Departments of Medical Biochemistry and Biophysics (P.R., L.L., C.R., Y.S., M.R., K.L.), Clinical Science/Obstetrics and Gynecology (P.L., U.O.), Radiation Sciences (K.B.), and Medical Biosciences (E.L.), Umeå University, SE-901 87, Umeå, Sweden
Address all correspondence and requests for reprints to: Dr. Kui Liu, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87, Umeå, Sweden. E-mail: kui.liu{at}medchem.umu.se.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
E-cadherin is a 120-kDa transmembrane molecule that plays a major role in epithelial cell adhesion. E-cadherin molecules can establish calcium-dependent homophilic interactions through their extracellular domain, and these bindings are stabilized by the association of the intracellular domain with -, ß-, and
-catenins, which are linked to F actin to form functional adherens junctions (8, 9). Due to the fact that expression of E-cadherin is frequently lost in human epithelial cancers such as bladder, lung, and pancreatic carcinomas, this molecule has been implicated as a tumor suppressor in carcinogenesis (10, 11, 12, 13, 14).
E-cadherin exhibits a distinct expression pattern in the development of ovarian carcinomas, however. Although healthy OSE cells covering the ovarian surface do not express E-cadherin, E-cadherin is expressed in OSE cells covering deep clefts, invaginations, and inclusion cysts of the ovaries (4, 15, 16), where early malignant changes are believed to take place (1, 2, 3, 5, 6, 7). E-cadherin is detected more frequently in OSE cells from patients with a family history of ovarian cancers (17). By mostly immunohistochemical analyses of ovarian tumor samples, E-cadherin has not been found in normal ovaries, but has been found constantly in benign, borderline, and malignant ovarian tumors of all stages, as well as in metastases from such ovarian tumors (4, 18, 19, 20). In vitro experiments have shown that introduction of E-cadherin expression into human OSE cells induces epithelial-differentiation markers associated with preneoplastic, metaplastic OSE, and OSE-derived primary carcinomas (16) and enhances tumorigenesis of OSE cells that lead to the development of invasive adenocarcinomas (2). Moreover, in immortalized mouse OSE cells transfected with E-cadherin, the cells exhibited higher proliferation rate and gained anchorage-independent growth ability, although they were not found to be tumorigenic in xenograft models with nude mice (Dr. Barbara Vanderhyden, personal communication).
Whether or not the expression of E-cadherin in OSE cells is functionally significant in the neoplastic transformation of the cells is still unknown. However, it would be intriguing to study whether the formation of intercellular E-cadherin homophilic binding may trigger proliferation signals in OSE and ovarian cancer cells. In the present study using the E-cadherin-expressing ovarian cancer cell line OVCAR-3, we have studied the activation of phosphatidylinositol 3-kinase (PI3 K) and MAPK pathways upon establishment of E-cadherin-mediated cellular contacts. The possible mechanisms by which Akt and MAPK are activated by E-cadherin were also investigated.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Using an E-cadherin-expressing ovarian cancer cell line, OVCAR-3, we adopted a well-established model for studying E-cadherin-mediated cell-cell junctions, in which the Ca2+-dependent E-cadherin-mediated adhesion was disrupted by EGTA and restored by physiological concentrations of Ca2+ contained in fresh media (22, 23). As shown in Fig. 2A, E-cadherin is expressed at sites of cell-cell contact in serum-starved OVCAR-3 cells before disruption by EGTA (Fig. 2A
, E-cadherin, Control). The anti-E-cadherin antibody we used for immunofluorescence studies recognizes the intracellular tail of the E-cadherin molecule, so the fluorescence signal seen actually indicates the location of the intracellular part of the E-cadherin molecule. As a parallel marker, ß-catenin, which links E-cadherin to the cytoskeleton, was found to be expressed in both the cell membrane area as well as in the cytoplasm (Fig. 2A
; ß-catenin, Control). The cell borders were also outlined by the tight junction protein zonula occludens-1 (ZO-1) (Fig. 2A
; ZO-1, Control). Addition of EGTA (4 mM) for 30 min substantially disrupted E-cadherin-mediated cellular adhesions, which was evidenced also by the disturbed expression of ß-catenin and ZO-1 along the cell contacts (Fig. 2A
, EGTA, 30 min). However, upon replenishment with fresh Ca2+-containing (1.8 mM) medium, the expression of E-cadherin was found to be relocated to sites of cell-cell adhesion, reflecting the reestablishment of homophilic binding between E-cadherin molecules (Fig. 2A
; E-cadherin restoring calcium 5 min). Expression of ß-catenin and ZO-1 was also restored at cell borders upon the reestablishment of cell-cell adhesion (Fig. 2A
; ß-catenin and ZO-1 restoring calcium 5 min).
