The tumorigenic potential of human CX-1 colon adenocarcinoma cells depends on carcinoembryonic antigen (CEACAM5) expression

It was shown that CEACAM5 can mediate cell-cell adhesion through homotypic and heterotypic interactions; however, its role in the expression of the malignant phenotype remains obscure. To study whether the formation of both primary tumors and metastases is directly related to the presence or absence of CEACAM5, we applied the antisense RNA strategy. By transfecting human CX-1.1 colon carcinoma cells with CEACAM5 antisense-expressing vector or with the vector itself, cell variants with a highly decreased expression of CEACAM5 were obtained. Profound differences in proliferative abilities among parental and obtained subclones of CX-1.1 cells were revealed when cells were implanted subcutaneously into nude mice. In contrast to their highly tumorigenic parental CX-1.1 cells (with high expression of membrane-bound and secreted CEACAM5), two subclones (3E and AS6Q) with substantially decreased expression of membrane-bound and secreted CEA showed a considerably diminished growth rate. Even more striking results were obtained with AS8Q cells, producing a residual amount of this glycoprotein. However, 3B cells (producing a large amount of secreted CEACAM5) did not differ significantly in their tumorigenic properties from CX-1.1 cells. Our experiments performed in nu/nu mice suggest that CEACAM5 supports the growth of primary tumors, but is not involved in the formation of metastases by colon cancer cells.     

Immunoperoxidase staining of carcinoembryonic antigen with monoclonal antibodies in adenocarcinoma of the colon

Mouse monoclonal antibodies to carcinoembryogenic antigen (CEA) obtained by the somatic cell hybridization technique of K?hler and Milstein were used in a modified enzyme bridge immunoperoxidase staining method. Both high and low affinity antibodies were tested and their staining properties compared with those of a commercial polyvalent rabbit antiserum. The staining pattern of neoplastic epithelial cells in all seven antibodies in samples of primary adenocarcinoma of the colon was similar, indicating that no gross differences were found in the exposure of the different antigenic determinants of CEA in formalin fixed tissue. The background staining of the monoclonal antibodies are negligible. It is concluded that monoclonal antibodies are superior to conventional antisera in immunoperoxidase staining of CEA.     

Immunoperoxidase staining of carcinoembryonic antigen with monoclonal antibodies in adenocarcinoma of the colon

Summary Mouse monoclonal antibodies to carcinoembryogenic antigen (CEA) obtained by the somatic cell hybridization technique of Köhler and Milstein were used in a modified enzyme bridge immunoperoxidase staining method. Both high and low affinity antibodies were tested and their staining properties compared with those of a commercial polyvalent rabbit antiserum. The staining pattern of neoplastic epithelial cells in all seven antibodies in samples of primary adenocarcinoma of the colon was similar, indicating that no gross differences were found in the exposure of the different antigenic determinants of CEA in formalin fixed tissue. The background staining of the monoclonal antibodies was negligible. It is concluded that monoclonal antibodies are superior to conventional antisera in immunoperoxidase staining of CEA.     

Glycoproteins from human colonic adenocarcinoma. Isolation and characterization of cell surface carcinoembryonic antigen from a cultured tumor cell line.

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.     

Impaired carcinoembryonic antigen release during the process of suramin-induced differentiation of the human colonic adenocarcinoma cell clone HT29-D4

