. Maintenance of the integrity of the p10/p14-binding region appears to be essential for cellular growth regulation by pVHL.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Von Hippel-Lindau (VHL) disease with pheochromocytoma in the Black Forest region of Germany: evidence for a founder effect.

We identified a germline missense mutation at nucleotide 505 (T to C) of the VHL tumor suppressor gene in 14, apparently unrelated, VHL type 2A families from the Black Forest region of Germany. This mutation was previously identified in two VHL 2A families living in Pennsylvania (USA). All affected individuals in the 16 families shared the same VHL haplotype indicating a founder effect. This missense mutation at codon 169 (Tyr to His) would probably cause an alteration in the structure of the putative VHL protein. The association of this distinct mutation with the pheochromocytoma phenotype in VHL may help to elucidate the genetic mechanism of carcinogenesis in this multi tumor cancer syndrome.

Adrenal Gland Neoplasms↗
PubMed1995

Detection of germline mutations in the von Hippel-Lindau disease gene by the primer specified restriction map modification method.

Von Hippel-Lindau disease (VHL) is an inherited disorder characterised by a predisposition to develop tumours in the eyes, central nervous system, kidneys, and adrenal glands. Recently the VHL gene was cloned and shown to be mutated in 75% of US and Canadian VHL families. To develop simple, rapid methods for the detection of mutations found in large numbers of affected people, we designed based on the primer specified restriction site modification method. These tests have proved useful in identifying asymptomatic mutated VHL gene carriers who have the nt 505 T to C mutation or the nt 686 T to C mutation. Together with an MspI digestion test which can detect a mutation hot spot in codon 238, polymerase chain reaction/restriction endonuclease based tests can now detect VHL mutations in more than 50% of VHL type 2 families.

Base Sequence↗
PubMed1995

Colocalization of the rat homolog of the von Hippel Lindau (Vhl) gene and the plasma membrane Ca++ transporting ATPase isoform 2 (Atp2b2) gene to rat chromosome bands 4q41.3-->42.1.

Using fluorescence in situ hybridization, we localized the rat homolog of the von Hippel-Lindau gene (Vhl) to rat chromosome band 4q41.3-->q42.1. We also mapped the gene encoding the plasma membrane Ca(++)-transporting ATPase isoform 2(Atp2b2) to the same chromosome subregion. These two genes together with Raf1 appear to be members of a large syntenic gene cluster that maps to human chromosome bands 3p25-->p26, mouse chromosome bands 6 C3-->E, and rat chromosome bands 4q41-->q42. Cytogenetic analysis of NRK 52E cells derived from immortalized normal rat kidney epithelial cells revealed an inverted duplication of the region containing this gene cluster.

Animals↗
PubMed1995

Genetics of renal-cell carcinoma and evidence for a critical role for von Hippel-Lindau in renal tumorigenesis.

Using a genetic linkage-based positional cloning approach the VHL gene was identified at chromosome 3p25.5. VHL is mutated in the germlines of affected individuals, and in VHL-associated tumors the mutation is almost always exposed by virtue of chromosomal deletion of the inherited wild-type allele. VHL is also frequently mutated in sporadic, nonpapillary RCC and in familial RCC. This was predicted because such tumors are histologically similar to VHL-associated renal tumors. Knowledge that VHL plays a critical role in sporadic RCC should aid in the future diagnosis and treatment of this malignancy. Detailed analyses of the biology of individual mutations will be required to determine whether the inherited VHL mutations or acquired sporadic mutations cause loss of protein function or have dominant-negative affects. However, the nature of the VHL protein is at present unclear and a complete understanding its function will only be expected after the cloning of the full-length gene.

Carcinoma, Renal Cell↗
PubMed1995

Expression of the Von Hippel-Lindau tumor suppressor gene, VHL, in human fetal kidney and during mouse embryogenesis.

