Biomedical subjects

K A Knudsen

Publications and source records attributed to K A Knudsen.

At least 37 records · Page 2Linked to original sources

PubMed1997

N-cadherin-catenin interaction: necessary component of cardiac cell compartmentalization during early vertebrate heart development.

During early heart development the expression pattern of N-cadherin, a calcium-dependent cell adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte differentiation. N-cadherin's adhesive activity is dependent upon its interaction with the intracellular catenins. An association with alpha-catenin and beta-catenin also is believed to be involved in cell signaling. This study details the expression patterns of alpha-catenin, beta-catenin, and gamma-catenin, during definition of the cardiac cell population as distinct compartments in the anterior regions of the chick embryo between stages 5 and 9. The restriction of N-cadherin/catenin localization at stage 5+ from a uniform pattern in vivo, to specific cell clusters that demarcate areas where mesoderm separation is initiated, suggests that the N-cadherin/catenin complex is involved in boundary formation and in the subsequent cell sorting. The latter two processes lead to the specification and formation of the somatic and cardiac splanchnic mesoderm. N-cadherin colocalized with alpha- and beta-catenin at the cell membrane before and during the time that its expression becomes restricted to the lateral mesoderm and continues cephalocaudad into stage 8. These proteins continue to colocalize in the myocardium of the tubular heart. Plakoglobin is not expressed in this region during stages 6-8, but is detected in the myocardium later at stage 13. The observed in vivo expression patterns of alpha-catenin, beta-catenin, and plakoglobin suggest that these proteins are directly linked with the developmental regulation of cell junctions, as cardiac cells become stably committed and phenotypically differentiated to eventually form a mature myocardium. The localization of N-CAM also was analyzed during these stages to determine whether the N-cadherin-catenin localization was unique or whether other cell adhesion molecules were expressed similarly. The results indicate that the unique pattern of N-cadherin expression is not shared with N-CAM. We also show that perturbation of N-cadherin using a function perturbing N-cadherin antibody (NCD-2) inhibits normal early heart development and myogenesis in a cephalocaudad, stage-dependent manner. We propose a model whereby myocardial cell compartmentalization also defines the endocardial population. The presence of beta-catenin suggests that a similar signaling pathway involving Wnt (wingless)-mediated events may function in myocardial cell compartmentalization during early vertebrate heart development, as in Drosophila contractile vessel development.

Animals↗

PubMed1997

N-cadherin promotes the commitment and differentiation of skeletal muscle precursor cells.

Cells with the potential to form skeletal muscle are present in the chick embryo prior to gastrulation. Muscle differentiation begins after gastrulation within the somites. The role of cadherin-mediated adhesion in the commitment and differentiation of skeletal muscle precursor cells was examined by analyzing the expression of cell-cell adhesion molecules in cultures of epiblast, segmental plate, and somite cells and by determining the effects of adhesion-perturbing antibodies on the accumulation of MyoD and sarcomeric myosin. Cultured primitive streak stage epiblast cells downregulate E-cadherin and upregulate N-cadherin. This switch in cadherin expression also occurs in vivo as epiblast cells enter the primitive streak. Although MyoD protein is present in cells with N- or E-cadherin, only cells with N-cadherin differentiate into skeletal muscle. In contrast to the primitive streak stage epiblast cells, prestreak epiblast cells maintain the expression of E-cadherin in vitro. While the majority of prestreak cells contain MyoD, only a few synthesize myosin. Treatment of primitive streak stage epiblast cells with function-perturbing antibodies to N-cadherin resulted in an inhibition of myosin accumulation and a decrease in the percentage of cells with MyoD. Segmental plate and somite cells are similar to primitive streak stage epiblast cells in that most differentiated into skeletal muscle when cultured in serum-free medium. While function-perturbing antibodies to N-cadherin inhibited the accumulation of myosin in these mesoderm cells, the number of MyoD positive cells was unaffected in somite cultures and only partially reduced in segmental plate cultures. These results suggest that N-cadherin-mediated cell-cell adhesion is involved in both the commitment of muscle precursors and their terminal differentiation.

Animals↗

PubMed1997

Developmental defects in mouse embryos lacking N-cadherin.