The establishment of a differentiated state of the human colic adenocarcinoma cell clone HT29-D4 can be obtained by two ways: 1) the removal of glucose and its replacement by galactose in the culture medium (Fantini et al.: Biology of the Cell 65:163-169, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose-containing medium (Fantini et al.: Journal of Biological Chemistry 264:10282-10286, 1989). We investigated the release of CEA in the culture medium of glucose-deprived HT29-D4 cells (HT29-D4-Gal) and studied its alteration in suramin-treated HT29-D4 cells (HT29-D4-S). The amount of CEA released in the medium in function of time in culture of undifferentiated HT29-D4-Glu cells was very low (5 to 8 ng/10(6) cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29-D4-Gal cells (380 ng/10(6) cells/24 hours after 9 days in culture). Surprisingly the amount of CEA released by differentiated HT29-D4-S cells remained very low and comparable with the one of HT29-D4-Glu cells. Moreover suramin, when added to CEA-producing HT29-D4-Gal cells, strongly inhibited its release. Radioiodination of cell surface proteins followed by immunoprecipitation using an anti-CEA monoclonal antibody showed the presence of a 180 kDa polypeptide, i.e., CEA, predominantly labeled in HT29-D4-Gal and -S cells. The total CEA cellular content was higher in HT29-D4-Glu and HT29-D4-S cells than in HT29-D4-Gal cells. When HT29-D4-Gal or -S cells were treated with the bacterial phosphatidylinositol phospholipase C (Pl-PLC) a similar level of CEA was released suggesting a similar type of CEA anchorage. The present data demonstrate that a decrease in CEA release (i.e., in HT29-D4-Glu and -S cells) corresponds to an increase in its overall cellular expression. These results are in favour of a regulatory mechanism, impaired by suramin, which determines the balance between the soluble and the membrane bound forms of CEA.     

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells.

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.     

Establishment and characterization of human pancreatic adenocarcinoma cell line SOJ producing carcinoembryonic antigen and carbohydrate antigen 19-9

A new tumor cell line derived from a human pancreatic exocrine adenocarcinoma was established in tissue culture and was transplantable in a nude mouse. In tissue culture, the neoplastic cells grew as epithelial-like, mucin-producing cells with a population doubling time of 50-70 hrs. Chromosomes ranged from 63 to 186 with a modal number of 77. Subcutaneous injection of 1 x 10(6) cultured neoplastic cells into nude mice resulted in tumor formation histologically closely resembling the original neoplasm. Ultrastructurally, the cell line showed characteristic ductal epithelium. Immunohistochemically, carcinoembryonic antigen (CEA). Carbohydrate Antigen 19-9 (CA19-9) and DU-PAN-2 antigen were demonstrated in the original tumor, the culture cells and the transplanted tumor. The cells secreted CEA (48.7 ng/1 x 10(5) cells/24 hrs) and CA19-9 (325 U/1 x 10(5) cells/24 hrs) in spent medium as well as sera of the nude mouse. This cell line has been passaged 30 times in vitro and maintained for more than one year. These characteristics will make the cell line SOJ a valuable tool in studying various aspects of biology of human pancreatic cancer.     

Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA)

Immobilized carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM alpha-methyl D-mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D-mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.     

Galectin-1 induces cell adhesion to the extracellular matrix and apoptosis of non-adherent human colon cancer Colo201 cells

To isolate cDNAs for molecules involved in cell adhesion to the extracellular matrix, expression cloning with non-adherent colon cancer Colo201 cells was carried out. Four positive clones were isolated and, when sequenced, one was found to be galectin-1, a beta-galactoside-binding protein. When cultured on fibronectin-, laminin-, and collagen-coated and non-coated dishes, the adherent galectin-1 cDNA-transfected Colo201 cells increased and spread somewhat. Immunofluorescence staining revealed that galectin-1 was expressed inside and outside of Colo201 cells. The adhesion was dependent on the carbohydrate-recognition domain of galectin-1 since lactose inhibited the adhesion and exogenously-added galectin-1 caused the adhesion. PD58059, an inhibitor of mitogen-activated protein kinase, or LY294002, a phosphoinositide 3-OH kinase inhibitor, decreased the adhesion. Furthermore, the expression of galectin-1 in Colo201 cells induced apoptotic cell death, while exogenously-added galectin-1 did not cause apoptosis. These results indicate that galectin-1 plays a role in both cell-matrix interactions and the inhibition of Colo201 cell proliferation, and suggest that galectin-1 expressed in cells could be associated with apoptosis.     

Effects of cholesterol and alfa-2-recombinant interferon on cell growth and cell antigen production in the HT 29 cells, an established cell line of human colon adenocarcinoma

HT 29 cells, an established cell line of human colon adenocarcinoma, were grown in RPMI 1640 medium without or with cholesterol at 25, 50, 100 micrograms/ml concentrations. In some experiments 100 or 200 U/ml alfa-2-A recombinant Interferon were added to the medium. Only in the case of the highest cholesterol concentration there was a reduced number of cells at confluence. Moreover, only the production of CEA increased in the presence of cholesterol. Interferon did not affect cell growth appreciably but stimulated CEA release into the medium during the first three days of culture. Morphological analysis of cells in the presence of cholesterol seems to indicate an attempt of the cells to differentiate.     

