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D R Critchley

Publications and source records attributed to D R Critchley.

At least 91 records · Page 5Linked to original sources
PubMed1986

Tumour basement membrane laminin in adenocarcinoma of rectum: an immunohistochemical study of biological and clinical significance.

Well-defined basement membrane laminin was seen in 98/158 (62%) rectal adenocarcinomas stained by an immunoperoxidase method. Only 27 (28%) patients with laminin-positive tumours developed distant metastases, compared with 39 (65%) patients with laminin-negative carcinomas. The corrected 5-year survival rates for patients with laminin-positive and laminin-negative tumours were 65% and 23%, respectively. Twenty-five out of 30 (83%) well-differentiated adenocarcinomas and only 3/15 (17%) poorly differentiated tumours contained basement membrane laminin, with moderately differentiated carcinomas showing intermediate laminin status (70/110, 64%, laminin positive). Forty-three of 60 (72%) of laminin-negative tumours had metastasized to regional lymph nodes. These data suggest that laminin may be a marker for differentiation. However, laminin status yields information about tumour behaviour which is not confined to stage and grade, and multivariate analysis shows that it is a better indicator of prognosis than tumour grade as assessed by conventional histology. Although laminin status alone is a less useful predictor of prognosis than Dukes' stage, a patient with a laminin-positive adenocarcinoma of rectum is 2.7 times as likely to survive 5 years than a patient with a laminin-negative tumour. Assessment of laminin status, together with Dukes' stage is, therefore, commended as a more precise and objective indicator of prognosis than histological degree of differentiation in colorectal carcinoma.

Adenocarcinoma↗
PubMed1986

Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.

Animals↗
PubMed1986

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity.

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.

Avian Sarcoma Viruses↗
PubMed1986

Variants of BALB/c 3T3 cells lacking complex gangliosides retain a fibronectin matrix and spread normally on fibronectin-coated substrates.

Evidence has accumulated that di- and trisialogangliosides are involved in the interaction of cells with fibronectin. We have therefore tested the ability of variants of BALB/c 3T3 deficient in such gangliosides to organize a fibronectin matrix and to spread on fibronectin-coated substrates. Whereas BALB/c 3T3 cells contained gangliosides GM3, GM1, and GD1a, direct chemical analysis showed that five out of six variants isolated contained no detectable GD1a. By the overlaying of thin layer chromatograms of cellular gangliosides with 125I-cholera toxin, these variants were also found to lack ganglioside GM1. In contrast, the sialogalactoprotein profile of these cells, analyzed using an 125I-ricin/SDS polyacrylamide gel overlay technique, was similar to that of the parent cell line. All variants organized an extensive fibronectin matrix comparable to that of BALB/c 3T3, as shown using either immunofluorescence or lactoperoxidase-catalyzed iodination. The variants could also spread on fibronectin-coated substrates and adopt a morphology similar to that of BALB/c 3T3 cells, with little or no difference in the concentration of fibronectin required for 50% cell spreading. Cell spreading of the variants was accompanied by the formation of focal contacts and microfilament bundles, in a manner closely resembling that seen with BALB/c 3T3 cells. Treatment of BALB/c 3T3 cells with neuraminidase, which converts much of the cellular GD1a to GM1, did not affect cell spreading on fibronectin. The results clearly demonstrate that complex gangliosides are not essential for retention of a fibronectin matrix or for spreading on fibronectin-coated substrates.

Actin Cytoskeleton↗
PubMed1986

Reevaluation of the role of gangliosides as receptors for tetanus toxin.

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Tris-saline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GD1b approximately equal to GT1b approximately equal to GQ1b greater than GD2 greater than GD3 much greater than GD1a approximately equal to GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.

Animals↗
PubMed1986

Comparison of the relative importance of tyrosine-specific vinculin phosphorylation and the loss of surface-associated fibronectin in the morphology of cells transformed by Rous sarcoma virus.

We have investigated the relative importance of tyrosine-specific phosphorylation of vinculin and the loss of surface-associated fibronectin in the maintenance of the rounded morphology characteristic of chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV). To address this question we have examined the interaction of CEF and RSV-CEF in vitro with exogenously added fibronectin in both 3-day culture experiments and short-term, 3-h spreading experiments. We report that the addition of human plasma fibronectin to cultures of RSV-CEF results in the restoration of a near-normal morphology, as has been described previously, with the added fibronectin incorporated into an extracellular matrix. However, the phosphotyrosine content of vinculin in these cells was unchanged from that of control, untreated RSV-CEF despite the change in morphology. In short-term spreading experiments RSV-CEF were unable to adopt a fully spread morphology on fibronectin substrates, with defects in the formation of adhesion plaques and microfilament bundles compared with untransformed CEF. pp60v-src was present in the newly formed adhesion plaques of RSV-CEF spreading on fibronectin substrates. The relevance of these results to the maintenance of the transformed phenotype is discussed.

