lewis-x-antigen and Colonic-Neoplasms

Topics: Adenomatous Polyposis Coli; Antigens, Neoplasm; Colonic Neoplasms; Glycolipids; Heterozygote; Humans; Intestinal Mucosa; Lewis X Antigen; Oncogenes

The metastatic potential of tumors is dependent on the cell to cell adhesion by cell surface carbohydrate antigens. Thus, expression of sialyl Lewis(a), which is one of the important molecules of cell surface carbohydrates, may serve as a prognostic marker of aggressive and metastasizing tumor growth. However, the prognostic value of sialyl Lewis(a) expression in colon cancer is still controversial.. In this study, we investigated the expression of sialyl Lewis(a) antigen in 233 colon cancer specimens from patients who were registered in a prospective adjuvant immunochemotherapy clinical trial. The clinical course and the prognosis of the patients were evaluated after all the immunohistochemical analyses had been performed.. Sialyl Lewis(a) expression levels were correlated with both overall survival (P = 0.0006) and disease-free survival (P = 0.004) in all patients with the log-rank test. This result could be assumed to have been influenced by the difference in the metastatic preponderance in a high versus low sialyl Lewis(a) expression in the tumor cells.. This prospective study in a randomized controlled trial suggests that sialyl Lewis(a) expression levels may serve as an indicator of the metastatic potential of colon cancer cells, which would strongly predict the prognosis.

Topics: Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Chemotherapy, Adjuvant; Colonic Neoplasms; Female; Humans; Immunochemistry; Lewis Blood Group Antigens; Lewis X Antigen; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Prospective Studies; Survival Rate

Sialyl Lewis x (sLe

Topics: Adenocarcinoma; Cell Adhesion; Cell Line, Tumor; Colonic Neoplasms; Humans; Lewis X Antigen; MAP Kinase Signaling System; MicroRNAs; Neoplasm Proteins; P-Selectin; RNA, Neoplasm; Sialyl Lewis X Antigen

To discover and confirm blood-based colon cancer early-detection markers.. We created a high-density antibody microarray to detect differences in protein levels in plasma from individuals diagnosed with colon cancer <3 years after blood was drawn (ie, prediagnostic) and cancer-free, matched controls. Potential markers were tested on plasma samples from people diagnosed with adenoma or cancer, compared with controls. Components of an optimal 5-marker panel were tested via immunoblotting using a third sample set, Luminex assay in a large fourth sample set and immunohistochemistry (IHC) on tissue microarrays.. In the prediagnostic samples, we found 78 significantly (t-test) increased proteins, 32 of which were confirmed in the diagnostic samples. From these 32, optimal 4-marker panels of BAG family molecular chaperone regulator 4 (BAG4), interleukin-6 receptor subunit beta (IL6ST), von Willebrand factor (VWF) and CD44 or epidermal growth factor receptor (EGFR) were established. Each panel member and the panels also showed increases in the diagnostic adenoma and cancer samples in independent third and fourth sample sets via immunoblot and Luminex, respectively. IHC results showed increased levels of BAG4, IL6ST and CD44 in adenoma and cancer tissues. Inclusion of EGFR and CD44 sialyl Lewis-A and Lewis-X content increased the panel performance. The protein/glycoprotein panel was statistically significantly higher in colon cancer samples, characterised by a range of area under the curves from 0.90 (95% CI 0.82 to 0.98) to 0.86 (95% CI 0.83 to 0.88), for the larger second and fourth sets, respectively.. A panel including BAG4, IL6ST, VWF, EGFR and CD44 protein/glycomics performed well for detection of early stages of colon cancer and should be further examined in larger studies.

Topics: Adaptor Proteins, Signal Transducing; Adenoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; CA-19-9 Antigen; Case-Control Studies; Colonic Neoplasms; Cytokine Receptor gp130; Early Detection of Cancer; ErbB Receptors; Female; Humans; Hyaluronan Receptors; Lewis X Antigen; Male; Middle Aged; Oligosaccharides; Protein Array Analysis; von Willebrand Factor

Circulation tumor cells (CTCs) in the bloodstream of early-stage cancer patients carry the important information about valuable biomarkers and biological properties of primary tumor. However, detection and capture of CTCs are challenging owing to their low concentrations. Traditional technologies have the limited detection sensitivity and the low capture efficiency. We, herein, report an effective approach to specifically bind and capture colon cancer HT29 cells by using multiple Sialyl Lewis X antibodies (aSlex)-conjugated PAMAM dendrimers. The conjugation was characterized by using atom force microscope, UV and fluorescence measurements. The capturing and regulating HT29 cells by the aSlex-coated dendrimer conjugate were analyzed by microscopy and flow cytometry. The results indicated that the conjugate showed the enhanced capture of HT29 cells in a concentration-dependent manner and the maximum capture efficiency of 77.88% was obtained within 1 h-exposure. G6-5aSlex-FITC conjugate showed capture efficiency better than FITC-G6-COOH-5aSlex conjugate. G6-5aSlex-FITC conjugate could specifically capture HT29 cells even when the target HT29 cells were diluted with the interfering cells (e.g., RBCs) to a low concentration. The capture resulted in a concentration-dependent restraint of the cell activity. In conclusion, the aSlex-coated dendrimer conjugate displayed the great potential in capturing and restraining colorectal CTCs in blood.

Topics: Antibodies; Colonic Neoplasms; Dendrimers; Drug Delivery Systems; HT29 Cells; Humans; Lewis X Antigen; Neoplastic Cells, Circulating

The histo blood group carbohydrate Sd(a) antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe(x), involved in metastasis. However, down regulation of sLe(x) expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sd(a) and to inhibit sLe(x) expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLe(x) synthesis in colon, resulted in a cumulative inhibition of sLe(x). In normal colon samples a significant relationship between sLe(x) expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLe(x) inhibition in vivo.

Topics: Adult; Aged; Cell Cycle Proteins; Cell Line, Tumor; Colonic Neoplasms; Female; Fucosyltransferases; Gastrointestinal Tract; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Lewis X Antigen; Male; Middle Aged; N-Acetylgalactosaminyltransferases; Nuclear Proteins; Sialyl Lewis X Antigen

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galβ1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carcinoembryonic Antigen; Cell Adhesion Molecules; Colon; Colonic Neoplasms; Galectin 3; Gastric Mucosa; Glycosylation; GPI-Linked Proteins; Humans; Intestinal Mucosa; Lectins, C-Type; Lewis Blood Group Antigens; Lewis X Antigen; Mannose; Mucin-1; Peanut Agglutinin; Receptors, Cell Surface

Glycosylation of the tumour cell surface is of importance in metastasis formation as indicated by lectin-binding studies. In particular, binding of the lectin HPA is associated with metastasis formation, both in clinical studies and in xenograft models of breast and colon cancer. Here we examined if there is an association between the HPA-positive glycotopes of metastasizing cancer cells and selectin-binding properties.. Glycotope expression of human breast and colon cancer cells (MCF7, T47D, HBL100, HT29, SW480) grown in culture and xenografted into SCID mice were investigated by histochemical analysis.. HPA binding was observed in metastasizing breast and colon cancers and not in non-metastasizing ones. In colon cancer, E-selectin binding and expression of the selectin ligands CD15s and CA19-9 was higher in metastatic HT29 than in non-metastatic SW480 cells, especially when cells were grown in vitro. In breast cancer, E-selectin binding, CD15s and CA19-9 expression were independent of the metastatic potential. P-Selectin binding was slightly higher in metastasizing breast cancer cells (MCF7, T47D) than in non-metastasizing HBL100 cells.. Binding to E-selectin and expression of E-selectin ligands of colon cancer cells grown in vitro is associated with metastasis formation in a xenograft model. However, analysis of selectin ligands is of limited predictive value for the metastatic potential of breast cancer cells in our xenograft model.

