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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas
Received October 21, 2005; accepted December 20, 2005.
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Abstract |
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, 1991; Gumbiner, 1996). In the epithelial layer, E-cadherins reside at the adherens junctions between tight junctions and desmosomes. The formation and maintenance of the tight junctions are influenced by the integrity of the adherens junctions (Takeichi, 1991). The opening and resealing of the tight junctions can be controlled by removing and restoring the extracellular Ca2+ (Gumbiner and Simons, 1986; Cereijido et al., 2000). This is presumably due to modulation of E-cadherin-mediated cell-cell adhesion at the junctions of the adherens. Ca2+ ions are necessary for the adhesive function of E-cadherin. Thus, E-cadherin has a key role in controlling the integrity of intestinal mucosa epithelium.
E-cadherin is a transmembrane glycoprotein that consists of an extracellular domain with five homologous repeats (EC1–5), a single transmembrane region, and a highly conserved cytoplasmic tail. The extracellular domains play important roles in organizing cadherin homophilic interactions and determining the specificity of cell-cell adhesion (Nose et al., 1990; Takeichi, 1990). EC1 and EC2 domains of cadherins have been studied extensively for their role in cell-cell adhesion (Nose et al., 1990; Shapiro et al., 1995; Nagar et al., 1996; Pertz et al., 1999; Shan et al., 2004). It has been suggested that a conserved His-Ala-Val (HAV) sequence found in the EC1 domain may be important for cadherin-cadherin interaction. Synthetic peptides containing an HAV sequence can interfere with cadherin-mediated cell-cell adhesion (Blaschuk et al., 1990a,b; Noe et al., 1999; Williams et al., 2000). We have shown that HAV peptides from EC1 of E-cadherin can effectively inhibit E-cadherin-mediated aggregation of bovine brain microvessel endothelial cells (BBMECs) (Lutz and Siahaan, 1997; Pal et al., 1997) and increase the paracellular porosity of Madin-Darby canine kidney (MDCK) cell monolayers (Makagiansar et al., 2001; Sinaga et al., 2002). Recent studies indicate that the trans-interactions between cadherins of opposing cells involve not only EC1-to-EC1 interaction but also interactions of multiple domains (Leckband and Sivasankar, 2000; Chappuis-Flament et al., 2001; Perret et al., 2004). Despite numerous studies to elucidate the mechanisms of E-cadherin-mediated cell-cell adhesion, the organization of E-cadherin interactions still remains unclear. The functions of each domain in the formation of cell-cell contact need further investigation.
One way to elucidate the mechanisms of E-cadherin-mediated cell-cell adhesion is by using peptides that are derived from E-cadherin sequence. The E-cadherin peptides can be used to evaluate the important regions of E-cadherin. Furthermore, peptides that can modulate E-cadherin-mediated cell adhesion may have potential application for modulation of the intercellular junctions of the biological barrier to improve permeation of large hydrophilic drugs via the paracellular pathway. Thus, there is a need to develop an assay that can be used to evaluate E-cadherin peptide in blocking cadherin-cadherin interactions. To date, there are a limited number of assays available to evaluate peptide activities in the context of whole live cells. One of these is measuring the transepithelial electrical resistance (TEER) or paracellular permeation of marker molecules of the cell monolayer upon modulation with peptides (Makagiansar et al., 2001; Sinaga et al., 2002). Although this method is well established, it is a time-consuming process to establish tight monolayers. In addition, due to the limited pore size (<11Å) of the tight junctions (Adson et al., 1994), there is a size limit for E-cadherin peptides that can be used. Thus, larger cadherin peptides (i.e., 10–24 amino acids) have difficulty in penetrating the tight junctions to reach the adherens junctions. Another method is the cell aggregation assay in which the extent of aggregation of single cells is evaluated as a function of inhibitor concentrations (Lutz and Siahaan, 1997; Pal et al., 1997; Noe et al., 1999; Renaud-Young and Gallin, 2002; Shan et al., 2004). Unfortunately, it is difficult to quantitate the effect of the peptides in inhibiting the E-cadherin-mediated cell adhesion in this assay.
Therefore, in the present study, we attempted to develop a novel adhesion assay to evaluate the activities of various compounds, including proteins, peptides, and peptidomimetics, in modulating E-cadherin-mediated cell-cell interactions. The assay is based on the homotypic adhesion of Caco-2 single cells to tissue culture plate-associated Caco-2 cells (i.e., attached cells). We found that the homotypic adhesion of Caco-2 cells was predominantly mediated by E-cadherin, by which the peptide activities in modulating the human E-cadherin-mediated cell-cell interactions can be evaluated in the context of live cells.
