Human MUC1 Carcinoma Antigen Regulates Intracellular Oxidant Levels and the Apoptotic Response to Oxidative Stress*

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 H 2 O 2 -induced intracel- lular 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 (1). In carcinoma cells, polarization of MUC1 is lost with high levels of expression over the entire cell surface (1). Esti-mates indicate that over 70% of newly diagnosed cancers aberrantly

The human DF3/MUC1 mucin-like transmembrane is normally expressed on the apical borders of secretory epithelial cells (1). In carcinoma cells, polarization of MUC1 is lost with high levels of expression over the entire cell surface (1). Estimates indicate that over 70% of newly diagnosed cancers aberrantly express MUC1 (2). The MUC1 proteins consist of an N-terminal ectodomain with variable numbers of 20-amino acid tandem repeats that are extensively modified with Olinked glycans (3,4). The C-terminal region includes a transmembrane domain and a 72-amino acid cytoplasmic tail. Following proteolytic cleavage, the Ͼ250-kDa ectodomain remains associated with the ϳ25-kDa C-terminal subunit at the cell surface. ␤-Catenin, a component of the adherens junction of mammalian cells, interacts directly with the MUC1 intracellular region (5). Other studies have shown that phosphorylation of MUC1 by glycogen synthase 3␤, c-Src, or the epidermal growth factor receptor contributes to regulation of the interaction between MUC1 and ␤-catenin (6 -8). More recent work has demonstrated that MUC1 colocalizes with ␤-catenin in the nucleus and that MUC1 induces transformation (9,10).
Normal cellular metabolism is associated with the production of reactive oxygen species (ROS). 1 Common forms of ROS include superoxide (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals, and nitric oxide. Mitogenic signals induced by certain growth factors and activated Ras are mediated by ROS production (11,12). Under nonphysiologic conditions, increases in ROS levels above the reducing capacity of the cell can cause damage to DNA, proteins, and lipids (13,14). 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 reductionoxidation (redox) state (15). In the absence of an adequate defense, cells respond to oxidative stress with the induction of apoptosis (14). Although few insights are available regarding mechanisms responsible for ROS-induced cell death, H 2 O 2 has been shown to activate topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (16). The p66 shc adaptor protein (17,18) and the p85 subunit of phosphatidylinositol 3-kinase (19) have also been implicated in the apoptotic response to H 2 O 2 .
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.
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 (5Ј-TCTAC-TCTGGTGCACAACGG-3Ј and 5Ј-TTATATCGAGAGGCTGCTTCC-5Ј) 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 ␤-actin were used as a control. The RNA was reverse transcribed and amplified using SuperScript One-Step RT-PCR with Plat-inum Taq (Invitrogen). Amplified fragments were analyzed by electrophoresis in 2% agarose gels.
Luciferase Reporter Assays-A fragment spanning the region from Ϫ1464 to ϩ24 of the human MUC1 gene (21) 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 H 2 O 2 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.
Measurement of ROS Levels-Cells were incubated with 10 M DCFH-AM (Molecular Probes) for 30 min at 37°C to assess H 2 O 2mediated oxidation to the fluorescent compound DCF (22). 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 (O 2 Ϫ ) levels, cells were incubated with 10 M hydroethidine (HE) (Polyscience Inc.) for 20 min at 37°C. O 2 Ϫ -mediated conversion of HE to ethidium was measured by excitation at 470 nm and emission at 590 nm (23).
Apoptosis Assays-Sub-G 1 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 (24).  (Fig. 1C). The kinetics, however, differed somewhat from that found in MCF-7 cells with maximal increases at 2 h and down-regulation to below base-line levels at 6 h (Fig. 1C). By contrast, similar studies with MUC1negative HCT116 cells demonstrated no detectable induction of MUC1 expression in response to H 2 O 2 treatment (data not shown). These findings indicate that MUC1-positive cells respond to oxidative stress with increases in MUC1 expression.

