Jian Ren

The DF3/MUC1 mucin-like, transmembrane glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts with the c-Src tyrosine kinase and thereby increases binding of MUC1 and beta-catenin. In the present work, coimmunoprecipitation studies demonstrate that MUC1 associates constitutively with the epidermal growth factor receptor (EGF-R) in human ZR-75-1 breast carcinoma cells. Immunofluorescence studies show that EGF-R and MUC1 associate at the cell membrane. We also show that the activated EGF-R phosphorylates the MUC1 cytoplasmic tail on tyrosine at a YEKV motif that functions as a binding site for the c-Src SH2 domain. The results demonstrate that EGF-R-mediated phosphorylation of MUC1 induces binding of MUC1 to c-Src in cells. Moreover, in vitro and in vivo studies demonstrate that EGF-R increases binding of MUC1 and beta-catenin. These findings support a novel role for EGF-R in regulating interactions of MUC1 with c-Src and beta-catenin.

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The cytoplasmic domain of MUC1 interacts with glycogen synthase kinase 3β (GSK3β) and thereby decreases binding of MUC1 and β-catenin. The present studies demonstrate that MUC1 associates with the c-Src tyrosine kinase. c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif located between sites involved in interactions with GSK3β and β-catenin. The results demonstrate that the c-Src SH2 domain binds directly to pYEKV and inhibits the interaction between MUC1 and GSK3β. Moreover and in contrast to GSK3β, in vitro andin vivo studies demonstrate that c-Src-mediated phosphorylation of MUC1 increases binding of MUC1 and β-catenin. The findings support a novel role for c-Src in regulating interactions of MUC1 with GSK3β and β-catenin. The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The cytoplasmic domain of MUC1 interacts with glycogen synthase kinase 3β (GSK3β) and thereby decreases binding of MUC1 and β-catenin. The present studies demonstrate that MUC1 associates with the c-Src tyrosine kinase. c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif located between sites involved in interactions with GSK3β and β-catenin. The results demonstrate that the c-Src SH2 domain binds directly to pYEKV and inhibits the interaction between MUC1 and GSK3β. Moreover and in contrast to GSK3β, in vitro andin vivo studies demonstrate that c-Src-mediated phosphorylation of MUC1 increases binding of MUC1 and β-catenin. The findings support a novel role for c-Src in regulating interactions of MUC1 with GSK3β and β-catenin. adenomatous polyposis coli glycogen synthase kinase 3β cytoplasmic domain glutathione S-transferase polyacrylamide gel electrophoresis monoclonal antibody epidermal growth factor receptor platelet-derived growth factor receptor phosphotyrosine β-catenin, a component of the adherens junctions of mammalian epithelial cells, binds directly to the cytoplasmic domain of the transmembrane E-cadherin protein that functions in Ca2+-dependent epithelial cell-cell interactions (1Takeichi M. Annu. Rev. Biochem. 1990; 59: 237-252Crossref PubMed Scopus (1113) Google Scholar). In turn, α-catenin binds to β-catenin and thereby links the complex to the actin cytoskeleton (2Jou T. Stewart D. Stappert J. Nelson W. Marrs J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5067-5071Crossref PubMed Scopus (305) Google Scholar). Formation of the cadherin-catenin complex is essential for adherens junction function (3Kawanishi J. Kato J. Sasaki K. Fujii S. Watanabe N. Niitsu Y. Mol. Cell. Biol. 1995; 15: 1175-1181Crossref PubMed Google Scholar). In the cytosol, β-catenin binds directly to the adenomatous polyposis coli (APC)1 tumor suppressor (4Rubinfield B. Souza B. Albert I. Muller O. Chamberlain S. Masiarz S. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1183) Google Scholar, 5Rubinfield B. Souza B. Albert I. Muller O. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 6Su L.-K. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1119) Google Scholar). Phosphorylation of APC and β-catenin by GSK3β increases the formation of APC-β-catenin complexes (7Rubinfield B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1310) Google Scholar) and targets β-catenin for ubiquitination and degradation by the 26 S proteosome (8Aberie H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2172) Google Scholar, 9Orford K. Crockett C. Jensen J. Weissman A. Byers S. J. Biol. Chem. 1997; 272: 24735-24738Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Solomon D. Sacco P. Roy S. Simcha I. Johnson K. Wheelock M. Ben-Ze'ev A. J. Cell Biol. 1997; 139: 1325-1335Crossref PubMed Scopus (131) Google Scholar). Cells that express certain APC mutants or are APC deficient thus exhibit increased levels of cytosolic β-catenin (11Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (957) Google Scholar). Other studies have shown that β-catenin forms complexes with members of the T-cell factor/leukocyte-enhancing factor (Tcf/LEF-1) family of transcription factors (12Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedi R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2605) Google Scholar, 13Huber O. Korn R. McLaughlin J. Ohsugi M. Hermann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (784) Google Scholar, 14Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1625) Google Scholar) and functions in the activation of gene expression (13Huber O. Korn R. McLaughlin J. Ohsugi M. Hermann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (784) Google Scholar, 14Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1625) Google Scholar, 15Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (447) Google Scholar). The finding that β-catenin and