Nonspecific Cross-reacting Antigen Research Articles

High Expression of Carcinoembryonic Antigen-Related Cell Adhesion Molecule(CEACAM) 6 In Primary Myelofibrosis

AbstractAbstract 4116 Introduction:Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, also known as non-specific cross-reacting antigen (NCA) or CD66c is a glycosylphosphoinositol (GPI)-linked cell surface protein and a member of the CEA family of proteins. Structurally, this protein shares close homology with CEACAM1, CEACAM7 and CEACAM8. Functionally, CEACAM6 has been implicated in cell adhesion, cellular invasiveness and metastatic behaviour of tumour cells. Expression of CEACAM6 protein has been found in a variety of normal human tissues, including myeloid cells. A key pathophysiological feature of primary myelofibrosis (PMF) is changes in the bone marrow micromilieu, progressive accumulation of connective tissue, pronounced neovascularisation and altered stromal cell adhesion. Consequently, CD34+ cells escape the bone marrow and seed extramedullarily. Being involved in cell adhesion, cellular invasiveness, angiogenesis, and inflammation – all key processes in the pathophysiology of PMF – we hypothesized that CEACAM6 might play an important role in these processes in patients with myelofibrosis. We have assessed gene expression of several CEA genes in patients with PMF and related neoplasms in order to elucidate the significance of CEACAM6 and other members of the CEA family of proteins in these disorders. Patients and Methods:Gene expression microarray studies have been performed on whole blood from control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54.675 probe sets (38.500 genes). Total RNA was purified from whole-blood and amplified to biotin-labeled aRNA and hybridized to microarray chips. Results:20.439, 25.307, 17.417, and 25.421 probe sets were identified to be differentially expressed between controls and patients with ET, PV, PMF, and CPMNs as a whole, respectively (false discovery rate (FDR) adjusted p values < 0.05). Several CEACAM-genes were significantly deregulated. In PMF patients, the CEACAM genes 1, 6 and 8 were significantly upregulated with the highest upregulation of CEACAM6 and CEACAM8 (fold change (FC) 12.5 and 14.0, respectively and FDR adjusted p values 7.71 × 10-7 and 1.48 × 10-5, respectively). Only the CEACAM21 gene was significantly downregulated (FC -1.3 and FDR adjusted p-value 4.14 × 10-7) whereas the other CEACAM-genes tested (3, 4, 5, 7, 19) displayed no significant changes as compared to controls. In ET patients, the CEACAM genes 3, 6, and 7 were significantly upregulated (FC 1.2, 1.8, and 1.1, respectively) and FDR adjusted p values < 0.03). The CEACAM21 gene was significantly downregulated (FC -1.3 and FDR adjusted p-value 0.0004). In PV patients, the CEACAM genes 1, 3, 6 and 7 were significantly upregulated (FC 1.7, 1.2, 1.7, and 1.1, respectively) and FDR adjusted p values 0.0002, 0.0009, 0.0002, and 0.03, respectively. The CEACAM21 gene was significantly downregulated (FC -1.3 and FDR adjusted p-value 4.14×10-5) All other CEACAM-genes showed no significant changes in either ET or PV as compared to controls.When comparing controls with non-PMF-patients, a significant upregulation of the CEACAM genes 1, 3, 6, and 7 were recorded in non-PMF patients (FC 1.5, 1.2, 1.7, and 1.1, respectively; FDR adjusted p values 0.001, 0.0002, 0.002, and 0.02, respectively). The CEACAM19 and CEACAM21 genes were significantly downregulated (FC - 1.1 and -1.4, respectively; adjusted p-values 0.008 and 3.46 × 10-8). Discussion and Conclusions:Using global gene expression profiling, we have found a pronounced deregulation of CEACAM genes, involving highly significant upregulation of the CEACAM genes 6 and 8 in PMF (FCs 12.0 and 14.0, respectively). Upregulation of CEACAM6 was seen in both ET, PV and PMF by far the highest levels being recorded in PMF-patients. Of note, significant upregulation of CEACAM8 (FC 14) was only seen in patients with myelofibrosis. The elevated expression of CEACAMs genes in ET, PV, and PMF may solely reflect neutrophil activation being most exaggerated in patients with PMF in whom the highest CEACAM6 and 8 expression patterns were recorded. Alternatively, the highly elevated gene expression of CEACAM6 and 8 in PMF are molecular markers of clonal expansion and myelofibrotic transformation, implying enhanced proteolytic activity and egress of CD34+ cells into the circulation. Disclosures:No relevant conflicts of interest to declare.

