Colon Adenocarcinoma Tissues Research Articles

Alterations of glycosidases in human colonic adenocarcinoma

Objectives: We have carried out a detailed study of some glycosidases in an attempt to explain the differential profile of enzyme activity between human colonic adenocarcinoma and normal mucosa. Design and Methods: Several glycosidase activities associated with human colonic adenocarcinoma and control tissues were submitted to a detailed structural and functional characterization. Results: Tumoral and control samples were assayed for β- d-galactosidase, β- d-glucuronidase, α- d-mannosidase, β-NAc- d-glucosaminidase and β-NAc- d-galactosaminidase activities. Tumoral tissue showed higher β- d-galactosidase, β-NAc- d-glucosaminidase, and β-NAc- d-galactosaminidase activities than control tissue. Glycosidases from tumoral and control tissues demonstrated no differences in optimum pH, subcellular distribution, pH and thermal stability. However, the kinetic analysis showed a statistically significant increased V max in tumoral colon with respect to the control for β- d-galactosidase, β-NAc- d-glucosaminidase, and β-NAc- d-galactosaminidase activities. The K m remained unaltered. Conclusions: The increased V max detected for some glycosidase activities in human colonic adenocarcinoma could correspond with a greater presence of enzyme proteins in the tumoral cells, and not to changes in protein and/or active site structure.

Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen.

Carcinomas of the gastrointestinal tract are not curable by standard therapies. Thus, new therapeutic approaches for this disease are needed. This study proposes the use of anti-Id mAb as Ag substitutes to induce anti-tumor immunity in gastrointestinal cancer patients. Recently, we have generated and characterized one monoclonal anti-Id antibody, designated 3H1 (Ab2), which mimics biologically and antigenically a distinct and specific epitope of the 180,000 m.w. carcinoembryonic antigen (CEA) primarily expressed in high density by human pancreatic and colonic tumor cells. This epitope is unique to CEA and not present on other CEA-related lower m.w. members of the Ag family also found on normal tissues. The antigenic determinant as defined by the mAb 8019 (Ab1) against which the Ab2, 3H1 was raised, is absent on normal adult tissues by immunoperoxidase staining and haematopoietic cells including granulocytes by flow cytometry analysis. Anti-Id (Ab2) 3H1 induced CEA-specific antibodies in mice and rabbits. The immune sera from both mice and rabbits competed with Ab1 for binding to the colon carcinoma cell line LS174T and inhibited the binding of radioiodinated Ab1 to Ab2. This indicates that anti-anti-Id (Ab3) in mice and rabbits share idiotopes with Ab1 (8019). Furthermore, monoclonal Ab3 that bind to CEA have been generated from mice immunized with 3H1. The Ab3 (both polyclonal as well as monoclonal) immunoprecipitated the same 180,000 m.w. CEA as Ab1 (8019) by Western blotting analysis and showed almost identical immuno-staining patterns as Ab1 on colonic adenocarcinoma tissue sections from several patients. Collectively these data suggest that Ab2 3H1 could potentially be used clinically as a network Ag for immunotherapy of patients with CEA positive tumors.

Radioimmunolocation of Metastases From Adenocarcinoma of the Colon: Using A Hepatoma Antibody

We have established a large library of monoclonal antibodies against a human hepatoma cell line called FOCUS. One such monoclonal antibody (SF–25) detects a 125–kilodalton cell surface antigen found on FOCUS cells. As both the liver and the colon are of endodermal origin, we examined the possibility of expression in colon adenocarcinomas. This antigen was found in all 23 colon adenocarcinoma tissues surgically obtained but was absent in the adjacent normal mucosal counterpart as determined by a direct radioimmunohistologic technique. In the present study, we have established a model for human metastatic colon adenocarcinoma using the LS 180 cell line. Athymic mice were further immunosuppressed by intravenous injection of anti–NK cell antibodies (antiasialo GM1). After 24 h, mice were injected with LS 180 cells either via the tail vein or into the spleen followed by splenectomy. Macroscopic pulmonary and lymphatic metastasis developed within 2–3 wk after injection of cells and 9 of 10 mice died with advanced metastatic disease 2–3 wk later. In addition, macroscopic hepatic metastases were evident in 4 of 5 mice 3–4 wk after intrasplenic injection. Both hepatic as well as pulmonary and lymphatic tumor spread was localized by nuclear imaging with 125I–SF–25. Furthermore, micrometastases were detected by autoradiography 5–10 days later. Monoclonal antibody SF–25 is a potential candidate for tumor localization and the experimental metastatic colon cancer animal model may be useful for treatment evaluation of monoclonal antibody SF–25 either alone or in combination with other monoclonal antibodies when conjugated to radionucleotides and chemotherapeutic agents.

Radioimmunolocation of Hepatic and Pulmonary Metastasis of Human Colon Adenocarcinoma

We have established a large library of monoclonal antibodies against a human hepatoma cell line called FOCUS. One such monoclonal antibody (SF-25) detects a 125-kilodalton cell surface antigen found on FOCUS cells. As both the liver and the colon are of endodermal origin, we examined the possibility of expression in colon adenocarcinomas. This antigen was found in all 23 colon adenocarcinoma tissues surgically obtained but was absent in the adjacent normal mucosal counterpart as determined by a direct radioimmunohistologic technique. In the present study, we have established a model for human metastatic colon adenocarcinoma using the LS 180 cell line. Athymic mice were further immunosuppressed by intravenous injection of anti-NK cell antibodies (antiasialo GM1). After 24 h, mice were injected with LS 180 cells either via the tail vein or into the spleen followed by splenectomy. Macroscopic pulmonary and lymphatic metastasis developed within 2-3 wk after injection of cells and 9 of 10 mice died with advanced metastatic disease 2-3 wk later. In addition, macroscopic hepatic metastases were evident in 4 of 5 mice 3-4 wk after intrasplenic injection. Both hepatic as well as pulmonary and lymphatic tumor spread was localized by nuclear imaging with 125I-SF-25. Furthermore, micrometastases were detected by autoradiography 5-10 days later. Monoclonal antibody SF-25 is a potential candidate for tumor localization and the experimental metastatic colon cancer animal model may be useful for treatment evaluation of monoclonal antibody SF-25 either alone or in combination with other monoclonal antibodies when conjugated to radionucleotides and chemotherapeutic agents.

