MUC1 is expressed at the apical surface of ductal epithelia of tissues, including breast, pancreas, airway, and the gastrointestinal tract, where its functions include lubrication and protection of the epithelia. In addition, roles for MUC1 have been suggested in both adhesive and antiadhesive properties of tumor cells, and extensive O-glycosylation of the MUC1 tandem repeat domain may contribute to these functions. Little information is available on the specific O-glycosylation of MUC1. One problem in identifying different MUC1 glycoforms has been that monoclonal antibodies raised against the MUC1 core protein recognize epitopes in the tandem repeat domain, which is often glycosylated to an extent that obscures these epitopes. We developed an epitope-tagged form of MUC1 that allowed the detection of multiple MUC1 glycoforms and established the presence of a number of important blood group and tumorassociated carbohydrate antigens on MUC1 expressed by two pancreatic tumor cell lines (Panc-1 and S2-013) and two colon tumor cell lines (Caco-2 and HT-29). Antigens detected include sialyl-Lewis a , sialyl-Lewis c , sialyl-Lewis x , and sialyl-Tn.The human epithelial mucin MUC1 (1-4) is a type 1 membrane-bound glycoprotein expressed by ductal epithelia of a number of organs including breast, pancreas, airway, and gastrointestinal tract (reviewed by Gum (5)). Its functions in these tissues include lubrication and protection of the epithelia, and roles in cell adhesion have been suggested (6 -8). There is great diversity in the post-translational processing of MUC1 by epithelial cells in different organs. Moreover, MUC1 is aberrantly expressed by tumors (9) with patterns of post-translational modifications that are different from corresponding normal cell types (10); several different glycoforms of MUC1 were originally described as "tumor-associated antigens." Although MUC1 has been the subject of a number of lines of research, little is known about the mechanisms that direct its diverse post-translational processing.Until recently, there has been little precise information available on the O-glycosylation of MUC1 with respect to blood group antigens. One report demonstrated sLe a 1 and sLe x epitopes on MUC1 core protein expressed in a pancreatic tumor cell line (11), and two reports showed that MUC1 was one of the proteins associated with SLEX antigen secreted by colon carcinoma cells (12,13). In addition, the glycosylation of MUC1 purified from a human mammary tumor cell line (T47D), solid tumor, and human breast milk has been described (14). A major problem in detecting and purifying different glycoforms of the MUC1 core protein is that monoclonal antibodies raised against the core protein bind epitopes in the tandem repeat domain. Many forms of MUC1 that are secreted by different adenocarcinomas are heavily glycosylated in this domain, which obscures the protein epitopes recognized by these anti-MUC1 antibodies. Polyclonal antibodies generated against the cytoplasmic tail (15) do not bind the secreted forms of the...

A number of hemostasis parameters were studied in a total of 63 patients undergoing cardiopulmonary bypass (CPB) for open heart surgery. In 33 patients fibrinogen, Factors II, V, VIII:C, X, XI, antithrombin, plasminogen, alpha 2-antiplasmin, and platelet counts were assayed before surgery, during maximal hypothermia, at the end of the bypass procedure, before and after protamine sulfate infusion, in the intensive care unit, and 48 hours postoperatively. All factors assayed decreased markedly when the patients were placed on the bypass machine, the drop fairly well paralleling the decrease in hematocrit. During bypass the factors remained low, although a slight tendency toward an increase was noted. Only platelet counts remained low with a decreasing trend until the end of bypass. In the intensive care unit a second decrease in fibrinogen, Factors II and V and antithrombin was noted. This drop was unrelated to four patients who experienced a greater blood loss during this time than the others. Forty-eight hours postoperatively, a marked increase could be found in all clotting factors and near normal levels were measured. Platelet counts remained low, however. The decrease in factors rarely dropped into a range where one would expect a compromised hemostasis (less than 30%). Although antithrombin levels decreased below 60%, no difficulties with heparinization were encountered. Several factors were assayed manually and by automated analyzer (Multistat III), and excellent correlations were found between both procedures. Also a good correlation was found between the activated whole blood clotting times and quantitative heparin assays. In 30 additional patients platelet function was studied before surgery, after thoracotomy, after heparin administration, after initiation of bypass, at maximal hypothermia, before and after protamine sulfate infusion, and 24 hours postoperatively. Platelet counts once again decreased as patients were placed on the CPB machine and remained low throughout the procedure. Mean platelet volumes were unchanged until protamine was given. At that time, a significant drop in mean platelet volume was recorded. Twenty-four hours postoperatively the volumes were normal again. Platelet aggregation studies were performed on a whole blood aggregometer using two concentrations of ADP, collagen, and ristocetin as aggregation inducers. A significant decrease in aggregability was seen when the patients were connected to the CPB apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)