Early Detection of Colorectal Cancer Metastasis and Relapse by Recognizing Nonspecific Cross-Reacting Antigen 2 in Commercial Carcinoembryonic Antigen Assays

Carcinoembryonic antigen (CEA),1 one of the first known tumor markers, is still the most useful clinical marker to aid in the diagnosis and monitoring of colorectal cancer. Many commercial assays are available for measuring CEA, but their practical use is complicated by the molecular heterogeneity of CEA. CEA is a heavily glycosylated protein of the immunoglobulin gene superfamily and has several related antigens such as nonspecific cross-reacting antigen (NCA), NCA-2, and normal fecal antigen. Previous investigations have indicated that some commercial assays for CEA cross-react with various CEA antigens owing to the epitope group specificity of the monoclonal antibodies (mAbs) employed in the assay (1)(2). NCA-2 is a member of the CEA gene family and is structurally most similar to CEA. It has been reported that CEA might be increased in the serum of some colorectal cancer patients, and the cross-reactivity to NCA-2 might be a beneficial characteristic of CEA assays with respect to their diagnostic sensitivity in colorectal cancer patients (3). A patient who had colorectal cancer and underwent a transverse colectomy at our hospital in 1996 suffered relapses of the cancer in 2000, 2002, and 2003 and had chemotherapy and surgical …

Early Detection of Colorectal Cancer Metastasis and Relapse by Recognizing Nonspecific Cross-Reacting Antigen 2 in Commercial Carcinoembryonic Antigen Assays

Carcinoembryonic antigen (CEA),1 one of the first known tumor markers, is still the most useful clinical marker to aid in the diagnosis and monitoring of colorectal cancer. Many commercial assays are available for measuring CEA, but their practical use is complicated by the molecular heterogeneity of CEA. CEA is a heavily glycosylated protein of the immunoglobulin gene superfamily and has several related antigens such as nonspecific cross-reacting antigen (NCA), NCA-2, and normal fecal antigen. Previous investigations have indicated that some commercial assays for CEA cross-react with various CEA antigens owing to the epitope group specificity of the monoclonal antibodies (mAbs) employed in the assay (1)(2). NCA-2 is a member of the CEA gene family and is structurally most similar to CEA. It has been reported that CEA might be increased in the serum of some colorectal cancer patients, and the cross-reactivity to NCA-2 might be a beneficial characteristic of CEA assays with respect to their diagnostic sensitivity in colorectal cancer patients (3). A patient who had colorectal cancer and underwent a transverse colectomy at our hospital in 1996 suffered relapses of the cancer in 2000, 2002, and 2003 and had chemotherapy and surgical …

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system.

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radioimmunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels.

Purification and characterization of a 95-kDa protein—carcinoembryonic antigen-related cell adhesion molecule 8—from normal human granulocytes

