cefoxitin and Colonic-Neoplasms

Resected carcinoma patients were immunized 3-5 times with ovine submaxillary gland mucin (OSM) containing predominantly sialylated Tn (sTn), completely desialylated ovine submaxillary gland mucin (dOSM) containing predominantly Tn, or 50% desialylated OSM containing Tn and sTn plus bacillus Calmette-Guerin (BCG) as an immunologic adjuvant. Pre- and postimmunization sera were quantified by ELISA, whole-cell ELISA, and immune stain dot blots. Fifteen of 17 patients produced IgG antibody titers from 40 to 5120 times more reactive with OSM and dOSM postimmunization. More importantly, these IgG antibodies reacted with LS-174T, a human colon carcinoma cell line. Significant DTH-like responses (1-17 cm) were observed in 15 of 17 patients; the strength of these responses was dependent on the presence or absence of sialic acid. Biopsies of these DTH-like reactions revealed infiltration with some CD8+ lymphocytes and mast cells. These results suggest that a single 9-carbon sugar can affect cellular immune responses to mucin antigens. It is thought that these large erythematous, nonindurated cellular reactions are antibody-mediated Arthus-like reactions. OSM, and especially dOSM, were also found to inhibit lymphocyte proliferation.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Antigens, Tumor-Associated, Carbohydrate; BCG Vaccine; Cancer Vaccines; Carcinoma; CD8-Positive T-Lymphocytes; Cell Proliferation; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Humans; Immunity, Cellular; Immunoglobulin G; Mast Cells; Mucins; Submandibular Gland; Swine; Tumor Cells, Cultured

During a 24-month period, 350 patients were prospectively studied in an effort to determine the perioperative factors in the development of infections after colon and rectal resections. All patients received standard mechanical bowel preparation; perioperative parenteral cefoxitin (group A) or preoperative oral neomycin and erythromycin, in addition to perioperative cefoxitin (Group B), were also given. Both groups were comparable with respect to age, sex, associated diseases, and primary diagnosis. Wound infections developed in nine of 169 (5%) group B patients and in 15 of 141 (11%) group A patients. Stratification by type of operative procedure revealed that the rectal resections involved the highest rate of infection in group A (22%) and in group B (11%). In patients requiring intraperitoneal colon resection, the rates of wound sepsis were similar (3% in both groups). Analysis of length of operation revealed that in operations lasting 215 minutes or more the infection rate was 12%; in those lasting less than 215 minutes the rate was 4%. Patients with rectal resection and operative times of 215 minutes or more had a wound infection rate of 19% compared to 2% (p less than 0.05) in those with shorter nonrectal operations. Group B patients with the longer rectal operations had lower infection rates (11%) than group A patients (27%), while there was no difference among those who had shorter operations. Intra-abdominal abscesses (p less than 0.01) and anastomotic dehiscence (p less than 0.05) were also significantly reduced in group B patients. Postoperative wound infection is associated with length of operation and location of colon resection and can be significantly lowered by a combination of oral and parenteral antibiotics.

Topics: Adult; Aged; Anti-Bacterial Agents; Cefoxitin; Colonic Diseases; Colonic Neoplasms; Escherichia coli Infections; Female; Humans; Male; Middle Aged; Prospective Studies; Random Allocation; Rectal Diseases; Rectal Neoplasms; Staphylococcal Infections; Surgical Wound Infection

Protein glycosylation is a diverse form of post-translational modification. Two to three consecutive O-linked N-acetylgalactosamines (Tn-antigens) are recognized by antibodies such as MLS128. MLS128 mAb inhibited cell growth and bound to a 110 kDa glycoprotein (GP) in LS180 and HT29 colon cancer cells. However, purification and identification of the 110 kDa GP was unsuccessful due to its low abundance. The present study used a highly sophisticated and sensitive mass spectrometry method to identify proteins immunoprecipitated with MLS128 and separated by two-dimensional gel electrophoresis. Three desmosome components were identified. Of these, desmocollin and desmoglein shared many similar characteristics, including molecular mass, pI, and potential Tn-antigen sites. Western blotting analyses of LS180 cell lysates revealed a common 110 kDa band recognized by MLS128 and anti-desmocollin, but not by anti-desmoglein. Immunofluorescence microscopy of LS180 cells revealed that desmocollin is membrane-bound, while desmoglein is primarily localized in the cytosol. Confocal microscopy demonstrated colocalization of the desmocollin-specific antibody with the MLS128 antibody on the cell membrane, suggesting that desmocollin may contain Tn-antigens recognized by MLS128. Treatment of LS180 cells with siRNA to knock down desmocollin expression or a desmocollin-specific antibody decreased cell viability, suggesting a critical role for this protein in cell growth and survival. N-glycosidase F digestion of the 110 kDa GP and desmocollin suggested that although both proteins contain N-glycosylation sites, they are not identical. These findings suggest that desmocollin colocalizes with the 110 kDa GP and that growth inhibition induced by the MLS128 antibody may be mediated through a mechanism that involves desmocollin.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Desmocollins; Glycoproteins; HT29 Cells; Humans; Microscopy, Confocal; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Tandem Mass Spectrometry

