PubMed Health⌕ Search

Biomedical subjects

F van Roy

Publications and source records attributed to F van Roy.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of eleven novel tumor-associated E-cadherin mutations. Mutations in brief no. 215. Online.

The cell adhesion molecule E-cadherin (CDH1; MIM# 192090) has been implicated in numerous cellular functions, ranging from controlling morphogenesis to suppressing tumor invasion. We describe 11 previously unreported somatic E-cadherin mutations in two subgroups of gastric and breast cancer showing markedly reduced homophilic cell-to-cell interactions. Using reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing of the entire coding region 5 mutations were detected in diffuse-type gastric cancer specimens. The sequence alterations include 3 missense mutations affecting exons 3, 10, and 12. Furthermore, two in-frame deletions were identified removing 63 and 9 base pairs from exon 4 and 5, respectively. In invasive Lobular breast cancer 6 E-cadherin mutations were detected after RT-PCR amplification and direct sequencing or using single strand conformation polymorphism (SSCP) analysis followed by sequencing. In addition to two nonsense mutations affecting exon 2, four out-of-frame deletions removing 115 base pairs (entire exon 2), 224 base pairs (entire exon 3), 8 base pairs from exon 12 or 1 base pair from exon 13 were seen. Our report confirms the general principle that in diffuse-type gastric cancer E-cadherin mutations result in structurally altered proteins with possible reduced adhesive functions whereas in invasive lobular breast carcinomas complete loss-of-function mutations are characteristic.

Breast Neoplasms↗
PubMed1999

The role of the E-cadherin/catenin adhesion complex in the development and progression of cancer.

The E-cadherin/catenin protein complex regulates the functional integrity of epithelia by mediating specific intercellular adhesion, Defects in the transmembrane E-cadherin protein play an important role in several human cancer types. E-cadherin-inactivating mutations were mainly found in sporadic lobular breast carcinoma and in both familial and sporadic diffuse gastric carcinoma. Armadillo proteins such as beta-catenin and p120ctn are complexed to the cytoplasmic tail of E-cadherin, whereas the vinculin-related alphaE-catenin protein forms a link to the actin cytoskeleton. The latter shows inactivating deletions in various tumor cell lines. Apparently, both E-cadherin and alphaE-catenin serve as tumor suppressor and invasion suppressor molecules. On the other hand, protein-stabilizing oncogenic mutations of beta-catenin were found at high frequency in particular human tumor types. Mutated beta-catenin protein is imported into the nucleus, and its binding to LEF/TCF transcription factors modulates transcription of intriguing target genes. Also p120ctn was recently found to arrive in the nucleus and to interact with a transcription factor. Furthermore, a wide variety of mechanisms have been described to regulate in a reversible way E-cadherin/catenin-mediated cell adhesion and differentiation. These phenomena appear to be crucial in human cancer development and progression.

Cadherins↗
PubMed1999

Plakophilin-3, a novel armadillo-like protein present in nuclei and desmosomes of epithelial cells.

We report on a novel Armadillo-like protein, termed plakophilin-3. The human protein, which is encoded by a 2.8 kb messenger RNA, has a predicted molecular mass of 87 kDa. The protein comprises 10 Armadillo-like repeats, preceded by an amino-terminal region of 293 amino acid residues and followed by a short carboxy-terminal region of 27 amino acid residues. Plakophilin-3 is classified as a member of the p120(ctn)/plakophilin subfamily of Armadillo proteins based on the number and organization of the Armadillo repeats and its high sequence similarity to other members of this family. CLUSTAL W alignment of p120(ctn)/plakophilin subfamily members showed the plakophilin-3 protein to be most similar to plakophilin-1 and -2. Western blot analysis of plakophilin-3 revealed expression in all epithelial cell lines tested but not in foreskin fibroblasts and various sarcoma-derived cell lines. This is unlike most other members of the p120(ctn)/plakophilin subfamily, which are widely expressed. By immunofluorescence, the plakophilin-3 protein was colocalized with desmoglein in desmosomes of epithelial cells. In addition, an intriguing speckle-like nuclear staining was observed. Hence, like plakophilin-1 and -2, plakophilin-3 displays a dual intracellular location, i.e. in the desmosomal plaque and in the nucleus. These results suggest the involvement of plakophilin-3 in both desmosome-dependent adhesion and signaling pathways. Furthermore, the human plakophilin-3 gene was mapped on the chromosomal locus 11p15 by fluorescent in situ hybridization.

