Biochemical characterization of a second family of human Ia molecules, HLA-DS, equivalent to murine I-A subregion molecules.

In mice, two families of structurally distinct Ia molecules, one designated I-A and the other I-E, have been identified and characterized. The HLA-DR molecules represent one family of human Ia molecules equivalent to the murine I-E molecules on the basis of amino acid sequence homology. We describe the isolation and biochemical characterization of a second family of human Ia molecules, designated HLA-DS for second D-region locus, equivalent to the murine I-A molecules. The human HLA-DS molecules consist of two polypeptide chains, DS alpha (37,000 mol wt) and DS beta (29,000 mol wt), with 73% amino acid sequence identity to the murine I-A molecules. Furthermore, the HLA-DS molecules are closely linked genetically to HLA-DR molecules, a situation analogous to that observed in mice. The similarity in molecular weights of the DR and DS molecules might explain why others have failed to identify the latter in man.

Lead inclusion bodies in osteoclasts.

Inclusion bodies occur frequently in the nuclei and rarely in the cytoplasm of osteoclasts in pigs with experimental lead poisoning. The light and electron microscope pictures of undemineralized sections are similar to those described for liver cord cells and renal tubular cells.

Functional organelles in prokaryotes: polyhedral inclusions (carboxysomes) of Thiobacillus neapolitanus.

The polyhedral inclusions of Thiobacillus neapolitanus have been isolated; they contain ribulose diphosphate carboxylase.

Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.

Highlights of protein structural analysis.

This article describes highlights of the state of the art in protein structural analysis, and comments on the current trends toward increased sensitivity and integrated isolation-structure methodologies.

Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression.

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region.

CAMK2γ in intestinal epithelial cells modulates colitis-associated colorectal carcinogenesis via enhancing STAT3 activation.

Inflammation is one of the major risk factors for cancer. Here, we show that calcium/calmodulin-dependent protein kinase II gamma (CAMK2γ) in intestinal epithelial cells (IECs) modulates inflammatory signals and promotes colitis-associated cancer (CAC) in mice. We have identified CAMK2γ as a downstream target of colitis-induced WNT5A signaling. Furthermore, we have shown that CAMK2γ protects against intestine tissue injury by increasing IEC survival and proliferation. Calcium/calmodulin-dependent protein kinase II gamma knockout mice displayed reduced CAC. Furthermore, we used bone marrow transplantation to reveal that CAMK2γ in IECs, but not immune cells, was crucial for its effect on CAC. Consistently, transgenic over-expression of CAMK2γ in IECs accelerated CAC development. Mechanistically, CAMK2γ in IECs enhanced epithelial signal transducer and activator of transcription 3 (STAT3) activation to promote survival and proliferation of colonic epithelial cells during CAC development. These results thus identify a new molecular mechanism mediated by CAMK2γ in IECs during CAC development, thereby providing a potential new therapeutic target for CAC.

Carbon cycling: the prokaryotic contribution.

Although the debate continues, the concept of global warming as a consequence of the increased production of 'greenhouse gases' via human activities is now widely accepted. The role of microbes, especially the prokaryotes, in the formation, trapping and retention of 'greenhouse gases' has, for the most part, been overlooked. The future requires that we pay close attention to these organisms for possible solutions to adverse global changes.

Demonstration of elevated anti-Lewis antibodies in sera of cancer patients using a carcinoembryonic antigen-polyethylene glycol immunoassay.

The serum levels of carcinoembryonic antigen (CEA) and CEA-binding proteins of 68 controls and 170 cancer patients were determined. The CEA levels were determined by a double-antibody radioimmunoassay using radiolabeled CEA, and the CEA-binding proteins were determined by an immunoassay utilizing radiolabeled CEA and polyethylene glycol. The major CEA-binding proteins in serum were anti-blood group antibodies as demonstrated by differential binding of serum proteins from A, B, or O positive individuals to radiolabeled CEA's which were previously shown to carry specific blood group determinants. No statistically significant differences were observed for the binding of control versus cancer patient sera to CEA-A (carrying blood group A1), except, as expected, A negative (O or B positive) individuals gave high binding to CEA-A, while A positive individuals gave low binding to CEA-A. Statistically significant differences were observed for controls versus cancer patients for binding to CEA-Lewisab (CEA-Leab). CEA-Leab-binding activity was higher in females and smokers in the control group, but this distinction was not found in the cancer patients. The high levels of anti-Leab antibodies may be explained in females by exposure to fetal antigens during pregnancies and in smokers or cancer patients by exposure to precursors to blood group substances. The sera of 7% of the patients with colonic carcinoma, 18% of the patients with breast carcinoma, and 23% of the patients with bronchogenic carcinoma bound more CEA-Leab than did the serum of the highest male control. A correlation between CEA-Leab-binding activity and the levels of serum CEA was found for patients with colonic carcinoma but was not significant for the groups with other cancers or the control group. Serial determinations of CEA-Leab-binding activity for 21 patients with bronchogenic carcinoma changed congruently with the serum levels of CEA in 12 cases. The significance of these results in terms of expression of immature blood group antigens, the subsequent production of antibodies against them, and the prognostic value of this response is discussed.