A 95-kDa protein was purified to homogeneity from granule extracts of normal human granulocytes. The column procedure consisted of Sephadex G-75, Mono-S cation exchange and Superdex HR 75 chromatography. The purified protein showed only one broad band at a molecular weight of 95 kDa when analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It reacted with polyclonal antibodies against carcinoembryonic antigen (CEA) and a specific monoclonal antibody against CD66b, but did not react with monoclonal antibodies against CD66acde and CD66c when analyzed by immunoblotting. The molecular weight of the protein shifted from 95 to 40 kDa on SDS-PAGE after deglycosylation. Tryptic peptide analysis by MALDI-Tof identified four peptides with spectra of m/ z matching the expected tryptic peptides from a CGM6 gene product. Furthermore, the nanoelectrospray mass spectrometry (MS/MS) analysis of the two selected tryptic peptides of the protein revealed two amino acid sequences corresponding to residues 79–98 and 199–207 of the CGM6 gene product. Based on this, and also on the immunochemical data, it is concluded that the purified 95 kDa is identical to carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) (nonspecific cross-reacting antigen (NCA)-95, CD67 and CD66b) and is a product of the CGM6 (W272) gene. We present, for the first time, a method for the purification of CEACAM8 from normal human granulocytes, which should be useful for further studies on its structure and functions. We also confirmed at the protein level that CEACAM8 is a product of the CGM6 (NCA–W272) gene.

Nail bed keratinocytes express an antigen of the carcinoembryonic antigen family.

Using a panel of monoclonal and polyclonal antibodies raised against human carcinoembryonic antigen (CEA) and CEA-related molecules, we detected strong expression of an antigen, with immunoreactivity consistent with non-specific cross-reacting antigen (NCA) (CD66c), in all of 26 normal human nail specimens obtained from various fingers and toes. In longitudinal sections, strong and constant expression of the NCA-like antigen was seen on keratinocytes distributed in the upper epithelial cell layers of the nail bed, while in transverse sections the expression was limited to the major central portions of the nail bed, but only where longitudinal epidermal ridges were observed. In the hyponychium and the ventral aspect of the proximal nail fold, the expression was weak or lacking. No expression was seen in the nail matrix, the nail plate, or the dorsal aspect of the proximal nail fold. The same results were obtained for all of the 26 nails studied. This report is the first to demonstrate that an antigen of the CEA family, with NCA-like immunoreactivity, is expressed in a specific subpopulation of keratinocytes in the nail bed. The specific expression pattern suggests that the antigen may play a part in adhesion of the nail plate to the nail bed.

The KOR-SA3544 antigen predominantly expressed on the surface of Philadelphia chromosome-positive acute lymphoblastic leukemia cells is nonspecific cross-reacting antigen-50/90 (CD66c) and invariably expressed in cytoplasm of human leukemia cells.

We previously reported a novel monoclonal antibody KOR-SA3544 which predominantly reacted with a surface antigen (sSA3544) expressed on Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL). In the present study, we demonstrate that the antibody specifically recognized nonspecific cross-reacting antigen (NCA)-50/90 (CD66c), one of the carcinoembryonic antigen (CEA)-related glycoproteins encoded by a member of the CEA gene family. In addition, we show that the SA3544 antigen (NCA-50/90) was invariably expressed in cytoplasm of all of the human leukemic cell lines examined (sSA3544-positive B-lymphoid two, sSA3544-negative T or B-lymphoid and non-lymphoid 24) regardless of the presence or absence of surface expression of this antigen. Immunoelectromicroscopic examination revealed that the cytoplasmic antigen was mainly present in granules in sSA3544-positive leukemia cells, whereas it was diffusely present in cytosol in sSA3544-negative leukemia cells. Thus, among members of the CEA family, NCA-50/90 was first demonstrated to be expressed not only on the surface of some leukemia cells, but also in cytoplasm of various types of leukemia cells.

Epitope mapping of monoclonal antibodies against N-domain of carcinoembryonic antigen

Monoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1–32, 1–32, and 33–59 of CEA, respectively, and that two discrete regions 1–32 and 60–93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27–29), eight residues (21, 27–29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA.

Immunohistochemical demonstration of carcinoembryonic antigen and related antigens in various cutaneous keratinous neoplasms and verruca vulgaris.