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Materials and Methods |
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Peptides
E-cadherin-derived peptides were synthesized with 9-fluorenylmethyloxycarbonyl-protected amino acid chemistry on 9-fluorenylmethyloxycarbonyl-PAL-PEG-PS resin (Applied Biosystems, Foster City, CA) using the automated peptide synthesis system (Pioneer; PerSeptive Biosystems, Framingham, MA). Cleavage of the peptides from the resin and removal of the protecting groups from the side chain were carried out using trifluoroacetic acid with appropriate scavengers. The crude peptides were purified by reversed-phase high-performance liquid chromatography using a C18 column with a gradient of solvent A [95%/5% = H2O (0.1% trifluoroacetic acid/acetonitrile)] and solvent B (100% acetonitrile). The purity of the peptide was analyzed by analytical high-performance liquid chromatography using an analytical C18 column. The identity of the synthesized peptide was confirmed by electrospray ionization-time of flight mass spectrometry. The peptides used in the present study were: Ac-[DRERIATYTLFSHAVSSNGNAVED]-NH2 (HAV24), Ac-[LFSHAVSSNG]-NH2 (HAV10), Ac-[SHAVSS]-NH2, Ac-[SHAVAS]-NH2, and Ac-[SHAASS]-NH2. These peptides were capped at their N and C termini with acetyl and amide groups, respectively.
Antibodies
Some of the antibodies were purchased from Sigma-Aldrich, including antiuvomorulin/E-cadherin monoclonal antibody (mAb) (clone DECMA-1), anti-pan cadherin mAb (clone CH-19), purified mouse IgG isolated from pooled normal mouse serum, and goat anti-mouse IgG (Fc-specific)-FITC antibody. Other antibodies were purchased from different vendors, including: mouse anti-human E-cadherin mAb (clone SHE78-7) from EMD Biosciences (San Diego, CA) and goat anti-N-cadherin polycolonal antibody (N-19) from Santa Cruz Biotechnologies (Santa Cruz, CA). DECMA-1 and SHE78-7 were used for inhibition of E-cadherin-mediated homotypic adhesion and for immunostaining of E-cadherin, respectively. CH-19 and mouse IgG were used as negative controls.
Homotypic Adhesion of Caco-2 Single Cells to Attached Cells
Preparation of Caco-2 Single-Cell Suspension. Caco-2 cells were cultured in 10% FBS-DMEM in a tissue culture flask. Highly confluent monolayers were avoided because it is difficult to prepare single-cell suspensions. After washing with HBSS(–) (i.e., HBSS without Ca2+ and Mg2+), the cells were treated with 2.5 mM EDTA in PBS for 1 to 2 min at room temperature (RT) to remove excess divalent cations. Then, the cells were washed with HBSS(–) to remove remaining EDTA and incubated with Ca2+- and Mg2+-free Earle's balanced salt solution (EBSS) for 1 h at 37°C in 5% CO2. Within 1 h, most of the cells were detached from the culture flask. For fluorescence labeling, the detached cells were incubated in 10 ml of solution of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) acetoxymethyl ester (AM; Invitrogen) with gentle shaking for 45 min at 37°C. The BCECF-AM solution was prepared by dissolving 50 µg of BCECF-AM in 50 µl of dimethyl sulfoxide followed by dilution to 10 ml with 0.1% BSA-containing DMEM. Long incubation was avoided because the amount of intracellular BCECF declined after incubation times of more than 45 min. The free BCECF was washed from the cell suspension three times with ice-cold PBS(–) (i.e., PBS without Ca2+ and Mg2+). The BCECF-labeled cells were resuspended in 0.1% BSA-DMEM at a density of 5 x 105 cells/ml. The cell suspension and inhibitor solutions were mixed in equal volumes. Due to this dilution, the inhibitor solution was prepared in a concentration that was twice the final concentration after mixing with the cells. For Ca2+-deficient samples, the BCECF-labeled Caco-2 single cells were suspended in Ca2+-free and 0.8 mM Mg2+-containing 0.1% BSA-EBSS at a density of 2.5 x 105 cells/ml. These single cells were added to the attached cells (see below) for evaluation of the cell adhesion properties.
