PubMed Health⌕ Search

Biomedical subjects

W Knudson

Publications and source records attributed to W Knudson.

At least 37 records · Page 2Linked to original sources
PubMed1996

CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural and biochemical analysis.

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.

Aggrecans↗
PubMed1995

Increased expression of CD44 in bovine articular chondrocytes by catabolic cellular mediators.

Bovine articular chondrocytes cultured in alginate beads were used to study the effect of catabolic cellular mediators on CD44 expression. Treatment with either the 29-kDa fragment of fibronectin or interleukin-1 alpha results in a time- and dose-dependent inhibition of proteoglycan synthesis as well as a stimulation in the expression of CD44 mRNA level as determined by semi-quantitative polymerase chain reaction following reverse transcription. No noticeable effect at 6 h was observed. By 24 h, the major CD44 product (CD44H) from fibronectin fragment-treated cultures showed an 8-fold increase; CD44H from interleukin-1 alpha-treated cultures showed a 6-fold increase as compared to control cultures. In addition, a minor band, determined to be an isoform of CD44, was also shown to be up-regulated by both mediators. Stimulation of CD44 mRNA via interleukin-1 was also evident by in situ hybridization studies of bovine as well as human articular cartilage in organ culture. The increased in CD44 mRNA is matched by an increase at the protein level as determined by Western blot analysis. The Western blot reveals a doublet protein band at 80-90 kDa that corresponds to the molecular mass of CD44H. Cultures incubated with fibronectin fragments for 24 h had an 8.0-fold increase in CD44, while a 6.6-fold was observed for interleukin-1 alpha. Fluorescein-conjugated hyaluronan binding and internalization studies indicate that the increase in CD44 protein, induced by interleukin-1 alpha, closely correlates with an increase in functional hyaluronan receptors present at the chondrocyte cell surface. Taken together these results indicate that conditions that up-regulate chondrocyte catabolism also up-regulate the expression of CD44, a cell surface hyaluronan receptor involved in hyaluronan endocytosis.

Animals↗
PubMed1995

Serum hyaluronic acid level as a predictor of disease progression in osteoarthritis of the knee.

OBJECTIVE: To investigate the prognostic value of serum hyaluronic acid (HA) and keratan sulfate (KS) levels in relation to tibiofemoral osteoarthritis (OA) of the knee. METHODS: Clinical and demographic data were collected on 94 patients. Radiographs were obtained at study entry and at 5-year followup. Disease progression was defined as 2 mm of joint space narrowing of any tibiofemoral compartment, and/or knee joint surgery during the study period. Serum HA and KS were measured and levels were correlated with entry data and disease progression. RESULTS: At entry, HA levels were significantly related to disease duration (P = 0.036), minimum joint space (P = 0.049), and previous surgery (P = 0.001). After these variables were taken into account, patients whose disease had progressed were shown to have had significantly higher levels of HA at baseline compared with those whose disease had not progressed (P = 0.019). However, there were no significant differences in levels of serum KS between those with and those without disease progression, at entry (P = 0.779) or at subsequent visits. CONCLUSION: These results suggest that serum HA levels predict disease outcome in OA of the knee and confirm that a single measurement of the serum level of KS is not useful as a prognostic marker in OA.

Age Factors↗
PubMed1993

Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes.

The capacity to assemble and retain a pericellular matrix is correlated with the expression of the cell surface binding sites specific for the extracellular matrix macromolecule hyaluronan. These binding proteins have been termed hyaluronan receptors. The lymphocyte-homing receptor CD44 may have identity with these hyaluronan receptors. To determine whether hyaluronan receptors function independently in this capacity for matrix assembly, mammalian cells were transfected with cDNA encoding the putative hyaluronan receptor CD44. After transfection with CD44 cDNA, COS cells gained the capacity to assemble hyaluronan-dependent pericellular matrices in the presence of exogenously added hyaluronan and proteoglycan. Thus, CD44 receptors do function as matrix-organizing, matrix-anchoring hyaluronan-binding proteins. In addition, the expression of CD44/hyaluronan receptors alone is sufficient to direct this matrix assembly. If matrix assembly is a function of cells in vivo that express hyaluronan receptors, this raises interesting possibilities for the role of the receptors in cell migration, when new extracellular matrix environments are encountered.

Animals↗
PubMed1993

Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis.

