CEA is the major PHA-L-reactive glycoprotein in colon carcinoma cell lines and tumors: relationship between K-ras activation and beta1-6 branching of N-linked carbohydrate on CEA

Previously we have shown that a positive correlation existed between the presence of beta1-6 branching of N-linked carbohydrate (detected as PHA-L reactivity) and the level of Ras activation in colon carcinoma cell lines. In these cell lines the major PHA-L-reactive species was found to be 180 kDa. Here we identified this species to be carcinoembryonic antigen (CEA) by demonstrating that: (a) CEA immunoreactivity and PHA-L reactivity colocalized on blots of crude cellular membranes from these cell lines, and that (b) immunoprecipitation of CEA resulted in quantitative coprecipitation of PHA-L reactivity at 180 kDa. Metabolic labeling of cell line HTB39 with [(3)H]mannose revealed that CEA was the predominantly labeled glycoprotein. This indicated that CEA was the major PHA-L-reactive species due its high level of expression. The amount of PHA-L reactivity present on CEA, expressed as the PHA-L/CEA ratio, was found to vary between cell lines. This ratio was found to correlate closely with the level of Ras activation in these cells. In cellular membrane isolated from primary colon carcinoma, the major PHA-L-reactive species was also 180 kDa. This reactivity colocalized with CEA immunoreactivity, indicating that the major beta1-6-branching glycoprotein in membranes from primary colon carcinoma was CEA. Similar to that seen in cell lines, the amount of PHA-L reactivity on CEA in human tumor samples varied, suggesting that a similar paradigm of Ras-induced expression of beta1-6 branching may occur in human colon carcinoma.     

Carcinoembryonic antigen expression level as a predictive factor for response to 5-fluorouracil in colorectal cancer

Carcinoembryonic antigen (CEA) expression has been shown to protect cancer cell lines from apoptosis and anoikis. The aim of this study was to further elucidate the role of CEA expression on resistance to anticancer drugs in human colorectal cancer (CRC). We transfected CEA negative CRC cell line SW742 as well as CHO cells to overexpress CEA and their chemoresistance were assessed by MTT assay. In comparison to the parental cell lines, transfected cells had significantly increased resistance to 5-fluorouracil (5-FU). The results also showed a direct correlation between the amount of cellular CEA protein and 5-FU resistance in CEA expressing cells. We found no significant difference in sensitivity to cisplatin and methotrexate between CEA-transfected cells and their counter parental cells. We also compared the association between CEA expression and chemoresistance of 4 CRC cell lines which differed in the levels of CEA production. The CEA expression levels in monolayer cultures of these cell lines did not correlate with the 5-FU resistance. However, 5-FU treatment resulted in the selection of sub-populations of resistant cells that displayed increased CEA expression levels by increasing drug concentration. We analyzed the effect of 5-FU in a 3D multicellular culture generated from the two CRC cell lines, LS180 and HT29/219. Compared with monolayer culture, CEA production and 5-FU resistance in both cell lines were stimulated by 3D growth. In comparison to the 3D spheroids of parental CHO, we observed a significantly elevated 5-FU resistance in 3D culture of the CEA-expressing CHO transfectants. Our findings suggest that the CEA level may be a suitable biomarker for predicting tumor response to 5-FU-based chemotherapy in CRC.     

Human colonic adenocarcinoma cells. I. Establishment and description of a new line.

A series of human colonic epithelial cell lines have been cultured from a single patient: LS-180 the original adenocarcinoma, LS-174T a trypsinized variant, and normal colonic tissue. The malignant cells, 20 to 40, mum in diameter and oval to polygonal, exhibited characteristics of normal colonic mucosal cells, namely, abundant microvilli prominent in secretory cells, and the presence of intracytoplasmic mucin vacuoles. The cultured adenocarcinoma cells, but not normal, demonstrated neoplastic properties by producing high levels of carcinoembryonic antigen (CEA) and by the ability to be propagated in hamster cheek pouches and in immunodeprived mice. The CEA production by the newly established line LS-180 released 900 times more CEA per cell into the culture medium and bore 30 times more cell-associated material than the established line, HT-29. These cell lines may permit detection of distinctive chemical, physiological, pharmacologic, and immunologic characteristics of neoplastic colonic cells.     

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.     

