Toxic effect of Ricinus lectin on hepatoma cells in relation to enzyme modification of the cell surface.

With regard to the toxic effects of Ricinus lectin, neuraminidase-treated hepatoma cells have been found to be the most sensitive, and untreated hepatoma cells the least. Cells treated with neuraminidase and galactose oxidase exhibited an intermediate sensitivity. At 37 degrees C, the number of Ricinus lectin molecules bound to untreated, neuraminidase-treated and neuraminidase and galactose oxidase-treated cells required to being about 30% toxicity within 2 h was 15 . 10(5), 7.5 . 10(5) and 11.5 . 10(5) molecules/cell, respectively. This difference was rather small and suggests that the additional binding sites exposed following enzyme treatment were as efficient in mediating lectin toxicity as those present before enzyme treatment. Positive cooperativity was observed during Ricinus lectin binding to enzyme-treated cells at 37 degrees C and the apparent association constant increased with the increase of binding site occupancy. The binding sites on enzyme-treated cells appeared to be homogeneous since under different physical conditions (4 degrees C) the shape of the Scatchard plot could be altered in such a way as to produce a single line of slope. In contrast to enzyme-treated cells, untreated cells did not exhibit a positive cooperative process either at 37 degrees C or at 4 degrees C. We found that the toxicity of Ricinus lectin paralleled the irreversible specific binding of lectin, suggesting that only this was able to mediate the toxic effect. Our results are discussed in terms of the possible entry into the cells of Ricinus lectin and this occurs more rapidly in enzyme-treated than in untreated cells. This difference agrees with the sequence of events proposed: (i) Binding of Ricinus lectin; (ii) Clustering of lectin binding sites; and (iii) Endocytosis.

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin.

Cell growth of tumour ascites cells was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2-100 micrograms/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. These results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells which represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the 14C-labelled asialofetuin degradation. We can conclude that Ricinus lectin present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.

Intracellular events are responsible for the differential expression of fibronectin on the fibroblast surface during chick embryo development.

We previously showed that differences in the adhesive behaviour of fibroblasts obtained from 8-day-old (8-day CEF) and 16-day-old chick embryos (16-day CEF) were not due to alterations of cell surface fibronectin receptors. Herein we show that fibronectin (FN) was expressed more rapidly on the 8-day CEF surface (30 min) than on the 16-day CEF surface (60 min). In order to elucidate the mechanism responsible for these differences in the expression of cell surface FN we investigated the biosynthesis and the post-translational modifications of FN in 8- and 16-day CEF. Pulse-chase experiments revealed that FN was processed more slowly to an endo-beta-N-acetylglucosaminidase H (endo H)-resistant form in 16-day CEF than in 8-day CEF, whereas the kinetic of FN biosynthesis was similar in both cell populations. This difference was not related to a differential retention of FN in endoplasmic reticulum (ER) as determined after saponin-permeabilization. These results suggested that the rate-limiting step in the transport of FN to the cell surface in 16-day cells occurred between the ER and the medial part of the Golgi apparatus. It seems that the delay in the processing of endo H-resistant N-glycans was sufficient to account for differences between 8- and 16-day CEF in the rate of surface expression of FN and CEF adhesion to a plastic substratum.

Changes in protein glycosylation during chick embryo development.

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to-N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo-beta-N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.

Correlation between the regeneration of cell surface glycoproteins and the cell re-adhesion to the substratum in trypsin-treated chick fibroblasts at various stages of embryo development.

The ability of fibroblasts from 8- to 16-day-old chick embryos to adhere to a substratum was altered by trypsin treatment. The consequences of this treatment were investigated on cell re-adhesion to the substratum and cell morphology in relation to the regeneration of cell surface glycoproteins as estimated by the incorporation of [3H]leucine and [14C]glucosamine. Cell re-adhesion, cell shape and restoration of cell surface glycoproteins of the fibroblasts from chick embryos were markedly alike for each stage of embryo development. Age-dependent differences were noted. The fibroblasts from 8-day-old embryos re-adhered progressively more rapidly than fibroblasts from 16-day-old embryos. The fibroblast morphology appeared to be dependent on the re-adhesion of cells to the substratum. Parallel to the re-adhesion, the cell surface glycoprotein recovery reached at least 90% in fibroblasts from 8-day-old embryos and only about 70% in fibroblasts from 16-day-old embryos after a 4 h culture as compared to the control cultures. These percentages coincided with 73% (fibroblasts from 8-day-old embryos) and 40% (fibroblasts from 16-day-old embryos) adhesion recovery. The results are discussed in terms of a possible mechanism for cell surface recovery.