Sensitive detection of carbohydrate determinants on carcinoembryonic antigen preparations by lectin and antibody binding using polyethylene glycol.

Blood group determinants have been found in five carcinoembryonic antigen (CEA) preparations with a lectin and antibody-binding assay using polyethylene glycol 6000 to separate free from bound radiolabeled antigen. The assay described gives excellent sensitivity for the binding or inhibition of binding of various lectins or antibodies to CEA. One of the CEA preparations investigated has an A1 determinant, another has a B determinant, and all have H, Lea, Leb, and MN blood group determinants. In addition, all of the preparations tested bound concanavalin A. These findings are consistent with the idea that incomplete or unexpected glycosylation patterns occur in glycoproteins produced by tumor cells. Since antibodies directed against blood group substances cross react with carbohydrate determinants on CEA, clinical determinations of CEA or anti-CEA levels in serum may be adversely affected.

In vitro and in vivo footprint analysis of the promoter of carcinoembryonic antigen in colon carcinoma cells: effects of interferon gamma treatment.

The analysis of the carcinoembryonic antigen (CEA) promoter in the colon carcinoma lines HT-29 and SW403, using HeLa as a control, was performed using gel mobility shift assays and in vitro and in vivo footprinting before and after IFN-gamma treatment. Using a 332-bp probe extending from the start of translation (+1 to -331), we detected 4-5 specific complexes that increased with intensity with IFN-gamma treatment as measured by a gel mobility shift assay. In contrast, no complexes were observed for probes covering the regions -500 to -1000 and -1000 to -1500. DNase I in vitro footprinting with the 332-bp probe revealed three footprints, FP-I to FP-III, none of which changed during the course of IFN-gamma treatment. Using probes corresponding to each footprint, 6-7 specific complexes were observed by gel mobility shift assays. Although minor changes were observed on IFN-gamma treatment, no consistent pattern was observed for all cell lines tested. Several of the proteins involved in the promoter complexes were identified by antibody super shifts, UV cross-linking, and Southwestern blotting. FP-I bound an Sp1-like protein, binding to a GT box sequence, and USF (upstream regulatory factor). FP-II and FP-III bound Sp1, binding through the consensus sequence for a GC box. Lower molecular weight complexes of an unknown nature were observed with sequence specificity for both single- and double-stranded DNA. DNase I in vivo footprints confirmed the boundaries of FP-I to FP-III and revealed a fourth but weaker footprint, FP-IV. The strongest in vivo footprints were observed for SW403 cells, with weaker and no footprints observed for HeLa cells, thus correlating with the degree of CEA transcriptional activity (HeLa cells make no CEA mRNA). DNase I hypersensitive sites correlated well with the boundaries of the footprints and also revealed activity around the start of transcription (-110). The specific pattern for DNase I hypersensitivity for Sp1 in the CEA promoter was the same as observed for the SV40 early promoter. In vivo footprinting with dimethyl sulfate revealed protein binding at the Sp1 consensus sequences in FP-II and FP-III and at the USF consensus sequence in FP-I. We conclude that in vivo footprinting is the most accurate predictor of the state of transcriptional activity of the CEA gene. It is also likely that Sp1 and USF play a major role in CEA transcriptional activation and that the majority of IFN-gamma effects are at the posttranscriptional level.

Amino-terminal sequence of a carcinoembryonic antigen-like glycoprotein isolated from the colonic lavages of healthy individuals.

A glycoprotein has been isolated from the colonic lavages of healthy individuals that is immunologically equivalent to carcinoembryonic antigen purified from tumor tissue. The NH2-terminal sequence of the glycoprotein from normal colon lavages is Lys-Leu-Thr-lle-Glu-Ser-Thr-Pro-Phe-(Asn)-Val-Ala-Glu-Gly-Lys-Glu-Val-(Leu,lle)-(Leu,lle)-(Leu,lle)-Val-(His,Arg?)-?-(Leu,lle). This is homologous to the NH2-terminal sequence of 23 of the first 24 amino acids of carcinoembryonic antigen isolated from tumor tissue.