BACKGROUND: Von Hippel-Lindau (VHL) disease is a familial cancer syndrome that has a dominant inherited pattern which predisposes affected individuals to a variety of tumours. The most frequent tumors are hemangioblastomas of the central nervous system and retina, renal cell carcinoma (RCC), and pheochromocytoma. The recent identification and characterization of the VHL gene on human chromosome 3p and mutational analyses confirms the VHL gene functions as a classical tumor suppressor. Not only are mutations in this gene responsible for the VHL syndrome, but mutations are also very frequent in sporadic RCC. MATERIALS AND METHODS: VHL expression in human kidney and during embryogenesis, was analyzed by in situ mRNA hybridization with 35S-labeled antisense VHL probes, derived from human and mouse cDNAs, on cryosections of human fetal kidney and paraffin sections of murine embryos. RESULTS: In human fetal kidney, there was enhanced expression of VHL within the epithelial lining of the proximal tubules. During embryogenesis, VHL expression was ubiquitous in all three germ cell layers and their derivatives. Expression occurred in the cerebral cortex, midbrain, cerebellum, retina, spinal cord, and postganglionic cell bodies. All organs of the thoracic and abdominal cavities expressed VHL, but enhanced expression was most apparent in the epithelial components of the lung, kidney, and eye. CONCLUSIONS: In human fetal kidney, the enhanced epithelial expression of the VHL gene is consistent with the role of this gene in RCC. There is widespread expression of the VHL gene during embryogenesis, but this is pronounced in areas associated with VHL phenotypes. These findings provide a histological framework for investigating the physiological role of the VHL gene and as basis for further mutational analysis.

Animals↗
PubMed1994

Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.

Mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) gene appear to be causal events for the majority of spontaneous clear-cell renal carcinomas. We now show that hypermethylation of a normally unmethylated CpG island in the 5' region provides another potentially important mechanism for inactivation of the VHL gene in a significant portion of these cancers. This hypermethylation was found in 5 of 26 (19%) tumors examined. Four of these had lost one copy of VHL while one retained two heavily methylated alleles. Four of the tumors with VHL hypermethylation had no detectable mutations, whereas one had a missense mutation in addition to hypermethylation of the single retained allele. As would be predicted for the consequence of methylation in this 5' CpG island, none of the 5 tumors expressed the VHL gene. In contrast, normal kidney and all tumors examined with inactivating VHL gene mutations but no CpG island methylation had expression. In a renal cell culture line, treatment with 5-aza-2'-deoxycytidine resulted in reexpression of the VHL gene. These findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers.

Adenocarcinoma, Clear Cell↗
PubMed1994

Deletion of two separate regions on chromosome 3p in breast cancers.

We have characterized the copy number of various loci on chromosome 3p in a series of breast cancers. To determine the precise region(s) involved, restriction fragment length polymorphism (RFLP) analysis for loss of heterozygosity (LOH) was performed using a panel of RFLP probes at 3p13-14, 3p21-22, and 3p24-26. The incidence of LOH at the three loci was 41, 32, and 45%, respectively. To validate the LOH data and to gain insights into the mechanisms resulting in LOH, chromosome 3 pericentromeric and 3p region-specific DNA probes were used to determine the DNA copy number by fluorescence in situ hybridization (FISH). Among 22 cases examined, 15 showed loss by both LOH and FISH, indicating that the dominant mechanism of LOH at 3p in breast cancer is a physical deletion. Two of the 22 cases showed loss by RFLP analysis but not by FISH, suggesting either mitotic recombination or loss and endoreduplication. In three cases, RFLP analysis indicated allelic imbalance, which was incorrectly interpreted as LOH, since a gain of one allele was suggested by FISH. By constructing a deletion map, we found that 2 separate regions, 3p13-14 and 3p24-26, were independently deleted in some breast cancers. Additionally, four cases had break points within the 3p24-26 region and one case had a homozygous deletion at 3p13, further supporting the hypothesis that there are tumor suppressor genes at both 3p13-14 and 3p24-26. Although high frequency of LOH was observed at the 3p21-22 region, there was no direct evidence supporting the existence of a breast cancer tumor suppressor gene there as opposed to codeletion with either the proximal or distal region.

Blotting, Southern↗
PubMed1994

The use of molecular genetic analysis in the diagnosis of renal cell carcinoma.

The most common genetic aberration seen in nonpapillary renal cell carcinoma is believed to be the loss or inactivation of allelic material on the short arm of chromosome 3 (3p). Two patients underwent nephrectomy at our institution, each initially receiving a histologic diagnosis of renal cell carcinoma. Molecular analysis of these tissues revealed no genetic deletion on 3p, prompting further histologic and immunohistochemical evaluation of the original specimens. These additional studies confirmed alternate histologies other than renal cell carcinoma in each case. These data suggest that the loss of genetic material on the short arm of chromosome 3 may be specific for nonpapillary renal cell carcinoma. Molecular analysis may prove to be a useful adjunct in establishing the diagnosis in renal neoplasms with equivocal histologies.

Adult↗
PubMed1994

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours.

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.

Alleles↗
PubMed1994

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.

Amino Acid Sequence↗