To investigate the functions of N-cadherin in vivo, we have mutated the gene encoding this adhesion protein in mice. Although N-cadherin is expressed at the time of gastrulation and neurulation, both neurulation and somitogenesis initiate apparently normally in homozygous mutant embryos. However, the resulting structures are often malformed. The somites of the mutant embryos are small, irregularly shaped, and less cohesive compared with those of their wild-type littermates, and the epithelial organization of the somites is partially disrupted. Undulation of the neural tube is also observed in the mutant embryos. Homozygous mutant embryos die by Day 10 of gestation. The mesodermal and endodermal cell layers of the yolk sac are separated in the mutants. The most dramatic cell adhesion defect is observed in the primitive heart; although myocardial tissue forms initially, the myocytes subsequently dissociate and the heart tube fails to develop normally. In vitro studies of cardiac myocytes derived from N-cadherin mutant embryos show that the cells can loosely aggregate and beat synchronously, demonstrating that electrical coupling can occur between N-cadherin-deficient cardiac myocytes. These results show that N-cadherin plays a critical role in early heart development as well as in other morphogenetic processes.

Animals↗

PubMed1997

Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues.

The differential diagnosis of pleural mesotheliomas and lung adenocarcinomas presents a continued challenge in the practice of surgical pathology. Paraffin immunohistochemistry (IHC) using different panels of antibodies can be helpful in some cases, but, as yet, no antigen is expressed specifically in mesotheliomas nor in adenocarcinomas. Using well characterized monoclonal antibodies (MAb) that recognized distinct mesenchymal and epithelial adhesion proteins, N-cadherin (13A9 MAb) and E-cadherin (E9 MAb), respectively, we found previously that in frozen-section IHC mesotheliomas and adenocarcinomas had distinct cadherin phenotypes: mesotheliomas were positive for N-cadherin, and lung adenocarcinomas were positive for E-cadherin. Using antigen-retrieval methods, we successfully extended our study to formalin-fixed, paraffin-embedded tissue sections. Tumors from 28 patients (14 originally diagnosed as mesotheliomas, and 14 diagnosed as adenocarcinomas) were stained with 13A9 MAb and E9 MAb. Review of hematoxylin-eosin sections excluded from analysis one case previously diagnosed as mesothelioma, which represented a hemangiopericytoma. Of the remaining 27 cases, 12 of 13 mesotheliomas were positive for N-cadherin and negative for E-cadherin. The exception was a multifocal microscopic papillary tumor of apparent mesothelial origin, which was negative for both N-cadherin and E-cadherin. Conversely, 13 of 14 adenocarcinomas were E-cadherin positive and N-cadherin negative except for one adenocarcinoma with focal N-cadherin expression. One case of a poorly differentiated adenocarcinoma invading skeletal muscle was negative for both 13A9 and E9. These studies confirmed the utility of the cadherin antibodies in distinguishing pleural mesotheliomas from lung adenocarcinomas. The reactivity of the cadherin-specific antibodies with antigens in paraffin sections make them powerful and reliable markers in the practice of diagnostic surgical pathology.

Adenocarcinoma↗

PubMed1997

Expression of E-cadherin and N-cadherin in surface epithelial-stromal tumors of the ovary distinguishes mucinous from serous and endometrioid tumors.

This study examines the expression of E-cadherin and N-cadherin in the most common epithelial tumors of the ovary. The homotypic interactions of distinctive members of the cadherin family of cell-cell adhesion molecules segregate cells into tissues during embryonic development, and their expression in tumors can be used to trace the histogenesis of tumor cells. Because the surface epithelium of the ovary is a modified mesothelium, we speculated that the expression of E (epithelial)-cadherin and N (neural, mesodermal)-cadherin may provide clues about the controversial origin of common epithelial ovarian tumors. Immunohistochemistry was performed in paraffin sections using well-characterized monoclonal antibodies to E- and N-cadherin and heat-induced antigen-retrieval methods. We found that serous and endometrioid tumors express both E- and N-cadherin. In contrast, mucinous tumors strongly express E-cadherin, but no N-cadherin. The presence of N-cadherin in serous and endometrioid tumors traces their origin to the mesoderm-derived ovarian surface epithelium. The absence of N-cadherin in mucinous tumors clearly distinguishes them from the former, suggesting histogenesis from a cell lineage other than the ovarian surface epithelium or aberrant differentiation mechanisms associated with neoplastic transformation.

Biomarkers↗

PubMed1997

Characterization of the interactions of alpha-catenin with alpha-actinin and beta-catenin/plakoglobin.

Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin and gamma-catenin (also known as plakoglobin), have been identified. beta-catenin or plakoglobin is associated directly with the cadherin; alpha-catenin binds to beta-catenin/plakoglobin and serves to link the cadherin/catenin complex to the actin cytoskeleton. The domains on the cadherin and betacatenin/plakoglobin that are responsible for protein-protein interactions have been mapped. However, little is known about the molecular interactions between alpha-catenin and beta-catenin/plakoglobin or about the interactions between alpha-catenin and the cytoskeleton. In this study we have used the yeast two-hybrid system to map the domains on alpha-catenin that allow it to associate with beta-catenin/plakoglobin and with alpha-actinin. We also identify a region on alpha-actinin that is responsible for its interaction with alpha-catenin. The yeast two-hybrid data were confirmed with biochemical studies.

Actinin↗

PubMed1997

N-cadherin protein distribution in normal embryos and in embryos carrying mutations in the homeobox gene Hoxa-4.

N-cadherin is a calcium-dependent adhesion molecule with a potential role in a variety of morphogenetic events. Although a dynamic pattern of expression in the mouse embryo has been suggested by in situ hybridization analysis, to date there has been no report of N-cadherin protein expression. In this immunohistochemical study we surveyed N-cadherin protein expression in the mid-late gestation mouse embryo utilizing a recently characterized monoclonal antibody. We found N-cadherin expression in a wide array of tissues, including the brain, the eye, various cranial ganglia, the spinal cord, spinal ganglia, somites, vertebral and limb cartilage and perichondria, the developing lung and kidney, the enteric nervous system, and germ cells. These results suggest that N-cadherin protein expression, as in the chick embryo, correlates with the segregation of cells and with organogenesis. As cadherins have been proposed as targets of vertebrate Hox genes, we also examined N-cadherin expression in two lines of Hoxa-4 mutant mice. We did not observe any alterations in N-cadherin expression in either Hoxa-4 null embryos or in transgenic embryos that overexpress Hoxa-4 in the mesenchyme of the gut. However, the partial overlap in expression between Hox genes and N-cadherin, and the likelihood of redundancy in the regulation of target genes, leaves open the possibility that cadherins are direct or indirect targets of Hox genes during mouse embryogenesis.

Animals↗

PubMed1997

Expression of P-cadherin identifies prostate-specific-antigen-negative cells in epithelial tissues of male sexual accessory organs and in prostatic carcinomas. Implications for prostate cancer biology.

Cadherins constitute a family of calcium-dependent cell-cell adhesion molecules the individual members of which are essential for the sorting of cells into tissues during development. In this study, we examined the expression of E-cadherin, N-cadherin, and P-cadherin in tissues obtained from radical prostatectomies. Epithelial cells of prostatic glands, ejaculatory ducts, and seminal vesicles expressed E-cadherin but not N-cadherin. P-cadherin was expressed in epithelial cells of the seminal vesicles and ejaculatory ducts. In the prostate it was limited to the basal cells of prostatic acini, glands with basal cell hyperplasia, and atrophic glands denuded of the luminal cells. All P-cadherin-positive cells were negative for prostatic-specific antigen. Prostatic cancers were mostly P-cadherin negative, but some tumors had P-cadherin-positive areas frequently located close to ejaculatory ducts and negative for prostatic-specific antigen. The mutually exclusive expression of P-cadherin and prostatic-specific antigen suggests that these proteins are involved in differential mechanisms of cell regulation in prostate cancer. P-cadherin may become a useful marker in the diagnosis and management of patients with prostate cancer and low levels of prostatic-specific antigen.

Animals↗

PubMed1996

Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells.

The cytokine tumor necrosis factor-alpha (TNF) increases the frequency of apoptosis in confluent renal epithelial LLC-PK1 cells, an effect that can be blocked by an anti-TNFR1 monoclonal antibody. However, there were no visible "holes" in the cell sheet as a result of TNF-induced apoptosis. Instead a striking tissue remodeling occurred in response to the TNF-induced apoptosis. Apoptotic cells became surrounded and engulfed by repositioned neighboring cells distributed in a distinct "rosette" pattern. The cadherin-catenin cell-cell adhesion molecules, the tight junction-associated protein ZO-1, and actin accumulated at the sites of contact between apoptotic and neighboring cells. Pretreatment with cytochalasin B prevented the accumulation of cadherins-catenins and ZO-1 at the sites of apoptosis and resulted in microscopic holes in the TNF-treated cell sheet. Our results indicate that a renal epithelium can accommodate an increased frequency of apoptosis and still maintain its integrity by mechanisms of tissue remodeling involving the cadherin-catenin adhesion molecules, tight junctional proteins, and actin filaments.