GSK3β interact with the cytoplasmic domain of the DF3/MUC1 mucin-like glycoprotein has supported the involvement of an additional pathway in β-catenin signaling (16Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). MUC1 is highly overexpressed by human carcinomas (18Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (502) Google Scholar). In addition, whereas MUC1 expression is restricted to the apical borders of normal secretory epithelial cells, MUC1 is aberrantly expressed by carcinoma cells at high levels throughout the cytoplasm and over the entire cell surface (18Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (502) Google Scholar, 19Friedman E.L. Hayes D.F. Kufe D.W. Cancer Res. 1986; 46: 5189-5194PubMed Google Scholar, 20Perey L. Hayes D.F. Maimonis P. Abe M. O'Hara C. Kufe D.W. Cancer Res. 1992; 52: 2563-3568PubMed Google Scholar). The MUC1 protein consists of an N-terminal ectodomain with variable numbers of 20-amino acid tandem repeats that are subject to extensive O-glycosylation (21Gendler S. Taylor-Papadimitriou J. Duhig T. Rothbard J. Burchell J.A. J. Biol. Chem. 1988; 263: 12820-12823Abstract Full Text PDF PubMed Google Scholar, 22Siddiqui J. Abe M. Hayes D. Shani E. Yunis E. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2320-2323Crossref PubMed Scopus (281) Google Scholar). The C-terminal region includes a transmembrane domain and a 72-amino acid cytoplasmic tail. MUC1 is subject to proteolytic cleavage and the large ectodomain containing the tandem repeats can remain complexed to the 25-kDa C-terminal subunit or undergo release from the cell surface (23Ligtenberg M. Buijs F. Vos H. Hilkens J. Cancer Res. 1992; 52: 223-232Google Scholar). β-catenin binds directly to MUC1 at a SAGNGGSSL motif in the cytoplasmic domain (16Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Similar SXXXXXSSL sites in E-cadherin and APC are responsible for β-catenin interactions (4Rubinfield B. Souza B. Albert I. Muller O. Chamberlain S. Masiarz S. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1183) Google Scholar, 5Rubinfield B. Souza B. Albert I. Muller O. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 6Su L.-K. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1119) Google Scholar). GSK3β also binds directly to MUC1 and phosphorylates serine in a DRSPY site adjacent to that for the β-catenin interaction (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). GSK3β-mediated phosphorylation of MUC1 decreases the association of MUC1 and β-catenin (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). The present studies demonstrate that the c-Src tyrosine kinase interacts directly with MUC1. A YEKV motif in the MUC1 cytoplasmic domain (CD) has been identified as a site for c-Src phosphorylation. The results demonstrate that c-Src regulates the interactions of MUC1 with GSK3β and β-catenin. Human ZR-75-1 breast carcinoma cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin, 100 units/ml penicillin, and 2 mml-glutamine. 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. Subconfluent cells were disrupted on ice in lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mmNaCl, 0.1% Nonidet P-40, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 1 mmdithiothreitol) for 30 min. Lysates were cleared by centrifugation at 14,000 × g for 20 min. Equal amounts of protein from cell lysates were incubated with normal mouse IgG, MAb DF3 (anti-MUC1) (18Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (502) Google Scholar), anti-c-Src (Upstate Biotechnology, Lake Placid, NY), or the rabbit anti-DF3-E antibody prepared against a peptide derived from the MUC1 extracellular domain (HDVETQFNQYKTEAAS). After incubation for 2 h at 4 °C, the immune complexes were precipitated with protein G-agarose. The immunoprecipitates were washed with lysis buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The immunoblots were probed with 500 ng/ml anti-MUC1 or 1 μg/ml anti-c-Src. Reactivity was detected with horseradish peroxidase-conjugated second antibodies and chemiluminescence (ECL, Amersham Pharmacia Biotech). The MUC1/CD(Y46F) and MUC1(Y46F) mutants were generated using site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) to change Tyr-46 to Phe. Kinase-inactive c-Src was similarly generated by mutation of Lys-295 to Arg (K295R) (24Kamps M.P. Sefton B.M. Mol. Cell. Biol. 1986; 6: 751-757Crossref PubMed Scopus (112) Google Scholar). Purified wild-type and mutant MUC1/CD proteins were incubated with 1.5 units of purified c-Src (Oncogene Research Products, Cambridge, MA) in 20 μl of kinase buffer (20 mm Tris-HCl, pH 7.6, 10 mmMgCl2, 5 mm dithiothreitol). The reaction was initiated by addition of 10 μCi [γ-32P]ATP. After incubation for 15 min at 30 °C, the reaction was stopped by addition of sample buffer and boiling for 5 min. Phosphorylated proteins were separated by SDS-PAGE and analyzed by autoradiography. Purified wild-type and mutant MUC1/CD proteins were incubated with 1.5 units of c-Src in the presence or absence of 200 μm ATP for 30 min at 30 °C. GST, GST-Src-SH3, GST-Src-SH3De90/92 (Ref. 25Shiue L. Zoller M. Brugge J. J. Biol. Chem. 1995; 270: 10498-10502Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, provided by Dr. J. Brugge, Harvard Medical School), GST-Src-SH2, or GST-β-catenin bound to glutathione beads was then added, and the reaction was incubated for 1 h at 4 °C. After washing, the proteins were subjected to SDS-PAGE and immunoblot analysis with the anti-MUC1/CD antibody that was generated against the cytoplasmic domain (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). In other studies, GST-MUC1/CD bound to glutathione beads was incubated with 1.5 units of