|
To study this question in greater depth, we have also evaluated the Akt kinase activities during the time course of the calcium switch in OVCAR-3 cells by measuring the enzymatic activity of immunoprecipitated P-Akt in an in vitro reaction system in which recombinant glycogen synthase kinase (GSK) was used as an Akt substrate. Thus, in accordance with the levels of activated Akt (Fig. 2B), calcium restoration also caused an increase in Akt kinase activity (Fig. 2C
; Akt activity, lanes 37). The increased Akt activity was caused by the increased amounts of P-Akt that could be immunoprecipitated (Fig. 2C
, IP-P-Akt).
Activation of Akt and MAPK by Formation of Cellular Contact Requires the Engagement of E-Cadherin
To study whether E-cadherin is essential for the activation of Akt and MAPK upon the reestablishment of cell-cell contacts, or whether the kinase activation was caused by other adhesion molecules, or even by the calcium ion in the changed culture media, we performed experiments with OVCAR-3 cells that were pretreated with the E-cadherin-neutralizing antibody SHE 787 (5 µg /ml). As shown in Fig. 3, serum-starved cells without any treatment (Fig. 3
, lane 1), or cells treated with EGTA alone (Fig. 3
, lane 2), exhibited low basal levels of P-Akt and P-MAPK. Similar to the data shown in Fig. 2B
, both Akt and MAPK were substantially activated by the reestablishment of cell-cell contacts (5 min after Ca2+ restoration; Fig. 3
, lane 3, P-Akt and P-MAPK). However, in cells that had been pretreated with E-cadherin-neutralizing antibody, activation of Akt and MAPK by Ca2+ restoration was greatly blocked (Fig. 3
, lane 4; P-Akt and P-MAPK), indicating that the activation of P-Akt and P-MAPK was substantially reduced by the blockage of E-cadherin homophilic binding. Suppression of Akt and MAPK activation was also seen with the E-cadherin-neutralizing antibody SHE 787 at concentrations of 10 and 20 µg/ml (data not shown). Similar effects on blockage of Akt and MAPK activation were also seen by the use of another widely used E-cadherin-neutralizing antibody HECD-1 (data not shown). Taken together, our results demonstrate that the formation of E-cadherin homophilic binding is responsible for the major part of the activation of Akt and MAPK in OVCAR-3 cells.
|
|
The upstream kinases that cause the activation of MAPK, MEK, and Raf were also studied. As shown in Fig. 4B, levels of phosphorylated MEK (P-MEK, lane 3) and Raf (P-Raf, lane 3) were elevated by Ca2+ restoration, indicating that Raf and MEK were activated by the formation of E-cadherin adhesions (Fig. 4B
, P-MEK and P-Raf, lane 3 vs. lanes 1 and 2). However, in OVCAR-3 cells that had been pretreated with E-cadherin-neutralizing antibody SHE 787 (5 µg/ml), the activation of MEK and Raf was largely suppressed (Fig. 4B
; P-MEK and P-Raf, lane 4 vs. lane 3). Similar inhibitory effects were also observed with higher concentrations of SHE 787 and with another E-cadherin-neutralizing antibody, HECD-1 (data not shown). Our data therefore suggest that E-cadherin activates MAPK by the classical Raf-MEK-MAPK pathway.