Increased UDP-GlcNAc: alpha-mannoside beta(1----4) N-acetylglucosaminyltransferase activity during chick embryo development.

In vitro assays for the beta-N-acetylglucosaminyltransferases (GlcNAcTase) were performed on crude microsomal fractions prepared from 8-day chick embryo fibroblasts (8-day-CEF) and 16-day chick embryo fibroblasts (16-day-CEF) using [3H]mannose-labeled GlcNAc beta 1----2 Man alpha 1----6 (GlcNAc beta 1----2 Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (Fuc alpha 1----6) GlcNAc-Asn and UDP-GlcNAc as substrates. 8-day-CEF synthesize preferentially triantennary complex type chains, whereas 16-day-CEF produce essentially tetraantennary complex type chains. Furthermore oligosaccharides containing the GlcNAc beta 1----4 Man alpha 1----3 Man sequence represent 90% of the structures found in 16-day-CEF versus 30% in 8-day-CEF, indicating an increase in beta-N-acetylglucosaminyltransferase IV activity during embryo development.

Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying beta (1-6)N-acetylglucosamine-branched N-linked oligosaccharides.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

Requirement of either the NH4Cl-sensitive or the cytochalasin D-sensitive pathway for ricin toxicity depends upon the enterocytic state of differentiation of HT-29 cells.

During the course of the present biochemical and ultrastructural studies, we found that the expression of either the undifferentiated or the differentiated HT-29 cell phenotype determined the intracellular fate of ricin. Although the recognition of ricin at the cell surface required interaction with the galactose-binding site on both cell populations, the lag time before ricin started to inhibit protein synthesis was longer in the differentiated than the undifferentiated cells. Dose-response studies and "time-addition" experiments performed with NH4Cl, which raises the pH of acidic vesicles and organelles, showed that ricin uptake as well as the movement of the toxin to the translocation site were affected in the differentiated cells. In contrast, NH4Cl acted on only post-internalization events in the undifferentiated cells. When the addition of cytochalasin D, an actin-depolymerizing drug, was staggered, the differentiated cells were found to be protected against ricin only during the very early stage of the internalization process. In contrast, the undifferentiated cells were protected during both the early and late stages of endocytosis. Moreover, electron microscopic examination showed that cytochalasin D altered the structure of the Golgi apparatus only in the undifferentiated cells. 3-Methyladenine, a specific inhibitor of the autophagic pathway, protected the undifferentiated and differentiated cells against ricin to about the same extent. We concluded that to enter the differentiated cells, ricin followed the classical endosome-Golgi pathway. In contrast, in the undifferentiated cells, ricin reaches the cytosol by two distinct routes: the minor one involves the endosome-Golgi pathway; the major one involves a cytochalasin D-sensitive pathway.

Integrins of the beta1 family influence keratinocyte-lymphocyte interaction.

Data from the literature indicate that ICAM-1 molecules play an important role in keratinocyte interactions with lymphocytes via the lymphocyte function-associated-1 lymphocyte-adhesion molecule. We examined the role of beta1 integrins in keratinocyte-lymphocyte adhesion under different activation conditions. Among the beta1 integrins expressed on keratinocytes and lymphocytes detected by indirect immunofluorescence microscopy and flow cytofluorometry, primarily the alpha2 and the alpha3 subunits on both cell types were involved in keratinocyte-lymphocyte adhesion. Moreover, the highest adhesion level was observed when both cell types were activated by IFN-gamma for keratinocytes and phorbol 12-myristate 13-acetate for lymphocytes, suggesting that the former involved the protein kinase C pathway. Keratinocyte activation, characterized by the expression of ICAM-1, a decrease of beta1 integrins, and the absence of alpha5beta1 integrin, was required for optimal lymphocyte adhesion. Thus, beta1 integrins remaining at the surface of IFN-gamma-treated keratinocytes could be activated by this cytokine, and could synergize with ICAM-1 and lymphocyte function-associated-1 molecules to consolidate keratinocyte-lymphocyte adhesion.

Altered collagen of human pathological fibroblasts impairs the synthesis of fibronectin.

