Molecular cloning of a gene for a member of carcinoembryonic antigen (CEA) gene family; signal peptide and N-terminal domain sequences of nonspecific crossreacting antigen (NCA)

A 2073-base pair DNA fragment containing a part of gene for a member of carcinoembryonic antigen (CEA) gene family, has been isolated from human DNA library after screening with 32P-labeled 53-mer oligodeoxyribonucleotide corresponding to N-terminal 18 amino acids of CEA gene family and cDNA encoding CEA (1,2). The fragment contains two exons; the one encodes the first 60% of signal peptide and the other the rest of it in addition to 107 amino acids which correspond to the N-terminal domain of CEA (1,2). Apparently, the second intron is inserted between the first and the second nucleotides of the codon for 108th amino acid. The presence of Ala instead of Val as the 21st amino acid of the N-terminal domain indicates that the exon encodes nonspecific crossreacting antigen (NCA).     

Long-range chromosomal mapping of the carcinoembryonic antigen (CEA) gene family cluster

A long-range physical map of the carcinoembryonic antigen (CEA) gene family cluster, which is located on the long arm of chromosome 19, has been constructed. This was achieved by hybridization analysis of large DNA fragments separated by pulse-field gel electrophoresis and of DNA from human/rodent somatic cell hybrids, as well as the assembly of ordered sets of cosmids for this gene region into contigs. The different approaches yielded very similar results and indicate that the entire gene family is contained within a region located at position 19q13.1-q13.2 between the CYP2A and the D19S15/D19S8 markers. The physical linkage of nine genes belonging to the CEA subgroup and their location with respect to the pregnancy-specific glycoprotein (PSG) subgroup genes have been determined, and the latter are located closer to the telomere. From large groups of ordered cosmid clones, the identity of all known CEA subgroup genes has been confirmed either by hybridization using gene-specific probes or by DNA sequencing. These studies have identified a new member of the CEA subgroup (CGM8), which probably represents a pseudogene due to the existence of two stop codons, one in the leader and one in the N-terminal domain exons. The gene order and orientation, which were determined by hybridization with probes from the 5' and 3' regions of the genes, are as follows: cen/3'-CGM7-5'/3'-CGM2-5'/5'-CEA-3'/5'-NCA-3'/5'-CGM1- 3'/3'-BGP-5'/3'- CGM9-5'/3'-CGM6-5'/5'-CGM8-3'/PSGcluster/qter.     

Multiple Smith-degradations of carcinoembryonic antigen (CEA) and of asialo CEA

Carcinoembryonic antigen (CEA) and asialo CEA were subjected to multiple Smith-degradation (i.e., for each degradation, application in sequence of periodate oxidation, borohydride reduction, and mild hydrolysis with acid; borohydride-t was substituted for unlabelled borohydride). High yields of modified glycoproteins were obtained at each stage. After three complete degradations and a further periodate-borohydride-t treatment, the carbohydrate content of CEA and of asialo CEA had decreased from 45–50% to 11–12% (i.e., 90% removal of carbohydrate). Glycerol was always one of the products obtained after each degradation, but threitol and erythritol were not detected. The second degradation caused a substantial loss of 2-acetamido-2-deoxyglucose, which is consistent with the location of some of this monosaccharide towards the terminal (non-reducing) end of the oligosaccharides. The “core” region of the oligosaccharides is composed of galactose, mannose, and 2-acetamido-2-deoxyglucose. After the fourth oxidation, 2-acetamido-2-deoxyglucose was 50–60% of the total content of residual carbohydrate. After the first degradation, there was a progressive loss in antigenic activity, but this was associated with a small amount of hydrolysis of the protein moiety of CEA.     

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

Summary Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.     

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.     

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.     

The carcinoembryonic antigen (CEA) contains multiple immunoglobulin-like domains

The amino acid sequences of human carcinoembryonic antigen deduced from the cDNA sequences have been analysed. This antigen contains seven extracellular domains (previously recognized three highly repetitive domains are further divided into A and B subdomains each) which are strikingly homologous to each other and to immunoglobulin variable regions, poly-Ig receptor and Thy 1.1. The N-terminal domain lacks immunoglobulin-like fold but the other six domains have, suggesting that the CEA belongs to immunoglobulin superfamily.     

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.     

Colorectal hyperplasia and dysplasia due to human carcinoembryonic antigen (CEA) family member expression in transgenic mice

CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer.     