Topics: Animals; Breast Neoplasms; CA-19-9 Antigen; Colonic Neoplasms; E-Selectin; Female; Glycosylation; Humans; Immunoenzyme Techniques; Lectins; Lewis X Antigen; Mice; Mice, Inbred BALB C; Mice, SCID; P-Selectin; Sialyl Lewis X Antigen

Colon cancer cells express the carbohydrate determinant sialyl Lewis(x), while they exhibit markedly decreased the expression of its sulfated derivative, sialyl 6-sulfo Lewis(x). In contrast, normal colonic epithelial cells strongly express sialyl 6-sulfo Lewis(x), but they virtually do not express sialyl Lewis(x). Impaired sulfation was therefore suggested to occur during the course of malignant transformation of colonic epithelial cells and was assumed to be responsible for the increased sialyl Lewis(x) expression in cancers. To elucidate the molecular biological background of the impaired sulfation in cancers, we studied the expression levels of mRNA for 6-O-sulfotransferase isoenzymes, PAPS synthases and transporters, and a cell membrane sulfate transporter, DTDST, in cancer tissues. The most striking decrease in cancer cells compared with nonmalignant epithelial cells was noted in the transcription of the DTDST gene (P = 0.0000014; n = 20). Most cultured colon cancer cells had a diminished DTDST transcription, which was restored when cultured with histone deacetylase inhibitors. Suppression of DTDST transcription under the control of a tet-off inducible promoter resulted in increased sialyl Lewis(x) expression and reduced sialyl 6-sulfo Lewis(x) expression. Unexpectedly, the growth rate of the cancer cells was markedly enhanced when transcription of DTDST was suppressed. These results show that the decrease in the transcription of the sulfate transporter gene is the major cause of decreased expression of sialyl 6-sulfo Lewis(x) and increased expression of sialyl Lewis(x) in colon cancers. The results also suggest that the diminished DTDST expression is closely related to enhanced proliferation of cancer cells.

Topics: Anion Transport Proteins; Blotting, Western; Butyrates; Cell Proliferation; Chromatin Immunoprecipitation; Colon; Colonic Neoplasms; Epigenesis, Genetic; Flow Cytometry; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Immunoenzyme Techniques; Lewis X Antigen; Middle Aged; Oligosaccharides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialyl Lewis X Antigen; Sulfate Transporters; Sulfotransferases

The purpose of the study was to develop a flow cytometric assay for quantitative determination of adhesive interactions of human endothelial cells (ECs) with tumor cells. EC lines established from human lymph node, appendix, lung, skin and intestine microvessels, labeled with PKH26-GL fluorescent dye, were grown to confluency in 24-well TC plates. Human colon adenocarcinoma cell suspension was overlaid onto labeled ECs, and allowed to adhere for 20 min at 4 degrees C under static conditions. Non-adhering cells were collected first, and adhering tumor cells together with ECs were detached from the culture plate. Collected cell fractions were evaluated by flow cytometry. Results were re-calculated as a ratio (R) of adhering colon carcinoma cells per one EC. We demonstrated that immortalized human microvascular ECs preserved their organ specificity. Colon carcinoma cells adhere preferentially to ECs of intestine origin. The immunofluorescent staining of adhering and non-adhering cancer cell subpopulations has revealed an augmented level of Lewis(x) antigen on adhering cancer cells. The organ specificity of endothelial cell interactions with colon carcinoma cells demonstrated in static conditions was verified and confirmed with flow adhesion assay. The method elaborated is suitable for quantifying of tumor cells adhering to ECs, with simultaneous evaluation of cell surface phenotypic markers of both partner cells participating in adhesive interactions. Validated by comparison to dynamic shear stress adhesion assay in blood flow reconstituted conditions this assay greatly facilitates evaluation of tumor cell-endothelial cell mutual interactions taking place during metastatic process.

Topics: Cell Adhesion; Cell Communication; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; Endothelial Cells; Flow Cytometry; Fluorescent Antibody Technique; Humans; Intestine, Small; Lewis X Antigen; Lung; Lymph Nodes; Neoplasms; Oligosaccharides; Sialyl Lewis X Antigen; Skin

We have previously demonstrated tumor-specific alpha1,2fucosylation, which is associated with resistance of tumor cells to anticancer treatment in human colorectal tumor tissues. By using the YB-2 monoclonal antibody, the resulting products have been identified as Y, Le(b), and H type 2 antigens in colorectal tumor tissues.. Immunohistochemical analyses of colorectal cancer tissues (74 specimens) were performed with a newly established mouse monoclonal antibody, YB-3 specifically recognizing H disaccharide (Fucalpha1,2Galbeta) structures, and anti-A, anti-B, YB-2, and anti-sialyl Lewis X (SLX) antibodies, together with the analyses of glycosyltransferases involved in the synthesis of ABH antigens in the same tissues.. The YB-3 antibody enabled us to detect colorectal tumors, particularly tumors in the distal large intestine and the rectum, with high sensitivity (74.3%) and specificity (100%). From immunohistochemical and enzymatic analyses of colorectal tissues, we found that once alpha1,2fucosylation had proceeded in tumor tissues, blood group A or B antigen was also synthesized in approximately half of the tissues of A or B blood type, but not in their normal tissues. A correlation of survival rate with immunostaining of tissues was found only by YB-3 antibody and not by anti-A, anti-B, or anti-SLX antibody.. As a predictor of postoperative prognosis of patients with colorectal cancer, immunodetection of alpha1,2fucosylated antigens with the YB-3 antibody seemed to be superior to blood groups A, B, or SLX antigen in colorectal tumor tissues.

Topics: ABO Blood-Group System; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Colonic Neoplasms; Disaccharides; Female; Forecasting; Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase; Glycosyltransferases; H-2 Antigens; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Male; Oligosaccharides; Prognosis; Rectal Neoplasms; Sialyl Lewis X Antigen

The carbohydrate determinants Sd(a) and sialyl Lewis x (sLex) both result from substitution of an alpha2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) beta1,4-linked to Gal and the second by an alpha1,3-linked fucose on N-acetylglucosamine. The Sd(a) antigen is synthesized by Sd(a) beta1,4-N-acetylgalactosaminyltransferase II (beta4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between beta4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether beta4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. beta4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sd(a) antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sd(a) antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, beta4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sd(a) and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of alpha1,3-FucTs on 3'-sialyllactosamine parallels that of beta4GalNAcT-II; and (iii) both beta4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription-polymerase chain reaction analysis reveals that the transcripts of beta4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3'-sialyllactosamine acceptors and that beta4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues.