Chronic inflammation and composition of the colon microbiota have been associated with colorectal cancer in humans. The human commensal enterotoxigenic Bacteroides fragilis (ETBF) is linked to both inflammatory bowel disease and colorectal cancer and, in our murine model, causes interleukin 17A (IL-17A)-dependent colon tumors. In these studies, we hypothesized that persistent colonization by ETBF is required for tumorigenesis.. We established a method for clearing ETBF in mice, using the antibiotic cefoxitin. Multiple intestinal neoplasia mice were colonized with ETBF for the experiment duration or were cleared of infection after 5 or 14 days. Gross tumors and/or microadenomas were then evaluated. In parallel, IL-17A expression was evaluated in wild-type littermates.. Cefoxitin treatment resulted in complete and durable clearance of ETBF colonization. We observed a stepwise increase in median colon tumor numbers as the duration of ETBF colonization increased before cefoxitin treatment. ETBF eradication also significantly decreased mucosal IL-17A expression.. The timing of ETBF clearance profoundly influences colon adenoma formation, defining a period during which the colon is susceptible to IL-17A-dependent tumorigenesis in this murine model. This model system can be used to study the microbiota-dependent and molecular mechanisms contributing to IL-17A-dependent colon tumor initiation.

Topics: Animals; Bacteroides fragilis; Carcinogenesis; Cefoxitin; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Enterotoxins; Humans; Mice

Anti-Tn antigen MLS128 monoclonal antibody was produced two decades ago by immunizing mice with "cancerous antigens" derived from LS180 colon cancer cells. Previous studies demonstrated that MLS128 bound to 110 kDa glycoprotein (GP) in colon cancer cells, thereby inhibiting cell growth. Extensive attempts have been made towards understanding the inhibitory action of MLS128 on colon cancer cell growth and solving the primary structure of 110 kDa GP. Since limited proteolysis of 110 kDa GP was observed in microdomain fractions that had been kept frozen for several years, susceptibility of 110 kDa GP to trypsin and other proteases as well as N-glycosidase F has been investigated. Furthermore, 110 kDa GP expression was examined in colon cancer cells independently cultured in Akiyama laboratory. In summary, 110 kDa GP contains N-glycans. It does not contain inter-disulfide bonds but appears to have intra-disulfides. It must contain multiple cleavage sites for trypsin and thermolysin since these proteases digested 110 kDa GP to MLS128-undetectable small fragments. It seems to contain cleavage sites for cathepsin D which could cause limited digestion. LS180 cells derived from Akiyama laboratory produced a limited proteolysis product-like 75 kDa GP. This study provides a structural basis for developing cancer diagnostics and therapeutics.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Blotting, Western; Colonic Neoplasms; Glycoproteins; HT29 Cells; Humans; Peptide Hydrolases; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Protein Binding; Trypsin

It has long been presumed, though with surprisingly little evidence, a competition between Core 1 Gal-transferase (C1GalT), Core 3 GlcNAc-transferase (C3GnT) and sialyl-transferase (ST6GalNAc-T) for elongation of O-linked mucin-type glycans initiated with GalNAcα-Ser/Thr. This study tested this presumption by selective suppression of one of these glycosyltransferases and then analysed the expressions of the enzymatic products of the other three glycosyltransferases. It was found that siRNA suppression of C1GalT markedly reduced the expression of Galβ1,3GalNAcα- (Core 1) and in the meantime increased the expressions of sialyl-GalNAcα- (sialyl-Tn), GalNAcα- (Tn) and GlcNAcβ1,3GalNAcα- (Core 3)-associated glycans in human colon cancer HT29 and SW620 cells. This supports a competitive modification of the GalNAcα-Ser/Thr between C1GalT, C3GnT and ST6GalNAc-T in O-glycan biosynthesis. As Tn, TF and sialyl-Tn are oncofetal antigens and are over-expressed in most human cancers, this information is useful for the development of glycosyltransferase-targeted therapeutic strategies for cancer treatment.