A carcinoembryonic antigen (CEA) binding protein from ascites influences CEA uptake by macrophages

A variant of CEA which is less readily endocytosed by macrophages has been isolated from malignant ascites. In vivo, CEA is cleared more slowly by the liver (t1/2 = 15.1 minutes) than CEAs isolated from hepatic metastases (t1/2 = 3.1 minutes). In vitro, rat and human Kupffer cells and rat alveolar macrophages endocytose this CEA less effectively. This slow clearing form of CEA is associated with a smaller (45kD) acidic glycoprotein (CORA) with which it forms a stable complex. CORA can be visualized on reducing gels but not on non reducing gels or by HPLC run under non reducing conditions. This suggests a non-covalent complex between the two glycoproteins. Analysis of protein conformation by circular dichroism revealed changes in the ascites CEA consistent with binding of CORA to the molecule. Western blot showed that CORA crossreacts with antisera to alpha 1-acid glycoprotein and double immunodiffusion demonstrated cross-reactivity but not identify. Sequencing of CNBr peptides showed sequence homology with alpha 1-acid glycoprotein but areas of unique sequence were also found. It is suggested that binding of CORA to CEA blocks the macrophage receptor binding of CEA.     

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.     

A carcinoembryonic antigen (CEA) specific monoclonal hybridoma antibody that reacts only with high-molecular-weight CEA

Summary A monoclonal antibody secreted by a hybridoma, formed by the fusion of a spleen cell from an immunized mouse and a myeloma cell, has been shown to react only with a form of carcinoembryonic antigen (CEA) with a molecular weight of 180,000 daltons. The monoclonal antibody precipitates a 180,000-dalton form of CEA from colorectal carcinoma cell lysates, purified iodinated CEA and iodinated CEA-S, but does not precipitate the cross-reactive antigen (NCA) from normal colon.     

Immunohistochemical detection of carcinoembryonic antigen (CEA) in non-cancerous and cancerous gastric mucosa

Carcinoembryonic antigen (CEA) was stained by the PAP immunoperoxidase method in cancerous and non-cancerous gastric mucosa of 40 patients (25 non-cancerous dyspeptic patients and 15 patients with gastric carcinoma). The pattern of CEA localization was apical or membranous-cytoplasmic and immuno-reactivity was mild (+), moderate (++) or intensive ( ). No CEA immunoreactivity was detected in normal gastric mucosa whereas it was marked in gastric mucosa of non-cancerous dyspeptic patients with chronic atrophic gastritis and dysplasia (intense). In patients with superficial gastritis and epithelial hyperplasia it was mild or absent. The CEA localization pattern was also apical in non-cancerous dyspeptic patients with microscopic changes, e.g. superficial or chronic atrophic gastritis, epithelial hyperplasia and dysplasia, and in non-cancerous mucosa and cancerous tissue of patients with well (G1) and moderately (G2) differentiated adenocarcinoma.     

A predicted three-dimensional structure for the carcinoembryonic antigen (CEA).

A three-dimensional model for the carcinoembryonic antigen (CEA) has been constructed by knowledge-based computer modelling. Each of the seven extracellular domains of CEA are expected to have immunoglobulin folds. The N-terminal domain of CEA was modelled using the first domain of the recently solved NMR structure of rat CD2, as well as the first domain of the X-ray crystal structure of human CD4 and an immunoglobulin variable domain REI as templates. The remaining domains were modelled from the first and second domains of CD4 and REI. Link conformations between the domains were taken from the elbow region of antibodies. A possible packing model between each of the seven domains is proposed. Each residue of the model is labelled as to its suitability for site-directed mutagenesis.     

Localization of mucinous-like carcinoma associated antigen (MCA) in breast pathology: comparison with carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA)

The topographic distribution of a mucinous-like cancer antigen (MCA) recognized by a monoclonal antibody b-12 (MAb b-12) was assessed in benign (38) and malignant (66) breast tissues. The reactivity of MAb b-12 showed a good selectivity for breast tissues, reacting both with normal tissues and breast cancer. The degree of MCA expression was evaluated in the various groups of breast pathology adopting quantitative criteria of assessment. With the criteria of evaluation adopted, strong staining was observed in 71.4% breast carcinomas. The most positive reaction was demonstrated in mucinous carcinoma. MCA distribution in breast tissue was compared with the distribution of two other antigens, CEA and TPA. Reactivity of MAb b-12 was higher than the reactivity shown by the anti-CEA and anti-TPA antibodies.