Differential regulation of carcinoembryonic antigen and biliary glycoprotein by gamma-interferon.

Carcinoembryonic antigen (CEA), biliary glycoprotein (BGP), and non-specific cross-reacting antigen (NCA) are three closely related cell surface glycoproteins induced by gamma-interferon (IFN-gamma) in colonic epithelial cells. Maximal induction of CEA by IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the colon carcinoma cell line HT-29 occurs at 5-6 days with maximal secreted levels at 14 ng/ml for IFN-gamma and 20 ng/ml for TNF-alpha. Cell viability was reduced to 67% of controls for TNF-alpha and to 36% for IFN-gamma. Dose-response curves showed maximal induction of CEA at 500 units/ml for TNF-alpha and at 200 units/ml for IFN-gamma. Combinations of the two lymphokines revealed that the CEA induction effects were additive and the cytotoxicity effects were synergistic. Northern blot analysis of HT-29 cells treated with IFN-gamma and probed with specific probes for BGP, CEA, and NCA showed a 2-fold increase in mRNA level for BGP, and a greater than 10-fold induction for CEA and NCA. Similar results were obtained for the SW403 cell line, but in the case of the LS174T cell line, CEA mRNA levels remained constant before and after IFN-gamma treatment, while BGP and NCA mRNA levels increased by 2-5-fold. Polymerase chain reaction analysis of the four alternatively spliced transcripts of BGP revealed no differential induction of one transcript over another by IFN-gamma. A comparison of the kinetics of induction of the mRNA levels for BGP and CEA by IFN-gamma in the HT29 cell line revealed a half-time of < 6 h for BGP and 48 h for CEA. The induction of CEA mRNA was completely inhibited with either cycloheximide (protein synthesis inhibitor) or actinomycin D (RNA synthesis inhibitor), but the induction of BGP mRNA was superinduced by cycloheximide. The difference in the kinetics of induction and effect of cycloheximide on CEA and BGP mRNAs suggest that the two genes are regulated differently in the same cell line. We conclude that the regulation occurs mainly at the posttranscriptional level for CEA and involves mRNA stability. BGP regulation may be more complex, involving transcriptional and posttranscriptional regulation, and more closely resembles the regulation of MHC class II mRNA by IFN-gamma in epithelial cells. The mRNA stability effects may be mediated by the dramatically different sequences present in the 3'-untranslated regions of CEA and BGP.

The 95-kilodalton membrane glycoprotein overexpressed in novel multidrug-resistant breast cancer cells is NCA, the nonspecific cross-reacting antigen of carcinoembryonic antigen.

Human breast carcinoma MCF-7/AdrVp cells display a novel multidrug resistance phenotype that is characterized by the overexpression of a 95-kDa membrane glycoprotein (p95) and by marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein MRP. p95 is also highly expressed in multidrug-resistant NCI-H1688 cells derived from a human small cell lung carcinoma. Deglycoslyated p95 from NCI-H1688 cells was isolated by two-dimensional gel electrophoresis and then digested with trypsin. The tryptic peptides were analyzed by mass spectrometry and microsequencing. These analyses identified p95 to be identical to NCA-90, the nonspecific cross-reacting antigen related to the carcinoembryonic antigen (CEA). Further confirmation that p95 is indeed NCA-90 was obtained by Northern and Western blot studies using probes or antibodies specific for p95, NCA-90, or CEA family members. Western blot studies also revealed that CEA itself is overexpressed in MCF-7/AdrVp cells compared to parental MCF-7/W cells. The enforced expression of NCA-90 protein in HeLa cells stably transfected with NCA-90 cDNA did not result in increased resistance of the transfected cells to daunorubicin or a decrease in daunorubicin accumulation in the transfected cells compared to cells transfected only with the expression vector. However, a recent report by H. Kawaharata et al. (Int. J. Cancer, 72: 377-382, 1997) of diminished accumulation, retention, and cytotoxicity of doxorubicin in EJNIH3T3 cells in which enforced expression of CEA was accomplished leaves open the possibility that the overexpression of CEA, possibly in combination with that of NCA-90, could account at least in part for the drug resistant phenotype displayed by MCF-7/AdrVp cells.

Correlation of DNA hypomethylation with expression of carcinoembryonic antigen in human colon carcinoma cells.

