In a 3D model of breast morphogenesis, CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) plays an essential role in lumen formation in a subline of the nonmalignant human breast cell line (MCF10A). We show that mammary carcinoma cells (MCF7), which do not express CEACAM1 or form lumena when grown in Matrigel, are restored to a normal morphogenic program when transfected with CEACAM1-4S, the short cytoplasmic isoform of CEACAM1 that predominates in breast epithelia. During the time course of lumen formation, CEACAM1-4S was found initially between the cells, and in mature acini, it was found exclusively in an apical location, identical to its expression pattern in normal breast. Lumena were formed by apoptosis as opposed to necrosis of the central cells within the alveolar structures, and apoptotic cells within the lumena expressed CEACAM1-4S. Dying cells exhibited classical hallmarks of apoptosis, including nuclear condensation, membrane blebbing, caspase activation, and DNA laddering. Apoptosis was mediated by Bax translocation to the mitochondria and release of cytochrome c into the cytoplasm, and was partially inhibited by culturing cells with caspase inhibitors. The dynamic changes in CEACAM1 expression during morphogenesis, together with studies implicating extracellular matrix and integrin signaling, suggest that a morphogenic program integrates cell-cell and cell-extracellular matrix signaling to produce the lumena in mammary glands. This report reveals a function of CEACAM1-4S relevant to cellular physiology that distinguishes it from its related long cytoplasmic domain isoform. mammary morphogenesis ͉ lumen formation

The cell adhesion molecule, carcinoembryonic Ag-related cellular adhesion molecule 1, shown by others to both activate and inhibit T cell proliferation, exhibits a reciprocal relationship to IL-2R expression over the time course of activation of PBMCs, and upon Ab ligation, inhibits both the production of IL-2 and cell proliferation. Carcinoembryonic Ag-related cellular adhesion molecule 1 associates with CD3 and is found in lipid rafts of PBMCs, is phosphorylated on the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of the -4L isoform, and associates with Src homology protein-1, providing an explanation for its inhibitory activity. When the ITIM-containing -4L and non-ITIM-containing -4S isoforms are transfected into Jurkat cells that produce, but do not depend on IL-2 for growth, both IL-2 production and cell proliferation are differentially inhibited, demonstrating that the two isoforms signal via different pathways. When the two isoforms are transfected into Kit-225 cells that depend on IL-2 for growth, IL-2Rβ and γ, but not α subunits are down-regulated, and the -4L, but not the -4S isoform inhibits cell proliferation by 6-fold in an IL-2 dose-response study.

CEACAM1-4S (carcinoembryonic antigen cell CEACAM14 (carcinoembryonic antigen cell adhesion molecule 1) is a type I membrane glycoprotein that mediates homotypic cell adhesion and belongs to the CEA gene family (1). It has been shown to play a role in bacterial and viral uptake (2-5), in insulin clearance (6), angiogenesis (7), NK and T-cell regulation (8 -11), and tumor suppression (12, 13). Despite its proposed function as a cell-cell adhesion molecule, CEACAM1 is often found on the lumenal surface of epithelial cells, suggesting the possibility that it also plays a role in cell polarization and lumen formation. In this respect, we have recently shown that CEACAM1 is expressed on the lumenal surface of normal mammary epithelial cells (14), and that in a three-dimensional model of mammary morphogenesis, blocking its expression with either an antisense gene or anti-CEACAM1 antibodies blocks lumen formation (15, 16), while breast cancer cells, which lack CEACAM1 and fail to form lumena in a three-dimensional culture, are restored to normal when transfected with the epithelial specific CEACAM1-4S isoform (16). CEACAM1 gene expression has been shown to be down-regulated early in colon cancer (12,17), and in a variety of cancer models, re-introduction of the CEACAM1 gene causes reversion of the malignant phenotype (18 -20). Although CEACAM1 is expressed in multiple isoforms with 1-4 Ig-like ectodomains and either long (72 amino acids) or short (12-14 amino acids) cytoplasmic domains, the short cytoplasmic domain isoforms predominate in epithelial cells. Because the cytoplasmic domain of this isoform is only 12-14 amino acids in length (the exact start of the domain is in doubt) it was originally thought not to play a role in signal transduction, but perhaps acted in a dominant negative fashion in the presence of the long cytoplasmic domain. However, we have shown that peptides or glutathione S-transferase fusion proteins from the short cytoplasmic domain bind directly to actin, tropomyosin, calmodulin, and more recently, to the annexin-2 p11 tetramer AIIt (21-23).In this study, we have used mutational analysis of the short cytoplasmic domain of CEACAM1 to reveal which residues are involved in actin interactions and lumen formation. The results of our studies show that mutants F454A and K456A greatly decrease or increase, respectively, actin interactions, whereas the F454A and the double mutant T457A,S459A greatly reduce lumen formation. Further analysis reveals that either Thr 457 or Ser 459 are phosphorylated during lumen formation. The studies that identified these key residues are the subject of this report. MATERIALS AND METHODS Construction of Baits for Yeast Two-hybrid ScreeningThe nucleotide and amino acid sequence for CEACAM1-4S is taken from NCBI entry number NM_001712, gi:4502404, the full-length coding sequence including the N-terminal signal * This work was supported by National Institutes of Health NCI Grant CA84202. The costs of publication of this article were defrayed in part by the payment of page ch...