Amino Acid Sequence↗
PubMed1999

Distinct deleted regions on chromosome segment 16q23-24 associated with metastases in prostate cancer.

Cadherins (CDH) are cell adhesion molecules and their dysfunctions have been implicated in the development of cancer metastases. Several cadherin genes are tandemly located on 16q, which is frequently deleted in prostate cancer. We therefore used 22 markers on 16q to localize important deleted regions in metastases of this tumor. We found 16q deletions in 24/32 (75%) tumors. All lymph node and brain metastases showed extensive deletions, while 52% of primary tumors displayed limited deletions. Commonly deleted regions (CDRs) on 16q23-24, CDR2 (D16S515-D16S516) and CDR4 (D16S520-D13S3028), were strongly associated with metastases and increased Gleason score. Reduced CDH1 (E-cadherin) expression was seen in 16/32 (50%) tumors, but the CDH1 gene is not within either of these two regions. Sequencing analysis for all 16 exons of the CDH1 gene did not reveal any mutations in 10 tumors, including three brain metastases with both 16q22.1 deletion and absent E-cadherin expression. Our results implicate other, yet unidentified genes on 16q23-24 to be the frequent targets of mutations and deletions in prostate cancer metastases.

Aged↗
PubMed1998

E-cadherin and metastasin (mts-1/S100A4) expression levels are inversely regulated in two tumor cell families.

Metastasin is putatively associated with cytoskeletal proteins and may influence cell motility, although its exact physiological role is not known. Because E-cadherin is an invasion suppressor molecule, and metastasin a metastasis-inducing molecule, we wondered which molecule was playing a dominant role in the balance that finally leads to noninvasiveness or invasiveness. The expression levels of E-cadherin and metastasin were monitored in two mouse tumor cell families and were found to be inversely regulated. Moreover, overexpression obtained via transfection of plasmids coding for either one of these two molecules abrogated expression of the other molecule as investigated via Northern and Western blotting experiments. Invasiveness was accordingly influenced. Expression levels of alpha- and beta-catenins were not influenced by up-regulated metastasin, but their intracellular distribution was disturbed. The present results suggest that metastasin-induced invasiveness of several malignant tumor cells is at least partially caused by down-regulation of E-cadherin.

Animals↗
PubMed1998

alpha-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.

alphaE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an alphaE-catenin-deficient colon carcinoma line with a series of alphaE-catenin mutant constructs. The results showed that the amino acid 326-509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326-509 internal domain was found to bind vinculin. When an NH2-terminal alphaE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the alphaE-catenin-deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the alphaE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia.

Binding Sites↗
PubMed1998

Chromosomal mapping of human armadillo genes belonging to the p120(ctn)/plakophilin subfamily.

Armadillo-like proteins are characterized by a series of armadillo repeats that are typically 42 to 45 amino acids in length. Three major subfamilies of Armadillo-like proteins can be distinguished on the basis of their number of repeats, their overall sequence similarity, and dispersion of the repeats throughout the protein. One of these is the p120(ctn)/plakophilin subfamily, which contains at least six members. We mapped the corresponding human genes by PCR on a monochromosomal cell hybrid mapping panel and by fluorescence in situ hybridization. The gene for plakophilin-1 (PKP1) was located at 1q32, the plakophilin-2 gene (PKP2) was located at 12p13, while the gene for p0071 was located at 2q23-q31. We confirmed the chromosomal localization of the p120(ctn) gene (CTNND1) at 11q11, the ARVCF gene at 22q11, and the delta-catenin/NPRAP gene (CTNND2) at 5p15. Although some of the Armadillo proteins are highly related to one another, the corresponding genes are dispersed throughout the human genome.