Growth-inhibitory effect of TGF-B on human fetal adrenal cells in primary monolayer culture

We examined the effects of transforming-growth factor-B (TGF-B) on growth ([3H]-thymidine uptake) and function (dehydroepiandrosterone sulfate [DHAS] and cortisol production) of human fetal zone adrenal cells. Results indicate that TGF-B significantly inhibits, in a dose-related manner, both basal and epidermal growth factor (EGF)-stimulated cell growth: IC50 = 0.1-0.25 ng/ml. EGF is ineffective in overcoming the inhibitory effect of TGF-B, suggesting a noncompetitive antagonism between the two factors. Also, the inhibitory effect of TGF-B is additive to that of adrenocorticotropic hormone (ACTH). On the other hand, TGF-B (1 ng/ml) does not significantly change basal or ACTH-stimulated DHAS or cortisol secretion. We conclude that, unlike its effect on other steroid-producing cells, TGF-B inhibits growth of fetal zone cells and does not appear to have a significant inhibitory effect on steroidogenesis.     

The effect of extracellular calcium on colonocytes: evidence for differential responsiveness based upon degree of cell differentiation

Calcium supplementation decreases the incidence of colon cancer in animal models and may prevent colon cancer in man. Potential mechanisms include binding of mitogens and direct effects of calcium on colonic epithelial cells. In this study, the effects of extracellular calcium on epithelial cell growth and differentiation were studied in three colon carcinoma and two colonic adenoma cell lines. The characteristics studied included morphology, cell cycle kinetics, [Ca²⁺]_IC (intracellular calcium concentration), proliferation, and expression of differentiation markers such as carcinoembryonic antigen (CEA) and alkaline phosphatase (AP). Sodium butyrate (NaB) and 1,25-dihydroxyvitamin D₃ were used as controls in the latter three assays as these two agents are known differentiating agents. Alteration of [Ca⁺²]_EC (extracellular calcium concentration) did not affect carcinoembryonic antigen (CEA) or alkaline phosphatase (AP) expression. NaB enhanced the expression of AP three-fold and CEA five-fold. This effect was augmented by increasing [Ca²⁺]_EC. The exposure of cells to 1,25-(OH)₂-Vitamin D₃ increased CEA but not AP. [Ca²⁺]_IC increased in response to 1,25-(OH)₂-vitamin D₃ and NaB but not with variation in [Ca²⁺]_EC. Increased [Ca²⁺]_EC inhibited proliferation of well-differentiated cells, but had no effect on poorly-differentiated cells. Morphological studies showed that extracellular calcium was necessary for normal cell—cell interactions. These studies have demonstrated direct effects of calcium on colonic epithelial cells which may contribute to the protective effects of dietary calcium against colon cancer. Loss of responsivess to the antiprotective effects of [Ca²⁺]_EC with de-differentiation suggests that calcium supplementation may be most beneficial prior to the development of neoplastic changes in colonic epithelium.     

Heterogeneity in antigenic expression and radiosensitivity in human colon carcinoma cell lines

Summary A panel of human colon carcinoma cell lines were characterized regarding both antigenic heterogeneity and variations in radiosensitivity. Monoclonal antibodies were used to study the expression of carcinoembryonic antigen (CEA), gastrointestinal cancer antigen (GICA or CA 19-9) and carcinoma-associated antigen (CA-50). Radiosensitivity was studied with the clonogenic survival technique. Three cell lines, LS 174T, HCTC, and SW 1116 stained positive for all three antigens. HT-29 was positive for CA 19-9 and CA-50 whereas Caco-2 was positive for CEA and CA 19-9. The cell lines SW 620 and LIM 1215 only stained positive for one of the antigens, CA-50 and CEA, respectively. In nearly all positive cases the stainings were very heterogeneous with mixtures of positive and negative cells. One exception was the HCTC cells which stained homogeneously for the CA 19-9 and CA-50 antigens. The neuroendocrinelike COLO 320 cells were negative in all cases. The radiosensitivity varied strongly between the cell lines with D_q-values between 0.8 and 1.9, extrapolation numbers between 2.0 and 4.7, D_o-values between 1.1 and 2.8. The surviving fraction at 2 Gy varied between 0.3 and 0.7 with HCTC as the most radiosensitive and HT-29 as the most radioresistant cell line. Thus, there were differences in antigenic expression and intrinsic radiosensitivity between the cell-lines and antigenic heterogeneities within each cell line. The analyzed panel of cell lines will be valuable in studies of dose-effect relations for monoclonal antibodies labeled with toxic radionuclides simulating both antigenic heterogeneity and variations in radiosensitivity.     