Carcinoembryonic antigen (CEA), which is a well-known marker for the normal sweat gland apparatus and its neoplasms in the skin, was recently demonstrated in sebaceous neoplasms. The aim of this study was to examine the expression of CEA and related antigens in the other cutaneous keratinous neoplasms and verruca vulgaris. Normal adult skin, squamous cell carcinoma (SCC), senile keratosis, Bowen's disease, basal cell carcinoma (BCC), seborrhoeic keratosis and verruca vulgaris were stained immunohistochemically with a panel of monoclonal and polyclonal antibodies that recognize different epitopes of CEA and related molecules. Localization of the antigens was compared with that of involucrin and proliferating cell nuclear antigen. The strongest expression of CEA-related antigens, other than non-specific cross-reacting antigen (NCA) -50/90, was seen in SCC and verruca vulgaris, while no detectable expression was seen in BCC. Senile keratosis, Bowen's disease and seborrhoeic keratosis showed the predominance of the CEA-related antigens over CEA weakly expressed. Strong expression of both CEA and NCA-50/90 was seen only in SCC. All the expressions were limited to the cells situated in the upper epidermal cell layers of the tumours, at the centre of tumour islands in SCC and along the pseudohorn cysts in seborrhoeic keratosis, where involucrin was coexpressed. We suggest that CEA and related antigens are not only markers for sweat gland differentiation in the skin, as currently accepted, but are also expressed in various cutaneous keratinous neoplasms and verruca vulgaris. The expression may correlate with the terminal differentiation of the tumour cells, the strong coexpression of CEA and NCA-50/90 may correlate with the malignant potential of the tumour types, and the mechanisms that control the expression of CEA and related antigens in the neoplasms may be similar to those operative in verruca vulgaris.

Transcriptional regulation of the non-specific cross-reacting antigen gene, a member of the carcinoembryonic antigen gene family up-regulated in colorectal carcinomas.

Human non-specific cross-reacting antigen (NCA), a close relative of the tumor marker human carcinoembryonic antigen (CEA), is also an in vitro homotypic intercellular adhesion molecule capable of inhibiting differentiation when expressed ectopically by myoblasts. Moreover, NCA appears to be overexpressed at the transcriptional level to a greater extent and more frequently in colorectal carcinomas than CEA. This study examines the transcriptional control mechanisms responsible for orchestrating NCA expression. The region within 284 bp upstream of the translational start site of the NCA gene was found to be capable of directing high levels of expression in functional promoter assays. Footprinting experiments identified three cis-acting elements and mobility-shift assays revealed that the first of these elements is bound by the upstream stimulating factors USF1 and USF2 while the other two are bound by the stimulatory proteins Sp1 and Sp3. No cis-acting elements corresponding to CEA footprint FP4 or the silencer CEA FP5 were detected in the NCA promoter, which may contribute to the differential expression of NCA versus CEA in tumorigenesis.

Restricted epitope of carcinoembryonic antigen (rCEA) may be a good marker for detecting precancerous lesions and early stage of colonic and gastric carcinomas

CEA is a member of a rather large family of glycoproteins found in a variety of normal tissues, including colon and gastric mucosa. Therefore, the clinical application of CEA detection in GI tissue sections for cancer diagnosis are limited. In this study, we report the normal and pathological GI tissues reactivity to a monoclonal antibody COL-l, which only reacts the restricted CEA antigen epitope (rCEA) but not cross reacts with nonspecific crossreacting antigens (NCA), including normal colorectal and gastric mucosa. MATERIALS and METHOD: All specimens were formalin fixed, parhffln embedded tissues obtained from surgical resection or biopsy. There are 180 cases of colonic tissues, including normal colonic mucosa, remote colonic mucosa at least 6 cm away from the cancer site. colorectal cancer and colonic polyps. 140 cases of gastric tissues, including normal gastric mucosa, chronic superficial gastritis, Chronic Atrophic Gastritis (CAG), with or without Intestinal Metoplasia (IM) or Dysplasia (DYS), and gastric carcinoma. LAB-SA (SP) method was applied for immunostaining. RESULTS:

Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells.