Topics: Antigens, Tumor-Associated, Carbohydrate; Cell Line, Tumor; Colonic Neoplasms; Galactosyltransferases; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mucins; Polysaccharides; RNA Interference

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Female; Glycopeptides; Humans; Male; Mice; Middle Aged; Neoplasm Staging; Neoplasms; Protein Binding

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

MLS128 is an anti-carbohydrate monoclonal antibody (mAb) that binds three or two consecutive Tn-antigens. MLS128 bound 110-210 kDa glycoproteins (GPs) and inhibited the growth of LS180 and HT29 colon and MCF-7 breast cancer cells. One possible mechanism of MLS128's inhibition of growth may be via insulin-like growth factor-I receptor (IGF-IR) down-regulation (Morita et al. BioScience Trends. 2009; 3:32-37). The current study examined the role of IGF-IR signaling in the growth of colon cancer cells and its possible interaction with MLS128-induced inhibition of cell growth in LS180, LS174T, and HT29 human colon cancer cells treated with MLS128 or anti-IGF-IR 1H7. Both MLS128 and 1H7 treatment significantly inhibited the growth of colon cancer cells. All three colon cancer cell lines expressed IGF-IR. Their growth was in part IGF-I dependent, but inhibition by MLS128 was independent of IGF-IR signaling. All of the colon cancer cell lines expressed an 110kDa GP for MLS128 binding, but MCF-7 cells expressed MLS128-detectable bands with higher molecular masses. 1H7 treatments caused down-regulation of IGF-IR but did not affect 110kDa GP levels. MLS128 treatments resulted in partial disappearance of the 110kDa band but did not affect IGF-IR levels. Western blotting analyses of colon and breast cancer cell lysates revealed that colon and breast cancer cells differed significantly in patterns of expression of growth-related molecules while colon cancer cells were similar but distinctive. In conclusion, MLS128 inhibited the growth of colon cancer cells by binding to the 110kDa GP receptor. Inhibition of growth by MLS128 did not appear to affect IGF-IR signaling and instead only affected other growth signaling pathways.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; HT29 Cells; Humans; Receptor, IGF Type 1; Signal Transduction

The Tn-antigen, GalNAcalpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Bacteriophages; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Fluorescent Antibody Technique; Glycopeptides; Humans; Immunoglobulin Variable Region; Peptide Library; Sequence Homology, Amino Acid; Single-Chain Antibodies; Surface Plasmon Resonance

The epithelial mucin MUC1 is a high molecular weight membrane glycoprotein frequently overexpressed and aberrantly glycosylated in adenocarcinoma. Mucins normally contain high amounts of O-linked carbohydrate structures that may influence immune reactions to this antigen. During malignant transformation, certain glyco-epitopes of MUC1, such as Tn-antigen, TF-antigen and their sialylated forms become exposed. The role of these glycan structures in tumor biology is unknown, but their presence is known to correlate with poor prognosis in several adenocarcinomas. We analyzed the potency of MUC1 containing Tn-antigens (MUC1-Tn) to target C-type lectins that function as carbohydrate recognition and uptake molecules on dendritic cells (DC). We identified the macrophage galactose type C-type lectin (MGL), expressed by both DC and macrophages, as the receptor for recognition and binding of MUC1-Tn. To validate the occurrence of MGL-MUC1 interactions in situ, we studied the binding of MGL to MUC1 in primary colon carcinoma tissue. Isolation of MUC1 out of colon carcinoma tissue showed strong binding activity to MGL. Interestingly, MGL binding to MUC1 was highly correlated to binding by the lectin Helix pomatia agglutinin (HPA), which is associated with poor prognosis in colorectal cancer. The detection of MGL positive cells in situ at the tumor site together with the modified glycosylation status of MUC1 to target MGL on DC suggests that MGL positive antigen presenting cells may play a role in tumor progression.

Topics: Acetylgalactosamine; Adenocarcinoma; Animals; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Cell Adhesion Molecules; CHO Cells; Colon; Colonic Neoplasms; Cricetinae; Cricetulus; Dendritic Cells; Endocytosis; Female; Glycosylation; Humans; Immunoglobulin G; Intestinal Mucosa; Lectins, C-Type; Mice; Mice, Inbred BALB C; Monocytes; Monosaccharides; Mucin-1; Mucins; Neoplasm Proteins; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Receptors, Cell Surface; Recombinant Fusion Proteins; Tandem Repeat Sequences

Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathology, since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes alpha-d-N-acetylgalactosamine (alphaGalNAc) containing epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc containing epitopes associated with cancer, the Tn (alphaGalNAc-Ser) and Forssman (alphaGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an additional sugar or amino acid in the alpha position. These structures provide the molecular basis for the use of HPA in metastasis research.