Topics: Caco-2 Cells; Carbohydrate Conformation; Carbohydrates; Cell Line, Tumor; Colon; Colonic Neoplasms; DNA, Complementary; Exons; Fucosyltransferases; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Lewis X Antigen; Models, Biological; N-Acetylgalactosaminyltransferases

To improve the survival rate of patients with colon cancer, liver metastases must be eradicated in a clinically occult state. This study was designed to find a predictor for potential liver metastases or micrometastases in colon cancer.. Peripheral blood samples and tumor specimens were obtained from 36 patients with colon cancers. The blood samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and the expression of sialylated carbohydrates was also investigated in the tumors immunohistochemically.. A carcinoembryonic antigen (CEA)-specific signal in the blood was detected in 9 of 12 (75%) patients with liver metastasis and in 8 of 24 (33%) patients without liver metastasis, respectively (P < 0.05). The positive rates of sialyl Lewis A (sLeA) and sialyl Lewis X (sLeX) were 36.3% and 40% in tumors without liver metastasis vs. 58.3% and 100% with liver metastasis, respectively. Within a year after surgery, liver metastases became clinically evident in three of the four patients without liver metastasis who showed a CEA-positive signal in their blood preoperatively and who had tumors with a strong expression of sLeX.. A combination of both markers may provide prognostic information for liver metastases in colon cancer.

Topics: Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lewis X Antigen; Liver Neoplasms; Oligosaccharides; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Sialyl Lewis X Antigen

Extranodal Hodgkin disease presenting as a primary localized neoplasm is uncommon, with rare case reports describing primary sites other than lymph nodes. The gastrointestinal tract is the most frequent site of involvement by extranodal Hodgkin disease, typically involving the stomach or small bowel. To date, we have been able to find only one fully documented case of Hodgkin disease of the sigmoid colon confirmed by immunohistochemical studies. We report a case of extranodal Hodgkin disease involving the transverse colon, presenting as inflammatory bowel disease and documented by light microscopic, immunohistochemical, cytogenetic, and molecular studies.

Topics: Adult; Colonic Neoplasms; Female; Hodgkin Disease; Humans; Immunohistochemistry; Ki-1 Antigen; Lewis X Antigen; Vimentin

Topics: Adenocarcinoma; Antigens, Neoplasm; Carcinoma, Papillary; Colonic Neoplasms; Humans; Immunohistochemistry; Lewis Blood Group Antigens; Lewis X Antigen; Rectal Neoplasms; Thyroid Neoplasms

Two anti-sialyl Lewis X (sLeX) monoclonal antibodies, mAb FH6 and mAb KM93, were analyzed by flow cytometry for their ability to bind to 16 human colon carcinoma cells. The binding profiles of these two anti-sLeX monoclonal antibodies did not correspond to each other. Three of the cell lines were reactive with mAb FH6 but not with mAb KM93. These three cell lines did not adhere to Chinese hamster ovary cells that were stably transfected with human E-selectin cDNA in an E-selectin-dependent manner. In contrast, almost all human colon carcinoma cell lines that bound to mAb KM93 adhered to cells that expressed E-selectin. These results suggest that a subtype of sLeX carbohydrate epitopes recognized by mAb FH6 do not always function as ligands for E-selectin.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Cell Adhesion; CHO Cells; Colonic Neoplasms; Cricetinae; E-Selectin; Epitopes; Humans; Lewis X Antigen; Transfection

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tumors produced by LS174T-HM7 cells, a subline of the human colon carcinoma LS174T with higher metastatic tendency and higher mucin production. A striking feature of the oligosaccharides of the LS174T-HM7 xenograft tumor mucin was a predominance of sulfated Lewis X determinants: HSO3-Galbeta1-4(Fucalpha1-3)GlcNAc. In addition to one previously known saccharide with one sulfated Lewis X determinant, the HM7 xenograft tumor mucin contained multiple novel structures containing one, two, or three sulfated Lewis X determinants. This determinant, known to act as a selectin ligand, has been found previously in minor saccharide components of human milk as well as mucins, but never before as a predominant structure in one mucin source.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Colonic Neoplasms; Humans; Lewis X Antigen; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Molecular Sequence Data; Mucins; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Alcohols; Tumor Cells, Cultured

To determine whether the Lewis enzyme responsible for the Lewis blood type antigens on erythrocytes synthesizes the Lewis antigens on normal cells and cancer cells in colon tissue, we performed genotyping of the Lewis gene by the PCR-RFLP method and by immunohistochemical staining of Lewis antigens and the Lewis enzyme with specific monoclonal antibodies (mAbs) in colon tissues obtained from 100 colon cancer patients. Five of the 100 patients were identified as homozygotes for the mutant Lewis gene, i.e., the le/le genotype that cannot encode functional Lewis enzyme. The cells in both the normal and cancerous regions of colon tissue from these five le/le patients were completely devoid of staining with mAbs against Lewis antigens with the type 1 chain, i.e., Lewis a, Lewis b, and sialyl Lewis a. In contrast, the cells in cancerous regions of the colon tissue of the 95 patients with the Le/Le or Le/le genotype positively stained with all three mAbs, anti-Lewis a, anti-Lewis b, and anti-sialyl Lewis a. The cells in the cancerous regions of the colon tissue of the five le/le patients stained with DU-PAN-2 mAb, whose recognizing epitope is known to be sialyl Lewis c, a precursor structure of sialyl Lewis a. By immunohistochemical staining with FTA 1-16 mAb, which is directed at the human Lewis enzyme, we were able to demonstrate for the first time that the enzyme is localized in the Golgi area of the colon epithelial cells of patients with the Le/Le or Le/le genotype. No staining was observed in the Golgi area of the cells of the patients with the le/le genotype. From these results, we conclude that individuals with the Le/Le or Le/le genotype possess a functional Lewis enzyme synthesizing fucosylated type-1 Lewis antigens in the Golgi apparatus of the colon epithelial cells, but that individuals with the le/le genotype are devoid of the Lewis enzyme in the Golgi apparatus, resulting in an inability to synthesize Lewis antigens with the type-1 chain, and that it is inappropriate to use CA19-9, whose antigenic epitope is defined as sialyl Lewis a, as a tumor marker in patients with the le/le genotype.