Adhesion of Caco-2 Single Cells and Attached Cells. Confluent Caco-2 cell monolayers were cultured in 10% FBS-DMEM in 48-well tissue culture-treated microplates (Corning, Acton, MA). The cell monolayers were washed twice with PBS(–) and then subjected to "Ca2+-deficient pretreatment" procedure to produce a cell layer called "attached cells." The Ca2+-deficient pretreatment procedure was done by incubating the monolayers in Ca2+-free PBS or EBSS (1 mM Mg2+, 0.1% BSA) for 2 h at 37°C in 5% CO2. After removing the pretreatment medium, the attached cells were incubated with 200 µl/well BCECF-labeled Caco-2 single-cell suspensions (5 x 104 cells/well) containing certain concentrations of antibodies or peptides. This mixture was incubated for 2 h (unless otherwise mentioned) at 37°C in 5% CO2.
To remove the unbound cells, the culture plates were flipped and softly tapped against a paper towel several times. To wash the remaining unbound cells, 500 µl of ice-cold PBS(+) (i.e., PBS containing 1 mM Ca2+, 1 mM Mg2+, and 0.1% D-glucose) was added to each well followed by flipping and softly tapping the plates; this procedure was repeated three times. We found that this washing procedure is crucial to distinguish the Ca2+-dependent cell adhesion from apparent adhesion at 0 mM Ca2+. In fact, we found that the use of a suction pump to remove the solution with unbound single cells leaves a significant amount of unbound cells in the wells (data not shown).
Then, 400 µl of lysis buffer (0.5% Triton X-100 and 2 mM EDTA in 0.1 M CHES, pH 9.5) was added to each well and incubated for 1 h at 37°C with gentle shaking. The solution from each well was subjected to centrifugation to remove the cell debris, and 150 µl of the supernatant was transferred to 96-well flat clear bottom (black) microplates (Corning). The fluorescence intensity of each well was measured in duplicate using a microplate fluorescence analyzer (Bio-Tek FL600) at 
excitation of 485 nm and emission of 530 nm. The fluorescence intensity was correlated with the number of bound cells using a standardization procedure given below.
Calibration of Fluorescence Intensity versus Cell Number. Because BCECF is a known substrate for multidrug resistance-associated protein (Draper et al., 1997; Bachmeier et al., 2004), the amount of BCECF in the cells decreases upon longer incubation time at 37°C. To calibrate the cell-associated fluorescence intensity at the duration of incubation, we separately measured the amount of BCECF remaining in the single cells versus incubation time. Based on these data, the fluorescence intensity was corrected for each cell adhesion experiment. The number of adherent cells (percentage of applied cells) was calculated using the amount of fluorescence from a determined number of cells. The data were also presented as Ca2+-dependent adhesion; this was calculated by subtracting the data obtained from cell adhesion in the absence of Ca2+. The relative activity of each peptide can be compared using a standard mAb DECMA-1; this mAb inhibits approximately 60% of Ca2+-dependent adhesion at 1:100 dilution of mAb concentration.
Heterotypic Adhesion of Molt-3 T Cells to Attached Caco-2 Cells
Heterotypic adhesion of Molt-3 human lymphoblastic T cells to Caco-2 cells was performed as described above with some modifications (Yusuf-Makagiansar et al., 2001a,b). In brief, Molt-3 T cells were activated with 0.2 µM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 16 h at 37°C in 5% CO2 before BCECF labeling. Caco-2 cell monolayers were incubated in 10% FBS-DMEM containing 100 IU/ml interferon-
(BD Biosciences) for 14 h at 37°C in 5% CO2 and then subjected to Ca2+-deficient pretreatment in PBS containing 0.1% BSA, 1 mM Mg2+, and 100 IU/ml interferon- for an additional 2 h. BCECF-labeled Molt-3 T cell suspensions (1 x 106 cells/ml in 0.1% BSA-RPMI 1640) were added (200 µl/well) to the attached Caco-2 cells and incubated in the presence or absence of mAb for 2 h at 37°C in 5% CO2. The washing procedure and determination of the number of adhesive cells were accomplished as described above.
Immunofluorescence Staining
Confluent Caco-2 cell monolayers were washed three times with PBS(+) and fixed in 4% formaldehyde for 20 min at RT. Some were then permeabilized with 0.5% Triton-X-100/PBS(+) for 15 min at 37°C. After blocking with 1% BSA- and 5% skim milk-containing PBS(+) (blocking buffer) for 60 min at RT, the cells were incubated with the primary antibody SHE78-7 (2 µg/ml in blocking buffer) for 60 min at 37°C. The cells were washed three times with 0.05% Tween 20-containing PBS and then were incubated with FITC-labeled goat anti-mouse IgG for 60 min at 37°C. The cells were washed three times with 0.05% Tween 20-containing PBS to remove the excess antibodies. The fluorescence was observed under a fluorescence microscope (Nikon, Tokyo, Japan) equipped with Flash Point software.