Topics: Acetylgalactosamine; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Colonic Neoplasms; Disaccharides; Epitopes; Female; Forssman Antigen; Humans; Lectins

The simple mucin-type truncated O-glycans Tn (GalNAc-O-Ser/Thr) and sialyl-Tn (STn) antigens are useful diagnostic markers for human colon cancer. We herein report the characterization of 1,2-dimethylhidrazine (DMH)-induced colon cancer in rats as a new model for the study of aberrant O-glycosylation products during carcinogenesis. Evaluated by immunohistochemistry, both anti-Tn and anti-STn MAbs revealed no staining of normal colonic mucosa. On the contrary, Tn and STn were expressed by the first lesions detected following carcinogen administration (aberrant crypt foci), observing the most intense and uniform pattern in crypts with severe dysplasia. Adenocarcinomas with non-secreting components showed moderately and strong stain, but mucin-secreting carcinomas were mildly stained. The biochemical characterization of soluble Tn glycoproteins from ascitic fluids of rats with colon cancer revealed that Tn is bearing high molecular weight glycoproteins (containing sialic acid and/or GlcNAc and GalNAc), which migrated as two major components (one of approximately 220 kDa and other>500 kDa). Evaluated by CsCl gradient ultracentrifugation and perchloric acid precipitation, it was shown that Tn is carried for mucins. These results indicate that Tn and STn are pre-cancerous biomarkers in colon of rats treated with DMH. This model of rat colon cancer could be useful to study in vivo the temporal sequence of molecular events responsible for the deregulation of O-glycosylation pathways during colon carcinogenesis, and could contribute to improve the evaluation of diagnostic and therapeutic strategies based on the utilization of Tn and STn antigens.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Colon; Colonic Neoplasms; Diagnosis, Differential; Dimethylhydrazines; Female; Glycoproteins; Immunohistochemistry; Mucins; Precancerous Conditions; Rats; Rats, Wistar

The Sialyl-Tn antigen (Sialyl alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells. Its presence is associated with a poor prognosis in patients with colorectal and other cancers. We previously reported that Sialyl-Tn expression in LSC human colon cancer cells could be explained by a specific lack of the activity of core 1 beta3-Gal-transferase (Brockhausen et al., Glycoconjugate J. 15, 595-603, 1998) and an inability to synthesize the common O-glycan core structures. To support this mechanism, or find other mechanisms to explain Sialyl-Tn antigen expression, we investigated the O-glycosylation pathways in clonal rat colon cancer cell lines that were selected for positive or negative expression of Sialyl-Tn antigen, and compared these pathways to those in normal rat colonic mucosa. Normal rat colonic mucosa had very active glycosyltransferases synthesizing O-glycan core structures 1 to 4. Several sialyl-, sulfo- and fucosyltransferases were also active. An M type core 2 beta6-GlcNAc-transferase was found to be present in rat colon mucosa and all of the rat colon cancer cells. O-glycosylation pathways in rat colon cancer cells were significantly different from normal rat colonic mucosa; for example, rat colon cancer cells lost the ability to synthesize O-glycan core 3. All rat colon cancer cell lines, regardless of the Sialyl-Tn phenotype, expressed glycosyltransferases assembling complex O-glycans of core 1 and core 2 structures (unlike human LSC colon cancer cells which lack core 1 beta3-Gal-transferase activity). It was the activity of CMP-sialic acid:GalNAc-mucin alpha6-sialyltransferase that coincided with Sialyl-Tn expression. Sialyl-Tn negative cells had a several fold higher activity of core 2 beta6-GlcNAc-transferase which synthesizes complex O-glycans that may mask adjacent Sialyl-Tn epitopes. The results suggest a new mechanism controlling Sialyl-Tn expression in cancer cells.