Topics: Antibodies, Monoclonal; Base Sequence; CA-19-9 Antigen; Carbohydrate Sequence; Colon; Colonic Neoplasms; Epitopes; Fucosyltransferases; Genotype; Humans; Immunohistochemistry; Intracellular Fluid; Lewis X Antigen; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Staining and Labeling

Patients with mucinous colon cancers often have a poor prognosis. The aim of this study was to determine whether metastatic potential depends on specific alterations in mucin-associated carbohydrate structures.. A quantitative scoring system was used to examine the expression of mucin-associated carbohydrates in paired human primary colon cancers and metastases and in cecal tumors and liver metastases from an animal model of metastasis. Adhesion of metastatic cells to basement membrane and endothelial ligands was examined.. Metastases expressed a decrease in mucin core structures Tn and T, a reciprocal increase in sialyl T and sialyl Tn, and an increase in peripheral sialyl Le(x) compared with the primary tumors from which they arose. Altered expression of sialylated mucin structures resulted from selective metastasis of cells that produce sialomucins. Antibodies to sialylated epitopes or desialylation inhibited adhesion of metastatic cells to basement membranes. Neutralizing antibody to endothelial-associated E-selectin (a ligand for sialyl Le(x)) inhibited adhesion of metastatic cells to cytokine-activated hepatic endothelial cells, and inhibition of sialomucin with antisense to the MUC2 gene inhibited adhesion to E-selectin.. Increased sialylation of mucin-associated carbohydrates is characteristic of colon cancer cells that are most likely to metastasize. Sialylated carbohydrate structures on mucin play a role in adhesive interactions involving both basement membrane and endothelial-associated ligands.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Base Sequence; Basement Membrane; Cell Adhesion; Colonic Neoplasms; Disease Models, Animal; E-Selectin; Endothelium; Humans; Immunohistochemistry; Lewis X Antigen; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Mucins; Neoplasm Metastasis; Neoplasm Transplantation; Sialoglycoproteins; Sialomucins

A secreted MUC1 mucin from the spent medium of the colon carcinoma cell line COLO 205 carrying sialyl-Lewis a and x epitopes (H-CanAg) was purified by trichloroacetic acid precipitation and Superose 6 gel filtration. The purified H-CanAg inhibited adhesion of the leukocyte cell line HL-60 to E-selectin transfected COS-1 cells or interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells. Sera from two patients with advanced colon carcinoma containing high concentrations of sialyl-Lewis a and x activity inhibited HL-60 cell adhesion to E-selectin-expressing COS-1 cells and IL-1 beta-activated endothelial cells. After affinity column absorption of the sialyl-Lewis a activity, the sera also lost most of their sialyl-Lewis x activity and at the same time their adhesion inhibitory effect. A large part of the sialyl-Lewis a/x activity in the two patients was found in fractions containing mucins having a MUC1 apoprotein, as shown by its size, and reactivity with the two anti-MUC1 apoprotein monoclonal antibodies, Ma552 and HMFG-2. The cell-adhesion inhibitory effect of the purified sialyl-Lewis a-carrying MUC1 mucin fraction from the sera of the two patients was stronger than that of smaller sized sialyl-Lewis a-carrying mucin-type glycoproteins also found in the patient sera. The MUC1 mucin fraction secreted by the COLO 205 cells and from the two sera were all shown to lack their C-terminal portion, in contrast to the MUC1 mucin from cells. It is hypothesized that sialyl-Lewis a- and/or x-containing mucins, especially MUC1, secreted by tumors can interact with E-selectin on endothelial cells and thus inhibit leukocyte adhesion.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Cell Adhesion; Colonic Neoplasms; Cytoplasm; E-Selectin; Endothelium, Vascular; Epitopes; HL-60 Cells; Humans; Leukocytes; Lewis Blood Group Antigens; Lewis X Antigen; Mucin-1; Tumor Cells, Cultured

Liver metastasis from colorectal carcinoma is an important problem in surgical treatment and profoundly affects the prognosis of patients. If it were possible to identify characteristic features in the primary lesion strongly related to liver metastasis, these could be used as prognostic markers for liver metastasis. To search for such features, the primary lesions of patients with colorectal carcinoma with liver metastasis were investigated.. Three groups of colorectal carcinoma were examined: Group A with synchronous liver metastases; Group B with only lymph node metastases without recurrence for 5 years; and Group C with recurrence of liver metastases. Groups A and B included 24 cases and Group C, 20. We focused on cancer cell morphology at the invasive front and expression of sialyl Lewis X (sialyl Lex) in the primary cancer.. At the invasive front in Group A it was frequently found that polygonal, not columnar, cancer cells with a single or solitary trabecular form with indistinct polarity, showed an infiltrative growth pattern. This type of morphology was termed "focal dedifferentiation" and graded four levels. Eleven of 24 cases (46%) had severe focal dedifferentiation in Group A, 1 of 24 (4%) in Group B, and 6 of 20 (30%) in Group C. Sialyl Lex staining was positive in 12 of 24 cases (50%) in Group A, in 3 of 24 cases (13%) in Group B, and in 7 of 20 cases (35%) in Group C in the primary carcinoma. In respect to the staining of (sialyl Lex) at focal dedifferentiation, it was positive in 17 of 24 cases (71%) in Group A, in 4 of 24 cases (17%) in Group B and in 11 of 20 cases (55%) in Group C. Focal dedifferentiation and sialyl Lex staining in the primary cancer showed a significant difference between Groups A and B. Sialyl Lex staining at focal dedifferentiation showed a significant difference between Groups A and B and Groups B and C. Other adhesion related molecules, sialyl LeA and CEA, showed no difference among Groups A, B, and C.. Both focal dedifferentiation and expression of sialyl Lex antigen in the primary lesion are considered good markers for assessing the metastatic proclivity of colorectal cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Oligosaccharides; Prognosis; Rectal Neoplasms; Sialyl Lewis X Antigen

The potential contribution of fucosyltransferases to the overexpression of sialyl-Le(x) antigen was investigated in the colon carcinoma cell line HT-29 and in human colon carcinoma tissue. In HT-29 cells as well as in normal or malignant colonic tissues Fuc-TIII, Fuc-TIV, Fuc-TVI but not Fuc-TV nor Fuc-TVII were detectable after RT-PCR. Sodium butyrate treatment of HT-29 cells increased (to about 200%) and DMSO treatment decreased (to about 20%) the expression of sialyl-Le(x). This modulation of sialyl-Le(x) was concomitant with the analogous increase/decrease of mRNA of Fuc-TIII but not Fuc-TIV. Fuc-TVI was not detectable by Northern blotting in HT-29 cells. In six human colon carcinomas which exhibited strong overexpression of sialyl-Le(x), the expression of Fuc-TIII-mRNA was the same or lower than in the corresponding normal colonic tissue. Thus Fuc-TIII expression may be affecting the expression of the sialyl-Le(x) moiety in HT-29 cells but not in human colon carcinoma tissue.

