Biomedical subjects

K F Becker

Publications and source records attributed to K F Becker.

At least 19 recordsLinked to original sources

PubMed2003

[Molecular carcinogenesis of the upper gastrointestinal tract].

Carcinomas of the upper gastrointestinal tract have been intensively studied for decades in order to identify markers for a) design of simple blood tests to detect presence or recurrence of the disease, b) prediction of therapy response, c) identification of molecular targets for novel therapies. These aims have not yet been fully reached by analysing single genes. However, some genes, including E-cadherin that is altered in sporadic and familial cases of gastric cancer and interleukin 1-beta whose polymorphisms together with Helicobacter pylori infection are associated with an increased risk for gastric cancer, may have the potential for clinical use. In this review we summarize current data for the single gene approach and provide an overview for recent results from cDNA microarray studies.

Adenocarcinoma↗

PubMed2001

Single amino acid substitutions in conserved extracellular domains of E-cadherin differ in their functional consequences.

The calcium-dependent homophilic cell adhesion molecule E-cadherin typically connects epithelial cells. The extracellular portion of the mature transmembrane protein consists of five homologous domains. The four sequences linking these domains contain the structural amino acid motif DXXD that is thought to be involved in direct calcium binding. In gastric cancer patients mutations affecting this motif between the second and third domain are frequently seen. In order to determine the functional significance of similar sequence alterations with regard to their location, we analyzed single amino acid substitutions changing the DXXD motif to DXXA in each linker region according to a mutation found in gastric cancer (D370A). The cDNA sequences coding for DQND, DVLD and DVND were changed (D257A, D479A, D590A, respectively) and stably expressed in E-cadherin negative MDA-MB-435S mammary carcinoma cells. We found that the D257A and D370A mutations result in abnormal protein localization, changes in the actin cytoskeleton, markedly reduced homophilic cell adhesion, and altered cell morphology. Unexpectedly, the tumor-associated D370A mutation but not the D257A mutation induced increased cell motility. The D479A mutation only had slight functional consequences whereas cells expressing the D590A mutant did not differ from cells expressing the wild-type molecule. Although the putative calcium binding motif DXXD is located at repetitive positions in the extracellular portion of E-cadherin, our results indicate that it has different functions depending on the location. Remarkably, tumor cells select for mutations in the most critical domains resulting both in loss of function (decreased cell adhesion) and in gain of function (increased cell motility). Since multiple DXXD motifs are typically seen in other cadherins, our structure-function study is relevant for this gene family in general.

Actins↗

PubMed2001

Highly specific tumor binding of a 213Bi-labeled monoclonal antibody against mutant E-cadherin suggests its usefulness for locoregional alpha-radioimmunotherapy of diffuse-type gastric cancer.

A monoclonal antibody (E-cadherin delta 9-1) directed against a characteristic E-cadherin mutation (in-frame deletion of exon 9), found in diffuse-type gastric cancer but not in any normal tissue, was conjugated with the high linear energy transfer alpha-emitter 213Bi and tested for its binding specificity in s.c. and i.p. nude mice tumor models. After intratumoral application in s.c. tumors expressing mutant E-cadherin, the 213Bi-labeled antibody was specifically retained at the injection site as shown by autoradiography. After injection into the peritoneal cavity, uptake in small i.p. tumor nodules expressing mutant E-cadherin was 17-fold higher than in tumor nodules expressing wild-type E-cadherin (62% injected dose/g versus 3.7% injected dose/g). 78% of the total activity in the ascites fluid was bound to free tumor cells expressing mutant E-cadherin, whereas in control cells, binding was only 18%. The selective binding of the 213Bi-labeled, mutation-specific monoclonal antibody E-cadherin delta 9-1 suggests that it will be successful for alpha-radioimmunotherapy of disseminated tumors after locoregional application.

Animals↗

PubMed2001

Gastric adenocarcinoma: pathomorphology and molecular pathology.

Two types of gastric adenocarcinoma can be distinguished histopathologically: the diffuse and the intestinal type. Molecular pathology supports this theory by showing differences in the genetic pathways of both tumor types. In addition to known pathomorphological factors of prognosis, e.g., depth of tumor infiltration, number of lymph node metastases and resection margins, a few genes have been suggested to have prognostic impact in gastric carcinoma. Clinically relevant molecules whose expression or structure is altered include the plasminogen activator (uPA) and its inhibitor PAI-1 (plasminogen activator inhibitor type 1), the cell cycle regulator cyclin E, epidermal growth factor (EGF), the apoptosis inhibitor bcl-2, the cell adhesion molecule E-cadherin, and the multifunctional protein beta-catenin. Gene amplification and protein overexpression of the growth factor receptors c-erbB-2 and K-sam may be prognostic factors for intestinal-type and diffuse-type gastric cancer, respectively. In addition, genetic instability is commonly seen. There has long been evidence for a genetic predisposition to gastric cancer by epidemiological studies and case reports. Very recently, germ line mutations of E-cadherin have been identified that are responsible for a dominantly inherited form of diffuse-type gastric cancer and could be used to identify individuals that are at high risk.