Armadillo Domain Proteins↗
PubMed1998

Molecular cloning of the human p120ctn catenin gene (CTNND1): expression of multiple alternatively spliced isoforms.

Catenins were discovered as proteins that are linked to the cytoplasmic domain of transmembrane cadherins. Among these junctional plaque proteins are several members of the Armadillo gene family: beta-catenin, plakoglobin, and p120ctn. Recently it became clear that some catenins also mediate nuclear signaling. We performed a detailed analysis of the human p120ctn gene (HGMW-approved symbol CTNND1) and its transcripts. The human p120ctn gene comprises 21 exons, potentially encoding up to 32 protein isoforms as products of alternative splicing. Human isoforms, designated 1 to 4, differ from each other by the start codon used. Additional isoforms are derived from combinations with alternatively used exons A (exon 18) and B (20), near the end of the open reading frame, and also with exon C (11) in the middle of the open reading frame. Hence, the longest isoform is of type 1ABC and comprises 968 amino acid residues. The functional consequence of the observed multitude of p120ctn splice variants awaits further study, but tissue-specific expression was obvious. Further, we demonstrate that the exon organization, which is not simply related to the Armadillo repeat structure, is very well conserved between the p120ctn gene and the related ARVCF gene, but not at all between these two genes and the beta-catenin or plakoglobin genes. The present data favor the concept that p120ctn is the prototype of a subfamily of Armadillo proteins, comprising ARVCF, p0071, delta-catenin/NPRAP, and plakophilins 1 and 2, that are more related to each other than to other Armadillo proteins.

Alternative Splicing↗
PubMed1998

Mutations of the human E-cadherin (CDH1) gene.

The cell-cell adhesion molecule E-cadherin is well known to act as a strong invasion suppressor in experimental tumor cell systems. Frequent inactivating mutations have been identified for the E-cadherin gene (CDH1) in diffuse gastric cancers and lobular breast cancers. To date, 69 somatic mutations have been reported comprising, in addition to few missense mutations, mainly splice site mutations and truncation mutations caused by insertions, deletions, and nonsense mutations. Interestingly, there is a major difference in mutation type between diffuse gastric and infiltrative lobular breast cancers. In diffuse gastric tumors, the predominant defects are exon skippings, which cause in-frame deletions. By contrast, most mutations found in infiltrating lobular breast cancers are out-of-frame mutations, which are predicted to yield secreted truncated E-cadherin fragments. In most cases, these mutations do occur in combination with loss of heterozygosity (LOH) of the wild-type allele. Inactivating germline mutations of E-cadherin were recently reported for families with early-onset diffuse gastric cancer. Also, at the early stages of sporadic lobular breast and diffuse gastric cancers, E-cadherin mutations were detected, suggesting loss of growth control by such mutations and defining E-cadherin as a true tumor suppressor for these particular tumor types.

Breast Neoplasms↗
PubMed1998

Mechanisms of downregulation of transfected E-cadherin cDNA during formation of invasive tumors in syngeneic mice.

Loss of E-cadherin expression has been observed both in experimental tumors and in human cancers and is related to invasiveness and poor differentiation. The E-cadherin-negative mouse mesenchymal tumor cell line MO4 was transfected with several plasmids expressing mouse E-cadherin cDNA. These plasmids differed from each other by the extent of E-cadherin-specific 3' untranslated region (UTR) sequences and by the use of different constitutive promoters. Transfectants were isolated that expressed functional E-cadherin in a homogeneous way. In syngeneic mice, such MO4-Ecad transfectants invariably produced malignant fibrosarcoma-like tumors, which were completely E-cadherin-negative at the protein level. Northern blotting revealed that E-cadherin mRNA expression was downregulated in some but not all MO4-Ecad tumors. Downregulation was caused by mRNA instability triggered by particular 3' UTR sequences. This in vivo downregulation of E-cadherin in malignant MO4-Ecad tumors turned out to be reversible and is likely to be mediated by host factors to be further identified.