Up-regulation of MUC1 Protein by Oxidative Stress-To
Oxidative Stress Induces MUC1 Transcription-To determine whether activation of MUC1 transcription contributes to up-regulation of MUC1 protein in the oxidative stress response, MUC1 mRNA levels were quantitated by RT-PCR. Treatment of MCF-7 cells with H 2 O 2 was associated with increases in MUC1 transcripts at 15 min ( Fig. 2A). Moreover, in concert with regulation at the protein level, MUC1 mRNA levels were increased through 45 min and then declined at 60 min ( Fig. 2A). As a control, there was little effect of H 2 O 2 on ␤-actin mRNA levels ( Fig. 2A). Treatment of ZR-75-1 cells with H 2 O 2 was also associated with increases in MUC1 transcripts (Fig. 2B). The increase in MUC1 transcripts was maximal at 1 h of H 2 O 2 exposure and was detectable in the absence of changes in ␤-actin mRNA levels (Fig. 2B). To assess the effects of H 2 O 2 on MUC1 gene transcription, MCF-7 cells were transfected to express a MUC1 promoter-Luc reporter and SV40-Renilla Luc constructs. Treatment with H 2 O 2 was associated with an increase in firefly, and not Renilla, luciferase activity, which was maximal at 45 min (Fig. 2C). In ZR-75-1 cells transfected with pMUC1-Luc and treated with H 2 O 2 , induction of firefly luciferase activity was maximal at 1 h (Fig. 2D). These findings demonstrate that H 2 O 2 activates MUC1 gene transcription and thereby increases MUC1 mRNA and protein levels.
MUC1 Regulates ROS Levels-To assess the role of MUC1 in response to oxidative stress, MUC1-negative HCT116 cells were transfected to stably express the empty vector or MUC1 (Fig. 3A). Expression of MUC1 in two separate isolates of stable HCT116 transfectants was somewhat lower than that found in MCF-7 cells ( Fig. 3A and data not shown). HeLa cells, which constitutively express MUC1 (6), were stably transfected to express MUC1 at relatively higher levels (Fig. 3A). Analysis of the HCT116 transfectants by flow cytometry demonstrated that MUC1 is expressed on the cell surface (Fig. 3B). The HeLa cells stably transfected with the MUC1 vector also demonstrated an increase in cell surface MUC1 expression (Fig. 3B). These findings indicate that, like endogenous MUC1, transfected MUC1 is expressed as a transmembrane glycoprotein.
To determine whether MUC1 affects ROS levels, cells were incubated with DCFH-AM, and H 2 O 2 -mediated oxidation of the fluorochrome was assayed by flow cytometry. The results demonstrate that, compared with HCT116 cells expressing the empty vector, MUC1-positive HCT116 cells exhibited substantially lower H 2 O 2 levels (Fig. 4A). Moreover, increased expression of MUC1 in HeLa cells resulted in marked decreases in H 2 O 2 levels (Fig. 4B). To extend this analysis, HCT116 cells were exposed to H 2 O 2 and then assayed for oxidation of DCFH-AM. Compared with HCT116/vector cells, which exhibited substantial increases in H 2 O 2 levels, expression of MUC1 was associated with attenuation of this response (Fig. 4C). The electrophoresis. C and D, MCF-7 (C) and ZR-75-1 (D) cells were transfected with pMUC1-Luc and SV40-Renilla luciferase and then exposed to 0.4 mM H 2 O 2 for the indicated times. Lysates were analyzed for firefly and Renilla luciferase activities. The results are expressed as the mean Ϯ S.D. of three separate experiments, each performed in triplicate, in which the ratio of firefly to Renilla luciferase activities is relative to that of control cells. HeLa/vector cells, which express endogenous MUC1, exhibited a less pronounced increase in H 2 O 2 levels compared with HCT116/vector cells (Fig. 4D). Moreover, HeLa cells transfected to express increased MUC1 levels showed an attenuated response to H 2 O 2 treatment (Fig. 4D). These findings demonstrate that MUC1 expression is associated with down-regulation of endogenous and induced intracellular H 2 O 2 levels.
Treatment of cells with H 2 O 2 is associated with mitochondrial dysfunction and thereby the generation of superoxide radicals (O 2 Ϫ ) (25). To assess the effects of MUC1 on O 2 Ϫ levels, the HCT116 cell transfectants were incubated with HE and then assayed by flow cytometry. The results demonstrate that O 2 Ϫ levels increase substantially after treatment of HCT116/ vector cells with H 2 O 2 (Fig. 5A). By contrast, this response to H 2 O 2 treatment was attenuated in HCT116/MUC1-A cells (Fig.  5A). Analysis of HE oxidation at different time points confirmed that MUC1 expression in HCT116/MUC1-A and HCT116/MUC1-B cells is associated with decreased O 2 Ϫ levels as compared with that in HCT116/vector cells (Fig. 5B). Treatment of HeLa/vector cells with H 2 O 2 also resulted in increased HE oxidation; this response was attenuated in HeLa/MUC1-A cells (Fig. 5C). These findings were confirmed at different time points in the HeLa/MUC1-B cells (Fig. 5D). Taken together with the DCF data, the results indicate that MUC1 expression attenuates H 2 O 2 -induced increases in intracellular oxidant levels. experiment show that, compared with HCT116/vector cells, SOD1 and SOD2 levels were increased up to 2.7-fold in the MUC1 transfectants (Fig. 6A). MUC1 expression was also associated with a 1.6 -2.2-fold increase in catalase levels (Fig.  6A). Notably, GPx levels were increased 6 -8-fold in the HCT116/MUC1 as compared with HCT116/vector cells (Fig.  6A). Immunoblotting for ␤-actin demonstrated equal loading of the lanes (Fig. 6A). Increased expression of MUC1 in HeLa cells was also associated with similar increases in SOD1, SOD2, catalase, and GPx levels (Fig. 6B). These findings demonstrate that MUC1 expression is associated with increases in antioxidant enzyme levels.