Adenocarcinoma↗

PubMed2001

Mutant cell surface receptors as targets for individualized cancer diagnosis and therapy.

The catalogue of gene alterations in human cancer is growing rapidly. Alterations in specific genes that play important roles in diverse cellular functions such as cell adhesion, signal transduction, differentiation, development or DNA-repair have been identified. Cancer-associated mutant cell surface molecules are very attractive candidates to target tumor cells because they offer the possibility of minimizing toxic effects to non-tumor cells. The cell adhesion molecule E-cadherin has been shown to play a major role in determining which of the two subtypes of gastric cancer, diffuse or intestinal type, develops. E-cadherin gene mutations typically affect the extracellular portion of the homophilic receptor and are frequently found in patients with diffuse-type tumors. Cancer-specific monoclonal antibodies against the E-cadherin mutational hot spot region are now available. In cell culture and in animal studies we have shown that mutation-specific antibodies exclusively target cells expressing abnormal E-cadherin. Those cells expressing the normal protein were not affected, demonstrating the specificity of our approach. After linking to toxins, drugs or radiolabeled mutation-specific antibodies could serve as very specific agents to treat small tumor deposits. Patients for this novel individualized cancer therapy can be identified within a day using routine immunohistochemistry of biopsies.

Cadherins↗

PubMed2000

Molecular analysis of E-cadherin and cadherin-11 in Wilms' tumours.

Different studies of Wilms' tumours have demonstrated a loss of heterozygosity (LOH) of chromosome 16q ranging from 17 to 25%. In order to search for a potential tumour suppressor gene on 16q, we chose the calcium-dependent cell adhesion molecules E-cadherin and cadherin-11 as candidate genes, which are both located on the long arm of chromosome 16. E-cadherin is known to be expressed in epithelial structures, whereas cadherin-11 is supposed to be expressed in mesenchymal structures and developing epithelium, including renal tubules. For the present study, fresh frozen tissue from 30 Wilms' tumours and corresponding non-tumour tissues were analysed. Single nucleotide polymorphisms of the E-cadherin and cadherin-11 genes were chosen and analysed for allelic inactivation by polymerase chain reaction (PCR) amplification and sequence analysis. Loss of expression of one E-cadherin allele was seen in 10% (2/20) of the informative cases. Two out of 11 informative cases (18%) showed loss of expression of one cadherin-11 allele. No length alterations of either the E-cadherin or the cadherin-11 messenger RNAs were identified using reverse transcription PCR and agarose gel electrophoresis in tumour tissue. Sequencing of the entire E-cadherin coding region in seven cases showed the wild-type sequence. These data imply that E-cadherin and cadherin-11 are not likely to play typical tumour suppressor roles in Wilms' tumour. Interestingly, the E-cadherin immunohistochemistry showed a deviation from the normal reaction pattern in 50% of the cases, with 27% (8/30) showing an apical or cytoplasmic reaction and 23% (7/30) being completely negative. Northern blot analysis revealed that the overall expression of cadherin-11 is much stronger than that of E-cadherin. In several cases, the expression levels of the two genes were inversely correlated, suggesting the existence of a regulatory mechanism. Analysis of differential expression of the various cadherins and their subsequent signal transduction pathways might contribute to a better understanding of the complexity of Wilms' tumour formation.

Adolescent↗

PubMed2000

The use of molecular biology in diagnosis and prognosis of gastric cancer.

The investigation of molecular and genetic changes in gastric cancer has brought new insights into the pathogenesis of the disease. Knowledge of the genetic abnormalities and altered molecules could be used for differential diagnosis in case of an unknown primary tumor, allows their evaluation as prognostic factors, and could open novel avenues for more specific clinical interventions. Clinically relevant molecules whose expression or structure is altered include the plasminogen activator and its inhibitor plasminogen activator inhibitor type 1, the cell cycle regulator cyclin E, epidermal growth factor, the apoptosis inhibitor bcl-2, the cell adhesion molecule E-cadherin, and the multifunctional protein beta-Catenin. In addition, genetic instability is commonly seen. Gene amplification and protein overexpression of the growth factor receptors c-erbB2 and K-sam may be prognostic factors for intestinal- and diffuse-type gastric cancer, respectively. There has long been evidence for a genetic predisposition to gastric cancer by epidemiological studies and case reports. Very recently, germ line mutations of E-cadherin have been identified that are responsible for a dominantly inherited from of diffuse-type gastric cancer and could be used to identify individuals that are at high risk. The clinical implications of the recent findings for diagnosis, prognosis, therapy, and risk assessment are discussed.