Human fibroblasts with mutated type I collagen have marked defective adhesive capacities on exogenous type I collagen and exogenous fibronectin in comparison to normal fibroblasts. This defective cell adhesion could be partly explained by the decreased level of cell surface receptors of the beta 1-integrin family, i.e., the alpha 2 integrin subunit for type I collagen and the alpha 5 integrin subunit for fibronectin, observed in pathological fibroblasts. However, it appeared that the presence of altered collagen interfered both with fibronectin biosynthesis and with its surface expression. Using a binding assay on immobilized fibronectin, we demonstrated that the mutated collagen had a weaker binding to fibronectin. In addition, the pathological fibroblasts plated on a mixture of normal exogenous type I collagen and fibronectin exhibited the same maximal level of adhesion as control fibroblasts. These results indicate that fibroblasts with the mutated collagen exhibit a decreased binding to normal fibronectin, a modification of synthesis and surface expression of fibronectin, and, finally, altered adhesive capacities.

Changes in cell-surface sialic acid content during chick embryo development.

The amount of cell-surface sialic acid found in the material released by trypsin from chick fibroblasts rose markedly from day 8 to day 16 of embryo development. The rise seemed to result from two processes: increased sialylation of N-linked carbohydrate chains, and enhancement of the amount of O-linked structures. Eight-day cells were more quickly detached from the substrate than 16-day cells, since in 8-day fibroblasts detachment of half the adherent cells only required 10 min, compared to 20 min in 16-day fibroblasts. Re-adhesion to the substrate was also faster for the younger fibroblasts, and 40% of the 8-day trypsin-treated cells re-adhered within 30 min compared to about 3 h for the same proportion of 16-day cells. The age-dependent differences in the amount of cell-surface sialic acid do not account for the differences in the adhesive capacity of embryo cells. The fact that neuraminidase did not affect cell detachment or re-adhesion indicates that cell-surface sialic acid does not play an important part in the adhesive capacity of embryo fibroblasts.

Age-related changes induced by tunicamycin in wheat germ agglutinin binding to chick embryo fibroblasts.

The specific roles of N-acetylglucosaminyl and sialyl residues were investigated in the binding of wheat germ agglutinin (WGA) to fibroblasts from 8- and 16-day chick embryos. Cells from the 8-day embryos exhibited two classes of WGA binding site, whereas fibroblasts from 16-day embryos only displayed one. Neuraminidase treatment of fibroblasts from 8-day embryos raised the number of WGA binding sites with a high affinity constant and reduced the number of sites with a low affinity constant. In 16-day cells, neuraminidase treatment reduced the number of WGA binding sites. The data presented here suggest that WGA binds to cell-surface glycoproteins containing sialic acid residues. This tendency was more marked in 16-day than in 8-day cells and corresponded to the finding that neuraminidase released more sialic acid from the surface of 16-day cells than of 8-day cells. Glycosylation of cell-surface N-linked glycoproteins did not seem essential in determining the WGA binding capacity, as shown by the effect of tunicamycin on fibroblasts from both 8- and 16-day embryos. In the absence of N-glycosylated binding sites, WGA bound to O-glycosylated structures and the older tunicamycin-treated embryo cells exhibited a larger number of WGA binding sites than the younger tunicamycin-treated cells, in relation to the increase of the amount of O-glycosylated structures during embryo development.

A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.

A simple and sensitive cell-cell adhesion microplate assay was established using the cytoplasmic fluorescent dye, calcein AM. The procedure involves three steps: the labeling of lymphocytes with an adequate concentration of calcein AM (20 microM) during a short incubation period (30 min); the adhesion of 2 x 10(5) labeled lymphocytes per well to confluent keratinocyte or fibroblast monolayers grown in microtiter plates for 90 min; and, finally, measurement of the fluorescent signal utilizing a new system of cold-light microfluorimetry (Rat, 1993). During the adhesion assay, the release of calcein from labeled lymphocytes is low and the method permits the detection of as few as 1000 adherent cells. This non-radioactive procedure takes less than 4 h to perform and has proven to be as accurate and reliable as the common method using radioactive isotopes. In addition to its simplicity, the use of a fluorescent molecular probe in conjunction with cold-light microfluorimetry (CLF) offers many advantages of safety and economy, and can readily be adapted to the different cell types that participate in cell-cell adhesion.

T lymphocytes from Sézary syndrome patients express beta1 integrins whose beta(1-6)-branched N-linked oligosaccharides reflect their adhesive capacity.

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

Activity of proximal promoter of the human beta(1)-integrin gene was increased in Sézary syndrome.