Although it is known that adhesion and antiadhesion are essential to the metastatic spread of tumor cells, little is known about the molecules that regulate these processes. Key words: selectin; metastasis; ICAM-1; pancreatic cancer; colon cancerHuman MUC1 1-4 is a large, type I transmembrane protein normally expressed on the apical surface of ductal epithelia. 5 MUC1 is synthesized as a single polypeptide chain but exists on the cell surface as a heterodimer. Proteolytic cleavage of the full-length protein results in 2 associated fragments: a large extracellular polypeptide containing the tandem repeat domain that can be released from the cell surface and a polypeptide consisting of the short extracellular domain, the transmembrane domain and the cytoplasmic tail that exists as an integral membrane protein. 6 -8 The tandem repeat domain of MUC1 is rich in serines and threonines and is heavily glycosylated with complex O-linked oligosaccharides. Fully processed forms of MUC1 produced by different secretory epithelia (e.g., pancreas, mammary gland, kidney, lung) are distinct in part because of differential glycosylation. 6,9 -12 Levels of expression and types of posttranslational modifications of MUC1 by transformed epithelial cells often differ from forms produced by corresponding normal epithelia. [13][14][15][16][17][18][19] The appearance of novel oligosaccharide structures on MUC1 expressed by tumors (compared with normal epithelia) or differential glycosylation at different positions on the MUC1 core protein may confer new properties of adhesion that contribute to the ability of tumor cells to metastasize. For example, certain glycoforms of MUC1 have been shown to associate with intracellular adhesion molecule 1 (ICAM-1), 20 and it is predicted that certain oligosaccharide structures present on MUC1, such as sialyl Lewis A (sLe a ) or sialyl Lewis X (sLe x ) 15,21 interact with different lectin-like molecules 22 that influence general properties of cell adhesion.There is also evidence for a seemingly opposing hypothesis: that MUC1 can have "antiadhesive" properties at the cell surface. Some allelic forms of MUC1 extend 200 -500 nm above the plasma membrane, 23 which implies that the extracellular domain of MUC1 can extend more than 150 -450 nm above the estimated length of other cell surface adhesion molecules. These physical properties, together with observed differences in morphologic properties of cells overexpressing MUC1, have led to the proposal of an antiadhesion function for MUC1. One line of supporting evidence comes from the normal cellular expression pattern: MUC1 is expressed at the apical surface of ductal epithelia, in contrast to adhesion molecules found on the basal and lateral surfaces that are in tight contact with surrounding cells or stromal elements. 24 Specific localization of MUC1 to the apical surface of the ductal lumen may contribute to establishment and maintenance of the zone of cell surface that is exposed to the lumen and is not in contact with adjoining cells. Loss of cellu...

MUC1 is expressed in ductal epithelial cells of a variety of tissues including breast, pancreas, airway, the gastrointestinal tract and in tumors deriving from these tissues. The human MUC1 gene product (Lan et al., 1990) is a polymorphic type I transmembrane molecule that consists of a large extracellular domain, a transmembrane domain and a cytoplasmic tail. One genetic polymorphism derives from a tandem repeat array of variable numbers of a highly conserved 20 amino acid repeat found in the extracellular domain. MUC1 is heavily O-glycosylated on the extracellular domain and glycosylation varies among different tissues and between normal and malignant cells. The following biological functions of MUC1 have been postulated from in vitro experiments using cultured cells or transfectants: (i) immunosuppressive activity of the soluble form of MUC1 mucin (Hayes et al., 1990); (ii) anti-adhesion activity, i.e., suppression of cell-cell (Ligtenberg et al., 1992;van de Wiel-van Kemenade et al., 1993) and cell-extracellular matrix (ECM) interactions (Wesseling et al., 1995) and (iii) ligands for selectins (Mannori et al., 1995). This evidence suggests that tumor cells expressing high levels of MUC1 may have increased invasive and metastatic potential. It is predicted that MUC1 may aid cellular detachment from the primary tumor and play a role in allowing the tumor cells to escape immune surveillance by blocking effectortarget cell interactions. Indeed, immunohistochemical results have suggested that MUC1 is highly expressed in invasive types of pancreatic, bile duct and breast cancers (Osako et al., 1993;Yamashita et al., 1993;Yonezawa et al., 1995). These effects may be due to the rigid and long extending structure of MUC1. It was demonstrated [based on light scattering and nuclear magnetic resonance ) studies on ovine submaxillary mucin] that O-linked carbohydrate side chains attached act primarily to expand and stiffen the peptide core. Upon the complete removal of the carbohydrate side chains, the extended structure characteristic of intact mucin-type glycoproteins collapses to chain dimensions typical of denatured globular proteins.Pancreatic cancer constitutes one of the major causes of cancerrelated death throughout the world: the 5-year survival rate is less than 5% because of difficulty in early detection, lack of effective treatment and limited knowledge of the biological characteristic of this type of cancer (Huguier et al., 1999). In an effort to understand biological characteristics of pancreatic cancer, we investigated the effect of overexpressing MUC1 on the malignant potential of human pancreatic cancer cells. The human pancreatic cancer cell line SUIT-2, which was derived from a patient's liver metastasis, is unusual among human tumor cell lines in that it exhibits spontaneous metastasis to lung from s.c. nude mouse xenografts (Iwamura et al., 1987;Taniguchi et al., 1992Taniguchi et al., , 1994. Twenty-eight clonal sublines of SUIT-2 were derived by in vitro methods and these clonal cell lines have di...

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 ␤-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAc␣2,6-(Gal␤1,3)GalNAc␣-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAc␣2,3Gal␤1,4(Fuc␣1,3)-GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumorassociated epitopes.