Apoptosis↗

PubMed2000

Tumor-derived mutated E-cadherin influences beta-catenin localization and increases susceptibility to actin cytoskeletal changes induced by pervanadate.

E-cadherin participates in homophilic cell-to-cell adhesion and is localized to intercellular junctions of the adherens type. In the present study, we investigated the localization of adherens junction components in cells expressing mutant E-cadherin derivatives which had been previously cloned from diffuse-type gastric carcinoma. The mutations are in frame deletions of exons 8 or 9 and a point mutation in exon 8 and affect the extracellular domain of E-cadherin. Our findings indicate that E-cadherin mutated in exon 8 causes beta-catenin staining at lateral cell-to-cell contact sites and, in addition, abnormally located beta-catenin in the perinuclear region. Moreover, the various mutant E-cadherin derivatives increased the steady-state levels of alpha- and beta-catenin and were found in association with these catenins even after induction of tyrosine phosphorylation by pervanadate. Sustained pervanadate treatment led, however, to rounding-up of cells and induction of filopodia, changes which were first detectable in cells expressing E-cadherin mutated in exon 8. The deterioration of the cell contact was not accompanied with disassembly of the E-cadherin-catenin complex. Based on these observations, we propose a model whereby in the presence of mutant E-cadherin tyrosine phoshorylation of components of the cell adhesion complex triggers loss of cell-to-cell contact and actin cytoskeletal changes which are not caused by the disruption of the E-cadherin-catenin complex per se, but instead might be due to phosphorylation of other signaling molecules or activation of proteins involved in the regulation of the actin cytoskeleton.

Actin Cytoskeleton↗

PubMed1999

Tumour-associated E-cadherin mutations alter cellular morphology, decrease cellular adhesion and increase cellular motility.

A major function of the cell-to-cell adhesion molecule E-cadherin is the maintenance of cell adhesion and tissue integrity. E-cadherin deficiency in tumours leads to changes in cell morphology and motility, so that E-cadherin is considered to be a suppressor of invasion. In this study we investigated the functional consequences of three tumour-associated gene mutations that affect the extracellular portion of E-cadherin: in-frame deletions of exons 8 or 9 and a point mutation in exon 8, as they were found in human gastric carcinomas. Human MDA-MB-435S breast carcinoma cells and mouse L fibroblasts were stably transfected with the wild-type and mutant cDNAs, and the resulting changes in localization of E-cadherin, cell morphology, strength of calcium-dependent aggregation as well as cell motility and actin cytoskeleton organization were studied. We found that cells transfected with wild-type E-cadherin showed an epitheloid morphology, while all cell lines expressing mutant E-cadherin exhibited more irregular cell shapes. Cells expressing E-cadherin mutated in exon 8 showed the most scattered appearance, whereas cells with deletion of exon 9 had an intermediate state. Mutant E-cadherins were localized to the lateral regions of cell-to-cell contact sites. Additionally, both exon 8-mutated E-cadherins showed apical and perinuclear localization, and actin filaments were drastically reduced. MDA-MB-435S cells with initial calcium-dependent cell aggregation exhibited decreased aggregation and, remarkably, increased cell motility, when mutant E-cadherin was expressed. Therefore, we conclude that these E-cadherin mutations may not simply affect cell adhesion but may act in a trans-dominant-active manner, i.e. lead to increased cell motility. Our study suggests that E-cadherin mutations affecting exons 8 or 9 are the cause of multiple morphological and functional disorders and could induce the scattered morphology and the invasive behaviour of diffuse type-gastric carcinomas.

Actins↗

PubMed1999

Functional loss of E-cadherin and cadherin-11 alleles on chromosome 16q22 in colonic cancer.