Topics: Amino Acid Sequence; Animals; Antigens, Tumor-Associated, Carbohydrate; beta-D-Galactoside alpha 2-6-Sialyltransferase; Carbohydrate Sequence; Cell Division; Colonic Neoplasms; Epithelial Cells; Gastric Mucosa; Glycosylation; Molecular Sequence Data; Mucins; N-Acetylglucosaminyltransferases; Polysaccharides; Rats; Reference Values; Sialyltransferases; Tumor Cells, Cultured

Epithelial mucins are present at the apical membranes of gastrointestinal epithelial cells or in their secretions. In this study, we examined the occurrence of peptide epitopes of the mucins MUC1 and MUC3 and of three mucin-associated glycotopes (TF, Tn, and s-Tn) in a series of colorectal tissue samples (normal colon, adenomas with different grades of dysplasia, carcinoma in situ, and invasive carcinomas). A new monoclonal antibody to a conformation-dependent peptide epitope of MUC1 was employed, which does not react with the fully glycosylated mucin as found in normal gastrointestinal mucosa. We found that adenomas acquired the ability to expose Tn, s-Tn, TF and MUC1 epitopes, and this correlated with increasing malignant potential. The secretory mucin, MUC3, revealed a different pattern: it was detectable in all sections, with maximum expression in adenomas and decrease in carcinomas. Most importantly, normal mucosa and benign lesions showed supra-nuclear and/or apical distribution of these antigens, but malignant lesions and lesions with a very high risk of malignancy revealed diffuse cytoplasmic and basolateral membrane localization. The immunohistological response to a combination of MUC1-related antibodies may assist in assessing the malignant potential and status of lesions of the colon.

Topics: Adenoma; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Carcinoma; Cell Membrane; Cell Transformation, Neoplastic; Colonic Neoplasms; Cytoplasm; Humans; Immunohistochemistry; Intestinal Mucosa; Intracellular Membranes; Mucin-1; Mucin-3; Mucins; Precancerous Conditions

The goal of this study was to determine if polypeptides that bind specifically to the carcinoma-associated Thomsen-Friedenreich (T) antigen could be isolated from a random peptide bacteriophage display library. T antigen is a carbohydrate antigen that is exposed and immunoreactive on the surfaces of most primary carcinomas and their metastases, while it is masked on normal cells. Tumor-specific surface carbohydrates are often used as markers of cell differentiation and play a role in cell aggregation, which is an important step in the metastatic process. Therefore, peptides that bind and mask T antigen may yield useful carbohydrate-specific probes and provide insight into carbohydrate-mediated tumor-cell aggregation. A 15-amino acid random peptide bacteriophage display library was screened for polypeptides that exhibited high specificity to two glycoproteins which display T antigen on their surfaces. The results suggest that synthetic peptides identified from the bacteriophage display library have high affinities (Kd approximately 1 microM) and specificities for proteins and human tumor cells which present T antigen. Thus, random bacteriophage peptide display libraries may be a rich source of sequences that bind to carbohydrate antigen structures.

Topics: Amino Acid Sequence; Animals; Antigens, Tumor-Associated, Carbohydrate; Carcinoma; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Humans; Inoviridae; Melanoma; Mice; Molecular Sequence Data; Peptide Library; Peptides; Tumor Cells, Cultured

Tn antigen is a glycosylated tumor associated antigen and a murine monoclonal antibody, MLS128, has been identified to react with it. The potential of MLS128 for the radioimmunoimaging of colorectal cancer was studied. MLS128 was labeled with radioiodine by the chloramine-T method or indium-111 (111In) by using isothiocyanatobenzyl EDTA, and was injected into nude mice bearing human colon cancer xenografts. Radiolabeled MLS128 showed a high and specific localization in xenografted tumor. At 48 h after injection, the %ID/g of 125I-labeled MLS128 in the tumor was 34.69, whereas that of isotype matched control antibody, FLOPC21, was 5.58 and the tumor-to-nontumor radioactivity ratios of 125I-labeled MLS128 reached to 4.56, 17.84 and 23.62 for the blood, liver and bone, respectively. 111In-labeled MLS128 showed similar results. High accumulation of MLS128 in xenografted tumors suggested that the monoclonal antibody MLS128 is promising for radioimmunoimaging of colorectal cancer.