Topics: Antibodies, Monoclonal; Blotting, Northern; Butyrates; Butyric Acid; Colonic Neoplasms; Dimethyl Sulfoxide; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Humans; Lewis X Antigen; Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Previously we have shown that high metastatic colonic carcinoma cells express relatively more lamp molecules and sialyl Le(x) structures on the cell surface than their corresponding low metastatic counterparts (Saitoh, O., Wang, W.-L., Lotan, R., and Fukuda, M. (1992) J. Biol. Chem. 267, 5700-5711). In the present study, we extended these findings by testing whether these high and low metastatic colonic carcinoma cells differ in their adhesion efficiency to E-selectin-expressing cells. First, it was found that the high metastatic cells, as compared to their low metastatic counterparts, bind more efficiently to activated human endothelial cells that express E-selectin. This was also true when the adhesion was tested for Chinese hamster ovary cells stably expressing E-selectin. In addition, it was found that the high metastatic cells also adhere more efficiently to mouse endothelioma cells after activation with interleukin-1 beta. It was also shown that the adhesion can be inhibited by soluble lamp-1 or soluble leukosialin that contain sialy Le(x) termini. The inhibition was not, however, observed when these soluble glycoproteins lack sialyl Le(x) structures. The results indicate that the efficiency of the E-selectin-mediated binding of colonic carcinoma cells to human and mouse endothelial cells correlates with the metastatic potential of the cells and suggest that this adhesive event may be one of the critical factors for the metastatic spread of tumor cells. Soluble forms of leukosialin or lamp-1 may be useful as therapeutic agents for the inhibition of E-selectin-mediated binding to tumor cells.

Topics: Animals; Antigens, CD; Base Sequence; Carcinoma; Cell Adhesion; Cell Adhesion Molecules; Colonic Neoplasms; DNA Primers; E-Selectin; Endothelium, Vascular; Humans; Leukosialin; Lewis X Antigen; Lysosomal Membrane Proteins; Membrane Glycoproteins; Mice; Molecular Sequence Data; Neoplasm Metastasis; Sialoglycoproteins

A human colon cancer-derived cell line, KC-1, was established from the surgical specimen of mucinous adenocarcinoma of the colon. The cells grew as monolayers, showing formation of irregular aggregation of cells and pleomorphic nuclei. The doubling time in vitro was 56.6 hours. The cells produced CEA, CA19-9 and sialyl SSEA-1(SLX). Chromosome numbers were distributed between 79 and 83 with many structural abnormalities. A point mutation of the Ki-ras gene in codon 61 (CAA-->CAT) was found. The cells have been subcultured 13 times during these three years. This cell line can be useful for investigations of colon cancer.

Topics: Adenocarcinoma, Mucinous; Aged; CA-19-9 Antigen; Carcinoembryonic Antigen; Cell Line; Colonic Neoplasms; Female; Humans; Lewis X Antigen; Point Mutation

Sialyl Le(a) antigen and sialyl Le(x) antigen are cancer-associated carbohydrate antigens. Previous immunohistologic and immunochemical studies have shown that these antigens are preferentially expressed in gastric cancer and colonic cancer and that they possibly are related to the metastatic potential of the cancer cells. The biosynthesis of these antigens is completed by fucosyltransferases, but it has not been reported how fucosyltransferases control the expression of these carbohydrate antigens concerning the invasive potential of the cancer.. The authors established an assay system for measuring the activity of alpha 1-->4 fucosyltransferase (sialyl Le(a) synthase) and alpha 1-->3 fucosyltransferase (sialyl Le(x) synthase) with a high-pressure liquid chromatography system (HPLC). The activity was measured in various parts of normal and cancerous gastric and colonic tissue and compared with the expression of sialyl Le(a), sialyl Le(x), Le(a), and Le(x) antigens determined in a solid-phase enzyme-linked immunosolvent assay (EIA).. Sialyl Le(a) synthase was detected in most normal or malignant mucosa of gastric and colonic tissues, regardless of anatomic locations. Sialyl Le(x) synthase activity generally was low in the normal gastric mucosa, whereas the activity was higher in 77% (7 of 9) of gastric cancer tissues than in corresponding normal tissues with enhanced expression of sialyl Le(x) antigen in most patients (5 of 7). In the large intestine, the activity of sialyl Le(a) synthase and sialyl Le(x) synthase was correlated. Although enhanced expression of sialyl Le(x) in colonic cancer was observed in 86% (12 of 14) of all patients, concomitant higher sialyl Le(x) synthase activity than that in normal tissue was observed in only 58% (7 of 12) of patients.. The expression of sialyl Le(a) and sialyl Le(x) antigens in the stomach and the colon was not controlled solely by fucosyltransferases but by a more complicated system involving other glycosyltransferases.

Topics: Adenocarcinoma; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; CA-19-9 Antigen; Colonic Neoplasms; Fucosyltransferases; Gangliosides; Gastric Mucosa; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Lewis Blood Group Antigens; Lewis X Antigen; Oligosaccharides; Stomach Neoplasms; Time Factors; Tumor Cells, Cultured

Lysosomal membrane glycoprotein (lamp)-1 and lamp-2 are the most abundant glycoproteins within the lysosomal membrane. A small amount of lamp-1 and lamp-2 molecules, however, can be present on the cell surface. We have shown previously that highly metastatic colonic carcinoma L4 cells express more lamp-1 and lamp-2 on the cell surface than low metastatic SP cells (Saitoh, O., Wang, W.-L., Lotan, R., and Fukuda, M. (1992) J. Biol. Chem. 267, 5700-5711). Since lamp-1 and lamp-2 are the major carriers for poly-N-acetyllactosamines that are able to display sialyl-Le(x) termini, we sought to determine if an increased amount of lamp-1 on the cell surface would lead to increased expression of cell surface sialyl-Le(x) determinants and to the increased adhesion of those cells to E-selectin. Expression of increased amounts of lamp-1 on the cell surface was achieved either by overexpression of lamp-1 or by expressing a mutant lamp-1 molecule preferentially at the plasma membrane, rather than in lysosomes. Cells that express variable amounts of cell surface lamp-1 were tested for their adhesion to activated endothelial cells or E-selectin expressing Chinese hamster ovary cells. The results clearly show that the extent of adhesion to E-selectin and cell surface sialyl-Le(x) determinants is proportional to the amount of cell surface lamp-1. Moreover, it was demonstrated that such adhesion can be inhibited by soluble lamp-1 generated from Chinese hamster ovary cells expressing sialyl-Le(x) structures. These results indicate that lamp-1 can efficiently present ligands for E-selectin and at the same time can be a useful reagent for inhibition of E-selectin (and possibly P-selectin)-mediated interaction.

