Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors.

Von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing to retinal and CNS hemangioblastomas, renal cell carcinoma (RCC), pheochromocytoma, and pancreatic tumors. Interfamilial differences in predisposition to pheochromocytoma reflect allelic heterogeneity such that there is a strong association between missense mutations and risk of pheochromocytoma. We investigated the mechanism of tumorigenesis in VHL disease tumors to determine whether there were differences between tumor types or classes of germ-line mutations. Fifty-three tumors (30 RCCs, 15 hemangioblastomas, 5 pheochromocytomas, and 3 pancreatic tumors) from 33 patients (27 kindreds) with VHL disease were analyzed. Overall, 51% of 45 informative tumors showed loss of heterozygosity (LOH) at the VHL locus. In 11 cases it was possible to distinguish between loss of the wild-type and mutant alleles, and in each case the wild-type allele was lost. LOH was detected in all tumor types and occurred in the presence of both germ-line missense mutations and other types of germline mutation associated with a low risk of pheochromocytoma. Intragenic somatic mutations were detected in three tumors (all hemangioblastomas) and in two of these could be shown to occur in the wild-type allele. This provides the first example of homozygous inactivation of the VHL by small intragenic mutations in this type of tumor. Hypermethylation of the VHL gene was detected in 33% (6/18) of tumors without LOH, including 2 RCCs and 4 hemangioblastomas. Although hypermethylation of the VHL gene has been reported previously in nonfamilial RCC and although methylation of tumor-suppressor genes has been implicated in the pathogenesis of other sporadic cancers, this is the first report of somatic methylation in a familial cancer syndrome.     

Expression of the PAX2 gene in human fetal kidney and Wilms' tumor.

We have examined the pattern of expression of the human PAX2 gene in Wilms' tumors and human fetal kidney by Northern blot and in situ hybridization. Human PAX2 encodes a paired box-containing protein and has a high degree of homology with mouse and Drosophila paired box genes. In situ hybridization analysis reveals that PAX2 is expressed in nephrogenic structures in fetal kidney and also in Wilms' tumors. This pattern of expression suggests that PAX2 may have a role in differentiation of tissues in the kidney. In fetal kidney, PAX2 expression rapidly attenuates following the initial differentiation, but no evidence of attenuation was found in Wilms' tumors. The timing of PAX2 expression is restricted to fetal development, although high levels of expression were also observed in nephrogenic rests of residual normal juvenile kidney tissue adjacent to a Wilms' tumor. Nephrogenic rests are the presumptive precursors of Wilms' tumor but are not necessarily neoplastic. The failure of PAX2 expression to attenuate in Wilms' tumors and nephrogenic rests may be associated with events leading to the onset of Wilms' tumor. By somatic cell hybrid mapping, the PAX2 gene was localized to chromosome 10q22.1-q24.3, although this region has not previously been implicated in Wilms' tumor.     

Mutations in the VHL tumor suppressor gene and associated lesions in families with von Hippel-Lindau disease from central Europe

von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management. Received: 10 November 1995 / Revised: 1 March 1996     

Comparative sequence analysis of the VHL tumor suppressor gene

Comparative genome analysis may provide novel insights into gene evolution and function. To investigate the von Hippel-Lindau (VHL) disease tumor suppressor gene, we sequenced the VHL gene in seven primate species. Comparative analysis was performed for human, primate, and rodent VHL genes and for a putative Caenorhabditis elegans VHL homologue identified by database analysis. The VHL gene has two translation initiation sites (at codons 1 and 54); however, the relative importance of the full-length translation product (pVHL30) and that translated from the second internal translation initiation site (pVHL19) is unclear. The N-terminal sequence of pVHL30 contains eight copies of a GXEEX acidic repeat motif in human and higher primates, but only three copies were present in the marmoset, and only one copy was present in rodent VHL genes. Evolutionary analysis suggested that the N-terminal repetitive sequence in pVHL30 was of less functional importance than those regions present in both pVHL30 and pVHL19. The VHL gene product is reported to form complexes with various proteins including elongin B, elongin C, VBP-1, fibronectin, Spl, CUL2, and HIF-1. Although most of the regions in pVHL that had been implicated in binding specific proteins demonstrated evolutionary conservation, the carboxy-terminal putative VBP-1 binding site was less well conserved, suggesting that VBP-1 binding may have less functional significance. Although an amino acid substitution (K171T) close to the pVHL elongin binding region was found in baboon, analysis of the structure of human pVHL suggested that this substitution would not interfere with pVHL/elongin C interaction. In general, there was a good correlation between the pVHL domains that demonstrated most evolutionary conservation and those that were most frequently mutated in tumors. Analysis of human/C. elegans conservation and human germline and somatic mutation patterns identified a highly conserved mutation cluster region between codons 74 and 90. However, this region is likely to be important for the structural integrity of pVHL rather than representing an additional protein binding domain.     

Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network

The von Hippel-Lindau (VHL) tumor suppressor gene is a protein interaction hub, controlling numerous genes implicated in tumor progression. Here we focus on structural aspects of protein interactions for a list of 35 experimentally verified protein VHL (pVHL) interactors. Using structural information and computational analysis we have located three distinct interaction interfaces (A, B, and C). Interface B is the most versatile, recognizing a refined linear motif present in 17 otherwise non-related proteins. It has been possible to distinguish compatible and exclusive interactions by relating pVHL function to interaction interfaces and subcellular localization. A novel hypothesis is presented regarding the possible function of the N-terminus as an inhibitor of pVHL function.     

Expression of the homeobox gene,Hox 2.1, during mouse embryogenesis

This article reviews recent studies on the expression of the homeobox gene, Hox 2.1, during mouse embryogenesis, using the technique of in situ hybridization. Differential hybridization of radiolabelled antisense versus sense strand RNA is first clearly detected in sections of 8.5 day post coitum (p.c.) early somite embryos. At 12.5 days p.c., higher levels of Hox 2.1 expression are seen in the spinal cord, extending into the base of the hind brain. Hybridization of antisense Hox 2.1 RNA is also seen in the spinal ganglia, in the nodose ganglia of the Xth cranial nerve (which contains derivatives of the neural crest arising from the posterior hind brain), and in the myenteric plexus. Mesodermal cells of certain visceral organs also express Hox 2.1 RNA, in particular the mesoderm of the lung, stomach and meso- and meta-nephric kidney. Comparison of the spatial domains of expression of mouse homeobox genes reveals a pattern consistent with the idea that they play a role in anteroposterior positional specification during embryogenesis.     

Expression of the Lingo/LERN gene family during mouse embryogenesis

We have analysed the expression during mouse development of the four member Lingo/LERN gene family which encodes type 1 transmembrane proteins containing 12 extracellular leucine rich repeats, an immunoglobulin C2 domain and a short intracellular tail. Each family member has a distinct pattern of expression in the mouse embryo as is the case for the related NLRR, FLRT and LRRTM gene families. Lingo1/LERN1 is expressed in the developing trigeminal, facio-acoustic and dorsal root ganglia. An interesting expression pattern is also observed in the somites with expression localising to the inner surface of the dermomyotome in the ventro-caudal lip. Further expression is seen in lateral cells of the hindbrain and midbrain, lateral cells in the motor horn of the neural tube, the otic vesicle epithelium and epithelium associated with the developing gut. Lingo3/LERN2 is expressed in a broad but specific pattern in many tissues across the embryo. Lingo2/LERN3 is seen in a population of cells lying adjacent to the epithelial lining of the olfactory pit while Lingo4/LERN4 is expressed in the neural tube in a subset of progenitors adjacent to the motor neurons. Expression of all Lingo/LERN genes increases as the embryo develops but is low in the adult with only Lingo1/LERN1 and Lingo2/LERN3 being detectable in adult brain.     

Expression of p53 during mouse embryogenesis.

By in situ hybridisation we have examined the expression of p53 during mouse embryogenesis from day 8.5 to day 18.5 post coitum (p.c.). High levels of p53 mRNA were detected in all cells of the day 8.5 p.c. and 10.5 p.c. mouse embryo. However, at later stages of development, expression became more pronounced during differentiation of specific tissues e.g. of the brain, liver, lung, thymus, intestine, salivary gland and kidney. In cells undergoing terminal differentiation, the level of p53 mRNA declined strongly. In the brain, hybridisation signals were also observed in postmitotic but not yet terminally differentiated cells. Therefore, gene expression of p53 does not appear to be linked with cellular proliferation in this organ. A proposed role for p53 in cellular differentiation is discussed.     

Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development.

Germ-line mutations of LKB1 and PTEN tumor suppressor genes underlie the phenotypically related Peutz-Jeghers syndrome (PJS) and Cowden disease (CD), respectively. To analyze possible developmental roles of PTEN and LKB1, we have studied their mRNA expression during mouse embryonic development (E7-17.5) by in situ hybridization. Ubiquitous expression of both genes during early stages (E7-11) became more restricted in later embryonic development (E15-19) where LKB1 and PTEN showed prominent overlapping expression in e.g. gastrointestinal tract and lung. In contrast, LKB1 was selectively expressed at high levels in testis and PTEN was prominently expressed in skin epithelium and underlying mesenchyme. These results indicate that LKB1 and PTEN display largely overlapping expression patterns during embryonic development. Moreover, a high expression of these genes was observed in the tissues and organs affected in PJS and CD patients and in PTEN+/- mice.     

The Expression of VHL (Von Hippel-Lindau) After Traumatic Spinal Cord Injury and Its Role in Neuronal Apoptosis

The VHL (Von Hippel-Lindau) gene is a tumor suppressor gene, which is best known as an E3 ubiquitin ligase that negatively regulates the hypoxia inducible factor. The inactivation of VHL gene could result in the abnormal synthesis of VHL protein, which is in contact with the development and occurrence of renal clear cell carcinoma. However, the expression and possible function of VHL in central nervous system (CNS) is still unclear. To examine the function of VHL in CNS injury and repair, we used an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed an important upregulation of VHL protein, reaching a peak at day 3 and then declined during the following days. Double immunofluorescence staining showed that VHL was co-expressed with neurons, but not with astrocytes and microglia. Moreover, we detected that active caspase-3 had co-localized with VHL in neurons after SCI. Additionally in vitro, VHL depletion, by short interfering RNA, significantly reduced neuronal apoptosis. In conclusion, these data suggested that the change of VHL protein expression was related to neuronal apoptosis after SCI.

     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.     

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.     

Expression of fascin-1, the gene encoding the actin-bundling protein fascin-1, during mouse embryogenesis

Fascin-1 is an actin-bundling protein that contributes to the architecture and function of cell protrusions and microfilaments in cell adhesion, interactions and motility. Fascin-1 has been studied in cultured cells and by biophysical methods, but little is known about its distribution and functions in vertebrate development. As a first step to understanding the role of fascin-1 in embryogenesis, we have characterised the expression pattern of fascin-1 by in situ hybridisation on whole-mount and sectioned mouse embryos from embryonic day (E)8.0-E16.5. Fascin-1 was widely expressed throughout the embryo and the developing nervous system and mesenchymal tissues represented major sites of expression. Intense signals were observed in different regions of the brain, in the spinal cord and retina, and the cranial and dorsal root ganglia (DRG) appeared strongly positive. This neural expression remained strong throughout development. Fascin-1 was also present in the developing somites. High expression was detected in branchial arches and limb bud mesenchyme. At later stages, fascin-1 was expressed in different muscles of the face, skeletal muscles of the body, and in smooth muscle layers of several organs. Limb tendons appeared strongly positive. There was weak expression in heart ventricles. These results show that fascin-1 is principally expressed in neural and mesenchymal derivatives during embryonic development.     

Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene

Regions of the mouse and human genomes with strong homology to the Drosophila engrailed gene have been identified by Southern blot analysis. One mouse engrailed-like region, Mo-en.1, has been cloned and partially sequenced; homology with the engrailed gene is localized to a 180 bp engrailed-like homeo box and 63 nucleotides immediately 3' to it. The protein sequence this region can encode includes 81 amino acids, of which 60 (75%) are identical with those of the putative translation product of the corresponding engrailed sequence. These data suggest that Mo-en.1 represents a mouse homolog of a gene of the Drosophila engrailed gene complex. Mo-en.1 has been mapped to chromosome 1, indicating it is not linked to other homeo box sequences that have been mapped in the mouse genome. Analysis of poly(A)+ RNA extracted from teratocarcinoma cells and whole mouse embryos demonstrates that the conserved homeo box region of Mo-en.1 is expressed differentially during mouse embryogenesis.