Topics: Animals; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Cell Line; Colonic Neoplasms; Humans; Iodine Radioisotopes; Mice; Mice, Nude; Radioimmunodetection; Radionuclide Imaging; Time Factors; Tissue Distribution; Transplantation, Heterologous; Tumor Cells, Cultured

The lectin GS I-A4 binds to terminal alpha-N-acetylgalactosaminyl (GalNAc) groups (which include the Tn antigen), but not to the closely related tumor-associated epitope, sialylated Tn antigen. The lectin also precipitates asialo OSM, but not its native sialylated form. Lectin histochemistry with human colonic tissues showed that GS I-A4 specifically stained specimens of colon cancer and colonic tissues from individuals with FAP; however, normal colonic tissues from patients without colonic disease were rarely stained with this lectin. Glycoconjugates bound by GS I-A4 were observed on the surface membranes of 2 human colon cancer cell lines, LS174t and SW1116, when fluorescein isothiocyanate (FITC)-conjugated GS I-A4 was used. GS I-A4 was toxic to these 2 human colon cancer cell lines in monolayer culture. A dose-response study conducted using 10-160 micrograms/ml, of GS I-A4 demonstrated significant dose-related toxicity against LS174t and SW1116 cells. At concentrations > 80 micrograms/ml, > 99% of LS174t and > 90% of SW1116 cells were killed. Four mM GalNAc specifically inhibited the cytotoxic effect of GS I-A4 (p < 0.001), whereas 4mM N-acetylglucosamine (GlcNAc) had no effect. Two other lectins that recognize terminal alpha-GalNAc residues, DBA and LBL, were significantly less cytotoxic to the colon cancer cells than GS I-A4. In the light of these findings, we speculate that GS I-A4 may have potential use as a diagnostic agent against colorectal cancer.

Topics: Acetylgalactosamine; Antigens, Tumor-Associated, Carbohydrate; Cell Division; Colon; Colonic Neoplasms; Epithelium; Epitopes; Glycoconjugates; Humans; Lectins; Plant Lectins; Sensitivity and Specificity; Tumor Cells, Cultured

Partially desialylated ovine submaxillary mucin plus an immunological adjuvant has been used by us to induce a humoral immune response to Tn antigen and sialylated Tn in colon cancer patients at risk of recurrence. Peripheral blood lymphocytes were purified from one of these patients with a high IgM titer reactive with Tn antigen transformed with Epstein-Barr virus and subsequently fused with a human-mouse heteromyeloma cell line, HMMA2.11TG/O. Clones were screened and subcloned using an enzyme-linked immunoassay and four stable IgM-secreting clones were tested for reactivity with a variety of natural and synthetic antigens, paraffin-embedded colon carcinomas and normal colonic mucosa to determine their specificity. Four human IgM monoclonal antibodies reactive predominantly with Tn antigen were produced and shown to react with a mucinous human colon carcinoma cell line, LS-174T, and with paraffin-embedded human colon carcinomas. We conclude that modified ovine submaxillary mucin is an effective vaccine for generating a humoral immune response directed to Tn antigen present on human colon cancer.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Colonic Neoplasms; Epitopes; Humans; Hybridomas; Immunoglobulin M; Immunohistochemistry; Mice; Mucins; Sheep; Sialic Acids; Submandibular Gland; Tumor Cells, Cultured; Vaccination

Using immunohistochemical techniques and whole-cell enzyme-linked immunosorbent assay, we have determined that monoclonal antibodies (mAbs) B72.3 and CC49, which are widely used in the diagnosis and treatment of several human epithelial cancers, are expressed in a transplantable rat colon carcinoma cell line, K12-TRb. MAbs B72.3 and CC49 react with tumor-associated glycoprotein-72 (TAG-72) which is a carcinoma mucin molecule expressed in colon, breast, pancreatic, ovarian, lung, and gastric cancers. The carbohydrate epitope for mAb B72.3 is sialylated Tn (sTn), whereas CC49 reacts with an unknown carbohydrate epitope. K12-TRb is a transplantable rat colon carcinoma cell line derived from a dimethylhydrazine tumor which grows as progressive tumors in syngeneic BD IX rats. We found that the carbohydrate epitopes for mAbs B72.3 and CC49, including sTn, were more tumor-restricted in the rat than in humans. The only binding these had mAbs to normal rat tissue was to small-intestinal mucosa. MAbs B72.3 and CC49 were radiolabeled with iodine-125 (125I) and injected intravenously into BD IX rats containing subcutaneously grown syngeneic K12-TRb tumors. Biodistribution experiments were conducted by dissecting groups of three rats on days 2, 4, 7, and 14 after injection of radiolabeled mAbs. These experiments confirmed that maBs B72.3 and CC49 localize to K12-TRb tumors in vivo, and that the higher affinity mAb CC49 localized better than mAb B72.3. Gamma-camera imaging of subcutaneous K12-TRb tumors was successfully performed using 125I-labeled mAb CC49. The importance of this model is that mAbs B72.3 and CC49, immunoconjugates of these mAbs, and vaccines containing their corresponding carbohydrate epitopes, including sTn, can be studied in a relevant immunocompetent syngeneic rat colon carcinoma model.