c-Src in the presence and absence of 200 μm ATP for 30 min at 30 °C before adding 0.1 mg of purified GSK3β (New England BioLabs) for an additional 1 h. Precipitated proteins were analyzed by immunoblotting with anti-GSK3β. ZR-75-1 or 293 cells were transiently transfected with pCMV, pCMV-MUC1, pCMV-c-Src (provided by Dr. R. Rickles, ARIAD Pharmaceuticals, Inc., Cambridge, MA) or pCMV-c-Src(K295R) using electroporation methods. Efficiency of transient transfections ranged from 40–50% of ZR-75-1 cells and 70–80% of 293 cells. Cell lysates were prepared at 48 h after transfection. To determine whether DF3/MUC1 forms a complex with c-Src, anti-MUC1 immunoprecipitates from lysates of human ZR-75-1 cells were analyzed by immunoblotting with anti-c-Src. The results demonstrate that c-Src coprecipitates with MUC1 (Fig.1 A, left). In the reciprocal experiment, analysis of anti-c-Src immunoprecipitates by immunoblotting with anti-MUC1 confirmed the association of MUC1 and c-Src (Fig. 1 A, right). Similar results have been obtained in human HeLa cells (data not shown). To assess whether the binding is direct, we incubated purified His-tagged MUC1 cytoplasmic domain (His-MUC1/CD) with a GST fusion protein that contains the c-Src SH3 domain. Analysis of the adsorbate to glutathione beads by immunoblotting with anti-MUC1/CD demonstrated binding of MUC1/CD to GST-Src SH3, and not GST or a GST-Src SH2 fusion protein (Fig.1 B). As an additional control, His-MUC1/CD was incubated with a GST fusion protein containing a mutated c-Src SH3 domain (GST-Src SH3De90/92) (26Weng Z. Rickles R. Feng S. Richard S. Shaw A. Schreiber S. Brugge J. Mol. Cell. Biol. 1995; 15: 5627-5634Crossref PubMed Scopus (110) Google Scholar). The finding that MUC1/CD binds to wild-type c-Src SH3 but not the mutant supported a direct interaction between MUC1 and c-Src (Fig. 1 C). To determine whether MUC1/CD is a substrate for c-Src, we incubated MUC1/CD with purified c-Src and [γ-32P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated c-Src-mediated phosphorylation of MUC1/CD (Fig. 2 A). Previous studies have demonstrated that GSK3β phosphorylates MUC1/CD on Ser at a DRSPYEKV site (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). As the adjacent YEKV sequence represents a consensus for c-Src phosphorylation, MUC1/CD was generated with a FEKV mutation (Fig. 2 B). Incubation of MUC1/CD(Y46F) with c-Src demonstrated a decrease in phosphorylation as compared with that found with wild-type MUC1/CD (Fig. 2 C). These findings indicate that c-Src phosphorylates MUC1/CD predominantly but not exclusively at the YEKV site. As the c-Src SH2 domain interacts with a preferred pYEEI sequence (27Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar), c-Src-mediated phosphorylation of YEKV in MUC1/CD provides a potential site for c-Src SH2 binding. To determine whether the c-Src SH2 domain binds to phosphorylated MUC1/CD, we incubated MUC1/CD with c-Src and ATP and then assessed binding to GST-Src SH2. The results demonstrate that GST-Src SH2 associates with phosphorylated but not unphosphorylated MUC1/CD (Fig. 2 D). Moreover, compared with MUC1/CD, there was substantially less binding of GST-Src SH2 to the MUC1/CD(Y46F) mutant that had been incubated with c-Src and ATP (Fig. 2 D). These results support c-Src-mediated phosphorylation of MUC1/CD and thereby a direct interaction of phosphorylated MUC1/CD with the c-Src SH2 domain. As the c-Src phosphorylation site on MUC1/CD resides next to the binding and phosphorylation site for GSK3β (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar), we asked if the interaction of MUC1/CD with c-Src affects that with GSK3β. GST-MUC1/CD was incubated with c-Src and ATP before addition of GSK3β. Analysis of proteins precipitated with glutathione beads demonstrated that c-Src-mediated phosphorylation of MUC1/CD is associated with a decrease in binding of MUC1/CD and GSK3β (Fig.3 A). To assess the effects of c-Src on the interaction of MUC1/CD and GSK3β in vivo, ZR-75-1 cells were transfected to express the empty vector or c-Src. Anti-MUC1 immunoprecipitates were analyzed by immunoblotting with anti-GSK3β. The results demonstrate that c-Src also decreases the interaction of MUC1 and GSK3β in vivo (Fig.3 B). These findings indicate that GSK3β interacts with MUC1/CD by a c-Src-dependent mechanism. Phosphorylation of MUC1 by GSK3β decreases binding of MUC1 to β-catenin in vitro and in cells (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). To determine whether c-Src-mediated phosphorylation of MUC1 affects the interaction of MUC1 with β-catenin, we incubated MUC1/CD with c-Src and ATP. Phosphorylated and unphosphorylated MUC1/CD were then incubated with GST or GST-β-catenin. Similar studies were performed with the MUC1/CD(Y46F) mutant. Analysis of proteins bound to glutathione beads by immunoblotting with anti-MUC1/CD demonstrated that c-Src-mediated phosphorylation of MUC1/CD increases binding of MUC1/CD to GST-β-catenin (Fig. 4 A). By contrast, there was no detectable binding of phosphorylated or unphosphorylated MUC1/CD to GST (Fig. 4 A). Studies performed with MUC1/CD(Y46F) demonstrated that c-Src-dependent phosphorylation of the YEKV site on MUC1/CD is necessary for the formation of MUC1/CD-β-catenin complexes (Fig. 4 A). To assess whether c-Src affects the interaction of MUC1 and β-cateninin vivo, MUC1-positive ZR-75-1 cells were transfected with pCMV or pCMV/c-Src. Anti-MUC1 immunoprecipitates prepared from the transfected cells were subjected to immunoblot analysis with anti-c-Src, anti-P-Tyr, and anti-β-catenin. The results demonstrate that c-Src associates with MUC1 in cells and induces tyrosine phosphorylation of