We hypothesize that a major factor regulating hepatic metastasis is the ability of CEA (carcinoembryonic antigen) producing colorectal carcinomas to activate Kupffer cells. CEA and NCA (nonspecific cross-reacting antigen) bind to an 80 kDa Kupffer cell receptor by the peptide sequence PELPK and stimulate cytokine production. Cytokines induce sinusoidal endothelial cells to express intercellular adhesion molecules and increase adhesion of the tumor cells and retention in the liver. In this study human Kupffer cells were activated in vitro with CEA, NCA, and the peptide PELPK. This resulted in release of IL-1beta, TNF-alpha and IL-6. CEA non-producing MIP-101 colon carcinoma cells labeled with 51Cr were incubated on monolayers of ECV-304 human umbilical vein endothelial cells treated with these Kupffer cell derived cytokines or with comparable recombinant human (rH) cytokines. Specific antibodies to the adhesion molecules ICAM-1, VCAM-1, E-selectin and beta2integrin were used to block their functions. A significant enhancement in the adhesion of colorectal carcinoma cells occurred when endothelial cells were stimulated with a very low concentration of Kupffer-cell derived cytokines. Activated endothelium demonstrated significant up-regulation primarily of ICAM-1. The adhesion was blocked by an antibody to ICAM-1. A combination of Kupffer-cell derived cytokines was more effective than IL-1beta or TNF-alpha alone. IL-6 alone did not influence adhesion under our conditions. Our results suggest a mechanism for CEA in the modulation of colorectal carcinoma adhesion to the hepatic endothelium and its enhancement of metastatic potential.

Hair cycle-dependent expression of a nonspecific cross reacting antigen (NCA)-50/90-like molecule on follicular keratinocytes.

We found a carcinoembryonic antigen (CEA)-related antigen to be strongly expressed on a subset of follicular keratinocytes in normal human skin. The antigen was characterized immunohistochemically using a panel of antibodies against human CEA and CEA-related molecules. The expression of the antigen was studied in different phases of the hair cycle as well as in different hair types. Immunohistochemically, the antigen resembled the nonspecific crossreacting antigen (NCA) NCA-50/90 rather than true CEA. Its expression was limited to the innermost cells of the lowest segment of hair follicles in the catagen/telogen phases, being detected only where the hair shaft was attached to the epithelial hair sac in these phases. The same results were obtained for all hair types, i.e. terminal, vellus and intermediate hair. Coexpression of the antigen with both involucrin and differentiation-associated cytokeratins was noted in the cells in additional studies attempting to identify the exact subpopulations of follicular keratinocytes expressing the antigen in comparison with the expression of other functional markers. However, involucrin and the cytokeratins were also expressed in the upper segments of anagen as well as catagen/telogen hair follicles. Our findings strongly suggest that an NCA-50/90-like molecule is expressed cyclically on the innermost cells in the lowest segment of the outer root sheath only in catagen/telogen hair follicles. The cyclical expression in this specific subset of follicular keratinocytes only, in which the epithelial hair sac is attached to the hair shaft, may be associated with the stability of the attachment through the adhesive or, conversely, the repulsive function of CEA-related molecules, both of which have recently been proposed.

Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.

Opacity (Opa) protein variation in Neisseria gonorrhoeae is implicated in the pathogenesis of gonorrhea, possibly by mediating adherence and entry of the bacteria into human tissues. One particular Opa protein mediates adherence to epithelial cells through cell surface proteoglycans. Recently, two other eukaryotic cell receptors for Opa proteins have been reported. These receptors are members of a subgroup of the carcinoembryonic (CEA) gene family that express CD66 antigens. CEA family members vary in their distribution in human tissues. In order to understand whether interactions between Opa and CEA-like molecules play any role in pathogenesis, we must investigate which CEA family members are able to serve as Opa receptors and which Opa proteins recognize CEA-like molecules. We therefore studied HeLa cells that were stably transfected with five different members of the CEA family, i.e., CEA, CEA gene family member 1a (CGM1a), CGM6, nonspecific cross-reacting antigen (NCA), and biliary glycoprotein a (BGPa). We infected these transfectants with all possible 11 Opa variants of gonococcal strain MS11 and determined the numbers of bacteria that were bound and internalized. To account for proteoglycan-mediated adherence, infection assays were also performed in the presence of heparin. Our results show that of the 11 Opa variants of MS11, the same 4 recognized CGM1a and NCA. CGM6, however, was not recognized by any Opa variant of MS11. CEA was recognized by at least 9 of 11 Opa variants, and the BGP transfectants specifically bound and internalized 10 of 11 Opa variants and also bound Opa-negative gonococci. Immunofluorescence experiments showed that clustering of CEA-like molecules occurred upon infection of HeLa transfectants with those Opa variants that interacted specifically with the CEA family member. Together these data show that CEA family members are differentially recognized by gonococcal Opa variants, suggesting that this phenomenon may contribute to cell tropism displayed by gonococci.