TGFbeta down-regulation of the CFTR: a means to limit epithelial chloride secretion

Transforming growth factor beta (TGFbeta) is a multifunctional cytokine with effects on many cell types. We recently showed that in addition to epithelial barrier enhancing properties, TGFbeta causes diminished cAMP-driven chloride secretion in colonic epithelia, in a manner that is p38 MAPK-dependent. In this study, we sought to further delineate the mechanism behind TGFbeta diminution of chloride secretion. Using colonic and kidney epithelial cell lines, we found that exposure to TGFbeta causes dramatic changes in the expression and localization of the apical membrane chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). In TGFbeta-treated colonic epithelia (T84 and HT-29), CFTR mRNA was significantly reduced 2-24 h post-cytokine exposure. At a time consistent with decreased colonic epithelial secretory responses (16 h), TGFbeta treatment caused diminished intracellular CFTR protein expression (confocal microscopy) and reduced channel expression in the apical membrane during stimulated chloride secretion (biotinylation assay). In comparison, polarized kidney epithelia (MDCK) treated with TGFbeta displayed similarly reduced secretory responses to cAMP stimulating agents; however, a perinuclear accumulation of CFTR was observed, contrasting the diffuse cytoplasmic CFTR expression of control cells. Our data indicate that TGFbeta has profound effects on the expression and subcellular localization of an important channel involved in cAMP-driven chloride secretion, and thus suggest TGFbeta represents a key regulator of fluid movement.     

A colonic tissue architecture assay applied to human colon carcinoma cells

Summary A two-component tissue architecture assay system has been devised that tests the ability of human colon carcinoma cells to conform to the specific three-dimensional cell-cell and cell-substratum interactions characteristic of normal colonic tissues. Dissociated fetal rat colonic cells (FRCC) were allowed to reaggregate in suspension with or without the addition of different proportions (0.1%, 1%, and 10% of the total cells) of the human colon carcinoma cell lines, SW-1222 and LS-174T. Cellular aggregates obtained after 36 hours’ incubation exhibited cell sorting by the formation of recognizable epithelial colonic crypt-like structures with glandular lumens in a mesenchyme-like background. Carcinoembryonic antigen (CEA)-positive SW-1222 cells in 10% mixed aggregates were organized into numerous well-formed glandular structures with a polarized apical distribution of CEA. LS-174T cells, on the other hand, were self-sorted but structurally disorganized with a continuous cell surface CEA distribution. Pure FRCC and mixed aggregates were implanted under the kidney capsules of Swiss nu/nu (nude) or CD-1 nu/nu mice and allowed to grow for a period of 7–10 days. Whereas the normal FRCC readily formed colonic tissue, the SW-1222 cells exhibited a capacity for differentiation into colonic crypts which became progressively less normal and more tumor-like as the proportion of carcinoma cells in the aggregates was increased. The LS-174T cells demonstrated poor differentiation at all concentrations. Cell surface levels of CEA and the CEA family member nonspecific crossreacting antigen (NCA), both overexpressed in colon cancer, were higher in LS-174T than in SW-1222 cells, whereas family member biliary glycoprotein (BGP), downregulated in colon carcinoma was higher in the SW-1222 cells. These results thus support the suggestion that deregulated expression of CEA family members can be involved in the ability of colonocytes to differentiate and conform to normal tissue architecture as assessed by the assay. The assay is therefore amenable to genetic analysis of normal and perturbed architectural phenotypes. This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. C. I. is a recipient of a studentship from “le Fonds pour la Formation de Chercheurs et l’Aide à la Recherche.”     

Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression

Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.     

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.     