Ligand-Independent Activation of Epidermal Growth Factor (EGF) Receptor Is Necessary for E-Cadherin-Mediated Akt and MAPK Activation
E-cadherin is an adhesion molecule with no reported enzymatic activities (8, 9), and Akt and MAPKs are known to be activated by various types of receptor protein tyrosine kinases (RPTKs) or protein tyrosine kinases (PTKs) (24). We therefore hypothesized that the E-cadherin activation of Akt and MAPK may be achieved by activation of certain PTKs or RPTKs. To address this question, we pretreated OVCAR-3 cells with a set of inhibitors that specifically inhibit various PTKs of interest, including Genistein (a general PTK inhibitor), PP1 (Src inhibitor), Tyrphostin AG 1478 (EGF receptor inhibitor), Tyrphostin AG 825 (ErbB-2 inhibitor), GTP-14564 (Kit, c-fms, and platelet-derived growth factor receptor ß inhibitor), and AGL 2263 (insulin receptor and IGF receptor inhibitor). Although most of the inhibitors did not appear to have any inhibitory effect on E-cadherin-mediated activation of Akt and MAPK, the general PTK inhibitor Genistein and the EGF receptor inhibitor Tyrphostin AG 1478 significantly suppressed the E-cadherin activation of Akt (Fig. 5A; P-Akt; lanes 4 and 7 vs. lane 2) and MAPK (Fig. 5A
; P-MAPK; lanes 4 and 7 vs. lane 2).
|
To prove the EGF receptor activation in OVCAR-3 cells before and after the reestablishment of E-cadherin homophilic binding, levels of phosphorylated tyrosine in immunoprecipitated EGF receptor were also measured. As shown in Fig. 6, phosphorylated tyrosine levels in the EGF receptor were elevated upon the establishment of E-cadherin homophilic binding in OVCAR-3 cells (Fig. 6
, lanes 37). However, the activation of EGF receptor by E-cadherin was temporary, which was seen from 130 min after calcium restoration (Fig. 6
, lanes 37). The activated EGF receptor level had decreased to basal levels at 60 min after calcium restoration, which was the longest time studied (Fig. 6
, lane 8). Nevertheless, no physical interaction between E-cadherin and EGF receptor was observed in our experimental system by performing immunoprecipitation and Western blot under different conditions (data not shown).
|
|
Determination of Downstream Effectors that Are Responsible for the E-Cadherin-EGF Receptor Activation of Akt and MAPK
Signals of the Rho family GTPases have been reported to be activated as epithelial cells reestablish contacts after chelation of extracellular calcium (22, 25, 26). As formation of E-cadherin homophilic ligation is known to regulate adhesive contacts through the activation of Rac signaling (25), by measuring levels of the active form of Rac (GTP.Rac) in OVCAR-3 cells, we have tried to determine whether E-cadherin activation of Akt and MAPK may be downstream of Rac activation. However, our data indicate that the E-cadherin activation of Akt and MAPK are not accompanied by Rac activation (data not shown). It is therefore unlikely that the activation of Akt and MAPK by E-cadherin-mediated adhesion is mediated by Rac activation.
We have also investigated molecules that function downstream of the EGF receptor, which may have different roles in the activation of Akt and MAPK upon the establishment of E-cadherin homophilic binding. Ras is a well-studied molecule that interacts with and activates Raf, which subsequently activates MEK, leading to MAPK activation in many cell types including tumor cells (24). By performing glutathione S transferase (GST)-Raf pull-down assay, we found that the reestablishment of E-cadherin binding did not lead to any change in Ras activation (data not shown). Thus, Ras is unlikely to be involved in the activation of Akt and MAPK in our system.
In addition, we have studied the activation of the adaptor protein SHC, which is known to act downstream of the EGF receptor, and can relay G protein-coupled receptor- and RPTK-induced signals (27, 28). However, our experiments showed that activation of Akt/MAPK and EGF receptor by E-cadherin-mediated cellular adhesion was not accompanied by further activated SHC (data not shown), indicating that the classic EGF receptor-SHC-MAPK pathway (27, 29) was not active in our cellular system. Nor did we see any inhibitory effects of two specific protein kinase C inhibitors, bisindolylmaleimide I and XI, on the E-cadherin-activated MAPKs (data not shown). This indicates that MAPK activation by E-cadherin-activated EGF receptor was not achieved via the known pathway of EGF receptor-phospholipase C-protein kinase C-MAPK (29).