Actins↗

PubMed1995

The differential expression of N-cadherin and E-cadherin distinguishes pleural mesotheliomas from lung adenocarcinomas.

Malignant mesotheliomas are highly aggressive tumors that develop most frequently in the pleura of patients chronically exposed to asbestos. The distinction between malignant mesotheliomas and tumors of epithelial origin, particularly peripheral lung adenocarcinoma, can be difficult despite the use of immunocytochemical markers and other diagnostic tools. During embryonic development the cadherin cell-cell adhesion molecules participate in the segregation of cells into different tissues. As a result of complex mechanisms of tissue selectivity, N-cadherin is expressed by the developing pleural mesothelial cells and E-cadherin is expressed by the epithelial cells of the lung. Thus, we postulated that N-cadherin could be used as a marker of mesothelial cells and mesothelial tumors, in contrast to adenocarcinomas of the lung that are tumors of epithelial origin. We studied the expression of N-cadherin, E-cadherin and two cadherin-associated proteins, alpha-catenin and beta-catenin, in 19 pleural mesotheliomas, 16 lung adenocarcinomas and in 2 mesothelioma cell lines using specific monoclonal antibodies and immunohistochemical methods. Our results show that all mesotheliomas express high levels of N-cadherin, regardless of their histological type, in contrast to lung adenocarcinomas which expressed E-cadherin but no N-cadherin. The cadherin-associated proteins, alpha-catenin and beta-catenin, were present in both mesotheliomas and adenocarcinomas. Our results show that pleural mesotheliomas can be distinguished from lung adenocarcinomas based on the differential expression of N-cadherin and E-cadherin, using specific monoclonal antibodies and immunocytochemistry.

Adenocarcinoma↗

PubMed1995

Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin.

Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E-, P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta-catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha-actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin-independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin.

Actinin↗

PubMed1995

Expression of isoforms of the neural cell adhesion molecule (NCAM) and polysialic acid during the development of the Bufo arenarum olfactory system.

The neural cell adhesion molecule (NCAM), a member of the immunoglobulin superfamily that promotes Ca(2+)-independent cell-cell adhesion, is expressed as various isoforms generated by alternative splicing. In this study, the expression of the 180 kDa isoform (180-NCAM), total NCAM (180, 140 and 120 kDa isoforms) and the polysialic acid moiety of NCAM (PSA) were analyzed during the development of the olfactory system of the toad Bufo arenarum using specific antibodies and immunofluorescence light microscopy. NCAM and PSA were not found in the ectodermal thickening corresponding to the olfactory placode at early larval stage (stage 17), but by stage 19, total NCAM, 180-NCAM and PSA were all expressed in the invaginating olfactory placode at the sites of cell-cell contact and in the differentiating olfactory epithelium. Later, NCAM isoforms and PSA were found also in the primary fibers of the olfactory nerve and in the olfactory bulb. However, the expression of 180-NCAM decreased near the end of larval development and was absent in post-metamorphic and adult animals. In contrast, total NCAM (representing 140 and/or 120 kDa isoforms) and PSA continued to be expressed in olfactory tissues of post-metamorphic and adult animals, consistent with the persistent neural plasticity of this tissue. Because 180-NCAM has been associated with non-proliferating neurons, its down-regulation in post-metamorphic and adult olfactory system may be associated with the regenerative capability and continuous cell turnover documented for this region in adult animals.

Animals↗

PubMed1994

N-cadherin involvement in cardiac myocyte interaction and myofibrillogenesis.

N-cadherin is a cell-surface, Ca(2+)-dependent adhesion molecule found in intercalated disks and extrajunctional sites in the myocardium. In this paper we show that antibodies specific for N-cadherin inhibit the contraction of both interacting and single myocytes isolated from embryonic chicken hearts. Quantitative electron microscopy revealed that anti-N-cadherin significantly decreases cell-cell contact between interacting myocytes, consistent with a role for N-cadherin as a cell-cell adhesion molecule. In addition, quantitative electron microscopy showed that anti-N-cadherin treatment of cardiomyocytes results in a significant reduction in the cytoplasmic area occupied by myofibrils, implying that N-cadherin also plays a role in myofibrillogenesis. We propose that N-cadherin, in addition to mediating cardiac myocyte interaction, promotes myofibril formation necessary for functional activity of the myocardium.