Topics: Animals; Antigens, CD; Base Sequence; Carbohydrate Conformation; Carbohydrate Sequence; Cell Adhesion; Cell Adhesion Molecules; CHO Cells; Colonic Neoplasms; Cricetinae; DNA; DNA Mutational Analysis; E-Selectin; Endothelium, Vascular; Flow Cytometry; Gene Expression; Gene Library; Humans; Interleukin-1; Lewis X Antigen; Lysosomal Membrane Proteins; Membrane Glycoproteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Tumor Cells, Cultured

A comparative immunohistochemical study was performed to analyse the expression of cancer-associated carbohydrate antigens by primary and metastatic lesions of colon cancer. We used monoclonal antibodies which reacted with Lea, Lex and Tn as well as their sialylated derivatives. Twenty-one primary lesions in patients without metastasis and 26 primary and metastatic lesions in patients with liver metastasis were studied. Sialyl Lea was expressed by 57% of the primary lesions of patients without metastasis, 65% of the primary lesions of patients with metastasis and 73% of their liver metastases. Sialyl Lex was expressed by 60% of the primary lesions of patients with and without metastasis as well as by approximately 80% of the liver metastases. Sialyl Lea and sialyl Lex showed strong expressions in the liver metastases, significantly greater than in the primary lesions. The findings indicate the increased expressions of sialyl Lea and sialyl Lex to be correlated with liver metastasis of colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; CA-19-9 Antigen; Colonic Neoplasms; Female; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Rectal Neoplasms

This report concerns the stepwise biosynthesis in vitro of Sialyl Lewis X, (SA-Le(x)), a carcinoembryonic antigen, in human colon carcinoma KM12 cells exhibiting different metastatic behaviors. The significance of SA-Le(x) has become even more apparent since the detection of its terminal epitope NeuAc(alpha 2-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, as the binding ligand of the selectin family member ELAM-1. The activity level of galactosyltransferase GalT-4 which catalyzes the formation of core nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) is very high in all the metastatic lines tested with highly metastatic lines (KM12-SM) exhibiting the highest activity. The same activity pattern for galactosyltransferase is also observed when tested with iLcOse5Cer (GlcNAc beta 1-3nLcOse4Cer), the precursor for polylactosamine glycolipid. Sialyltransferase SAT-3 which catalyzes the formation of LM1 (NeuAc alpha 2-3nLcOse4Cer), the precursor for SA-Le(x), is also present in all the metastatic cell lines although the activity levels are much lower compared to galactosyltransferase. The fucosyltransferase FucT-3, which catalyzes the formation of R'-Gal-Fuc(alpha 1-3)GlcNAc-R linkage, is active with both nonsialylated substrate, nLcOse4Cer, and sialylated substrate, LM1 (NeuAc alpha 2-3nLcOse4Cer) with the formation of either Le(x) (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer) or SA-Le(x) (NeuAc alpha 2-3nLcOse4Cer). However, the sialylated substrate LM1 is preferred to enzymatic activity since it exhibited lower Km (46 microM) than that of nLcOse4Cer (67 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Colonic Neoplasms; Fucosyltransferases; Galactosyltransferases; Humans; Kinetics; Lewis X Antigen; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Monoclonal antibody AM-3 (MAb AM-3) raised against a sialomucin from human colorectal carcinoma has previously been shown to define a carbohydrate epitope, which is detectable by immunocytochemistry on all investigated colonic carcinomas and is expressed in correlation with the grade of dysplasia in colonic adenomas (Hanski et al., J. Clin. Pathol., 43: 379-385, 1990). Epitope analyses in solid-phase enzyme immunoassays revealed that AM-3 antibody recognizes the sialylated Lewis(x) sequence on a branched O-linked glycan and its reductively cleaved alditol from human amniotic mucins. In comparative binding and binding inhibition studies MAbs AM-3 and CSLEX1 displayed reciprocal affinities to mucins versus gangliosides. Correspondingly, the weaker binding activities of AM-3 versus CSLEX to III3-alpha Fuc-IV3-alpha NeuAc-nLcOse4-Cer or to monogangliosides from human granulocytes were measured. Gangliosides from a human colon carcinoma were recognized by MAb CSLEX1 exhibiting a broader specificity to various sialyl-Lewis(x) antigens and by MAb FH6 reactive to sialyl-dimeric Lewis(x) antigen, but not by MAb AM-3. In conclusion, MAb AM-3 is distinguished from other sialyl Lewis(x)-specific MAbs by its selective reactivity to mucin-carried epitopes on the monomeric antigen.

Topics: Adenoma; Animals; Antibodies, Monoclonal; Biomarkers, Tumor; Birds; Carbohydrate Sequence; Cattle; Colon; Colonic Neoplasms; Female; Humans; Lewis X Antigen; Milk, Human; Molecular Sequence Data; Mucins; Oligosaccharides; Pancreatic Neoplasms; Precancerous Conditions; Sheep

The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAc alpha 2-3Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta 1-3Gal beta 1-4Glc-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an alpha 1-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-substituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4 Glc-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Brain; Carbohydrate Conformation; Carbohydrate Sequence; Carcinoembryonic Antigen; Cell Line; Chick Embryo; Chromatography, Affinity; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Epitopes; Fucosyltransferases; Humans; Immunoenzyme Techniques; Kinetics; Lewis X Antigen; Molecular Sequence Data; Substrate Specificity

Colorectal primary carcinomas and metastases from 20 Dukes' stage C or D patients were examined for the immunohistochemical localization and contents of various fucosylated N-acetyl-lactosamine oligomers by specific monoclonal antibodies (MAbs). MAbs used were SH1, specific for Lewis X antigen; FH4, specific for dimeric Lewis X antigen; FH6, specific for sialyl-dimeric Lewis X antigen; and KH1, specific for Lewis Y-Lewis X antigen. The distribution of the carbohydrate antigens identified by these MAbs was heterogeneous within the primary tumor as well as within the metastatic lesion. Examinations of serial sections indicated that areas within an individual tumor which were stained with one MAb were not always reactive with the other MAbs, although these four MAbs identify closely related structures. The degree of MAb reactivity with carcinoma sections was classified by percentage positive carcinoma cells, and primary tumors and metastases from the same patients were compared. An equivalent or higher proportion of carcinoma cells in the metastatic lesions were reactive with MAb FH6 than in the primary colon carcinomas, but each correlation was not seen with the other MAbs. Electrophoretic separation of tumor tissue extracts followed by staining with these MAbs revealed that a component having an approximate molecular weight of 1,000,000 is the major site for the binding of MAbs, FH6, FH4, and KH1. The electrophoretic mobility of the antigenic molecule on polyacrylamide gels as shown by direct MAb bindings was slightly different from that of a major sialomucin revealed by wheat germ agglutinin in the same tissues. MAb FH6 binding to a high molecular weight component was eliminated by prior treatment of the glycoprotein with mild acid or sialidase to remove sialic acid. Simultaneously, binding of MAb SH2, specific for dimeric Lex antigen, to this component increased. An extract was prepared from a liver metastasis, and high molecular weight components were isolated by gel filtration and then fractionated by DEAE-cellulose ion exchange chromatography. A fraction eluted from DEAE-cellulose between 0.10-0.25 M sodium chloride contained most of the MAb FH6 reactivity, as shown by antibody affinity chromatography. These results support a hypothesis that high molecular weight glycoproteins produced by colorectal carcinoma tissues are heterogeneous with regard to their carbohydrate chains and their antigenic structures may change during tumor progression.