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Results |
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, 1991; Le et al., 1999). Addition of Caco-2 single cells to the Caco-2 attached cells showed strong homotypic cell-cell adhesion compared with untreated Caco-2 cell monolayers (Fig. 1). The extent of homotypic adhesion was influenced by the duration of treatment of Caco-2 cell monolayers with Ca2+-deficient medium. The homotypic adhesion was enhanced by increasing the pretreatment time of the confluent monolayers; the increase in cell-cell adhesion reached a plateau at pretreatment times beyond 2 h. Thus, 2-h pretreatment was used throughout this study.
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Next, we examined the effect of incubation time on Caco-2 single-cell adhesion to treated or untreated cells (Fig. 3). In this experiment, the effect of BCECF efflux (Draper et al., 1997; Bachmeier et al., 2004) has been compensated as described under Materials and Methods. In general, the number of adherent single cells increased with time of incubation. However, the treated Caco-2 cells have stronger adhesion properties than those of untreated monolayers. The single-cell adhesion was rapidly increased between 0 to 4 h incubation time and plateaued between 4 to 8 h incubation time. This result is consistent with the relocalization of E-cadherins to the cell surface of Caco-2 attached cells. Because the efflux of BCECF was very apparent between 4- and 8-h time points (data not shown), we used 2-h incubation time for evaluating the effect of E-cadherin peptides on the single-cell adhesion.
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Involvement of E-Cadherin in Caco-2 Single-Cell Adhesion to Attached Cells. To confirm the involvement of E-cadherin in the Caco-2 single-cell adhesion to attached cells, the effect of Ca2+ concentration on the magnitude of cell-cell adhesion was evaluated (Fig. 4). In this experiment, EBSS with various concentrations of Ca2+ (0–2 mM) was used instead of DMEM containing 1.8 mM Ca2+. Our preliminary evaluation showed that EBSS supplemented with 0.8 mM Mg2+ and 1.8 mM Ca2+ produced an extent of cell adhesion similar to that with DMEM (data not shown). This result confirms that the difference in composition other than Ca2+ in EBSS and DMEM does not significantly influence the cell adhesion properties. The number of adherent cells was largely dependent on the Ca2+ concentrations and was increased at higher Ca2+ concentration. These results indicate that the homotypic cell adhesion process uses Ca2+-dependent mechanisms, which are normally mediated by E-cadherin homophilic interactions.
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), the results indicate that the cell-cell adhesion process is predominantly mediated by E-cadherin homophilic interactions.
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To further confirm the involvement of E-cadherin in the cell adhesion process, the effect of extracellular Ca2+ concentrations on the inhibitory effect of DECMA-1 mAb was examined (Fig. 6A). In addition, the heterotypic adhesion of Molt-3 human lymphoblastic T cells to the Caco-2 attached cells was examined (Fig. 6B). As expected, the homotypic adhesion of Caco-2 cells was significantly inhibited by DECMA-1 mAb at Ca2+ concentration of 1.8 mM. However, DECMA-1 could not inhibit the E-cadherin-mediated cell adhesion in the absence of Ca2+. The heterotypic adhesion of Molt-3 T cells to the Caco-2 attached cells was not inhibited by DECMA-1 mAb, suggesting that this heterotypic cell adhesion was not mediated by E-cadherin. Molt-3 T cells have been shown to express N-cadherin but not E-cadherin (Makagiansar et al., 2002). These results support the idea that the homotypic adhesion of Caco-2 single cells to attached cells is mediated mainly by E-cadherin homophilic interactions.