Using an assay based on the binding of a carcinoembryonic antigen (CEA)-specific monoclonal antibody, we have examined the expression of carcinoembryonic antigen genes in human colon tumor and normal fibroblast cell lines. CEA expression was not detectable in the normal fibroblast cell lines, whereas varying levels of high CEA expression were found in the colon tumor cell lines LS-174T, GEO, and WIDR. We have used a 550-base pair CEA probe derived from cloned complementary DNA to carry out Southern analysis of the DNA isolated from the normal and colon tumor cell lines. At high stringency, the CEA probe detected seven BamHI fragments in all DNAs analyzed. At low stringency, however, 14 BamHI fragments ranging from 1.5 to 23 kilobases were detected. Results of the Southern analysis demonstrate no amplification or rearrangement of the CEA genes in tumor cells. We used methylation-sensitive restriction endonucleases, HpaII and HhaI, to compare the degree of methylation of CEA family of genes in normal and colon tumor cell lines. Our results demonstrate that the CEA family of genes exists in a state of hypermethylation in the normal cell lines. In contrast, the CEA gene(s) are relatively hypomethylated in the tumor cell lines, suggesting a correlation between the state of methylation and degree of expression of the CEA gene(s). A comparison of the state of methylation of the CEA gene(s) in cells before and after treatment with the gamma-interferon (which up-regulates CEA steady-state mRNA levels) showed no detectable difference in the degree of DNA methylation. The segments of CEA genes that are hypermethylated in normal cells, but are hypomethylated in tumor cells, were also identified. Thus, these studies may help identify the sites of methylation that are crucial for the control of CEA gene regulation.

Expression of complementary DNA and genomic clones for carcinoembryonic antigen and nonspecific cross-reacting antigen in Chinese hamster ovary and mouse fibroblast cells and characterization of the membrane-expressed products.

Complementary DNA clones coding for both carcinoembryonic antigen (CEA), a well characterized colonic tumor marker, and nonspecific cross-reacting antigen (NCA), a related antigen, were expressed in Chinese hamster ovary (CHO) cells and L-cells (mouse fibroblasts). A genomic clone coding for CEA was also expressed in CHO cells. Positive clones were identified by fluorescence flow cytometry and enzyme-linked immunosorbent assay. Membrane location of the recombinant CEA and NCA was confirmed by indirect immunofluorescence labeling of the transfectants, followed by visualization under a fluorescence microscope. The apparent molecular weight of the expressed CEA and NCA were 180,000 and 96,000, respectively, for both cell lines, as determined by immunoblot analysis. The CEA and NCA expressed on CHO cells were sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the CEA and NCA proteins on L-cells were resistant to removal by PI-PLC. Unlike NCA, which contains three methionine residues, the only methionine in CEA is in the C-terminal hydrophobic domain. This domain in CEA was shown to be removed and replaced by a phosphatidylinositol glycan (PI-G) anchor (Hefta et al., Proc. Natl. Acad. Sci. USA, 85: 4648-4652, 1988). The recombinant CEA from both CHO cells and L-cells could be labeled with [3H]-ethanolamine (a component of the PI-G anchor) but not with [35S] methionine, whereas the recombinant NCA could be labeled with both [3H]ethanolamine and [35S]methionine. The labeling studies and PI-PLC treatment results are consistent with the CEA and NCA expressed on CHO cells possessing a PI-G anchor. The CEA expressed on the L-cell transfectants may contain a PI-G anchor which is resistant to cleavage by PI-PLC. In addition, the membrane-bound and secreted levels of CEA from the CHO and L-cell transfectants were determined.

Modulation of carcinoembryonic antigen messenger RNA levels in human colon carcinoma cells by recombinant human gamma-interferon.