MUC1 (Fig. 4 B, left panel). In addition, c-Src expression induced the interaction of MUC1 and β-catenin (Fig. 4 B, left panel). The finding that the MUC1 C-terminal subunit and not the large ectodomain is subject to tyrosine phosphorylation is consistent with an interaction between c-Src and MUC1/CD (Fig. 4 B, left panel). To confirm these findings, we performed immunoprecipitation studies with the anti-DF3-E antibody that was generated against the extracellular region of the C-terminal subunit. Immunoblot analysis of the precipitates demonstrated c-Src-mediated phosphorylation of the MUC1 C-terminal subunit and increased binding of MUC1/CD to β-catenin (Fig. 4 B, middle panel). By contrast, expression of a kinase-inactive c-Src(K295R) mutant resulted in less phosphorylation of MUC1 on tyrosine as compared with the control (Fig.4 B, middle panel). Moreover, expression of c-Src(K295R) was associated with a decreased interaction between MUC1/CD and β-catenin (Fig. 4 B, middle panel). To extend these findings, MUC1-negative 293 cells (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar) were transfected to express MUC1 or MUC1(Y46F) in which the CD YEKV site has been mutated to FEKV. There was a low but detectable level of MUC1 binding to endogenous c-Src (Fig. 4 C, left panel). Moreover, cotransfection of MUC1 and c-Src was associated with increased formation of MUC1-c-Src complexes (Fig. 4 C,middle panel). Cotransfection of MUC1 and c-Src was also associated with increased tyrosine phosphorylation of MUC1 and binding of MUC1 and c-Src (Fig. 4 C, middle panel). By contrast, cotransfection of MUC1(Y46F) and c-Src resulted in little binding of these proteins (Fig. 4 C, middle panel). Moreover, there was little if any tyrosine phosphorylation of MUC1(Y46F) (Fig. 4 C, middle panel). Importantly, cotransfection of MUC1 but not MUC1(Y46F) with c-Src induced the binding of MUC1 and β-catenin (Fig. 4 C,middle panel). These findings demonstrate that c-Src-mediated phosphorylation of the MUC1 YEKV site increases the interaction of MUC1 and β-catenin in cells. The present findings thus demonstrate that signaling of β-catenin and the MUC1 carcinoma-associated protein is regulated by the c-Src tyrosine kinase. Previous studies have shown that β-catenin interacts with the cytoplasmic domain of MUC1 and that GSK3β inhibits the formation of MUC1/CD-β-catenin complexes (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). By contrast, the present work supports a model in which c-Src phosphorylates MUC1/CD and promotes the interaction of MUC1/CD and β-catenin. The c-Src kinase functions in signaling pathways activated by heterotrimeric G protein-coupled receptors (28Malarkey K. Belham C. Paul A. Graham A. Mclees A. Scott P. Plevin R. Biochem. J. 1995; 309: 361-375Crossref PubMed Scopus (266) Google Scholar) and neuronal ion channels (29Holmes T. Fadool D. Levitan I. J. Neurosci. 1996; 16: 581-590Crossref Google Scholar, 30Yu S. Yeh C. Sensi S. Gwag B. Canzoniero L. Farhangrazi Z. Ying H. Tian M. Dugan L. Choi D. Science. 1997; 278: 114-117Crossref PubMed Scopus (536) Google Scholar, 31van Hoek M. Allen C. Parsons S. Biochem. J. 1997; 326: 271-277Crossref PubMed Scopus (20) Google Scholar). c-Src also participates in the transduction of signals from the epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R) and other receptor tyrosine kinases (32Biscardi J. Tice D. Parsons S. Adv. Cancer Res. 1999; 76: 61-119Crossref PubMed Google Scholar). The available evidence indicates that c-Src phosphorylates the EGF-R and thereby contributes to mitogenesis and transformation (33Maa M. Leu T. McCarley D. Schatzman R. Parsons S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6981-6985Crossref PubMed Scopus (290) Google Scholar). Mitogenesis induced by PDGF is also positively regulated by c-Src-mediated phosphorylation of the PDGF-R (34Hansen K. Johnell M. A. C. U. C. C. L. EMBO J. 1996; 15: PubMed Scopus Google Scholar). Other of c-Src that and have with the actin cytoskeleton (32Biscardi J. Tice D. Parsons S. Adv. Cancer Res. 1999; 76: 61-119Crossref PubMed Google Scholar). These findings have provided support for the involvement of c-Src in the of cell and The present studies extend these findings by that the MUC1 carcinoma-associated is also a substrate for c-Src and that interaction of MUC1 with GSK3β and β-catenin are regulated by c-Src-dependent Brugge for and Rickles for and Hilkens for

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies. The mucin 1 (MUC1) oncoprotein is overexpressed in human carcinomas and, like NF-kappaB, blocks cell death and induces transformation. The present studies show that MUC1 constitutively associates with NF-kappaB p65 in carcinoma cells. The MUC1 COOH-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-kappaB p65 and, importantly, blocks the interaction between NF-kappaB p65 and its inhibitor IkappaBalpha. We show that NF-kappaB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-kappaB-mediated transcriptional activation. Studies in nonmalignant epithelial cells show that MUC1-C interacts with NF-kappaB in the response to tumor necrosis factor-alpha stimulation. Moreover, tumor necrosis factor-alpha induces the recruitment of NF-kappaB p65-MUC1-C complexes to NF-kappaB target genes, including the promoter of the MUC1 gene itself. We also show that an inhibitor of MUC1-C oligomerization blocks the interaction with NF-kappaB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-kappaB p65 promoter occupancy and expression of NF-kappaB target genes. These findings indicate that MUC1-C is a direct activator of NF-kappaB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-kappaB pathway.