Several studies have suggested that chondrocytes must have the capacity to internalize and degrade extracellular hyaluronan. In the present study we show direct evidence that hyaluronan is, in fact, endocytosed by chondrocytes and that the endocytosis is mediated via cell surface CD44/hyaluronan receptors. Cultures of bovine articular chondrocytes as well as rat chondrosarcoma chondrocytes were incubated with either fluorescein- or 3H-labeled hyaluronan. Intense binding and accumulation of labeled hyaluronan was visualized by fluorescence microscopy or bright-field/dark-field microscopy following autoradiography. Cell surface hyaluronan was removed with either trypsin or Streptomyces hyaluronidase in order to distinguish and quantify intracellular endocytosed hyaluronan. Labeled hyaluronan was visualized within small discrete intracellular vesicles distributed throughout the cytoplasm. Binding and endocytosis of fluorescein- or 3H-labeled hyaluronan was totally blocked by the addition of excess unlabeled hyaluronan or hyaluronan hexasaccharides, competitive inhibitors of hyaluronan/hyaluronan receptor interactions. Binding and endocytosis was also blocked by the addition of anti-CD44 monoclonal antibodies. Characterization of endocytosed 3H-labeled hyaluronan demonstrated that a significant portion of the hyaluronan was degraded by both the bovine articular and rat chondrosarcoma chondrocytes. Interestingly, a higher proportion of bound hyaluronan was internalized by the bovine chondrocytes. Therefore, hyaluronan receptor-mediated endocytosis and degradation of hyaluronan may provide a critical link to the maintenance and homeostasis of cartilage tissue.

Animals↗
PubMed1993

Hyaluronan-binding proteins in development, tissue homeostasis, and disease.

The high molecular weight glycosaminoglycan hyaluronan plays an important role in tissue remodeling during development, normal tissue homeostasis, and disease. The interaction of hyaluronan with matrix hyaluronan-binding proteins and cell-surface hyaluronan receptors regulates many aspects of cell behavior such as cell migration, cell-cell adhesion, and cell differentiation. Hyaluronan-binding proteins have been grouped together as a family termed hyaladherins--further subdivided in matrix and cell-surface hyaladherins (receptors). Specific hyaluronan-hyaladherin interactions that affect cell behavior are the focus of this review. Both clearance and turnover of hyaluronan involve hyaluronan receptor-mediated endocytosis. Pericellular matrix assembly and retention on many cells, especially chondrocytes, are mediated by hyaluronan receptors, in coordination with other matrix hyaladherins. Hyaluronan can also have an independent, direct effect on cell-to-cell adhesion as well as migration, again mediated by specific cell-surface hyaluronan receptors. This is especially apparent in tumor cells, where metastatic potential is correlated with hyaluronan receptor expression. As migrating cells encounter new environments enriched in matrix hyaladherins, the capacity for matrix assembly may terminate cell migration. Thus, the temporal/spatial deposition of particular matrix hyaladherins also serves as signals or matrix cues to alter cell behavior.

Animals↗
PubMed1992

Seven different assays of hyaluronan compared for clinical utility.

To compare six assays of hyaluronan (hyaluronic acid; HYA) in serum, developed in different laboratories, we analyzed 10 samples from each of three groups: healthy persons, patients with primary biliary cirrhosis, and patients with rheumatoid arthritis. All the assays are based on the use of affinity proteins specific for HYA, prepared from cartilage or brain tissue, and are analogous to RIA or enzyme immunoassay techniques. The assay results were of the same magnitude. Although statistical analysis indicated that the methods in some cases deviated significantly from one another, this variation was less than the physiological variation in the healthy population. Therefore, the results of clinical investigations in which the various methods have been used are comparable. The analyses have high specificity and sensitivity for primary biliary cirrhosis but are somewhat less suitable for detecting rheumatoid arthritis. A seventh laboratory, which obtained antibodies to HYA, used these in an RIA to analyze a separate series of serum specimens. Results were in agreement with those obtained by one of the other assays.

Animals↗
PubMed1991

Solutol HS 15, nontoxic polyoxyethylene esters of 12-hydroxystearic acid, reverses multidrug resistance.

A recently developed non-ionic surfactant called Solutol HS 15 (poly-oxyethylene esters of 12-hydroxystearic acid), with low toxicity in vivo, was shown to reverse completely the multidrug resistance of KB 8-5 and KB 8-5-11 human epidermoid carcinoma cells in vitro but did not potentiate drug toxicity in drug-sensitive KB 3-1 cells. At a concentration of 10% of its own IC50 (mean concentration of drug that causes 50% inhibition of cell growth compared to controls), Solutol HS 15 produced a 35-, 28-, and 42-fold reduction in the resistance of KB 8-5-11 cells to colchicine, vinblastine, and doxorubicin, respectively. Solutol HS 15 was relatively much more potent than the prototypic reversing agent, verapamil, for reversing colchicine resistance, compared to the ability of each agent to reverse colchicine resistance, compared to the ability of each agent to reverse vinblastine resistance. Like verapamil, Solutol HS 15 promoted a 50-fold accumulation of rhodamine 123 in KB 8-5-11 cells, as measured by flow cytometry. Also, Solutol HS 15 and verapamil reduced the efflux of rhodamine 123 from KB 8-5-11 cells previously loaded with rhodamine 123 to a similar low rate. Solutol HS 15 did not affect the transport of alanine or glucose into KB 8-5-11 cells, indicating that its effect upon membrane active transport is not entirely nonspecific. Considering their different structure and different relative potency for reversing colchicine resistance, Solutol HS 15 and verapamil probably reverse multidrug resistance by different mechanisms. Solutol HS 15 merits consideration as a potential therapeutic agent because of its effectiveness for reversing multidrug resistance in vitro and its low toxicity in vivo.