Animals↗
PubMed1998

Dysregulation of the E-cadherin/catenin complex by irreversible mutations in human carcinomas.

The different proteins of the E-cadherin/catenin cell-cell adhesion complex are believed to play a predominant role in carcinogenesis. Aberrant expression of these proteins has been found in many different human carcinomas, indicating abnormal regulation. In general, inactivating mutations of the human E-cadherin gene are rare; they are, however, highly frequent in infiltrating lobular breast carcinomas and in diffuse gastric carcinomas. These mutations mostly occur in combination with loss of heterozygosity (LOH) of the wild-type allele. Mutations were found at very early non-invasive stages, thus associating E-cadherin mutations with loss of growth control and defining E-cadherin as a real tumour suppressor for these particular tumour types. Defects affecting both alleles of the alpha E-catenin gene have been found in different human carcinoma cell lines, resulting in the loss of E-cadherin-mediated cell-cell adhesion. Mutations of the beta-catenin gene in colon tumours and melanomas were found to result in an accumulation of the protein in the cytosol. Upon translocation to the nucleus, this beta-catenin enhances TCF/LEF-dependent transcriptional activity. This suggests that mutated beta-catenin can act as an oncogene in these particular tumour types. The multiple interaction partners of beta-catenin are known to be involved in signal transduction, actin organization, protein phosphorylation or transcriptional regulation. This makes this protein an intriguing alternative target for either activation or inactivation in human cancer types characterized by frequent E-cadherin or APC deficiencies.

Breast Neoplasms↗
PubMed1997

Molecular cloning of an alternative human alphaE-catenin cDNA.

The cytoplasmic protein alpha-catenin plays a crucial role in E-cadherin mediated cell-cell adhesion by binding E-cadherin to the cytoskeleton via beta- or gamma-catenin and actin. Functional loss of one of these interacting components leads to decreased cell-cell adhesion, and therefore to loss of epithelial integrity. Northern analysis revealed two distinct alphaE-catenin transcripts in different cell lines, whereas apparently only one protein is expressed. Because of the biological importance of this protein we sought to molecularly characterize the differences between the two observed transcripts. cDNA cloning and sequence analysis revealed the earlier described 3.4 kb alphaE-catenin transcript and an alphaE-catenin transcript of approximately 3.8 kb. This larger transcript contains a 321 bp extension in the 3'UTR sequence, which probably arises as a result of alternative polyadenylation. Considering the presence of AU-rich sequences in the extension, it may be involved in mRNA stability.

Base Sequence↗
PubMed1997

E-cadherin gene alterations are rare events in thyroid tumors.

Reduced expression of E-cadherin has been associated with loss of differentiation and/or increased invasiveness and metastatic ability in several types of neoplasms. Rare studies on thyroid tumors have shown minimal or absent expression in undifferentiated carcinomas and a variable degree of immunoreactivity/mRNA expression in follicular and papillary carcinomas. We studied immunohistochemically 2 follicular adenomas, 8 follicular carcinomas, 18 papillary carcinomas and 3 poorly differentiated carcinomas to evaluate E-cadherin expression. In 7 papillary carcinomas, lymph node metastases were also studied. The E-cadherin gene was analysed in 27 tumors by PCR/SSCP followed, whenever appropriate, by DNA sequencing. LOH at the E-cadherin locus was assessed in 16 tumors. Reduced and heterogeneous expression of E-cadherin was found in primary and metastatic papillary carcinomas, whereas follicular carcinomas showed moderate to strong homogeneous immunoreactivity, and poorly differentiated carcinomas showed no or very faint immunoreactivity. The only case harbouring a (missense type) mutation of the E-cadherin gene was a papillary carcinoma exhibiting immunoreactivity in the primary tumor as well as in metastasis. LOH was found in a follicular adenoma and in a poorly differentiated carcinoma without evidence of mutation in the remaining allele. Our results show that irreversible alterations (allele loss or mutation) of the E-cadherin gene are infrequent in thyroid tumors. The reduced and heterogeneous expression of E-cadherin in thyroid carcinomas, particularly of the papillary histotype, appears to reflect transcriptional regulation or post-transcriptional modulation rather than structural abnormalities.