The DF3/MUC1 transmembrane oncoprotein is aberrantly overexpressed by most human carcinomas. Certain insights are available regarding a role for MUC1 in intracellular signaling; however, no precise function has been ascribed to this molecule. The present results demonstrate that MUC1 expression is up-regulated by oxidative stress and that this response is mediated by activation of MUC1 gene transcription. A role for MUC1 in the oxidative stress response is supported by the demonstration that MUC1 expression is associated with attenuation of endogenous and H2O2-induced intracellular levels of reactive oxygen species (ROS). MUC1-dependent regulation of ROS is mediated at least in part by up-regulation of anti-oxidant enzyme (superoxide dismutase, catalase, and glutathione peroxidase) expression. In concert with these findings, we show that the apoptotic response to oxidative stress is attenuated by a MUC1-dependent mechanism. These results support a model in which activation of MUC1 by oxidative stress provides a protective function against increased intracellular oxidant levels and ROS-induced apoptosis. The DF3/MUC1 transmembrane oncoprotein is aberrantly overexpressed by most human carcinomas. Certain insights are available regarding a role for MUC1 in intracellular signaling; however, no precise function has been ascribed to this molecule. The present results demonstrate that MUC1 expression is up-regulated by oxidative stress and that this response is mediated by activation of MUC1 gene transcription. A role for MUC1 in the oxidative stress response is supported by the demonstration that MUC1 expression is associated with attenuation of endogenous and H2O2-induced intracellular levels of reactive oxygen species (ROS). MUC1-dependent regulation of ROS is mediated at least in part by up-regulation of anti-oxidant enzyme (superoxide dismutase, catalase, and glutathione peroxidase) expression. In concert with these findings, we show that the apoptotic response to oxidative stress is attenuated by a MUC1-dependent mechanism. These results support a model in which activation of MUC1 by oxidative stress provides a protective function against increased intracellular oxidant levels and ROS-induced apoptosis. The human DF3/MUC1 mucin-like transmembrane is normally expressed on the apical borders of secretory epithelial cells (1Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (499) Google Scholar). In carcinoma cells, polarization of MUC1 is lost with high levels of expression over the entire cell surface (1Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (499) Google Scholar). Estimates indicate that over 70% of newly diagnosed cancers aberrantly express MUC1 (2Greenlee R.T. Murray T. Bolden S. Wingo P.A. CA-Cancer J. Clin. 2000; 50: 7-33Crossref PubMed Scopus (3968) Google Scholar). The MUC1 proteins consist of an N-terminal ectodomain with variable numbers of 20-amino acid tandem repeats that are extensively modified with O-linked glycans (3Gendler S. Taylor-Papadimitriou J. Duhig T. Rothbard J. Burchell J.A. J. Biol. Chem. 1988; 263: 12820-12823Abstract Full Text PDF PubMed Google Scholar, 4Siddiqui J. Abe M. Hayes D. Shani E. Yunis E. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2320-2323Crossref PubMed Scopus (280) Google Scholar). The C-terminal region includes a transmembrane domain and a 72-amino acid cytoplasmic tail. Following proteolytic cleavage, the >250-kDa ectodomain remains associated with the ∼25-kDa C-terminal subunit at the cell surface. β-Catenin, a component of the adherens junction of mammalian cells, interacts directly with the MUC1 intracellular region (5Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Other studies have shown that phosphorylation of MUC1 by glycogen synthase 3β, c-Src, or the epidermal growth factor receptor contributes to regulation of the interaction between MUC1 and β-catenin (6Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (224) Google Scholar, 7Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 8Li Y. Ren J. Yu W.-H. Li G. Kuwahara H. Yin L. Carraway K.L. Kufe D. J. Biol. Chem. 2001; 276: 35239-35242Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). More recent work has demonstrated that MUC1 colocalizes with β-catenin in the nucleus and that MUC1 induces transformation (9Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 187-193Crossref PubMed Scopus (70) Google Scholar, 10Li Y. Liu D. Chen D. Kharbanda S. Kufe D. Oncogene. 2003; 22: 6107-6110Crossref PubMed Scopus (171) Google Scholar). Normal cellular metabolism is associated with the production of reactive oxygen species (ROS). 1The abbreviations used are: ROS, reactive oxygen species; RT-PCR, reverse transcription PCR; Luc, luciferase; SOD, superoxide dismutase; GPx, glutathione peroxidase; HE, hydroethidine; DCF, dichlorodihydrofluorescein; DCFH-AM, 5-(and -6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate.1The abbreviations used are: ROS, reactive oxygen species; RT-PCR, reverse transcription PCR; Luc, luciferase; SOD, superoxide dismutase; GPx, glutathione peroxidase; HE, hydroethidine; DCF, dichlorodihydrofluorescein; DCFH-AM, 5-(and -6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. Common forms of ROS include superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radicals, and nitric oxide. Mitogenic signals induced by certain growth factors and activated Ras are mediated by ROS production (11Sundaresan M. Yu Z.-X. Ferrans V. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2311) Google Scholar, 12Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1431) Google Scholar). Under nonphysiologic conditions, increases in ROS levels above the reducing capacity of the cell can cause damage to DNA, proteins, and lipids (13Croteau D. Bohr V. J. Biol. Chem. 1997; 272: 25409-25412Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 14Berlett S. Stadtman E. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2783) Google Scholar). To prevent damage associated with increases in ROS, aerobic cells have developed enzymatic (superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)) and non-enzymatic (glutathione and thioredoxin) defense mechanisms to balance the reduction-oxidation (redox) state (15Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (997) Google Scholar). In the absence of an adequate defense, cells respond to oxidative stress with the induction of apoptosis (14Berlett S. Stadtman E. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2783) Google Scholar). Although few insights are available regarding mechanisms responsible for ROS-induced cell death, H2O2 has been shown to activate topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (16Li T. Chen A. Yu C. Mao Y. Wang H. Liu L. Genes & Dev. 1999; 13: 1553-1560Crossref PubMed Scopus (148) Google Scholar). The p66 shc adaptor protein (17Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1472) Google Scholar, 18Nemoto S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (736) Google Scholar) and the p85 subunit of phosphatidylinositol 3-kinase (19Yin Y. Terauchi Y. Solomon G. Aizawa S. Rangarajan P. Yazaki Y. Kadowaki T. Barrett J. Nature. 1998; 391: 707-710Crossref PubMed Scopus (151) Google Scholar) have also been implicated in the apoptotic response to H2O2. The present studies demonstrate that MUC1 expression is activated by oxidative stress. The results also demonstrate that MUC1 regulates intracellular oxidant levels and attenuates the apoptotic response to oxidative stress. Cell Culture—Human breast (MCF-7, ZR-75-1), colon (HCT116), and cervical (HeLa) carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). MCF-7, HCT116, and HeLa cells were grown in Dulbecco's modified Eagle's medium (high glucose; Cellgro) supplemented with 10% heat-inactivated fetal calf serum, and 2 mm l-glutamine. ZR-75-1 cells were cultured in RPMI 1640 medium (Cellgro) supplemented with 10% fetal calf serum and 2 mm l-glutamine. Cells were treated with H2O2 (Sigma). Immunoblot Analysis—Cells were lysed in ice-cold lysis buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) for 30 min. Lysates were cleared by centrifugation for 20 min at 4 °C as described (20Yin L. Ohno T. Weichselbaum R. Kharbanda S. Kufe D. Mol. Cancer Ther. 2001; 1: 43-48PubMed Google Scholar). Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-DF3/MUC1 (1Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (499) Google Scholar), anti-SOD1 (Santa Cruz Biotechnology), anti-SOD2 (Upstate Biotechnology, Inc.), anti-catalase (Sigma), anti-GPx (MBL Medical and Biological Laboratories) or anti-β-actin (Sigma). The antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL, Amersham Life Sciences). Reverse Transcription Polymerase Chain Reaction (RT-PCR)—Total cellular RNA was extracted in Trizol, dissolved in RNase-free water, and incubated for 10 min at 55 °C. MUC1-specific primers (5′-TCTACTCTGGTGCACAACGG-3′ and 5′-TTATATCGAGAGGCTGCTTCC-5′) were designed to span a region within genomic DNA that contains two introns, resulting in the amplification of a 489-bp fragment from RNA and a 738-bp fragment from genomic DNA. RNA-specific primers for human β-actin were used as a control. The RNA was reverse transcribed and amplified using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen). Amplified fragments were analyzed by electrophoresis in 2% agarose gels. Luciferase Reporter Assays—A fragment spanning the region from –1464 to +24 of the human MUC1 gene (21Gaemers I. Vos H. Volders H. van der Valk S. Hilkens J. J. Biol. Chem. 2001; 276: 6191-6199Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) was ligated in the KpnI and BglII sites of the firefly luciferase pGl3-Basic vector (Promega). The resulting plasmid was designated pMUC1-Luc. Cells were transfected with a mixture of pMUC1-Luc and SV40-Renilla Luc (5:1) constructs (Promega) in the presence of LipofectAMINE for 14 h. After washing and incubation for an additional 24 h, the cells were treated with H2O2 and then lysed in Passive Lysis Buffer (Promega). Lysates were analyzed for firefly and Renilla luciferase activities using the Dual Luciferase Reagent Assay Kit (Promega). Luminescence was measured in a luminometer. Stable Transfectants—HCT116 and HeLa cells were transfected with pIRESpuro2 or pIRESpuro2-MUC1 as described (8Li Y. Ren J. Yu W.-H. Li G. Kuwahara H. Yin L. Carraway K.L. Kufe D. J. Biol. Chem. 2001; 276: 35239-35242Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and selected in the presence of 0.4 μg/ml puromycin (Calbiochem-Novabiochem). Measurement of ROS Levels—Cells were incubated with 10 μm DCFH-AM (Molecular Probes) for 30 min at 37 °C to assess H2O2-mediated oxidation to the fluorescent compound DCF (22LeBel C. Ischiropoulos H. Bondy S. Chem. Res. Toxicol. 