The genetic changes that occur in cancer, whether these be mutations or alterations in levels of gene expression, become evident as changes in the phenotype of a specific cell type. In characterizing these phenotypic changes in malignancy, it is therefore important to work with the appropriate cells or cell lines. Breast cancers show the phenotype of the luminal epithelial cell (1), which can be cultured from milk, and cell lines have been developed from these milk cells which retain the luminal phenotype (2). One of these cell lines (MTSV1-7) has been used to look at the effect of overexpression of various oncogenes and proto-oncogenes on the behavioral properties of this cell type (3, 4).Overexpression of the c-ErbB2 receptor has been observed in a proportion of breast cancers and found to correlate with a poor prognosis (5), making signaling from this receptor an important parameter for investigation. To study the function of c-ErbB2 in human mammary epithelial cells, the receptor was overexpressed in MTSV1-7 cells to produce the ce-1 cell line (3). Unlike other receptors in the ErbB family, the c-ErbB2 homodimer has no known ligand, although c-ErbB2 can function as a heterodimeric receptor for the heregulin family of ligands with c-ErbB3 or c-ErbB4 or for EGF with c-ErbB1 (6 -9). Signaling from c-ErbB2 in overexpressing cells is, however, thought to be constitutive, operating through autophosphorylation of the homodimer, which forms because of overexpression (10).To look for genes whose expression is reversibly regulated by c-ErbB2 signaling as a homodimer, we have down-regulated c-ErbB2 phosphorylation using an antibody that has been shown to inhibit signaling from the receptor in breast cancer cell lines (11). In the humanized form (12), the antibody is under investigation in the clinic for the treatment of breast cancer (13). cDNAs prepared from ce-1 cells, treated or untreated with the antibody 4D5, were used to differentially screen filters from a fetal brain library using a computerized analysis (14), and a partial clone was isolated representing a novel sequence. Clones covering the full-length sequence (6.4 kb) 1 of the novel PLU-1 gene 2 were subsequently isolated by screening a breast cancer cDNA library.

Rabbit antiserum against a synthetic peptide corresponding to a tandemly repeated amino acid sequence in the human intestinal mucin apoprotein MUC2 was used in immunoprecipitation to study the biosynthesis of MUC2 in the colon-carcinoma cell line LS 174T. Under non-reducing conditions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electrophoresis after reduction, the larger band migrated to the same position as the smaller band and was interpreted as a putative disulphide-bond-stabilized dimer. Pulse-chase experiments showed only the monomer after 5 min and the appearance of the putative dimer after 30 min. The MUC2 apoprotein was also precipitated by antisera against the HF-deglycosylated peptides of the two highly glycosylated domains of the 'insoluble' mucin complex of rat small intestine [Carlstedt, Herrmann, Karlsson, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, [18771-18781]. Endoprotease Lys-C cleavage of the immunopurified apoprotein gave a large fragment of about 250 kDa that was detected by both the antiserum against the MUC2 tandem repeat and one of the glycopeptide antisera. This supports the view that the 'insoluble' mucin of rat small intestine is encoded by the Muc2 gene, as recently indicated by a partial cDNA sequence [Hansson, Baeckström, Carlstedt and Klinga-Levan (1994) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation starts after dimerization. The results indicate that the biosynthetic pathway of the MUC2 mucin may be similar to that of the von Willebrand factor with which MUC2 shares sequence similarities at its C- and N-termini.

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The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a mucin-like glycoprotein carrying the carcinoma-associated sialyl-Lewis a carbohydrate epitope. In an attempt to identify its apoprotein, an NH2-terminal peptide sequence was obtained from purified L-CanAg. In all interpretable positions, this sequence showed 100% identity to the NH2-terminal of human CD43 (leukosialin, sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only identified in leukocytes and other hematopoietic cells. An antiserum against deglycosylated L-CanAg and an anti-CD43 antiserum both immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor, from COLO 205 cells as well as from the known CD43-expressing cell line HL-60. Results from immunoprecipitations following pulse-chase experiments and tunicamycin treatments were in agreement with earlier studies on the CD43 precursor. RNA blot analysis confirmed the expression of CD43 by the COLO 205 cell line, whereas three other colon carcinoma cell lines were negative. The glycosylation-dependent monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to bind L-CanAg, probably due to its much more extensive glycosylation. We conclude that L-CanAg is the secreted extracellular domain of a novel glycoform of CD43 and that CD43, if expressed in other carcinoma cells, may have escaped notice in studies relying on glycosylation-dependent monoclonal antibodies against leukocyte CD43.

CD43 is an abundant transmembrane sialoglycoprotein in leukocyte-type cell lines, but it has also been suggested to be present in colon adenomas and colon carcinomas. We have now shown that CD43 is expressed in a variety of cell lines of different origins (CaSKI, A549, 293, MTSV1-7, MCF7, HT-1080, Jurkat, K562, COLO 205, HT-29, Caco-2, DLD-1 and SW480). The level of expression of CD43 mRNA was analyzed by reverse transcriptase-polymerase chain reaction and that of the protein by immunoprecipitation and Western blot, flow cytometry and confocal microscopy using two monoclonal anti-CD43 antibodies (L10 and 4D2). As all cell lines expressed CD43, it is suggested that CD43 has a more fundamental function than previously believed and thus cannot be considered only as a specific leukocyte marker.

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of gamma-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel gamma-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for gamma-secretase. However, the intracellular gamma-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without gamma-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.

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A secreted MUCI mucin from the spent medium of the colon carcinoma cell line COLO 205 carrying sialyl-Lewis a and x epitopes (H-CanAg) was purified by trichloroacetic acid precipitation and Superose 6 gel filtration.The purified H-CanAg inhibited adhesion of the leukocyte cell line HL-60 to E-selectin transfected COS-1 cells or interleukin-1 p (IL-1 p)-activated human umbilical vein endothelial cells. Sera from two patients with advanced colon carcinoma containing high concentrations of sialyl-Lewis a and x activity inhibited HL-60 cell adhesion to E-selectinexpressing COS-1 cells and IL-1 p-activated endothelial cells. After affinity column absorption of the sialyl-Lewis a activity, the sera also lost most of their sialyl-Lewis x activity and at the same time their adhesion inhibitory effect. A large part of the sialyl-Lewis a/x activity in the two patients was found in fractions containing rnucins having a MUCl apoprotein, as shown by its size, and reactivity with the two anti-MUCI apoprotein monoclonal antibodies, Ma552 and HMFG-2. The cell-adhesion inhibitory effect of the purified sialyl-Lewis a-carrying MUCI mucin fraction from the sera of the two patients was stronger than that of smaller sized sialyl-Lewis a-carrying mucin-type glycoproteins also found in the patient sera. The MUCl mucin fraction secreted by the COLO 205 cells and from the two sera were all shown to lack their C-terminal portion, in contrast to the MUCl mucin from cells. It is hypothesized that sialyl-Lewis a-and/or x-containing mucins, especially MUCl, secreted by tumors can interact with E-selectin on endothelial cells and thus inhibit leukocyte adhesion.