Animals↗

PubMed1993

P- and E-cadherin are in separate complexes in cells expressing both cadherins.

E- and P-cadherin are members of a family of calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, the transmembrane cadherins self-associate, while intracellularly, they interact with the actin-based cytoskeleton. Several intracellular proteins, collectively termed catenins, are tightly associated with E- and P-cadherin. These proteins appear to link the cadherin to the cytoskeleton and have been proposed to be involved in concentrating cadherins at cell-cell adherens junctions. In this paper we report the production of monoclonal antibodies against both alpha- and beta-catenin and use these antibodies to show that in cells simultaneously expressing two different cadherins, E-cadherin and P-cadherin, each cadherin appears to be present in a separate cadherin/catenin complex.

Antibodies, Monoclonal↗

PubMed1993

Rhabdomyosarcoma-derived cell lines exhibit aberrant expression of the cell-cell adhesion molecules N-CAM, N-cadherin, and cadherin-associated proteins.

Many cancer cells show aberrant adhesion properties that likely contribute to tumorigenesis, invasion, and metastasis. Here, we examine three MyoD-expressing, rhabdomyosarcoma-derived human cell lines (RD, A-204, and HS 729) for their expression of the neural cell adhesion molecule (N-CAM), N-cadherin, and the cadherin-associated proteins, alpha-catenin, beta-catenin, and plakoglobin, using specific antibodies and immunoblotting and immunocytochemical methods. Normally, during the formation of skeletal muscle, both N-CAM and N-cadherin are expressed and participate in mediating myoblast adhesion accompanying cell fusion. RD cells express N-CAM, N-cadherin, and the cadherin-associated proteins; however, N-CAM is expressed as a highly sialylated isoform that functions poorly in promoting Ca(2+)-independent cell aggregation. HS 729 cells express N-cadherin and its associated intracellular proteins but have no detectable N-CAM. A-204 cells express no detectable N-CAM or N-cadherin but do express cadherin-associated proteins. Thus, all three rhabdomyosarcoma cell lines exhibit some abnormality in the expression of adhesion molecules known to participate in skeletal myogenesis; however, no common defect was observed that might be considered as a characteristic marker for rhabdomyosarcomas.

Cadherins↗

PubMed1992

Plakoglobin, or an 83-kD homologue distinct from beta-catenin, interacts with E-cadherin and N-cadherin.

E- and N-cadherin are members of a family of calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, the transmembrane cadherins self-associate, while, intracellularly, they interact with the actin-based cytoskeleton. Several intracellular proteins, collectively termed catenins, have been noted to co-immunoprecipitate with E- and N-cadherin and are thought to be involved in linking the cadherins to the cytoskeleton. Two catenins have been identified recently: a 102-kD vinculin-like protein (alpha-catenin) and a 92-kD Drosophila armadillo/plakoglobin-like protein (beta-catenin). Here, we show that plakoglobin, or an 83-kD plakoglobin-like protein, co-immunoprecipitates and colocalizes with both E- and N-cadherin. The 83-kD protein is immunologically distinct from the 92-kD beta-catenin and, because of its molecular mass, likely represents the cadherin-associated protein called gamma-catenin. Thus, two different members of a plakoglobin family associate with N- and E-cadherin and, together with the 102-kD alpha-catenin, appear to participate in linking the cadherins to the actin-based cytoskeleton.

Animals↗

PubMed1991

Colocalization of N-CAM and N-cadherin in avian skeletal myoblasts.

The cell-cell adhesion molecules, N-CAM and N-cadherin, have been shown previously to mediate myoblast interaction during cell fusion accompanying skeletal myogenesis. To study the localization of both molecules in fusion-competent myoblasts, we used antigen-specific primary antibodies and a double-labeling preembedding immuno-electron microscopy technique. Ultrastructural observations and quantitative analysis of the results reveal that N-CAM and N-cadherin frequently colocalize in clusters on the myoblast plasma membrane. The data provide morphological evidence that the two adhesion glycoproteins cooperate in mediating myoblast interaction during myoblast fusion.

Animals↗

PubMed1991

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris.

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.

Animals↗