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Inhibition of Cell-Cell Adhesion by HAV Peptides. HAV peptides were also used to inhibit the Caco-2 single-cell adhesion to attached cells. HAV peptides were derived from the conserved HAV motif at the EC1 domain of E-cadherin. Previously, we have shown that these peptides block E-cadherin-mediated cell-cell adhesion of BBMEC and MDCK cells (Lutz and Siahaan, 1997; Pal et al., 1997; Makagiansar et al., 2001; Sinaga et al., 2002). The long HAV24 and HAV10 peptides showed significant and dose-dependent inhibitory activities to almost the same extent as DECMA-1 mAb (Fig. 7B). On the other hand, the shorter hexapeptides (SHAVSS, SHAVAS, and SHAASS) did not inhibit the Caco-2 single-cell adhesion (Fig. 7, A and B). The hexapeptides SHAVSS and SHAVAS have also been shown to modulate the intercellular junctions of the MDCK cell monolayers (Makagiansar et al., 2001). These results suggest that the longer HAV peptides bind to E-cadherins and block the E-cadherin-mediated cell-cell adhesion. Furthermore, this assay can be used to evaluate molecules (peptides, protein, and small molecules) that modulate E-cadherin-mediated cell-cell adhesion.
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Discussion |
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The assay involves E-cadherin-mediated homotypic adhesion of Caco-2 single cells to Caco-2 attached cells. The attached cells were derived from Caco-2 cell monolayers that were treated by extracellular Ca2+ depletion. This pretreatment produces partial relocalization of E-cadherins from the intercellular junctions to the cell surface without detaching the cells from the plate. Our hypothesis is that incubation of Caco-2 cell monolayers with Ca2+-free medium would disrupt the Ca2+-dependent E-cadherin homophilic interaction at the adherens junction; this disruption would be followed by internalization and recycling of E-cadherins to the cell membrane (Kartenbeck et al., 1982, 1991; Le et al., 1999). Thereby, some of E-cadherins would be redistributed to the cell surface of the attached cells and become accessible for binding with E-cadherins from Caco-2 single cells. It should be noted that we could not exclude the possibility that the tight junctions were disrupted by the treatment with Ca2+-deficient medium, and this may allow access of the primary antibody to the intracellular space between the cells. Mg2+ ions were included in the pretreatment medium to prevent the release of the attached cells from the plate; this is based on the fact that divalent cations are required for cell attachment to the plate via integrin-extracellular matrix adhesion process. The redistribution of the E-cadherins was evident from the E-cadherin immunolocalization study using anti-E-cadherin mAb (Fig. 2). Addition of Caco-2 single cells to the attached cells produced Ca2+-dependent cell-cell adhesion. The unbound single cells can be washed out to determine the amount of bound single cells. The rate and amount of the single-cell adhesion are dependent on the duration of pretreatment of the cell monolayer with Ca2+-deficient medium (Figs. 1 and 3). The single-cell adhesion can be inhibited by anti-E-cadherin mAb (DECMA-1) to the extracellular domain but not mAb (CH-19) to the cytoplasmic domain (Fig. 5). The antibody inhibition of cell adhesion was also dependent on Ca2+ concentration (Fig. 6A). These results suggest that the homotypic adhesion of Caco-2 single cells to the attached cells is predominantly mediated by E-cadherins.
Ca2+ ions have an essential role to provide rigidity to the extracellular domain of E-cadherin to form a rod-like conformation (Pokutta et al., 1994; Nagar et al., 1996). This rod-like structure is crucial for the adhesive functions of cadherins (Ozawa et al., 1990; Takeichi, 1991; Pokutta et al., 1994; Pertz et al., 1999). E-cadherins of a cell form a lateral dimer (cis-dimer) at the extracellular domain before the cis-dimers from opposing cells form trans-oligomers that are responsible for cell-cell adhesion (Brieher et al., 1996; Yap et al., 1997; Takeda et al., 1999; Ozawa, 2002). Pertz et al. (1999) have demonstrated that the cis- and trans-dimer formations of the EC1-EC2 domain of E-cadherin were observed at Ca2+ concentrations of 50 to 1000 µM and >1000 µM, respectively. This is consistent with our current assay, in which the homotypic adhesion of Caco-2 single cells to attached cells is Ca2+-dependent (Fig. 4). There is some extent of cell adhesion in the absence of Ca2+, the level of which varies in each experiment probably depending on the efficacy of Ca2+-deficient pretreatment and the extent of washing process. The presence of cell adhesion in the absence of Ca2+ may be due to the Ca2+-independent cell-cell adhesion, presumably mediated by Ca2+-independent cell adhesion molecules such as nectins (Shimizu and Takai, 2003) or integrins. The Ca2+-dependent homotypic cell adhesion was significantly inhibited by anti-E-cadherin mAb DECMA-1 in a concentration-dependent manner; however, DECMA-1 mAb could not completely inhibit the Ca2+-dependent adhesion. This is presumably due to the presence of other Ca2+-dependent cell adhesion molecules such as desmogleins and desmocollins (Burdett, 1998; Garrod et al., 2002). It was interesting to find that DECMA-1 mAb did not block the homotypic adhesion of Caco-2 cells in Ca2+-free medium where E-cadherins did not form trans-oligomers (Ozawa et al., 1990; Takeichi, 1991; Pokutta et al., 1994; Pertz et al., 1999) and the heterotypic adhesion of Molt-3 T cells to Caco-2 attached cells; Molt-3 T cells did not express E-cadherin (Makagiansar et al., 2002) (Fig. 6). Taken together, these results suggest that the major contribution for the cell adhesion process in this assay is the E-cadherin homophilic interactions. Therefore, this assay was used to evaluate the activity of E-cadherin peptides.