Proteins of the cadherin family regulate cellular adhesion and motility and are believed to act as tumour suppressors. Previous studies have identified frequent mutation and allelic inactivation of the E-cadherin (cadherin-1) locus in diffuse gastric cancer. At least two other cadherin genes, P-cadherin (cadherin-3) and OB-cadherin (cadherin-11), have been mapped close to the E-cadherin gene on chromosome 16q22. As this region of the genome is frequently deleted in malignancy, multiple cadherin loci may be affected by losses of chromosome 16q22. The expression of mRNA transcripts from polymorphic alleles of the E-cadherin and cadherin-11 genes was examined in 30 cases of colonic, gastric, and renal carcinoma. In gastric cancer, loss of expression of one allele was restricted to the E-cadherin locus, whilst in renal carcinoma neither locus was affected. In colonic cancers, loss of expression of one E-cadherin allele was detected in 5 of 22 cases, whilst loss of a cadherin-11 allele was seen in 5 of 23 cases. This functional loss of cadherin gene expression may be due to gene deletion, inactivation or recombination. As no evidence of cadherin gene mutation was observed in the remaining transcripts, we can conclude that these two genes are only indirectly involved in the pathogenesis of colorectal cancer.

Blotting, Northern↗

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

Loss of immunohistochemical E-cadherin expression in colon cancer is not due to structural gene alterations.

E-cadherin, a transmembrane cell adhesion molecule, has been observed to have an altered pattern of immunoreactivity in several types of carcinomas. In lobular breast cancer, loss of immunoreactivity has been shown to be due either to out-of-frame deletions or to nonsense mutations of the E-cadherin gene. We analysed 29 cases of completely resected colon carcinoma with immunohistochemistry using the HEC-D1 antibody. Normal protein expression similar to that in the adjacent nonmalignant mucosa was seen in 6 cases, whereas 23 tumours had reduced or absent E-cadherin expression. In the 8 cases with no expression of E-cadherin revealed by immunohistochemistry, the entire E-cadherin cDNA sequence was analysed. In these cases, sequence analysis failed to reveal any cDNA mutations despite the negative immunohistochemistry. Possible explanations for this discrepancy include regulatory defects in the E-cadherin promoter, abnormalities at the translation or protein processing levels and mutations in other parts of the gene that were not investigated by the cDNA analysis (e.g. intronic sequences), which could play a role in causing abnormal processing of the E-cadherin protein.

Adult↗

PubMed1999

Diffuse type gastric and lobular breast carcinoma in a familial gastric cancer patient with an E-cadherin germline mutation.

E-Cadherin alterations have been reported frequently in sporadic diffuse type gastric and lobular breast carcinomas. Germline mutations of this gene have been identified recently in several gastric cancer families. We analyzed seven patients with a family history of the disease who had diffuse type gastric cancer diagnosed before the age of 45 for germline mutations in CDH1, the gene encoding the E-cadherin protein. We identified a frameshift mutation in exon 3 in one patient with a strong family history of gastric cancer. The same germline mutation was found in the patient's mother, who had metachronous development of lobular breast and diffuse type gastric carcinomas. Immunohistochemistry for E-cadherin protein expression revealed an abnormal staining pattern in both of these tumors, suggesting complete inactivation of the cell adhesion molecule. Thus, our finding suggests that besides diffuse type gastric cancer, lobular breast carcinomas may be associated with germline CDH1 mutations.

Adult↗

PubMed1999

Analysis of E-cadherin in diffuse-type gastric cancer using a mutation-specific monoclonal antibody.

In-frame deletions from the E-cadherin mRNA, coding for a homophilic cell adhesion molecule, are characteristic for diffuse-type gastric carcinomas. Using immunohistochemical analysis the mutant form cannot be distinguished from normal E-cadherin, making results difficult to interpret. In this study, a rat monoclonal antibody, designated E-cad delta 9-1, was generated against a peptide spanning the fusion junction region between exons 8 and 10. This new epitope is present in an E-cadherin variant that lacks exon 9 from the mRNA due to different splice-site gene mutations. Using Western blotting and immunohistochemistry of E-cadherin-transfected cells, we demonstrate that E-cad delta 9-1 specifically reacts with E-cadherin lacking exon 9 but not with the wild-type protein. No immunoreactivity was observed in 31 nontumorous and embryonal tissues analyzed. In gastric carcinoma specimens known to express mutant E-cadherin mRNA lacking exon 9, E-cad delta 9-1 targets exclusively tumor cells in routine formalin-fixed and paraffin-embedded material from biopsies, primary tumors, and lymph node metastases. In a retrospective series of 172 diffuse-type gastric carcinomas expressing E-cadherin, E-cad delta 9-1 reacted with 22 tumors (13%). This new tumor marker-monoclonal antibody system could open novel avenues for selective diagnosis and specific therapy of a subgroup of diffuse-type gastric cancer patients.