1992; 5: 227-231Crossref PubMed Scopus (2231) Google Scholar). Fluorescence of oxidized DCF was measured at an excitation wavelength of 480 nm and an emission wavelength of 525 nm using a flow cytometer (BD Biosciences). For the assessment of superoxide (O2-) levels, cells were incubated with 10 μm hydroethidine (HE) (Polyscience Inc.) for 20 min at 37 °C. O2--mediated conversion of HE to ethidium was measured by excitation at 470 nm and emission at 590 nm (23Benov L. Sztejnberg L. Fridovich I. Free Radic. Biol. Med. 1998; 25: 826-831Crossref PubMed Scopus (426) Google Scholar). Apoptosis Assays—Sub-G1 DNA content was assessed by staining ethanol-fixed and citrate buffer-permeabilized cells with propidium iodide and monitoring by flow cytometry (BD Biosciences). Chromatin condensation was assessed by staining cells with ethidium bromide and counting the number of cells with bright orange areas in their nuclei as described (24McGahon A.J. Martin S.J. Bissonnette R.P. Mahboubi A. Shi Y. Mogil R.J. Nishioka W.K. Green D.R. Methods Cell Biol. 1995; 46: 153-185Crossref PubMed Scopus (520) Google Scholar). Up-regulation of MUC1 Protein by Oxidative Stress—To assess the effects of oxidative stress on MUC1 expression, human MUC1-positive MCF-7 cells were exposed to 0.4 mm H2O2 as a of Lysates of the cells were analyzed by with The results demonstrate that MUC1 levels at min of H2O2 MUC1 expression was up-regulated min and then at min of H2O2 a of the with anti-β-actin demonstrated of the To these findings, MCF-7 cells were treated with of H2O2 for 30 min. The results show that over a of to increases in MUC1 expression were at mm and at mm H2O2 of human MUC1-positive ZR-75-1 cells with H2O2 was also associated with increases in MUC1 expression The however, from that in MCF-7 cells with increases at 2 and to levels at studies with cells demonstrated no induction of MUC1 expression in response to H2O2 These indicate that MUC1-positive cells respond to oxidative stress with increases in MUC1 expression. Oxidative MUC1 activation of MUC1 transcription contributes to up-regulation of MUC1 protein in the oxidative stress MUC1 levels were by of MCF-7 cells with H2O2 was associated with increases in MUC1 at min in concert with regulation at the protein MUC1 levels were increased min and then at min a was of H2O2 on β-actin levels of ZR-75-1 cells with H2O2 was also associated with increases in MUC1 The in MUC1 was at 1 of H2O2 and was in the absence of in β-actin levels To assess the effects of H2O2 on MUC1 gene MCF-7 cells were transfected to express a MUC1 and SV40-Renilla Luc with H2O2 was associated with an in and luciferase which was at min In ZR-75-1 cells transfected with pMUC1-Luc and treated with induction of firefly luciferase was at 1 These demonstrate that H2O2 MUC1 gene transcription and thereby increases MUC1 and protein MUC1 ROS assess the role of MUC1 in response to oxidative cells were transfected to express the vector or MUC1 of MUC1 in two of was that in MCF-7 cells and HeLa cells, which express MUC1 (6Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (224) Google Scholar), were transfected to express MUC1 at levels of the by flow cytometry demonstrated that MUC1 is expressed on the cell surface The HeLa cells transfected with the MUC1 vector also demonstrated an in cell surface MUC1 expression These indicate endogenous transfected MUC1 is expressed as a transmembrane To MUC1 ROS levels, cells were incubated with DCFH-AM, and H2O2-mediated oxidation of the was by flow The results demonstrate with cells the MUC1-positive cells H2O2 levels increased expression of MUC1 in HeLa cells in in H2O2 levels To this cells were exposed to H2O2 and then for oxidation of with cells, which increases in H2O2 levels, expression of MUC1 was associated with attenuation of this response The cells, which express endogenous a in H2O2 levels with cells HeLa cells transfected to express increased MUC1 levels an attenuated response to H2O2 These demonstrate that MUC1 expression is associated with of endogenous and induced intracellular H2O2 of cells with H2O2 is associated with and thereby the of superoxide (O2-) M. K. T. D. G. J. Immunol. 2002; PubMed Scopus Google Scholar). To assess the effects of MUC1 on levels, the cell were incubated with HE and then by flow The results demonstrate that levels of cells with H2O2 this response to H2O2 was attenuated in cells of HE oxidation at that MUC1 expression in and cells is associated with levels as with that in cells of cells with H2O2 also in increased HE this response was attenuated in cells These were at in the cells with the DCF the results indicate that MUC1 expression attenuates H2O2-induced increases in intracellular oxidant MUC1 of enzymatic mechanisms that intracellular oxidant levels are mediated by SOD, catalase, and J. E. Free Radic. Biol. Med. 2001; PubMed Scopus Google Scholar). To MUC1 expression of these anti-oxidant from the were to with anti-SOD1 and and The results of a show with cells, and levels were increased to in the MUC1 MUC1 expression was also associated with a in levels levels were increased in the as with cells for β-actin demonstrated of the expression of MUC1 in HeLa cells was also associated with increases in catalase, and levels These demonstrate that MUC1 expression is associated with increases in anti-oxidant enzyme MUC1 the to Oxidative Stress—To MUC1 regulates the response to oxidative and cells were for induction of apoptotic cells with DNA. The results demonstrate that H2O2-induced apoptosis is attenuated in MUC1-positive as with cells A and The apoptotic response to H2O2 was also attenuated by increased expression of MUC1 in HeLa cells and of the induction of ethidium bromide staining of A and and cells and demonstrated bright orange areas of in which apoptotic from was ethidium bromide staining of cells or MUC1 cells These demonstrate that MUC1 expression is associated with an attenuated apoptotic response to oxidative attenuates the apoptotic response to oxidative stress. and and HeLa and cells the vector