Carcinoma, Squamous Cell↗
PubMed1991

Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix.

In this study, we have examined the capacity of various cell types, which express cell surface hyaluronan receptors, to organize a chondrocyte-like pericellular matrix when given chondrocyte-derived extracellular matrix macromolecules exogenously. The assembly of a pericellular matrix was visualized by a particle exclusion assay. Without the addition of exogenous macromolecular components, none of the cell types studied exhibited significant pericellular matrices extending from their plasma membranes. However, upon the addition of high molecular weight hyaluronan in combination with aggregating cartilage proteoglycan monomers, large pericellular matrices were formed within two hours of incubation. No pericellular matrices were formed if these macromolecular components were added separately at equivalent concentrations or if the components were added in the presence of hyaluronan hexasaccharide, a competitive inhibitor of hyaluronan interaction with cell surface hyaluronan receptors. Fully assembled pericellular matrices could also be displaced by the subsequent addition of hyaluronan hexasaccharides. Nonliving, glutaraldehyde-fixed cells, which retained functional hyaluronan receptors, maintained the capacity to assembly pericellular matrices with exogenous components, in serum-containing or serum-free medium. Cells that were incubated with exogenous matrix macromolecules for 24 h, followed by a chase incubation in medium minus the exogenous macromolecules, continued to maintain the matrix for up to 6 h on live cells and more than 24 h on glutaraldehyde-fixed cells. Cell types that did not express hyaluronan receptors were not capable of organizing such pericellular matrices when incubated with these exogenous components. These findings suggest that cells expressing hyaluronan receptors have a significant capacity to organize their immediate extracellular environment via hyaluronan-hyaluronan receptor interactions. Possible physiological functions for this type of matrix organizing capacity are discussed.

Animals↗
PubMed1990

Similar epithelial-stromal interactions in the regulation of hyaluronate production during limb morphogenesis and tumor invasion.

Changes in extracellular hyaluronate occur during the onset of cell migratory stages of development, wound healing, regeneration, and tumor invasion. During development, the production of hyaluronate, which is spatially and temporarily patterned, is regulated, in part, by epithelial-mesenchymal interactions, as demonstrated in the developing limb (Knudson, and Toole (1988) Biochem, Int., 17, 735). Analogous regulatory interactions occur during tumor invasion. One of us (Knudson, W. et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 6767) has shown that several human carcinoma cells interact with normal human fibroblasts in co-culture to effect the stimulation of hyaluronate production. This type of interaction in vivo may account for the large accumulations of hyaluronate often associated with invasive tumors. Heterologous coculture experiments were performed to determine whether carcinoma cells and embryonic epithelial cells express a common regulatory mechanism to effect the stimulation of hyaluronate production by stromal cells. Human LX-1 lung carcinoma cells or human HCV-29T bladder carcinoma cells cultured together with chick embryo limb bud mesoderm synthesized 2- to 4-fold more hyaluronate than the sum of that produced by carcinoma and mesoderm cultures grown separately. Co-cultures of chick embryo limb bud epithelial cells with adult human skin fibroblasts also synthesized 1.5- to 2.5-fold more hyaluronate. The increase in hyaluronate in these co-cultures was not due to a stimulation of cell proliferation and was additive to the effect of fetal bovine serum. The results suggest a common mechanism of epithelial-stromal interaction in the regulation of hyaluronate production during embryonic development and tumor invasion.

Animals↗
PubMed1990

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells.

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.

Carcinoma, Transitional Cell↗
PubMed1989

The role and regulation of tumour-associated hyaluronan.