Recombinant human interferons have recently been shown to enhance tumor antigen expression, including carcinoembryonic antigen (CEA), on the surface of human carcinoma cells, which results in an increase in the targeting of antitumor monoclonal antibodies (MAb) in vivo. We report here the effect of recombinant human gamma-interferon (HuIFN-gamma) on the expression of human CEA and its related transcripts in several human colon carcinoma and normal human fibroblast cell lines. The colon tumor cell lines HT-29, WiDr, and LS-174T were each shown to express different constitutive levels of CEA glycopeptide, as measured by the binding of the CEA-specific MAb COL-4. Treatment with HuIFN-gamma enhanced the level of binding of COL-4 in total cell extracts of HT-29 and WiDr cells 2.5- and 6.5-fold, respectively. Using a CEA complementary DNA probe, this increase in MAb binding was shown to be accompanied by a 6- to 11-fold increase in the steady state levels of three CEA transcripts with sizes of 4.2, 3.5, and 2.8 kilobases. On the other hand, HuIFN-gamma treatment had no effect on the level of COL-4 binding or expression of CEA transcripts in LS-174T colon carcinoma cells, which are high constitutive expressors of CEA glycoprotein. Normal human fibroblast cell lines MRC-5 and WI38 had no detectable cytoplasmic CEA glycopeptide levels nor did they contain detectable levels of CEA mRNA, either before or after treatment with HuIFN-gamma. In contrast, HuIFN-gamma induced the de novo expression of the normal major histocompatibility complex class II antigen, HLA-DR, on HT-29 and WiDr colon cancer cells as well as the two fibroblast cell lines. Treatment of the LS-174T cell line with HuIFN-gamma did not result in the induction of class II HLA-DR antigen. These observations suggest that some common factors may be involved in the regulation of the CEA and class II histocompatibility genes. In addition, the demonstration that HuIFN-gamma enhances CEA expression in some carcinoma cell lines but fails to induce de novo expression of CEA transcripts in fibroblasts supports the potential application of HuIFN-gamma in enhancement of tumor targeting of antitumor MAbs and adds to our understanding of the mechanism of gamma-interferon-mediated up-regulation of some tumor antigens.

Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping.

Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.

Testis imaging with 111In-labeled anticarcinoembryonic antigen monoclonal antibody: identification of carcinoembryonic antigen in normal germ cells.

A monoclonal antibody to carcinoembryonic antigen (CEA) labeled with 111In (Indacea) was used to image tumors in patients with colorectal cancer. The anti-CEA antibody has a high affinity (2.6 X 10(10) M-1) for CEA and does not cross-react with normal cross-reacting antigen, biliary glycoprotein-1, or tumor-extracted, CEA-related antigen. During the course of these studies, it was noted that a significant number of male patients (20 of 27, 74%) showed uptake of Indacea in the testes. In order to determine if the Indacea uptake was specific, 20 testicular specimens were analyzed by immunohistological methods using five different anti-CEA CEA monoclonal antibodies recognizing five different epitopes on CEA. In 18 cases (90%) germ cells were uniformly stained by all five antibodies. Fresh frozen testis tissue was homogenized in water and precipitated with 1.0 M perchloric acid. The supernatant contained a CEA-like material as measured by an enzyme immunoassay specific for CEA. The same supernatant was radiolabeled with 125I and immunoprecipitated with anti-CEA monoclonal antibodies. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the immunoprecipitates revealed a single species (Mr 180,000) which was indistinguishable from CEA. This study documents the first description of CEA in the germ cells of normal testis. The CEA in the testis was accessible to circulating monoclonal antibodies in the majority of male patients tested.

Identification and characterization of new antigenic fragments related to carcinoembryonic antigen in adult feces.

Antigens in human adult feces related to carcinoembryonic antigen (CEA) were analyzed with respect to their molecular masses, CEA domain compositions, and N-terminal amino acid sequences. By avoiding perchloric acid treatment, new fecal antigens related to CEA were identified. The fecal antigens revealed by Western blot were M(r) 78,000, 70,000, 60,000, 50,000, 44,000, 36,000, 33,000, and 25,000 and a species M(r) less than or equal to 14,000. Unlike native CEA, all of the fecal antigens were very poorly soluble in perchloric acid and did not bind to concanavalin A, suggesting that they had undergone significant deglycosylation in the digestive tract. The major fecal antigens were purified by immunoaffinity chromatography and their N-terminal amino acid sequences determined. FA78, FA60, FA33, and the M(r) less than or equal to 14,000 antigen had the N-terminal amino acid sequence of the CEA N-domain, and FA44 and FA25, the sequence of the CEA A2 domain. The CEA domain compositions of the fecal antigens were investigated by probing them with anti-CEA monoclonal antibodies of known domain specificities. The N-terminal amino acid sequences, immunoreactivities with anti-CEA monoclonal antibodies, and apparent molecular masses of the fecal antigens allowed the following domain assignments (based on CEA as N-A1B1-A2B2-A3B3): FA78, N-A1B1-A2B2-A3B3; FA60, N-A1B1-A2B2; FA44, A2B2-A3B3; FA33, N-A1B1; and FA25, A2B2. The M(r) less than or equal to 14,000 antigen was shown to be the N-domain of CEA or nonspecific cross-reacting antigen. FA36 was assigned the N-AB domain structure of nonspecific cross-reacting antigen. The results suggested that FA78, FA60, FA44, FA33, and FA25 were degradation products (including deglycosylation and proteolysis) of CEA and that FA36 was a degradation product of nonspecific cross-reacting antigen.