Among the many downstream effectors of EGF receptor, we found that GRB-associated binder 1 (Gab1) was activated concurrently with the establishment of E-cadherin-mediated cellular adhesion. As shown in Fig. 8A, in a similar experimental setup, as indicated by the phosphorylated tyrosine levels of immunoprecipitated Gab1, Gab1 was rapidly activated by 1 min after the replenishment of fresh calcium, and this activation reached its peak level at 215 min and then decreased gradually over a period of 90 min (Fig. 8A
; P-tyrosine; lanes 39). As a control, the activation of Gab1 was comparable to its activation by treatment with 30% fetal bovine serum for 30 min (Fig. 8A
, lane 10). The total Gab1 levels are also shown in Fig. 8A
as internal controls (Fig. 8A
; Gab 1).
|
Suppression of E-Cadherin Function Leads to Reduced Cell Proliferation and Viability
To study the physiological functions of E-cadherin-mediated cell-cell adhesion and the E-cadherin activation of PI3 K and MAPK pathways, we performed the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) cell viability and proliferation assay. As shown in Fig. 9, compared with the control group of cells, incubation of cells with the E-cadherin-neutralizing antibody SHE 787 (5 µg/ml) stopped the cells from proliferating after 24 h of incubation, and there was a markedly reduced cell viability during an incubation period of 4872 h (Fig. 9
; E-cad Ab). Similarly, the PI3 K inhibitor LY294002 (50 µM) was found to dramatically decrease the viability of the cells (Fig. 9
; LY294002), and the MEK 1 inhibitor PD98059 (50 µM) was found to stop the cells from proliferation (Fig. 9
; PD98059). Thus, our data indicate further that E-cadherin adhesion as well as E-cadherin-mediated activation of PI3 K and MAPK pathways may be important for proliferation and survival of ovarian cancer cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because uncontrolled cellular proliferation is known as a prerequisite for tumor development (24), and to investigate whether the conversion between disruption and reestablishment of E-cadherin-mediated cell-cell adhesion may trigger intracellular activation of proliferation and survival signals, we have adopted a well-established calcium-restoration model (22, 23) and have demonstrated a rapid activation of Akt and MAPK upon reestablishment of E-cadherin-mediated adhesion in ovarian cancer cells. Our data show that it is not only the establishment of cell-cell adhesions but also the E-cadherin molecules that have an indispensable role in this process, as antibodies that can neutralize E-cadherin function abolished the activation of Akt and MAPK to a large extent. Our results therefore suggest that in ovarian cancer cells, E-cadherin may serve not only as an intercellular adhesion molecule, but also as an upstream regulator that triggers downstream kinase activation.
By performing signal transduction studies, we have investigated the pathways by which the establishment of E-cadherin homophilic binding activates Akt and MAPK. We have shown that E-cadherin-activated Akt can be blocked completely by PI3 K inhibitors, indicating that Akt is activated by PI3 K. We have also shown that activation of MAPK by E-cadherin-mediated cell-cell adhesion is mediated through the Raf-MEK-MAPK cascade. However, Ras, the molecule that interacts with and phosphorylates Raf in the classic MAPK pathway in many other cell types (24), was not activated by E-cadherin homophilic binding. Thus, we assume that there are other signal transduction pathways upstream of the Raf-MEK-MAPK pathway that participate in the activation of these molecules.
Because E-cadherin is known to activate the Rho family of GTPases for the regulation of adhesion properties (22, 25, 26), we have also investigated whether the key molecule for such activation, activation of Rac, is activated concurrently with activation of Akt and MAPK when E-cadherin homophilic binding was established in OVCAR-3 cells. We found that Rac is not activated when Akt and MAPK are activated by the establishment of E-cadherin-mediated cell-cell adhesion, suggesting that the activation of Rac and Akt/MAPK by E-cadherin are achieved through different pathways.
E-cadherin is a well-known adhesion molecule, which does not exhibit any enzymatic activity in its short intracytoplasmic tail (8, 9). Thus, the activation of Akt and MAPK by E-cadherin-mediated cell-cell adhesion is most likely mediated by activation of other signal transduction systems, especially the PTKs and RPTKs. We have demonstrated in the current study that E-cadherin-mediated cell-cell adhesion can trigger a ligand-independent activation of EGF receptor, which may subsequently lead to activation of Akt and MAPK. However, using the immunoprecipitation-western blot approach, we failed to detect any interaction between E-cadherin and EGF receptor, suggesting the existence of intermediate molecules between E-cadherin and EGF receptor, which relay signals initiated by intercellular adhesion to intracellular proliferation signal activation. We have also investigated more closely the signaling molecules that are downstream of the E-cadherin-mediated EGF receptor activation, which is responsible for the activation of Akt and MAPK. Our data indicate that Gab 1 was activated during the calcium switch procedure in OVCAR-3 cells, and it is probably important for activation of the downstream Akt and MAPK by EGF receptor.