Topics: Animals; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Carcinoma; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Humans; Immunohistochemistry; Mice; Mucins; Neoplasms, Experimental; Radionuclide Imaging; Rats; Rats, Inbred Strains; Sialoglycoproteins; Transplantation, Isogeneic

The susceptibility to natural-killer-cell lysis and expression of histo-blood-group antigens of 2 clones from a rat colon adenocarcinoma, of variants derived from them and of 17 human colon carcinoma cell lines were assessed in an attempt to determine if the major glycosidic tissue antigens of epithelial cells could influence the NK susceptibility of tumor target cells of epithelial origin. The rat REGb clone, which is relatively NK-sensitive, expressed higher levels of precursor structures T and Tn and lower levels of H antigenic determinants than the PROb clone, which displays higher resistance to NK-cell lysis. Cell variants were obtained from these 2 clones; it appeared that whether the cell variants were selected on the basis of expression of a blood-group antigenic determinant or on the basis of altered susceptibility to NK-cell lysis, there was a link between increased resistance and higher expression of cell-surface A and H histo-blood-group antigens, or conversely, between increased sensitivity and higher expression of precursor structures. Similar conclusions were obtained upon study of the human cell lines, since a significant correlation was found between the level of expression of T or Tn antigens and sensitivity to NK-cell lysis. A significant relationship was found between the expression of Lewis antigens and increased resistance to NK-cell-mediated cytotoxicity.

Topics: ABO Blood-Group System; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Blood Group Antigens; Colonic Neoplasms; Cytotoxicity, Immunologic; Glycosylation; Humans; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Lewis Blood Group Antigens; Tumor Cells, Cultured

The Tn, sialosyl-Tn, and T antigens are carbohydrate-associated antigens that represent initial steps in mucin O-linked glycosylation. Previous immunohistochemical studies have shown that these three antigens are rarely, if ever, expressed in normal colonic mucosa; however, most colonic cancerous tissues express these structures. Little is known about the factors that control the expression of these antigens in colonic tissues or cell lines. One hypothesis is that cancers have increased levels of the glycosyltransferase activities responsible for synthesizing these antigens.. The current study analyzed antigen expression by immunohistochemistry and glycosyltransferase enzyme activities for Tn, sialosyl-Tn, and T antigens in colonic tissues and cell lines to (1) compare values between normal and cancerous tissues and (2) correlate these results with tumor stage, histologic findings, and location.. All nine colonic cancer cell lines expressed Tn antigen; sialosyl-Tn and T antigens were expressed by the more mucin-producing cell lines. Sialosyl-Tn transferase activity was higher in the more mucinous cell lines; T transferase activity was higher in those with less mucin. In paired specimens of normal and cancerous tissues, levels of each of the three glycosyltransferases were similar. In cancerous tissues, enzyme activity did not correlate with tumor location, stage, or histologic type. There also was no correlation between glycosyltransferase activity and expression of the relevant antigen.. Thus, because normal and malignant colonic tissues have comparable levels of Tn, sialosyl-Tn, and T glycosyltransferases, the absence of these antigens in normal mucosa apparently is related to other factors such as antigen masking.

Topics: Aged; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Colonic Neoplasms; Glycosylation; Glycosyltransferases; Humans; Immunoenzyme Techniques; Male; Middle Aged; Mucins; Tumor Cells, Cultured

Sialosyl-Tn antigen and its immediate precursor, Tn antigen, are carbohydrate structures associated with the earliest steps of mucin O-linked glycosylation. Both antigens have been shown previously to be highly sensitive and specific markers of colorectal cancer. One hundred and three colorectal polyps (79 adenomatous; 24 hyperplastic) were examined for expression of Tn antigen using vicia villosa isolectin B4, and for sialosyl-Tn antigen by monoclonal antibody TKH2. Tn antigen was expressed by all of the polyps studied. Sialosyl-Tn, on the other hand was expressed weakly by a few cells in 7 of 24 (29%) hyperplastic polyps. Among the adenomatous polyps, 56% expressed sialosyl-Tn and expression correlated with larger adenoma size, greater villous component, and more severe grades of dysplasia. In individuals with two or more synchronous adenomas, the level of sialosyl-Tn expression within an adenoma was associated with the severity of cytological atypia. All the adenomas that contained a focus of invasive carcinoma expressed sialosyl-Tn. These results indicate that colorectal polyps manifest incomplete glycosylation, exposing antigens in the innermost region of mucin oligosaccharides. In addition, the correlation of sialosyl-Tn antigen expression with the adenoma-carcinoma sequence may make this a useful marker for studying malignant progression in the colon.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Colonic Neoplasms; Humans; Immunoenzyme Techniques; Intestinal Polyps; Mucins; Rectal Neoplasms

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.