Animals↗
PubMed1985

Isolation of variants of BALB/c 3T3 cells defective in complex ganglioside biosynthesis.

In an attempt to clarify the relationship between altered glycosphingolipid metabolism and other aspects of the transformed phenotype, we have isolated variants of BALB/c 3T3 cells (clone A31) which are defective in synthesis of the more complex gangliosides (GM2, GM1 and GD1a). The selection protocol was based on the specificity of cholera toxin for ganglioside GM1, and the ability to lyse cells which bound toxin using anti-toxin and complement. Following treatment of cells with ethane methane sulfonate (EMS), populations resistant to lysis were obtained after 5-6 rounds of selection, despite a low and rather variable killing efficiency (75-95%). Five out of six clones isolated from such populations showed reduced toxin-binding capacity and loss of gangliosides more complex than GM3, as determined by metabolic labelling with [1-14C] palmitate. An identical phenotype was displayed by a variant isolated from a non-mutagenized population of cells. The phenotype remained stable for several months in culture and for over at least 40 cell doublings. Ganglioside nomenclature is according to Svennerholm [24].

Animals↗
PubMed1985

Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization.

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.

Animals↗
PubMed1984

Laminin and fibronectin in rectal adenocarcinoma: relationship to tumour grade, stage and metastasis.

Using an immunoperoxidase procedure, we have examined the distribution of laminin and fibronectin in normal human large intestinal mucosa and in 50 cases of rectal adenocarcinoma for which extensive clinical follow up was available. In normal tissue, laminin staining was largely restricted to basement membranes, including that underlying the epithelial cells, whereas fibronectin was found in both basement membranes and surrounding connective tissue. In rectal carcinomas, basement membrane-like staining for laminin associated with tumour cells was found in only 27 out of the 50 cases studied. Statistical analysis showed that the presence of laminin-containing basement membranes was correlated with low histological grade (well-differentiated tumours), but not with stage (progression through the bowel wall and the development of lymph node metastases) and, in a highly significant way, with a reduced incidence of distant metastases and increased patient survival. Although fibronectin was found in tumour cell basement membranes where these were present, it was also found in the stroma of all 50 tumours. There was no apparent correlation between the presence of stromal fibronectin and grade, stage or development of metastases. Finally, attention is drawn to some of the technical difficulties in detecting basement membrane antigens in formalin-fixed tissue, the material most frequently available for retrospective study.

Adenocarcinoma↗
PubMed1983

Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells.

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.

Animals↗
PubMed1983

Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin.

We used cholera toxin, which binds exclusively and with a high affinity to the ganglioside GM1, as a probe to investigate the distribution of this glycolipid on the surface of mouse lymphocytes. When lymphocytes are incubated with cholera toxin (or its B subunit) and then sequentially with horse anti-toxin and FITC-swine anti-horse Ig at 37 degrees C, the cholera toxin-ganglioside GM1 complex is redistributed to a cap at one pole of the cell. The capping of cholera toxin-GM1 complexes is slower than the capping of surface-Ig complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM1, like surface-Ig capping, is an energy-dependent process and is inhibited by sodium azide, low temperatures, or cytochalasin B, but is unaffected by demecolcine. An affinity-purified antibody against alpha-actinin was used to examine the distribution of this cytoskeletal component during the capping process. 88% of the cells that had a surface Ig cap displayed a co-cap of alpha-actinin, and 57% of the cells that had a cholera toxin-GM1 cap displayed a co-cap of alpha-actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of alpha-actinin co-caps. We conclude that capping of a cell-surface glycolipid is associated with a reorganization of the underlying cytoskeleton. The implications of such an association are discussed in the context of current models of the mechanism of capping.

Actinin↗
PubMed1982

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

Animals↗
PubMed1982

Lack of correlation between metastasis of human rectal carcinoma and the absence of stromal fibronectin.

In a retrospective study we have used an immunoperoxidase procedure to localize the glycoprotein fibronectin in human rectal carcinomas, concentrating on tumour invading thick-walled extramural veins. Fibronectin was present in 29 out of 38 cases, in connective tissue stroma, and was not in direct association with the tumour cells, except in areas of necrosis. We found no correlation between the presence or absence of stromal fibronectin and (1) the degree of cellular differentiation within the tumour, (2) tumour progression (Dukes' classification) (3) the subsequent development of metastases and (4) patient longevity. OUr results do not support the conclusions from in vitro studies (Smith et al., 1979) that the metastatic potential of carcinomas may be partly determined by the ability of tumour cells to synthesize pericellular fibronectin.

Adenocarcinoma↗