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First published online 21 September 2004
doi: 10.1242/jcs.01350

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Expression of sialyl-Tn epitopes on ß1 integrin alters epithelial cell phenotype, proliferation and haptotaxis

¹ INSERM U601, Institut de Biologie, 9 quai Moncousu, 44093 Nantes CEDEX, France
² INSERM U533, Faculté des Sciences, 2 rue de la Houssinière, 44322, Nantes CEDEX 3, France

View larger version (51K): [in a new window]   Fig. 1. ST6GalNAc I transfectants express cell surface STn epitopes. (A) Cell surface glycosylation was analyzed by flow cytometry using the anti-STn mAbs TKH2 and B72.3 as well as the PNA and JAC lectins that recognize preferentially the O-glycan core 1. Control clones B10 and F4 correspond to parental and mock-transfected cells, respectively. Clones C2 and G9 are ST6GalNAc I transfectants. The log of fluorescence intensities in arbitrary units is plotted against cell number. Fluorescence intensities from negative controls performed in the presence of either an irrelevant antibody or FITC-labeled streptavidin alone are shown superimposed in dotted lines. (B) Sialyltransferase activities from total cell extracts were assayed using fetuin as acceptor and CMP-[14C]NeuAc as sugar donor as described in Materials and Methods. Specific activities are given as pmol/h/mg total proteins of [14C]NeuAc transferred. (C) Expression of STn epitopes was observed on fixed and permeabilized cells by confocal microscopy using the TKH2 antibody on the C2 clone.

View larger version (92K): [in a new window]   Fig. 2. ST6GalNAc I overexpression alters cell shape. (A) Phase contrast microphotographs of STn-control cells and of STn+ ST6GalNAc I transfectants illustrating morphological differences between the two cell types. (B,C) Quantitative analysis of morphological parameters was performed on three STn-control clones and three STn+ clones. Cell area and perimeter were acquired for each cell type and the shape factor (f) was calculated as described in Materials and Methods. Error bars represent the standard deviation for 100 cells. (D) Rhodamine-labeled phalloidin was used to detect polymerized actin on fixed and permeabilized cells cultivated on glass lamellae. (E) Focal contacts were visualized using an anti-vinculin mAb. Pictures from representative fields are shown.

View larger version (79K): [in a new window]   Fig. 3. The ability of ST6GalNAc I transfectants to repair a scratch wound and to proliferate is impaired. (A) Photomicrographs were taken 24 hours after performing scratch wounds on confluent cell monolayers of a STn- and a STn+ clone. (B) Wound repair was quantified by image analysis on the STn-control clones B10 and F4 (blue symbols) and the STn+ clones C2 and G9 (red symbols). The wound breadth was measured at different time points after wounding, and results represent the mean of three independent experiments. Mean speed of wound repair, 13.3±1.1 and 6.28±1.2 µm/hour for STn- and STn+ cells, respectively (P<0.001). (C) Cell proliferation was measured by a neutral red uptake assay as described in Materials and Methods. Error bars represent the standard error of triplicates within one out of two experiments.

View larger version (67K): [in a new window]   Fig. 4. The ability of ST6GalNAc I transfectants to migrate but not to adhere on fibronectin is impaired. (A) The motility of STn- and STn+ cells on various extracellular matrix components was quantified by image analysis in a phagokinetic track assay. The extracellular matrix components coated on wells of culture plates were BSA, fibronectin (FN), collagen (C) I, collagen IV, collagen VI and hyaluronic acid (HA). All were used at 10 µg/ml except for hyaluronic acid, which was used at 5 µg/ml. Error bars represent the standard error of three experiments. The differences in migration between STn+ and STn-cells on fibronectin and hyaluronic acid were significant (P<0.001 and P<0.05, respectively); the difference on collagen was not significant. (B) Representative fields of phagokinetic tracks on fibronectin from one STn- and one STn+ clone. (C) Inhibition by soluble fibronectin of the STn- and STn+ cells' motility on coated fibronectin in a phagokinetic track assay. Treated cells vs control cells: P<0.001. (D) Motility on fibronectin of the STn+ transfectant clones compared with that of the STn-control clones quantified by a phagokinetic track assay. Error bars represent the standard deviation of the mean from ten independent experiments. STn+ vs STn-: P<0.001. (E) Motility on fibronectin (10 µg/ml) of STn+ and STn-cells in a transwell assay was quantified by image analysis. Error bars represent the standard error of the mean of three experiments (P<0.001 between STn- and STn+ cells). (F) Adhesion on fibronectin coated at different concentrations of STn-(blue circles) and STn+ (red circles) cells measured at 15 minutes and 40 minutes after seeding. Error bars represent the standard deviation of the mean from eight values. In all experiments, the values obtained for the two STn-control clones (B10 and F4) and the two STn+ ST6GalNAc I transfectants (C2 and G9), respectively, were pooled.

View larger version (20K): [in a new window]   Fig. 5. STn epitopes are carried by various glycoproteins, among which is the integrin ß1 subunit. (A) Total Triton X-100 cell extracts of the STn-clones (B10 and F4) and of the STn+ clones (C2 and G9) were submitted to SDS-PAGE in nonreducing conditions and to western blotting. Blots were probed with the anti-STn mAbs TKH2 and B72.3 and the anti-ß1 mAb KMI6. (B) Total cell extracts were immunoprecipitated with the anti-STn TKH2, and immunoprecipitates were submitted to western blotting using either the anti-ß1 KMI6 or the anti-STn TKH2. The bands marked by a star correspond to the immunoprecipitating immunoglobulin.

View larger version (16K): [in a new window]   Fig. 6. Treatment with anti-STn and anti-integrin restores the motility of ST6GalNAc I transfectants on fibronectin. Motility was assessed by a phagokinetic track assay on immobilized fibronectin. Before seeding, cells were incubated with antibodies for 30 minutes at 37°C. (A) Cells were treated with the anti-STn mAb TKH2 and the irrelevant control IgG1 at the optimal concentration of 1 µg/ml. The values correspond to the mean ± standard deviation of five independent experiments. (B) Cells were pretreated with the anti-5 and ß1 integrin subunits 5H10-27 and KMI6, respectively, at the concentrations that gave maximal effect (25 µg/ml and 5 µg/ml, respectively). Values represent the mean ± standard deviation of three independent experiments.

View larger version (80K): [in a new window]   Fig. 7. Treatment with an activator of RhoA restores motility on fibronectin and actin polymerization of ST6GalNAc I transfectants. (A) STn-control cells and STn+ ST6GalNAc I transfectants were pretreated for 30 minutes with TATRhoAVal-14 protein (V14), a RhoA activator at 6 µg/ml, before being seeded on immobilized fibronectin at 10 µg/ml (FN) or on hyaluronic acid at 5 µg/ml (HA). Motility was tested in a phagokinetic track assay, and results from one representative experiment out of two are shown. Error bars correspond to the standard deviation of the mean of triplicates. (B) Quantitative analysis of morphological parameters was performed on STn- and STn+ cells transduced with the constitutively active TAT-RhoAVal-14 protein (V14), with the inactive TAT-RhoA (N19) or untreated (controls). Cell area and perimeter were acquired for each cell type and the shape factor (f) was calculated as described in Materials and Methods. Error bars represent the standard deviation for 100 cells. (C) Staining of actin stress fibers was performed using rhodamine-labeled phalloidin on fixed and permeabilized STn- and STn+ cells cultivated on glass lamellae. Cells were treated with 6 µg/ml TAT-RhoAVal-14 protein (TAT-RhoAVal-14 protein) for 16 hours before fixation.

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