Promotion of differentiation in human colon carcinoma cells by vasoactive intestinal polypeptide

The effects of vasoactive intestinal polypeptide (VIP) and dibutyryl cyclic adenosine 3':5'monophosphate (dbcAMP) on two human colon carcinoma cell lines, HCT 116 and GEO, were investigated. VIP and dbcAMP inhibited the growth of both cell lines in monolayer culture in a dose-dependent manner. Within 6 h of treatment with 1 mM dbcAMP or 0.3 microM VIP, numerous mucin-like droplets were secreted by GEO cells. VIP and dbcAMP also increased carcinoembryonic antigen (CEA) secretion. In both cell lines, a 9-fold increase in conditioned medium CEA levels was observed at 1 mM dbcAMP and a 2.6-fold increase at 1.5 microM VIP. Time- and concentration-dependent evaluation in cAMP levels were elicited by VIP in the two cell lines. Immunocytochemical studies for cell-surface glycoprotein detection in GEO cells showed that VIP induced a morphological and functional organization of mucin-secreting cells. These results indicate that VIP and dbcAMP have antiproliferative and strong differentiation-promoting effects in colon cancer cells. This is the first report of VIP-induced mucin secretion in colon tumor cells.     

Synthesis and secretion of colonic glycoproteins: Evidence for shedding in vivo of low molecular weight membrane components

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [³H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.     

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer-associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin-type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAc alpha 2----6Ga1NAc seemed to be involved in the epitope.     

Development of a novel method to evaluate sialylation of glycoproteins and analysis of gp96 sialylation in Hela,SW1990 and A549 cell lines

Background

Glycoproteins play a critical role in the cellular activities of eukaryotes. Sialic acid is typically the outermost monosaccharide of glycolipids and glycoproteins, and is necessary for normal development.

Results

A strategy based on avidin–biotin affinity was established to enrich sialylated glycoproteins from HeLa cervical carcinoma, SW1990 pancreatic adenocarcinoma, and A549 lung adenocarcinoma cells. Using HPLC–MS/MS, western blot, real-time PCR, and enzyme-linked immunosorbent assay, gp96 was identified in all three cell lines. No significant difference in the protein expression of gp96 was detected at the whole cell level, but the amount of biotinylated gp96 in SW1990 cells was 30–40 % lower than that in A549 and HeLa cells, and the amount of sialylated gp96 in SW1990 cells was 30 % lower than that in A549 and HeLa cells. Immunoblotting results showed that the expression of sialyltransferase proteins in the total cell lysates from HeLa and A549 cells were higher than that in SW1990 cells.

Conclusions

We established a new method for investigating the expression and sialylation of glycoproteins using metabolic labeling, click chemistry, and avidin–biotin affinity. We successfully used this method to purify sialylated glycoproteins from cancer cell lines. Our results showed that the levels of gp96 sialylation varied across different cancer cell lines, and this may be because of differences in sialyltransferase expression.     

The DHE cell line as a model for studying rat gastro-intestinal mucin expression: effects of dexamethasone

The expression of mucin genes was evaluated in rat intestinal cell lines in order to establish an in vitro model for investigating the regulation of intestinal mucin expression in this species. Two rat intestinal cancer cell lines (DHE, LGA) and three nontumoral rat intestinal cell lines (IEC6, IEC17, IEC18) were screened. The mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes was studied by semiquantitative RT-PCR, real-time RT-PCR and Northern-blot analysis. Results were correlated with immunohistochemical expression of rat gastric and intestinal mucin proteins, and secretion of glycoconjugates was examined by enzyme-linked lectin assay. We showed that mRNA of rMucl and rMuc2 were constitutively expressed in all IEC cell populations but periodic acid Schiff staining of these cells did not reveal the presence of glycoproteins. DHE cells expressed rMuc1-5AC mRNA and LGA expressed the same mucins but the level of rMuc4 was much lower. Mucin mRNA expression also differed in relation with the length of cultivation. Immunocytochemical studies revealed the presence of gastric and intestinal mucins in the two tumoral cell lines. Functional experiments showed that bethanechol, A23187 and PMA stimulated release of glycoconjugates in DHE but not in LGA cells. Treatment of DHE cells with dexamethasone (10(-7) mol/l) enhanced rMuc2 mRNA but decreased rMuc1 and rMuc5AC mRNA. Real-time RT-PCR showed that the expression of rMuc1 and rMuc5AC genes was reduced by more than tenfold after 24 h. The increased expression of rMuc2 gene was confirmed by Northern blot analysis. In conclusion, DHE cells provide a valuable cellular model for research on rat mucin secretion and expression.