Yongqing Li

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

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts directly with beta-catenin, a component of the adherens junction of mammalian epithelial cells. The present results demonstrate that MUC1 associates with protein kinase Cdelta (PKCdelta). A TDR sequence adjacent to the beta-catenin binding motif in the MUC1 cytoplasmic domain functions as a site for PKCdelta phosphorylation. We show that phosphorylation of MUC1 by PKCdelta increases binding of MUC1 and beta-catenin in vitro and in vivo. The functional significance of the MUC1-PKCdelta interaction is further supported by the demonstration that mutation of the PKCdelta phosphorylation site abrogates MUC1-mediated decreases in binding of beta-catenin to E-cadherin. We also show that the stimulatory effects of MUC1 on anchorage-independent growth are abrogated by mutation of the PKCdelta phosphorylation site. These findings support a novel role for PKCdelta in regulating the interaction between MUC1 and the beta-catenin signaling pathway.

Here we identify the BAP1 and BAP2 genes of Arabidopsis (Arabidopsis thaliana) as general inhibitors of programmed cell death (PCD) across the kingdoms. These two homologous genes encode small proteins containing a calcium-dependent phospholipid-binding C2 domain. BAP1 and its functional partner BON1 have been shown to negatively regulate defense responses and a disease resistance gene SNC1. Genetic studies here reveal an overlapping function of the BAP1 and BAP2 genes in cell death control. The loss of BAP2 function induces accelerated hypersensitive responses but does not compromise plant growth or confer enhanced resistance to virulent bacterial or oomycete pathogens. The loss of both BAP1 and BAP2 confers seedling lethality mediated by PAD4 and EDS1, two regulators of cell death and defense responses. Overexpression of BAP1 or BAP2 with their partner BON1 inhibits PCD induced by pathogens, the proapoptotic gene BAX, and superoxide-generating paraquat in Arabidopsis or Nicotiana benthamiana. Moreover, expressing BAP1 or BAP2 in yeast (Saccharomyces cerevisiae) alleviates cell death induced by hydrogen peroxide. Thus, the BAP genes function as general negative regulators of PCD induced by biotic and abiotic stimuli including reactive oxygen species. The dual roles of BAP and BON genes in repressing defense responses mediated by disease resistance genes and in inhibiting general PCD has implications in understanding the evolution of plant innate immunity.

The Arabidopsis BAP1 gene encodes a small protein with a C2-like domain. Here we show that the BAP1 protein is capable of binding to phospholipids in a calcium-dependent manner and is associated with membranes in vivo. We identify multiple roles of BAP1 in negatively regulating defense responses and cell death in Arabidopsis thaliana. The loss of BAP1 function confers an enhanced disease resistance to virulent bacterial and oomycete pathogens. The enhanced resistance is mediated by salicylic acid, PAD4 and a disease resistance gene SNC1. BAP1 is also involved in the control of cell death, which is suggested by an altered hypersensitive response to an avirulent bacterial pathogen in the bap1 loss-of-function mutant. BAP1 overexpression leads to an enhanced susceptibility to a virulent oomycete, suggesting a role for BAP1 in basal defense response. Furthermore, the BAP1 protein probably functions together with an evolutionarily conserved C2 domain protein BON1/CPN1 to negatively regulate defense responses in plants.