Our results are supported by a previously published report that establishment of E-cadherin-mediated adhesion can activate Akt in dog kidney epithelial cells (MDCK) (30). While this paper was being revised, a study from Shen and Kramer (31) reported that E-cadherin can physically interact with and activate the EGF receptor in squamous cell carcinoma, which in turn leads to the activation of MAPK as well as elevation of the level of the antiapoptotic molecule Bcl-2. Although these results were found in cell lines other than ovarian cancer cells, they do indicate, together with our work, that E-cadherin-mediated cellular adhesion is capable of activating cellular survival pathways.
Compared with other types of tumors, ovarian epithelial cancers are characterized by greater local tumor growth initially, which is followed by spreading into the peritoneal cavity only at later stages of tumor progression (3, 4). E-cadherin may therefore play an important adhesive role in maintaining tissue integrity in this process (4, 15). From the results described in the current study, we propose that the concurrent establishment of E-cadherin-mediated cellular adhesion and activation of proliferation signals for the cells may enhance the transformation from a single layer of normal epithelial cells to neoplastic cells, as well as the development of a tumor tissue mass. Thus, distinct from its role as a tumor suppressor in other types of human epithelial cancers, E-cadherin may function as a tumor enhancer in the development of ovarian epithelial cancers.
A natural question that arises from the current study is what the difference is between ovarian epithelial cancers and other types of epithelial cancers, such as lung, bladder, and pancreatic cancers, in which E-cadherin has been well identified as a tumor suppressor. Although we do not yet understand the in-depth mechanisms behind this phenomenon, we have hypothesized that the substantial hormonal influences on the development and progression of ovarian cancers may drive E-cadherin to play an extraordinary role in the development of ovarian epithelial cancers. However, we have some preliminary data indicating that treatment of OVCAR-3 cells with hormones, including progesterone, estradiol, FSH, LH, or GnRH, does not alter the expression level of E-cadherin (data not shown). Moreover, in our screening of expression levels of E-cadherin, EGF receptor, P-Akt, P-MAPK, as well as urokinase plasminogen activator, matrix metalloproteinase 2, and matrix metalloproteinase 9 (as metastasis markers) in various types of ovarian cancer samples, we did not see any correlation in expression patterns, where, for example, activation of Akt and MAPK in tumor samples was activated in proportion to the degree of overexpression of E-cadherin. Our explanation for this result is either that Akt or MAPK activation is not proportional to the E-cadherin expression level, or the phosphorylation status of Akt and MAPK had been disturbed due to improper handling during the procedures of surgical removal and storage of patient samples. Thus, mechanisms of how E-cadherin activates Akt and MAPK in vivo remain elusive at this stage.
To our knowledge, this study is the first to report that in ovarian cancer cells, E-cadherin-mediated cellular adhesion is capable of activating proliferation and survival signals including EGF receptor, PI3 K (Akt), and MAPK pathways. We have also demonstrated that inhibition of E-cadherin function can lead to reduced cell proliferation and viability. Our results are supported by data obtained from another E-cadherin expressing ovarian cancer cell line CaOV-3, where we have seen almost identical results on the activation of Akt and MAPK via ligand-independent activation of EGF receptor by E-cadherin-mediated adhesion. This suggests that the E-cadherin-mediated activation of survival signals may be a common feature of this adhesion molecule in ovarian cancers. However, at the current stage of the study, we still do not know why E-cadherin shows a different role in ovarian cancers; nor do we know the detailed mechanisms of how E-cadherin activates downstream signals. Questions raised from this study remain to be investigated further by screening for molecules that interact with E-cadherin in ovarian cancer cells and tumor samples. Signal transduction studies, including the use of dominant negative and/or constant active DNA constructs of various kinases, and RNA interference approaches will help to determine the signaling pathways through which Akt and MAPK are activated by E-cadherin in ovarian cancer cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immediately after surgical removal, the ovaries or ovarian tumor samples were transported to the Department of Pathology, dissected, and frozen in liquid nitrogen and kept at 80 C until the time of analysis. The samples were homogenized in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 137 mM sodium chloride, 15% glycerol (vol/vol), 1% Nonidet P-40, 20 mM sodium fluoride, 10 mM sodium phosphate (NaPPi), 25 mM ß-glycerolphosphate, 2 mM benzamidine, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM sodium vanadate, and 2 mM phenylmethylsulfonylfluoride. Protein concentrations were measured and 50 µg of total proteins were loaded onto SDS-PAGE gels and blotted for determination of E-cadherin and ß-actin levels.