Animals↗

PubMed1999

Rapid detection of mutated E-cadherin in peritoneal lavage specimens from patients with diffuse-type gastric carcinoma.

Tumor cells in abdominal lavage specimens from patients with gastric carcinoma strongly predict subsequent peritoneal metastasis and poor prognosis. Reverse transcription (RT)-polymerase chain reaction (PCR) detection of wild-type E-cadherin has been claimed to be superior to conventional cytology for the detection of patients who subsequently develop peritoneal metastases. The present study tested this hypothesis and determined whether or not the detection of mutated, tumor-specific E-cadherin messenger RNA in abdominal lavage specimens serve as a useful diagnostic tool. Preoperative lavage specimens from 52 patients with diffuse-type gastric carcinoma and from 5 patients with benign disease were analyzed by conventional cytology and by RT-PCR for amplification of E-cadherin. Tumor cells were detected by cytology in 8 (15.3%) of the 52 patients with gastric cancer. The E-cadherin was detected in all 57 samples by RT-PCR. Two of these had abnormal E-cadherin amplification products confirmed to be mutations by direct sequencing, which were identical in the primary tumors. These findings suggest that the detection of wild-type E-cadherin is not sufficiently tumor specific. Also, for diffuse gastric carcinomas with confirmed E-cadherin mutations, detection of mutant E-cadherin by RT-PCR is a potentially valuable method for tumor cell detection in lavage specimens.

Ascitic Fluid↗

PubMed1999

[Novel mutation-specific monoclonal E-cadherin antibodies make possible allele differentiation at the protein level in tumors].

Somatic deletion mutations in the cell adhesion molecule E-cadherin are present in almost 50% of diffuse type gastric cancer. We recently generated monoclonal antibodies against an in-frame deletion of exon 9. The aim of this study was to generate and characterize monoclonal antibodies against the second mutational hot spot, in-frame deletions of exon 8. Lou/C rats were immunized using a KLH-coupled peptide that represents a unique sequence generated by fusion of exon 7 and exon 9 from an E-cadherin deletion mutation lacking exon 8. Hybridoma supernatants were tested in a solid-phase immunoassay using BSA-coupled peptide. Positive reacting hybridomas were confirmed by Western Blots, FACS analysis, and immunohistochemistry of E-cadherin negative carcinoma cells that had been transfected with mutant and wild-type E-cadherin cDNA, respectively. In addition, routine formalin fixed and paraffin embedded tissues from gastric cancer patients were analyzed using both mutation-specific and commercial monoclonal antibodies against E-cadherin, including HECD-1 and AEC. Two hybridoma supernatants, termed E-cad delta 8-1, were selected that reacted with the mutant peptide used for immunization and gave strong signals in Western Blot and FACS analysis with cells expressing mutant E-cadherin lacking exon 8. Wild-type protein expressing cells only reacted with the commercial antibodies but not with the two selected hybridoma supernatants. In contrast to AEC, monoclonal antibody HECD-1 did not react with exon 8 deleted E-cadherin, suggesting that the previously unknown epitope for this often used monoclonal antibody is located at least in part within exon 8. Four gastric cancer specimens known to express mutated E-cadherin mRNA strongly reacted with both mutation-specific supernatants and with AEC monoclonal antibody but not with HECD-1. Taken together, we succeeded in generating monoclonal antibodies reacting with mutant E-cadherin protein lacking exon 8. Furthermore, using both HECD-1 and the new mutation-specific antibodies E-cadherin immunoreactivity can for the first time be evaluated in an allele-specific manner in archival tissues.

Animals↗

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↗

PubMed1997

Cut out or poke in--the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease.

The optical micromanipulation systems UV(ultraviolet)-Laser Microbeam and Optical Tweezers Trap, already proven to be powerful tools for 'non-contact' micro-manipulation of gametes, cells and organelles, have now made their way into the nanocosmos of genes and molecules. Force measurements of DNA transcription have been performed and selective DNA molecule micromanipulation gives insight into single molecule behaviour. Retrievement of selected single cells without contamination is an import prerequisite for further processing with modern methods of molecular biology. Laser micro-dissection allows to precisely eliminate any unwanted material or to isolate pieces of chromosomes or single cells of interest with high accuracy and efficiency. This enables the cell or chromosome specific molecular analysis of genes and genetic defects underlying disease, such as cancer or infection. This review article gives an overview of current topics of laser microbeam application in biological or medical research and advanced molecular diagnosis.

Animals↗