or MUC1 were treated with mm H2O2 for h. The cells were with ethidium bromide to assess A and condensation as by the presence of bright orange areas in the nuclei apoptotic from and the results are expressed as the of apoptotic cells of for and of MUC1 in to Oxidative are to function in the of epithelial and the transmembrane that are at the cell surface a protective The transmembrane also function in the presence of in the MUC1 is expressed at the cell surface as a of the >250-kDa N-terminal ectodomain and the ∼25-kDa transmembrane C-terminal The of the MUC1 ectodomain and the resulting that the contributes to the of the ectodomain also to The available however, provides few insights the function of MUC1 in The present results indicate that MUC1 is in the response of cells to oxidative stress. a of in the ROS can damage DNA, proteins, and lipids (13Croteau D. Bohr V. J. Biol. Chem. 1997; 272: 25409-25412Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 14Berlett S. Stadtman E. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2783) Google Scholar). the presence of ROS-induced damage can in the activation of cell mechanisms (14Berlett S. Stadtman E. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2783) Google Scholar). results demonstrate that MUC1 expression is activated by of cells to H2O2 as a of by of a luciferase of the MUC1 ROS transcription of the MUC1 In concert with this ROS was also associated with increases in MUC1 and MUC1 of MUC1 expression was in response to ROS and to levels within on the cell These indicate that carcinoma cells respond to oxidative stress with a activation of MUC1 expression. MUC1 of MUC1 in the oxidative stress response the activation of to of the function of MUC1 as an intracellular molecule. To the role of we expressed MUC1 in carcinoma cells that or (HeLa) MUC1 of the oxidation of DCFH-AM to the that MUC1 endogenous intracellular H2O2 increases in H2O2 levels in response to H2O2 were attenuated in MUC1 of the and HeLa In concert with the demonstration that increases in H2O2 levels cause and the of M. K. T. D. G. J. Immunol. 2002; PubMed Scopus Google Scholar), we that oxidation of HE is increased in MUC1 expression was associated with the attenuation of H2O2-induced H2O2 is cell expression of the MUC1 ectodomain to intracellular ROS is that the transmembrane contributes to a that regulates levels of MUC1 of present results demonstrate that MUC1 increases expression of the anti-oxidant that intracellular H2O2 In mammalian cells, is to H2O2 by the in the and the in in and were in the MUC1 H2O2 is to and in by the P. A. G. R. I. P. PubMed Scopus Google Scholar), H2O2 to in a that glutathione to The present results demonstrate that MUC1 expression is also associated with increases in and is that increased expression of these anti-oxidant in MUC1-positive cells at least in to the attenuation of endogenous and H2O2-induced oxidant The present studies the that MUC1 regulates or the non-enzymatic mechanisms in the signals that expression of SOD, catalase, and as enzymatic of the ROS response are is the mechanisms that intracellular ROS studies have shown that p66 shc regulates oxidant levels in mammalian cells S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (736) Google Scholar, M. Giorgio M. A. S. A. E. E. M. V. S. P. Lanfrancone L. Pelicci P.G. Oncogene. 2002; PubMed Scopus Google Scholar). In the protein increases H2O2 and to oxidative stress by expression S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (736) Google Scholar). Other work has demonstrated that the and are activated by oxidative stress and that these proteins intracellular oxidant levels P. H. S. Weichselbaum R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, C. Ren Kharbanda S. A.J. K. Kufe D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, C. Y. Kufe D. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). to MUC1 interacts with the p66 shc or MUC1 by a is normally expressed at the apical borders of epithelial cells (1Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (499) Google Scholar). the polarization of MUC1 expression is lost in carcinoma cells that aberrantly the protein in the and over the entire cell surface (1Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (499) Google Scholar, J. J. P. J. A. van der Valk M. J. 1984; PubMed Scopus Google Scholar). MUC1 is also expressed in the nucleus in a with β-catenin (9Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 187-193Crossref PubMed Scopus (70) Google Scholar, 10Li Y. Liu D. Chen D. Kharbanda S. Kufe D. Oncogene. 2003; 22: 6107-6110Crossref PubMed Scopus (171) Google Scholar) or Y. Yu W.-H. Ren J. L. Chen W. Kharbanda S. M. Kufe D. Mol. Cancer Res. 2003; 1: Google Scholar). on the present of MUC1 the apical borders of the a defense against ROS for carcinoma cells have this by MUC1 to a of oxidative or forms of stress. In this the present studies show that MUC1 to oxidative stress. results support a model in which expression of MUC1 by carcinoma cells oxidant levels and thereby attenuates the apoptotic response to oxidative stress. MUC1 ROS-induced apoptosis by a in to or of effects on oxidant MUC1 also to by the response to oxidative and of stress. In this of MUC1 is to transformation as assessed by growth and Y. Liu D. Chen D. Kharbanda S. Kufe D. Oncogene. 2003; 22: 6107-6110Crossref PubMed Scopus (171) Google Scholar). the to MUC1 expression by carcinoma cells in to human MUC1-positive is The present findings, however, the that a protective function of a to regulation of intracellular oxidant levels and the apoptotic stress the of