Human biliary glycoprotein (BGP)1 is a member of the CEA gene family, a gene cluster on chromosome 19, including CEA, BGP, nonspecific cross-reacting antigen, and pregnancy-specific glycoprotein (1-5). BGP was first described as a CEA cross-reacting substance in bile (6) and was localized to the cell surface of bile canaliculi in the liver and gallbladder mucosa (7). Molecular cloning studies on BGP (8, 9) revealed that it exists in alternatively spliced isoforms from a single gene (9). BGP has a transmembrane domain and, depending on alternative splicing, a long (71 amino acids) or short (10 amino acids) cytoplasmic domain. The long form can be phosphorylated by protein kinases (10) after activation by antibody binding (11) We have previously shown that type II interferon, interferon-␥ (IFN-␥), is capable of up-regulating both the BGP and CEA genes, but by different mechanisms (22). CEA is slowly induced by IFN-␥ over a period of 24 -72 h, requiring new protein synthesis, while BGP is rapidly induced from 4 -24 h, without significant inhibition by cycloheximide (22). Rapid induction of gene expression by IFN-␥ is known to be mediated by GAS (for a review, see Ref. 23), found in a variety of genes, including interferon regulatory factor-1 (IRF-1). IRF-1, in turn, can activate a large number of genes by binding to the interferon-stimulated response element (ISRE). ISRE was previously identified for genes activated by type I interferons, IFN-␣ and IFN-␤. Thus, gene activation by IRF-1 is a major crossover pathway for gene activation by type I and II interferons. The mRNA for IRF-1 has a short half-life (30 min) (24) and is rapidly down-regulated by IRF-2 by competing for the same ISRE-binding site (25,26). Because of their potent effects on cell growth, IRF-1 has been termed a tumor suppressor gene and IRF-2 an oncogene (27).In this report, we show that the BGP promoter has an ISRE that is specifically activated by IRF-1 after treatment of colorectal or HeLa (cervical) cells with IFN-␥. IRF-1 mRNA is superinduced in these cells by cycloheximide, and a major species of BGP mRNA is inhibited by cycloheximide. We also show that both USF and Sp1 bind to another footprint in the BGP promoter and that an Sp1-like protein is induced by IFN-␥ in HT-29 cells. MATERIALS AND METHODS Cell Culture and TreatmentThe colon carcinoma cell lines HT-29 and SW403 and the cervix carcinoma cell line HeLa were obtained from American Type Culture Collection. The cells (0.5 ϫ 10 6 cells/ml) were suspended in 20 ml of medium in 75-cm 2 flasks, allowed to reach semiconfluency (3 days), and

CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cellcell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the Lisoform (GST-Cyto-L) bound poorly to F-actin in a cosedimentation assay, the S-isoform fusion protein (GSTCyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K D ‫؍‬ 7.0 M). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 ؋ 10 ؊8 and 1.0 ؋ 10 ؊7 M for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 ؋ 10 ؊8 and 1.3 ؋ 10 ؊7 M for GSTCyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K D values of 1.3 ؋ 10 ؊5 and 1.8 ؋ 10 ؊5 M, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin. CEACAM1 1 (biliary glycoprotein, CD66a) is a member of the carcinoembryonic antigen (CEA) family, which in turn belongs to the Ig superfamily (1-4). Alternative splicing of the transcripts of a single gene results in expression of at least four CEACAM1 isoforms (5, 6), all of which contain a transmembrane region, followed by a 74-amino acid long (CEACAM1-L) or a 14-amino acid short (CEACAM1-S) cytoplasmic domain. CEACAM1 is a highly glycosylated type 1 transmembrane protein expressed on the surface of epithelial, endothelial, and granulocytic cells (7). The human as well as the rat and mouse isoforms of CEACAM1 have been characterized as homotypic cell adhesion molecules (8, 9). Murine CEACAM1 also functions as a receptor for murine hepatitis virus (10), whereas human CEACAM1 can bind bacterial membrane proteins from Escherichia coli, Salmonella typhimurium, or Neisseria gonorrhoeae (11,12). CEACAM1 expression is down-regulated in human colon (13) and prostate (14) cancer and in 30% of breast cancers (15). Transfection of rat CEACAM1-L into a human tumorig...