Significantly increased levels of the glycosaminoglycan hyaluronan are often associated with human and animal tumours. In the rabbit V2 carcinoma elevated levels of tumour-associated hyaluronan are also closely correlated with invasiveness. We have therefore initiated studies to better define the role and regulation of hyaluronan synthesis in tumour tissues. In cell culture many tumour cell types have reduced capacities to synthesize hyaluronan even when derived from tumours enriched in hyaluronan. We showed that several of these same cells can nevertheless stimulate hyaluronan synthesis by normal fibroblasts. In the LX-1 human lung carcinoma cell line this stimulatory potential resides in a membrane-bound, heat-sensitive, lipophilic, cell surface glycoprotein. These data suggest that production of tumour-associated hyaluronan occurs via tumour-stromal cell interactions. We recently demonstrated that some human tumour cells also possess unoccupied, high affinity, cell surface binding sites for hyaluronan which may allow tumour cells to interact directly with hyaluronan-enriched extracellular matrices. This interaction may in turn allow tumour cells to use hyaluronan as a support for adhesion and locomotion. The spatial organization of hyaluronan could then function to guide tumour cells into surrounding stroma. We attempted to visualize this spatial deposition of hyaluronan in situ within frozen sections of human tumour tissue using a morphological probe that specifically recognizes hyaluronan. Hyaluronan appears most prominently in the partially degraded connective tissue.

Animals↗
PubMed1989

Accumulation of hyaluronate in human lung carcinoma as measured by a new hyaluronate ELISA.

We have developed a new ELISA to quantify hyaluronate. This ELISA takes advantage of an anti-keratan sulfate antibody to differentiate between the coated aggregating rat chondrosarcoma proteoglycan which captures the hyaluronate and the keratan sulfate-bearing aggregating proteoglycan added subsequently. Absorbance in this assay was shown to be linear to the logarithmic concentration of hyaluronate in the range of 15 to 1000 ng/ml. Pre-treatment of hyaluronate with papain or protease did not interfere with its quantification; in contrast, pre-treatment with 0.1N NaOH significantly interferes with the subsequent measurement of the hyaluronate molecules. The size of the hyaluronate molecule was found to be an important factor in quantification: only large size hyaluronate molecules could be quantified accurately. The ELISA was used to show that human lung carcinomas contain 2 to 500 times as much hyaluronate as normal lung tissue from the same patient.

Antibodies, Monoclonal↗
PubMed1988

Membrane association of the hyaluronate stimulatory factor from LX-1 human lung carcinoma cells.

LX-1 human lung carcinoma cells interact with human fibroblasts in culture to cause an increase in hyaluronate production (Knudson et al: Proceedings of the National Academy of Sciences of the United States of America 81:6767, 1984). It is shown here that a similar increase in hyaluronate production also occurs when membranes derived from LX-1 cells, or detergent extracts thereof, are added to cultures of the human fibroblasts. However, no stimulation occurs when membranes or extracts from fibroblasts are added to cultures of the LX-1 cells. The hyaluronate stimulatory factor present in the detergent extracts is a heat- and trypsin-sensitive protein, requires more than 12 h for its action on fibroblasts, causes an elevation in hyaluronate synthetase activity in membranes derived from the fibroblasts, and can be reconstituted into artificial lipid vesicles. Thus, it is concluded that the stimulatory factor is a membrane-bound protein present on the surface of the LX-1 cells and that it interacts with fibroblasts to induce increased hyaluronate synthesis.

Glucuronosyltransferase↗
PubMed1987

The cell surface hyaluronate binding sites of invasive human bladder carcinoma cells.

High-affinity, cell surface binding sites for hyaluronate were demonstrated on highly invasive human bladder carcinoma cells. These binding sites were shown to be specific for hyaluronate, saturable and exhibit a Km of 0.94 x 10(-9) M and a Bmax of 65 ng hyaluronate/10(6) cells. The binding of [3H]hyaluronate to a fixed cell-affinity column was competed with unlabeled hyaluronate and hyaluronate-hexasaccharide but not with hyaluronate-tetrasaccharide, chondroitin sulfate, heparin or non-sulfated dextran. Pre-treatment of cells with protease destroyed the binding activity whereas pretreatment with Streptomyces hyaluronidase to reveal occupied binding sites had no effect. No hyaluronate-binding activity was observed on normal human fibroblasts.

Binding, Competitive↗
PubMed1985

Loss of hyaluronate-dependent coat during myoblast fusion.

Cultured myoblasts were found to exhibit extensive, Streptomyces hyaluronidase-sensitive pericellular coats as revealed by exclusion of particles (fixed red blood cells). These coats are not discernible subsequent to fusion of the myoblasts to form myotubes. The myoblasts contained 2.5 times more hyaluronate attached to their cell surface than myotubes when the data was expressed per unit of protein, but no change in hyaluronate was evident on a per DNA basis. Hyaluronidase activities in the cultures were equivalent when expressed per unit of protein. We conclude that, although the myotubes accumulate larger amounts of protein than myoblasts, there is no compensatory increase in hyaluronate.

Animals↗