Adenocarcinoma, Follicular↗
PubMed1997

An efficient and reliable multiplex PCR-SSCP mutation analysis test applied to the human E-cadherin gene.

The invasion suppressor gene E-CADHERIN (CDH1) is downregulated in a large variety of human carcinomas. Up to now, mutational analysis of the CDH1 gene has been described for 325 tumors derived from only four different tissue types. A simple but sensitive mutation detection assay is needed to screen many more tumor types, possibly bearing E-cadherin inactivating mutations. For that purpose, we developed a multiplex PCR-SSCP analysis for all 16 CDH1 exons. Ease of experimentation was combined with reliable sensitivity. Indeed, the present multiplex analysis reduces the number of manipulations to 50%, while the mutation detection turned out to be highly efficient and sensitive.

Base Sequence↗
PubMed1997

E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis.

In breast cancer, inactivating point mutations in the E-cadherin gene are frequently found in invasive lobular carcinoma (ILC) but never in invasive ductal carcinoma (IDC). Lobular carcinoma in situ (LCIS) adjacent to ILC has previously been shown to lack E-cadherin expression, but whether LCIS without adjacent invasive carcinoma also lacks E-cadherin expression and whether the gene mutations present in ILC are already present in LCIS is not known. We report here that E-cadherin expression is absent in six cases of LCIS and present in 150 cases of ductal carcinoma in situ (DCIS), both without an adjacent invasive component. Furthermore, using mutation analysis, we could demonstrate the presence of the same truncating mutations and loss of heterozygosity (LOH) of the wild-type E-cadherin in the LCIS component and in the adjacent ILC. Our results indicate that E-cadherin is a very early target gene in lobular breast carcinogenesis and plays a tumour-suppressive role, additional to the previously suggested invasion-suppressive role.

Breast Neoplasms↗
PubMed1996

E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain.

We have analysed a series of 49 human breast cancers for mutations in the entire coding region plus flanking intron sequences of the E-cadherin gene. The tumours included 41 infiltrating lobular carcinomas, two infiltrating ducto-lobular carcinomas and six infiltrative ductal carcinomas. In the lobular carcinomas 23 different somatic mutations were detected, of which seven were insertions, 11 deletions, two nonsense mutations and three splice site mutations. The other tumours showed no detectable E-cadherin mutations. All the frameshift and nonsense mutations are expected to generate a secreted E-cadherin fragment instead of a transmembrane protein with cell adhesion activity. The majority of the mutations (21 of 23) were found in combination with loss of heterozygosity of the wild type E-cadherin locus (16q22.1), a hallmark of classical tumour suppressor genes. The mutations were scattered over the whole coding region and no hot spots could be identified. All mutations described here were previously unreported. In conclusion, we have identified up to now E-cadherin mutations in 27 of 48 (56%) infiltrating lobular breast carcinomas and in 0 of 50 breast cancers of other histopathological subtypes. These data provide strong evidence that frequent E-cadherin mutations are involved in the particular etiology of sporadic lobular breast cancers.

Binding Sites↗
PubMed1996

Genomic organization of the human beta-catenin gene (CTNNB1).

The cytoplasmic beta-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human beta-catenin gene (CTNNB1) by analysis of cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 bp and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of beta-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5' end and 766 at the 3' end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3' UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5'-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NF kappa B, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5'-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line.

Alternative Splicing↗