Topics: Acetylgalactosamine; Animals; Antibodies, Monoclonal; Antibody Specificity; Antigen-Antibody Reactions; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Binding Sites, Antibody; Colonic Neoplasms; Humans; Immunoglobulin G; Mice; Mice, Nude; Monosaccharides; Mucins; Tumor Cells, Cultured

Mucin from xenografts of LS174T human colon cancer cells was treated with anhydrous HF for 1 h at 0 degree C to give a product (HFA) with over 80% of the glucosamine and hexose removed, but retaining some galactosamine, and for 3 h at room temperature to give a product (HFB) devoid of carbohydrate. Rabbit antibodies against HFA bound to HFA much more than to HFB, and bound to native mucin to an intermediate extent. Antibodies to HFB bound to HFB more than to HFA, and did not bind to native mucin. Both HFA and native mucin bound a number of lectins, but HFB did not. By SDS/polyacrylamide-gel electrophoresis and size-exclusion h.p.l.c., native mucin and HFA are of apparent molecular mass greater than 400 kDa, whereas HFB is heterogeneous and of low molecular mass. On Western blots, antibody to HFA detected both high-molecular-mass mucin and a 90 kDa protein in homogenates of LS174T cells. Antibody to HFB detected a major 70 kDa band as well as higher-molecular-mass species. In tissue sections of normal colon and colon cancers, antibody to HFA showed both cytoplasmic and extracellular staining, whereas antibody to HFB generally stained only cytoplasmic antigens. These results indicate that anti-HFB antibody is specific for apo-mucin, whereas anti-HFA antibody is specific for GalNAc-apo-mucin.

Topics: Animals; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Colonic Neoplasms; Gastric Mucins; Hydrofluoric Acid; Mice; Molecular Weight; Mucins; Peptides

Mucin glycoproteins are major secretory products of the colon and contain O-linked oligosaccharides synthesized on a polypeptide backbone. The initial step in the synthesis of O-linked oligosaccharides is the addition of N-acetylgalactosamine to serine or threonine residues forming the Tn antigen. This substance can then receive additional carbohydrate residues such as sialic acid to form sialosyl-Tn antigen, or galactose to form T antigen. In the colon, the T antigen is an oncodevelopmental cancer-associated antigen but little is known about Tn and sialosyl-Tn expression. The present comparative immunohistochemical study was performed to analyze the expression of these antigens in fetal, normal adult, and malignant colorectal tissues with an aim toward elucidating whether Tn and sialosyl-Tn are also oncodevelopmental colon cancer-associated antigens and to gain insight into the earliest steps of mucin glycosylation in colonocytes. We used three reagents to detect Tn antigen (two monoclonal antibodies ETn1.01 and CU-1, and one lectin Vicia villosa), two reagents to detect sialosyl-Tn (monoclonal antibodies TKH2 and B72.3) and one to detect T antigen (monoclonal antibody AH9-16). Except for occasional reactivity with VVA and CU-1, cells of normal colonic mucosa did not express Tn, sialosyl-Tn, or T antigens. However, in the transitional mucosa immediately adjacent to cancer, all three antigens were expressed (ranging from 35 to 67% of cases depending upon the reagent). In colon cancers, the percentage of cases expressing each antigen were as follows: Tn 72-81%, sialosyl-Tn 93-96%, and T 71%. Unlike T antigen, which was preferentially expressed by moderately well- and well-differentiated adenocarcinomas, both Tn and sialosyl-Tn antigens were expressed by most histological subsets of colon cancers, including poorly differentiated adenocarcinomas and mucinous (colloid and signet ring cell type) carcinomas. The majority of cancers expressed both Tn and sialosyl-Tn, usually in association with T antigen. Only one cancer lacked all three antigens. Fetal colonic mucosal cells expressed all three antigens, particularly in goblet cell mucin. These results indicate that like T antigen, Tn and sialosyl-Tn are oncodevelopmental cancer-associated antigens in the colon. Moreover, Tn and sialosyl-Tn antigens appear to be useful markers of poorly differentiated adenocarcinomas and mucinous carcinomas: two histological subsets that often fail to express other cancer-associ

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Colon; Colonic Neoplasms; Fetus; Glycosylation; Humans; Intestinal Mucosa