Toxoplasma gondii RON4 binds to heparan sulfate on the host cell surface

Toxoplasma gondii rhoptry neck protein 4 (TgRON4) is a component of the moving junction, a key structure for host cell invasion. We previously showed that host cellular β-tubulin is a binding partner of TgRON4 in the invasion process. Here, to identify other binding partners of TgRON4 in the host cell, we examined the binding of TgRON4 to components of the host cell surface. TgRON4 binds to various mammalian cells, but this binding disappeared in glycosaminoglycan- and heparan sulfate-deficient CHO cells and after heparitinase treatment of mammalian cells. The C-terminal half of TgRON4 showed relatively strong binding to cells and heparin agarose. A glycoarray assay indicated that TgRON4 binds to heparin and modified heparin derivatives. Immunoprecipitation of T. gondii-infected CHO cell lysates showed that TgRON4 interacts with glypican 1 during Toxoplasma invasion. This interaction suggests a role for heparan sulfate in parasite invasion.     

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids

Summary The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogeneous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.     

Antiproliferative effects of nitrosulindac on human colon adenocarcinoma cell lines

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colon cancer, but their use is limited by toxicity in the gastrointestinal tract. The coupling of a nitric oxide-releasing moiety to NSAIDs strongly reduces these side effects. We demonstrated that the NO-releasing sulindac (nitrosulindac) has much more potent effects on colon adenocarcinoma cell lines compared to sulindac. Moreover, it could inhibit the growth of cells in soft agar experiments, demonstrating the antineoplastic activity at low concentration of nitrosulindac. However, this reduction in the growth of colon cancer cells seemed to be independent of the classical apoptosis pathway and could be explained by a cytostatic effect. Nitrosulindac caused a light perturbation of the cell cycle parameters not linked to a modification of the levels of p21 or the proliferating cell nuclear antigen. Moreover, neither sulindac, nor nitrosulindac, were able to inhibit the NF-kappa B pathway. These data suggested that nitrosulindac could be a better solution compared to other NSAIDs in the treatment of colon cancer.     

Role of protein kinase C in transforming growth factor-beta 1 induction of carcinoembryonic antigen in human colon carcinoma cells.

Transforming growth factor-beta 1 (TGF-beta 1) regulates the expression of the carcinoembryonic antigen (CEA) gene family in the human colon carcinoma cell line Moser. The mechanisms through which it acts, however, are unknown. In this communication, several lines of evidence are presented to show that the induction of CEA expression and secretion (collectively called CEA responses) by TGF-beta 1 is associated with protein kinase C (PKC) pathway of signal transduction. Treatment of intact cells with the PKC-specific inhibitor calphostin C down-modulated cellular PKC phosphotransferase activity and blocked the induction of the CEA responses by TGF-beta 1. Depletion of PKC by treatment of intact cells with phorbol ester also blocked the action of TGF-beta 1. The induction of the CEA responses by TGF-beta 1 was also blocked by the protein kinase inhibitor 1-(isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which also inhibited cellular PKC activity. However, TGF-beta 1 did induce the CEA responses in intact cells treated with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), the calmodulin-dependent phosphodiesterase inhibitor calmidazolium, the diacylglycerol kinase inhibitor R59 022, and the G-protein inhibitors cholera toxin and pertussis toxin. Treatment of intact cells with TGF-beta 1 induced a rapid and transient increase in PKC phosphotransferase activity. TGF-beta 1, however, was unable to induce PKC enzymatic activity in cells pretreated with calphostin C. Therefore, it is concluded that TGF-beta 1 regulates the CEA responses through a signal transducing pathway associated with PKC.     

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10^− 8 M, 12-24 h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.     

Keratin and carcinoembryonic antigen (CEA) in human melanoma cells.

Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin.     

Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface

Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.