Induction of carcinoembryonic antigen expression in a three-dimensional culture system.

MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture. However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures. To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process.

Induction of nonspecific cross-reacting antigen mRNA by interferon-gamma and anti-fibronectin receptor antibody in colon cancer cells.

Nonspecific cross-reacting antigen (NCA), a member of the carcinoembryonic antigen (CEA) family, shows increased expression levels in colorectal cancer tissues. To elucidate the mechanism, we observed the effect of interferon (IFN)-gamma on the expression level of NCA mRNA in colon cancer cell lines by quantitative reverse transcriptase-polymerase chain reaction assay. IFN-gamma induced NCA mRNA in three of four cell lines tested. The effect of anti-fibronectin receptor (FnR) antibody on the expression of NCA mRNA was then examined in the same manner. Colo201 and DLD-1 cells showed an increased expression level of NCA mRNA after stimulation with the antibody. On flow cytometry, FnR was expressed in only two, Colo201 and DLD-1, of the five cell lines tested. These findings indicate that IFN-gamma and anti-FnR antibody induce NCA mRNA in cultured colon cancer cell lines, suggesting that inflammatory response and cell-to-extracellular matrix interaction may be related to the increased expression of NCA mRNA in colorectal cancers in vivo.

Cis-acting elements required for expression of the nonspecific cross- reacting antigen gene in colorectal carcinoma

BACKGROUND & AIMS: The nonspecific cross-reacting antigen (NCA) is a cell adhesion molecule, and the messenger RNA for NCA is overexpressed in 92% of colorectal carcinomas. The aim of this study was to determine the cis-acting elements that may be responsible for the expression of NCA.METHODS: Deletion mutants of the 5' flanking sequence and first intron were ligated into chloramphenicol acetyltransferase expression vectors, transfected into Chinese hamster ovary (CHO), DiFi, and HT-29 human colorectal carcinoma cells, and BxPC-3 and MDAPanc-28 human pancreatic carcinoma cells. The amount of acetylated chloramphenicol was determined to show the presence and activity of cis-acting sequences.RESULTS: The 5' flanking sequence functions as a promoter in all of cell lines and contains negative regulatory and enhancer sequences. The minimal promoter is active in Chinese hamster ovary and HT-29, though not in MDAPanc-28 cells. The first intron contains a silencer capable of suppressing a heterologous promoter.CONCLUSIONS: The results show cis-acting sequences within and 5' to the NCA gene, which appear to play a role in the expression of this gene in malignant tissues. Some of these sequences function in a cell type-specific manner. Further studies of these elements may provide insight into the mechanisms of the abnormal growth patterns of malignant cells.(Gastroenterology 1997 Mar;112(3):776-82)

A colonic tissue architecture assay applied to human colon carcinoma cells.

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.

Role of nonspecific cross-reacting antigen, a CD66 cluster antigen, in activation of human granulocytes.

Nonspecific cross-reacting antigen (NCA) is the name of a family of highly glycosylated bacterial-binding receptors found on human granulocytes and other tissues. These glycoproteins are members of the immunoglobulin supergene family and are related structurally to carcinoembryonic antigen. In this study, we demonstrate that ligation of granulocyte NCA results in the activation of the cells, as measured by degranulation and the flux of intracellular calcium. These studies further the proposition that NCA has a function in the immune response of granulocytes against bacterial infections.

Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.