MUC1 Increases Expression of Anti-oxidant Enzymes-The
MUC1 Inhibits the Apoptotic Response to Oxidative Stress-To determine whether MUC1 regulates the response to oxidative stress, H 2 O 2 -treated HCT116/vector and HCT116/ MUC1 cells were assayed for induction of apoptotic cells with sub-G 1 DNA. The results demonstrate that H 2 O 2 -induced apoptosis is significantly attenuated in MUC1-positive as compared with MUC1-negative HCT116 cells (Fig. 7, A and B). The apoptotic response to H 2 O 2 was also attenuated by increased expression of MUC1 in HeLa cells (Fig. 7, C and D). As confirmation of the induction of apoptosis, ethidium bromide staining of H 2 O 2 -treated HCT116/vector (Fig. 8, A and B) and HeLa/vector cells (Fig. 8, C and D) further demonstrated bright orange areas of condensed chromatin in nuclei, which distinguishes late apoptotic from necrotic cells. Notably, there was little if any detectable ethidium bromide staining of untreated control cells or H 2 O 2 -treated MUC1 expressing cells (Fig. 8). These findings  7. MUC1 attenuates induction of cells with sub-G 1 DNA by oxidative stress. HCT116 (A and B) and collectively demonstrate that MUC1 expression is associated with an attenuated apoptotic response to oxidative stress.

DISCUSSION
Activation of MUC1 in Response to Oxidative Stress-The heavily glycosylated mucins are believed to function in the protection of epithelial surfaces. Secreted mucins and the transmembrane mucins that are tethered at the cell surface form a protective mucous barrier. The transmembrane mucins may also function in signaling the presence of adverse conditions in the extracellular environment. MUC1 is expressed at the cell surface as a heterodimer of the Ͼ250-kDa glycosylated Nterminal ectodomain and the ϳ25-kDa transmembrane Cterminal subunit. The extensive O-glycosylation of the MUC1 ectodomain and the resulting rod-like structure that extends beyond the glycocalyx probably contributes to the mucous barrier. Shedding of the ectodomain may also contribute to mucous formation. The available information, however, provides few if any insights into the function of MUC1 in stress-induced signaling mechanisms.
The present results indicate that MUC1 is involved in the response of cells to oxidative stress. As a consequence of prooxidant conditions in the extracellular milieu, ROS can damage DNA, RNA, proteins, and lipids (13,14). Moreover, the presence of excessive ROS-induced damage can result in the activation of cell death mechanisms (14 (27), whereas GPx converts H 2 O 2 to H 2 O in a reaction that oxidizes glutathione to its disulfide form. The present results demonstrate that MUC1 expression is also associated with increases in catalase and GPx levels. Thus, it is likely that increased expression of these anti-oxidant enzymes in MUC1-positive cells contributes, at least in part, to the attenuation of endogenous and H 2 O 2induced oxidant levels.
The present studies do not exclude the possibility that MUC1 regulates other enzymes or the non-enzymatic mechanisms involved in maintaining redox balance. Moreover, the specific MUC1-mediated signals that regulate expression of SOD, catalase, and GPx as major enzymatic effectors of the ROS response are presently not known. Indeed, little is known about the signaling mechanisms that control intracellular ROS levels. Recent studies have shown that p66 shc regulates oxidant levels in mammalian cells (18,28). In addition, the forkhead FKHRL1 protein increases H 2 O 2 scavenging and resistance to oxidative stress by increasing catalase expression (18). Other work has demonstrated that the c-Abl and Arg tyrosine kinases are activated by oxidative stress and that these proteins regulate intracellular oxidant levels (29 -31). Further experiments will be needed to determine whether MUC1 signaling interacts with the p66 shc , FKHRL1, or c-Abl/Arg pathways.
Does MUC1 Expression by Human Carcinomas Confer a Survival Advantage?-MUC1 is normally expressed at the apical borders of glandular epithelial cells (1). By contrast, the polarization of MUC1 expression is lost in carcinoma cells that aberrantly overexpress the protein in the cytoplasm and over the entire cell surface (1,32). MUC1 is also expressed in the nucleus in a complex with ␤-catenin (9, 10) or ␣-catenin (33). Based on the present results, positioning of MUC1 along the apical borders of the normal ductal epithelium could provide a defense against ROS generated, for example, during inflammatory conditions. Conversely, carcinoma cells may have exploited this mechanism by overexpressing MUC1 to achieve a survival advantage under conditions of oxidative or other forms of stress. In this context, the present studies show that MUC1 confers resistance to oxidative stress. Our results support a model in which expression of MUC1 by carcinoma cells decreases oxidant levels and thereby attenuates the apoptotic response to oxidative stress. Alternatively, MUC1 may attenuate ROS-induced apoptosis by a mechanism in addition to or independent of its effects on oxidant levels. MUC1 may also contribute to survival by blocking the necrotic response to oxidative and other types of stress. In this regard, overexpression of MUC1 is sufficient to confer transformation as assessed by anchorage-independent growth and tumorigenicity (10).
Whether the survival advantage attributable to MUC1 expression by carcinoma cells in vitro applies to human MUC1-positive tumors is not yet clear. The present findings, however, provide the first evidence that links a protective function of a mucin to regulation of intracellular oxidant levels and the apoptotic stress response.