Cells and Culture Conditions
OVCAR-3 cells were provided by Dr. Karin Sundfeldt of Göteborg University, Sweden, and were routinely maintained at 37 C in DMEM/F12 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere of 5% CO2 and 95% air. For all experiments, the cells were grown as a confluent monolayer to optimize cell-cell contacts and to minimize the influence of integrin-extracellular matrix interactions (33).
The OVCAR-3 cells were serum starved overnight, and E-cadherin-mediated cell-cell contacts were disrupted by the addition of EGTA to the medium to a final concentration of 4 mM, for 30 min at 37 C. Thereafter, intercellular interactions were allowed to reestablish in the presence of fresh calcium-containing medium (final concentration of CaCl2, 1.8 mM) (22, 23). At different time points (190 min) after calcium restoration, the cells were harvested, lysed in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM ß-glycerol phosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% Nonidet P-40, 1 mM dithiothreitol, 5 mM benzamidine, 1 mM phenylmethylsulfonylfluoride, 250 µM sodium vanadate, 2 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin, on ice for 20 min, and centrifuged in a refrigerated microcentrifuge at 14,000 rpm for 15 min. Supernatants were collected and prepared for further analysis.
Reagents, Antibodies, and Immunological Detection Methods
Specific kinase inhibitors Tyrphostin AG 1478, GTP-14564, AGL 2263, Tyrphostin AG 825, and Genistein were purchased from Calbiochem (San Diego, CA), and PP1 was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Antibodies against E-cadherins, ZO-1 and ß-catenin, were purchased from BD Transduction Laboratories (San Jose, CA). Neutralizing antibodies to E-cadherin (SHE 787 and HECD-1) were purchased from Takara (Shiga, Japan) and were used for antibody inhibition experiments. Rabbit polyclonal antibodies against p44/42 MAPKs (MAPK, or ERK 1 and 2), phosphorylated p44/42 MAPK (Thr202/Tyr204, p-MAPK), Akt (PKB), phosphorylated Akt (Ser473), and Gab 1 were from Cell Signaling Technologies (Beverly, MA). Rat monoclonal phospho-Raf-1 (Ser338) and EGF receptor-neutralizing antibody (LA1) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse monoclonal antibody against Raf-1 (E-10) and antiphosphorylated tyrosine (pY99) were obtained from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA). Antimouse and antirabbit secondary antibodies coupled to horseradish peroxidase were from Bio-Rad Laboratories, Inc. (Hercules, CA). Fluorochrome-conjugated antibodies for immunofluorescence were obtained from Zymed Laboratories, Inc. (South San Francisco, CA).
Akt Kinase Assay
Akt kinase assay was performed using the kinase assay kit from Cell Signaling Technologies (Beverly, MA). Briefly, Akt was precipitated using an immobilized monoclonal antibody that preferably recognizes phosphorylated Akt at Serine 473. After washing, ATP and recombinant GSK were added to the reaction mixture and the kinase reaction was run at 30 C for 30 min. The reaction mixture was then heated at 95 C for 5 min before being run in a SDS-PAGE gel and blotted against an antibody recognizing phosphorylated GSK (Cell Signaling Technologies).
Immunofluorescence Microscopy
OVCAR-3 cells were grown on glass coverslips until multicell clusters were formed, washed with PBS, and fixed in 4% paraformaldehyde for 20 min at room temperature. After being permeabilized with 0.5% Triton X-100 for 10 min at room temperature, the cells were washed with PBS and incubated with mouse anti-E-cadherin antibody (which recognizes the intracellular part of E-cadherin) (1:100) for 1 h at 37 C. As secondary antibody, FITC antimouse IgG (H+L) antibody was used (1:50) (Zymed Laboratories). Coverslips were examined under a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY) at a x63 magnification using appropriate filters. Images were captured using a Leica DMLB (Leica Corp., Deerfield, IL) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA).