Topics: Adult; Aged; Antibodies, Monoclonal; Carbohydrate Sequence; Chromatography, Ion Exchange; Colonic Neoplasms; Female; Glycoproteins; Humans; Lewis X Antigen; Macromolecular Substances; Male; Middle Aged; Molecular Sequence Data; Molecular Weight; Mucins; Neoplasm Metastasis; Rectal Neoplasms; Regression Analysis; Sialic Acids

The epidermal growth factor (EGF) receptor in two colon carcinoma lines and in one vulval carcinoma line tested contains carbohydrate determinants that are recognized by monoclonal antibodies to tumor-associated antigens. These antibodies are directed to sialylated Lea and to difucosylated structures of the Y type. Cell lines which react with these antibodies express these antigens on their surface glycolipids and glycoproteins, including the EGF receptor. These unusual carbohydrates are absent in EGF receptors from normal untransformed cells, and from tumor cells which do not express these specific antigens. Although EGF receptor represents only 0.1-2% of total plasma membrane proteins of antigen-positive carcinomas, it accounts for 20-80% of total protein-associated sialylated Lea/Y type of nonsecreted carbohydrates present in these cells. The results of cell-binding, immunoprecipitation, and Western blot analyses of the antigen-positive carcinomas indicate that sialylated Lea/Y type of antigenicity is intrinsic to the EGF receptors of these cells, and that the antigen is present in receptors from both over-expressing and normal-expressing carcinomas.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Carbohydrates; Carcinoma; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Epitopes; ErbB Receptors; Female; Glycolipids; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Melanoma; Membrane Proteins; Mice; Uterine Cervical Neoplasms; Vulvar Neoplasms

The epithelial expression of carbohydrate antigen, stage-specific embryonic antigen 1 (SSEA-1) was examined immunohistochemically in noncancerous specimens from patients with familial polyposis coli, and compared with the colorectal epithelia from patients with sporadic colorectal cancer. In mucosa remote from carcinoma of sporadic cases, SSEA-1 was expressed only faintly in the lower crypts. In mucosa adjacent to carcinoma of sporadic cases, SSEA-1 was expressed not only in the lower crypts but also in the upper crypts. These results corresponded to those observed in the authors' previous study. In the flat mucosa of familial polyposis coli cases, SSEA-1 was detected not only in the lower crypts, but also in both upper crypts and the surface epithelium in contrast with the flat mucosa of sporadic cases. The staining pattern in the upper crypts of the flat mucosa of familial polyposis coli cases was very similar to that of the mucosa adjacent to carcinoma of sporadic cases, but was stronger and more diffuse in the surface epithelium. In microscopic adenomas, SSEA-1 was expressed diffusely. These results demonstrate that the flat mucosa of patients with familial polyposis coli shows preneoplastic changes similar to those in the mucosa adjacent to carcinoma of sporadic cases, and that SSEA-1 is related to adenoma formation in the early stage of carcinogenesis in the colorectum. In addition, the results suggest that immunohistochemical studies of flat mucosa may be useful for the early detection of high-risk individuals in a familial polyposis coli family.

Topics: Cell Transformation, Neoplastic; Colonic Neoplasms; Colonic Polyps; Glycolipids; Humans; Intestinal Mucosa; Lewis X Antigen

SSEA-1 antigen (stage-specific embryonic antigen-1) has been shown to be a series of carbohydrate antigens having type-2 chain and X-hapten structures. These carbohydrate antigens are remarkably accumulated in various human cancer tissues, and activity secreted into the blood stream. Frequently SSEA-1 antigens are further modified with fucoses or sialic acid in human cancer tissues, thus forming various subgroups of antigens such as fucosyl SSEA-1, sialyl SSEA-1 or polyfucosylated antigens. Many monoclonal antibodies are established which can discriminate each subgroup of antigens. Assay systems for these antigens in the sera of cancer patients have been developed using these monoclonal antibodies. Sialyl SSEA-1 is especially elevated in the sera of patients with adenocarcinoma of the lung. The antigens detected with these monoclonal antibodies are mucin-like glycoproteins(cancer-associated mucin, CAM). Various types of cancer-associated mucins can be characterized by respective monoclonal antibodies. It is therefore possible to classify cancer-associated mucins according to the structure of their carbohydrate side chains using these monoclonal antibodies.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Carbohydrates; Colonic Neoplasms; Glycolipids; Humans; Lewis X Antigen; Lung Neoplasms; Mice; Mucins; Stomach Neoplasms

Colorectal carcinomas are composed of heterogeneous cell subpopulations which may be instrumental in conferring metastatic potential and therapeutic refractoriness to these tumours. To assess cellular heterogeneity, the expression has been examined of two oncodevelopmental antigens, carcinoembryonic antigen (CEA) and stage-specific embryonic antigen 1 (SSEA-1), by double immunofluorescence microscopy on 11 human colorectal carcinomas. Although both antigens were expressed in each tumour, their regional and cellular locations differed considerably. SSEA-1 expression was rarely expressed in poorly differentiated cancers but was enhanced with increasing degrees of differentiation. CEA expression was independent of histological differentiation. SSEA-1 was expressed with similar frequency in cell membranes, cytoplasm, and glandular contents regardless of degree of differentiation. Cytoplasmic staining with CEA however, was limited to more poorly differentiated tumours. In normal mucosa remote from the tumours and transitional mucosa adjacent to them, SSEA-1 stained only a few lower crypts whereas CEA stained a majority of both upper and lower crypts. Although biochemical studies have indicated that the SSEA-1 epitope may reside on CEA molecules, the fact that colon cancer tissues express these two antigens quite heterogeneously suggests differences in antigenic processing which may be dependent upon the degree of cellular differentiation.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoembryonic Antigen; Cell Transformation, Neoplastic; Colonic Neoplasms; Glycolipids; Humans; Intestinal Mucosa; Lewis X Antigen; Neoplasm Staging; Rectal Neoplasms; Reference Values

The distribution in paraffin fixed human tissues of a carbohydrate antigen defined by two monoclonal antibodies raised against human granulocytes has been studied by means of an immunoperoxidase technique. In addition to granulocytes, the antigen has been detected in adult tissues on identifiable cell types of the stomach, kidney, adrenal medulla, and brain and on the mucins of the gastrointestinal tract and other secretions. In fetal tissue, epithelial cells of the alimentary tract, lung, brain, and kidney express the antigen. Adenocarcinoma of the colon, stomach, breast, and lung are stained strongly, as are other types of lung cancer. The monoclonal antibodies give a staining pattern similar but not identical to other monoclonal antibodies raised against granulocytes or neoplastic cell lines which recognise the antigen 3-fucosyl N-acetyllactosamine. The use of antibodies against this oncofetal antigen in the study of differentiation and as tumour markers is discussed.