In this assay, HAV peptides were used to inhibit the homotypic adhesion of Caco-2 single cells to attached cells. HAV peptides had been shown to inhibit E-cadherin-mediated aggregation of BBMEC (Lutz and Siahaan, 1997; Pal et al., 1997) and to increase paracellular porosity of MDCK cell monolayers (Makagiansar et al., 2001; Sinaga et al., 2002). We also have discovered ADT peptides from another region of EC1 domain, called a bulge region, that can modulate E-cadherin-mediated cell-cell adhesion in MDCK cell layers (Sinaga et al., 2002). Consistent with our previous studies, HAV10 and HAV24 peptides inhibited homotypic Caco-2 cell adhesion in a dose-dependent manner (Fig. 7). As in our BBMEC aggregation assay (Lutz and Siahaan, 1997), HAV24 peptide was more potent than HAV10 peptide in the present assay. This may be due to the propensity to preserve the conformation of the HAV motif in longer peptides (HAV24) than in smaller peptides (HAV10). Although hexapeptides SHAVSS and SHAVAS could lower the TEER values of MDCK cell monolayers (Makagiansar et al., 2001; Sinaga et al., 2002), they did not block the E-cadherin-mediated homotypic adhesion of Caco-2 cells in this study (Fig. 7). In our recent study, these hexapeptides could not modulate the paracellular porosity of Caco-2 cell monolayers nor block the resealing of intercellular junctions loosened by EDTA chelation (A. Calcagno and T. J. Siahaan, unpublished data). This could be due to several factors. First, the hexapeptides may not maintain the active conformation of the HAV motif; thus, they have a low binding selectivity for E-cadherin. Second, there is a possibility of subtle differences in E-cadherin in MDCK and Caco-2 cells that contributes to the binding properties between the hexapeptides and E-cadherin. In the present assay, peptides had free access to the E-cadherin-E-cadherin interaction sites between the single cells and the attached cells. Thus, the different activity of the same HAV hexapeptides in Caco-2 cells and MDCK cells is likely not due to the limited accessibility of peptides to the binding sites but probably to the species differences in binding selectivity of E-cadherin. We have shown that the HAV sequence and its flanking residues are important for the binding selectivity of HAV-peptides (Lutz and Siahaan, 1995, 1997; Lutz et al., 1995; Pal et al., 1997; Makagiansar et al., 2001; Sinaga et al., 2002). Using this novel assay, the optimization of HAV peptides as well as the exploration of other active sequences in E-cadherin are now under investigation.
In conclusion, we have established a novel adhesion assay based on the homotypic adhesion of Caco-2 single cells to attached cells. The predominant involvement of E-cadherin was confirmed. Therefore, this assay can be used to evaluate the activities of compounds of interest, including mAb, peptides, or peptidomimetics, in modulating the E-cadherin-mediated cell-cell adhesion in the context of live cells. It was also suggested that the HAV motif in the EC1 domain of E-cadherin is at least in part involved in E-cadherin-mediated cell-cell adhesion. The concept of the present adhesion assay may be applicable to other cell lines such as MDCK cells and also to heterotypic adhesion between different species-originated cells.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: HAV, His-Ala-Val; BBMEC, bovine brain microvessel endothelial cell; MDCK, Madin-Darby canine kidney; TEER, transepithelial electrical resistance; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; RT, room temperature; EBSS, Earle's balanced salt solution; BSA, bovine serum albumin; BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; AM, acetoxymethyl ester; CHES, 2-(cyclohexylamino)ethanesulfonic acid.
Address correspondence to: Dr. Teruna J. Siahaan, Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66049-3729. E-mail: siahaan{at}ku.edu
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3). Significant differences versus no pretreatment (0 min); **, P < 0.01.