GST-Raf Pull-Down Assay for Measurement of Active Ras
The GST-Raf-RBD construct was kindly provided by Dr. Bengt Hallberg (Umeå University, Umeå, Sweden). The construct was expressed in BL21 Escherichia coli and isolated with glutathione-agarose (Amersham Biosciences, Uppsala, Sweden) according to manufacturers instructions. GST-Raf-RBD fusion protein (30 µg) bound to glutathione-agarose was added to each sample of cellular lysates (500 µg) and incubated on a rotator for 1 h at 4 C. The beads were then washed three times with the lysis buffer, and associated proteins were separated by 12% SDS-PAGE and blotted with an antibody against Ras (BD Transduction Laboratories) to indicate levels of active Ras. As an internal control, levels of total Ras in the corresponding lysates were also measured.
p21-Activated Kinase (PAK) Cdc42/Rac1-interactive binding domain (CRIB) Pull-Down Assay for Measurement of Active Rac
The PAK-CRIB GST fusion protein construct (a kind gift from Dr. J. Chernoff, Fox Chase Cancer Center, Philadelphia, PA) was expressed in BL21 E. coli and isolated with glutathione-agarose (Amersham Biosciences). GST fusion protein (50 µg) bound to glutathione-agarose was added to each sample of OVCAR-3 lysate (200 µg) and incubated on a rotator for 1 h at 4 C. The beads were then washed three times with lysis buffer and separated in a 12% SDS-PAGE gel. Western blot was performed with an anti-Rac monoclonal antibody (Upstate Biotechnology, Inc.) to indicate active forms of Rac. As an internal control, levels of total Rac in the corresponding lysates were also measured.
MTT Assay
Briefly, 2 x 105 OVCAR-3 cells were seeded in 24-well plates and incubated with or without E-cadherin-neutralizing antibody SHE 787 or mouse IgG control (5 µg /ml), LY294002 (50 µM), or PD98059 (50 µM) for 24, 48, and 72 h at 37 C in DMEM/F12 culture media supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 µg/ml streptomycin, in a humidified atmosphere of 5% CO2 and 95% air. The standard MTT assay was performed as previously described (34). Values at 0 h was defined as 1.0, and fold inductions/reductions at different time points were used to calculate the mean values ± SD.
Data Analysis
All experiments were repeated at least three times. For Western blot figures, a representative blot from three independent experiments is depicted. In addition, normalized values of Western blot as determined by densitometric analyses showing the mean ± SD from three independent experiments were also included, where the band of the highest density in a Western blot was defined as 1.0, and from this value all other values of the same blot were calculated. Note that the relative density representing the expression level of a molecule is only comparable to other time points or treatments within the same set of experiments. The relative densities are not comparable among measurements of different molecules or among different experiments. Data were analyzed by ANOVA. Differences among groups were calculated by Tukeys multiple-comparison test, and a difference was considered to be significant with P < 0.05.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Professor Tor Ny (Umeå University, Umeå, Sweden) for his support on this manuscript; Dr. Karin Sundfeldt (Göteborg University, Göteborg, Sweden) for providing OVCAR-3 cells; Dr. Bengt Hallberg (Umeå University) for providing the GST-Raf-RBD construct; and Dr. J. Chernoff (Fox Chase Cancer Center) for providing the PAK-CRIB GST construct.
![]() |
FOOTNOTES |
---|
Present address of M.R.: Department of Medical Biochemistry, University of Kuopio, POB 1627, 70211 Kuopio, Finland.
First Published Online May 31, 2005
1 P.R., L.L., and C.R. contributed equally to this study.
Abbreviations: CRIB, cdc42/Rac1-interactive binding domain; EGF, epidermal growth factor; GSK, glycogen synthase kinase; GST, glutathione-S-transferase; MEK, MAPK kinase; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide; OSE, ovarian surface epithelial; PAK, p21-activated kinase; P-Akt, phosphorylated Akt; PI 3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; P-MAPK, phosphorylated MAPK; PTK, protein tyrosine kinase; RPTK, receptor protein tyrosine kinase.
Received for publication September 1, 2004. Accepted for publication May 25, 2005.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|