Topics: Adrenal Medulla; Animals; Antibodies, Monoclonal; Antigens; Antigens, Neoplasm; Brain; Colonic Neoplasms; Digestive System; Epitopes; Fetus; Granulocytes; Humans; Immunoenzyme Techniques; Kidney; Lewis X Antigen; Lung Neoplasms; Mice; Mice, Inbred BALB C; Oligosaccharides

Topics: Adenocarcinoma; Antigens, Neoplasm; Colonic Neoplasms; Female; Fetus; Glycolipids; Humans; Intestinal Mucosa; Lewis X Antigen; Precancerous Conditions; Pregnancy

The expression of carbohydrate antigen 19-9 (CA 19-9) and stage-specific embryonic antigen 1 (SSEA-1) in various human colorectal epithelia was examined by an immunohistochemical method. In mucosa remote from the carcinoma, CA 19-9 was not expressed, whereas SSEA-1 was only faintly expressed in lower crypts in all cases. In mucosa adjacent to the carcinoma, CA 19-9 was weakly expressed in upper crypts in 20% of the cases, whereas SSEA-1 was expressed not only in lower crypts in all cases but also in upper crypts in 93.3% of the cases. In adenoma, CA 19-9 was expressed in 80.6% of the cases, and SSEA-1 was expressed in all cases. The expression of both antigens was to some extent related to the degree of cellular atypia. In focal carcinoma in adenoma, CA 19-9 was strongly and diffusely expressed in 50% of the cases, and SSEA-1 was strongly and diffusely expressed in all cases. In advanced carcinoma, CA 19-9 was homogeneously or heterogeneously expressed in 82.2% of the cases, and SSEA-1 was homogeneously or heterogeneously expressed in all cases, but lower intensity of SSEA-1 staining was associated with a decrease in the degree of carcinoma differentiation. These results show that the expression of both CA 19-9 and SSEA-1 changes along with neoplastic transformation and progression in the colon and rectum. Immunohistochemical studies of SSEA-1 in flat colorectal mucosa might be a useful approach for detecting foci with preneoplastic change in the general population, whereas those of SSEA-1 and CA 19-9 could be a useful method for detecting focal carcinoma in adenoma.

Topics: Adenoma; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Carcinoma; Colon; Colonic Neoplasms; Glycolipids; Humans; Intestinal Mucosa; Lewis X Antigen; Rectal Neoplasms; Rectum

The expression of the carbohydrate structure defined by monoclonal antibody to murine stage-specific embryonic antigen 1 (SSEA-1) was examined, using immunofluorescence, in formalin-fixed, paraffin-embedded sections of normal fetal and adult human colon and human colonic adenocarcinoma. SSEA-1 was expressed in all human colonic adenocarcinoma tissues examined, although in some cases the staining was heterogeneous. In normal human colonic mucosa, under the conditions used, faint staining was seen in the lower crypts and in only 26% of the crypts examined. When human fetal colon was tested, SSEA-1 was expressed in much larger amounts and in over 50% of all crypts. Transitional mucosa, immediately adjacent to human colonic adenocarcinomas, was also tested, and in this case, increased SSEA-1 expression was seen not only in the lower crypts but also in the upper crypts and surface epithelium. These results show that the increased expression of SSEA-1 in human colonic adenocarcinoma is an oncodevelopmental marker for this cancer. In addition, the results suggest that increased expression of SSEA-1 may be a preneoplastic change in human colon.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antigens, Neoplasm; Colon; Colonic Neoplasms; Fluorescent Antibody Technique; Glycolipids; Humans; Intestinal Mucosa; Lewis X Antigen; Reference Values

Distribution patterns of specific fucose-containing antigens having X determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) as well as the di- or trimeric X determinants (Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4[Fuc alpha 1----3]-GlcNAc) in the developing human embryo and fetus and in human cancer have been examined using immunohistological techniques. Tissue sections were stained with monoclonal antibody FH3, which defines X determinant, and with monoclonal antibody FH4, which defines di- or trimeric X determinant. The following general trends in the expression of the antigens defined by FH3 and FH4 have been observed: (a) A well-organized, orderly appearance and disappearance of the antigens was observed during the histogenesis of various epithelia of gastrointestinal and other organs. The developmental stage exhibiting the maximum antigen expression is different for each organ. (b) The X determinant defined by FH3 was expressed approximately 2 wk earlier than the di- or trimeric X determinant defined by FH4, and the antigen defined by FH4 regressed more rapidly and more completely than the X determinant defined by FH3 on further development of epithelial tissue. Thus, expression of the FH4 antigen is highly limited to specific types of cells in newborn and adult epithelial tissues. (c) The antigen defined by FH4 was strongly expressed in the majority of tubular and papillary adenocarcinoma of stomach, adenocarcinoma of colon, and infiltrating ductal carcinoma of breast and its metastatic lesions. No antigen was found in poorly differentiated stomach adenocarcinoma, squamous lung carcinoma, and many other types of tumors from ovary, testis, prostate, skin, and muscle. The presence of the antigen defined by FH4 is therefore limited to carcinoma of the stomach, colon, and breast and can be regarded as a retrograde expression of the antigen to a certain stage of fetal development in which expression of this antigen was maximal.

Topics: Adult; Antibodies, Monoclonal; Antigens, Neoplasm; Breast Neoplasms; Bronchi; Cerebrosides; Colonic Neoplasms; Digestive System; Embryo, Mammalian; Female; Fetus; Glycolipids; Histocytochemistry; Humans; Infant, Newborn; Kidney Neoplasms; Lewis X Antigen; Pregnancy; Stomach Neoplasms

A monoclonal antibody defining the Lewis blood group determinant was used to immobilize antigen in sera of patients with adenocarcinoma of the gastrointestinal tract, and a second radiolabeled antibody, which defines a gastrointestinal cancer-associated antigen (GICA), was used to detect the immobilized antigen. With this approach, elevated antigen levels were found in 34 of 49 (69%) of sera from patients with advanced colorectal carcinoma as compared with 9 of 292 (3%) of sera from patients with non-malignant gastrointestinal diseases and of healthy donors. For early primary colorectal carcinoma, the combination of anti-Lewis and anti-GICA monoclonal antibodies was more sensitive in detecting GICA than using anti-GICA antibody alone. Double determinant radioimmunoassay revealed the glycolipid determinant lacto-N-fucopentaose (LNF) III circulating in colorectal carcinoma patients' sera. 53% of patients older than 65 years had elevated levels of the LNF III determinant compared to none of age-matched, apparently healthy donors or patients with benign gastrointestinal tract lesions, and 18% of patients with inflammatory gastrointestinal tract diseases. In younger patients, the differences were less marked. Our results suggest the potential usefulness of Lewis and LNF III determinants as markers for the detection of gastrointestinal tract malignancies.

Topics: Adult; Aged; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Colonic Neoplasms; Gastrointestinal Diseases; Gastrointestinal Neoplasms; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Mice; Mice, Inbred BALB C; Middle Aged; Oligosaccharides; Rectal Neoplasms

Topics: Adenocarcinoma; Antigens, Neoplasm; Carbohydrate Sequence; Colonic Neoplasms; Erythrocytes; Glycolipids; Haptens; Humans; Immunochemistry; Lewis X Antigen