Changes in beta1-integrin expression have been involved in abnormal cellular interactions between malignant lymphocytes from Sézary (Sz) patients and keratinocytes. In this paper, we compare the activity of both distal and proximal promoters of the beta1-integrin gene in malignant lymphocytes from Sz patients with human normal lymphocytes. Activity of both beta1-integrin promoters was also analysed in human normal keratinocytes. Northern blot analysis shows that beta1-integrin mRNA expression is higher in malignant Sz lymphocytes than in normal lymphocytes. CAT assays show that the activity of proximal beta1-integrin promoter is markedly increased (up to 6-fold) in malignant lymphocytes from Sz patients, in comparison to normal lymphocytes. These results suggest that changes in activity of the proximal promoter of beta1-integrin subunit could be, in part, responsible for the abnormal cellular interactions between malignant lymphocytes and keratinocytes observed in Sz syndrome.

Lectin activities of cytokines and growth factors: function and implications for pathology.

The discovery that certain cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain, which is localized opposite to the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase involved in receptor phosphorylation and in specific signal transduction. It is expected that blood accumulation of free or membrane-bound glucan ligands of cytokines may dramatically perturb their endogenous function inducing specific immunodeficiencies.

Alterations of the glycan moiety of human alpha 1-acid glycoprotein in late-term pregnancy.

The carbohydrate moiety of purified alpha 1-acid glycoprotein (AGP) from healthy male adults (AGPn) and late-term pregnant women (AGPp) was analysed. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate before and after N-glycanase treatment showed that AGPp had a slightly higher molecular mass due to an enriched carbohydrate moiety. BIO-GEL P-4 and Concanavalin A (Con A)-Sepharose chromatography of the oligosaccharides released by hydrazinolysis and fractionated by high-voltage electrophoresis indicated a progression towards Con A-unbound oligosaccharides and towards larger glycans in pregnancy. Carbohydrate analysis of purified AGPp and AGPn and of the most increased oligosaccharide fraction (F4A) evidenced a decrease in the fucosyl molar ratio and a slight increase in the galactosyl, N-acetyl-glucosaminyl and N-acetyl neuraminyl ratios. These results suggest that AGP contains more highly branched oligosaccharides and/or additional N-acetyllactosamine-type oligosaccharides in pregnancy.

Human keratinocyte models: Assessment of cell adhesion and dermotoxicity using fluorescent probes.

To assess the molecular and cellular events that occur in the skin during biological and pharmaco-toxicological processes, we developed different in vitro models. Two major systems are described: (1) relatively simple ones such as normal human keratinocytes (NHK) grown in monolayer or continuous culture of spontaneously immortalized keratinocyte cells, the HaCaT cell line. This cell line forms a monolayer and displays the same phenotypic morphology, pattern of differentiation markers as NHK. (2) More complex models such as NHK multilayers differentiated on a synthetic porous membrane. Indeed, NHK grown at the air-liquid interface of culture inserts may undergo epidermal differentiation in 21 days (Noël-Hudson et al., 1995a). Under the same culture conditions, no stratification of the HaCaT cell line was obtained. NHK and/or HaCaT monolayers were used to study the cell surface molecules involved in heterologous cell interactions, and to estimate the cytotoxic effects of different compounds through a sensitive fluorimetric microtitration assay. When cell adhesion was measured with calcein-AM labelled lymphocytes, it appeared that lymphocytes display the same behaviour towards NHK or HaCaT cells. The importance of the activation status of each cell and the involvement of alpha(2) and alpha(3)beta(1) integrins in lymphocyte-keratinocyte interactions were demonstrated. Likewise cytotoxicity of SDS and DNP was easily and rapidly detected with calcein-AM and Alamar blue probes. Skin models in combination with fluorescent probes offer promising alternatives for assessing cell interactions as well as cytotoxic effects.

Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol.

A three-dimensional culture of human keratinocytes exposed at the air-liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, (35)S-methionine incorporation, IL1-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the apical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD(50) values (40 J/cm(2)) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.

Use of fluorescent probes to assess the early sulfhydryl depletion and oxidative stress induced by mechlorethamine in human bronchial epithelial cells.

In this study, we report in vitro methods using fluorescent probes to assess thiol depletion and the oxidative stress induced by mechlorethamine (HN2), a nitrogen mustard, on a human bronchial epithelial cell line (16HBE14o-). Monobromobimane (mBBr) and 2',7'-dichlorofluorescin-diacetate (H(2)DCF-DA) were respectively used to monitor the intracellular thiol and peroxide levels. Fluorescent measurements were realized on gated live cells with a flow cytometer and a microspectrofluorimeter. Results clearly show that HN2 induced an early reduction of free sulfhydryl groups inside the cell correlated with an increase in the intracellular peroxides content. HN2 effects were time and dose dependent. The use of these fluorescent probes provide a useful and rapid methods for future screening of protective molecules against the early sulfydryl depletion and oxidative stress induced by HN2 on human airway epithelium.