Differentiation of human tumors from nonmalignant tissue by natural‐abundance 13C NMR spectroscopy

High-quality, high-resolution, proton-decoupled natural-abundance 13C NMR spectra have been obtained in vitro at 100.6 MHz from unprocessed human pathology specimens of tumors and adjacent nonneoplastic control tissues from lung, colon, and prostate. In these preliminary studies, specific molecular parameters were identified from the spectra that distinguished neoplastic from nonneoplastic tissue of a given organ in all sites studied. The NMR results were congruent with data derived from histochemical and biochemical examinations of the tissues and with previous studies using non-NMR methods. In particular, a comparison of the spectra of prostatic adenocarcinoma with that of adjacent hyperplastic tissue revealed the following differences: The tumors contained (1) larger amounts of triacylglycerols, (2) smaller amounts of citrate, and (3) acidic mucins. These transformation-associated deviations from the normally high amounts of citrate and low amounts of lipids in the prostate are consistent with an alteration in either the concentration or the activity of ATP-citrate lyase in the tumors. The 13C NMR spectra of colonic adenocarcinoma tissue showed that this tumor type contained (1) smaller signals from triacylglycerols, (2) larger signals from phospholipids and lactate, and (3) decreased lipid fatty acyl chain saturation, when compared to spectra from adjacent normal colon. Colloid carcinoma, another variant of colonic carcinoma, showed prominent 13C resonances from glycoproteins, which were absent from the spectra of normal colon, and from spectra from the more common pattern of colonic adenocarcinoma. Smaller 13C NMR signals from mucins and other proteins, and the presence of triacylglycerol signals distinguished poorly differentiated lung carcinoma and from nonmalignant lung tissue. These results indicate that natural-abundance 13C NMR spectroscopy may constitute a unique, nondestructive method, for the simultaneous measurement of a large number of tissue metabolites and structural components of significance to the study and diagnosis of a wide range of human tumors.

Characterization and reactivity of a monoclonal antibody that recognizes a 76 kilodalton human colon tumor antigen

A monoclonal antibody (MAb 76) was produced by immunizing mice with a soluble cytoplasmic protein fraction from a human adenocarcinoma of the colon. MAb 76 showed specific immunoreactivity against a 76 kDa protein in immunoblot studies using total colon tumor cytosol proteins. Immunoprecipitation of phosphorylated cytosolic protein products with MAb 76 and subsequent analysis on SDS containing polyacrylamide gels revealed a single 38 kDa band, indicating that the 76 kDa antigen is associated with a 38 kDa phosphoprotein species. Indirect immunofluorescence analysis of primary tumor specimens and human colon tumor cell lines showed positive immunoreactivity with 6 7 human colon adenocarcinoma tissues and 15 18 human colon tumor cell lines. MAb 76 was unreactive with normal colon, liver and lung specimens from human, mouse and hamster. The epitope-bearing monomer detected by MAb 76 is immunologically conserved in a high percentage of colon tumor cells and tissues and may represent a cellular product that is characteristic of the transformed colon cell phenotype.

Synthesis of type 1 and 2 lacto series glycolipid antigens in human colonic adenocarcinoma and derived cell lines is due to activation of a normally unexpressed beta 1—3N-acetylglucosaminyltransferase.

Human colonic adenocarcinoma tissue and derived cell lines have been characterized by an abundance of different type 1 and 2 lacto series glycolipid antigens which are either low or not found in normal colonic mucosa. The enzymatic basis for the expression of contrasting glycolipid compositions between adenocarcinomas and normal colonic mucosa, as well as between derived cell lines, has been studied. The following results were of particular interest. (i) Abundant activities of beta 1----4galactosyltransferase associated with synthesis of both lactosylceramide and lactoneotetraosylceramide, beta 1----3galactosyltransferase for synthesis of lactotetraosylceramide, and an alpha 1----3/4fucosyltransferase responsible for synthesis of Lex and Lea antigens were found in normal colonic mucosa or in a normal mucosal epithelial cell line HCMC, or in both. Variable levels of these activities were found in adenocarcinoma tissues and in various established adenocarcinoma cell lines. In striking contrast, significant activity of a beta 1----3N-acetylglucosaminyltransferase responsible for synthesis of lactotriaosylceramide (Lc3) was found in various cases of colonic adenocarcinoma and cell lines, but was undetectable in normal colonic epithelial cells. (ii) In situ transfer of galactose to Lc3 was performed on histologic sections by preincubation of the tissue with acceptor glycolipid followed by incubation with UDP-galactose. The biosynthesized glycolipid was revealed by indirect immunofluorescence with the monoclonal antibody 1B2 which defines lactoneotetraosylceramide antigen. In these studies, histologic sections prepared from frozen normal proximal colon tissue were shown to lack native type 2 chain structures. However, transfer of galactose from UDP-galactose could be demonstrated in the epithelial cells of normal proximal colon after incorporation of Lc3 into the membranes, indicating the ability of normal colonic epithelial cells to synthesize type 2 chain core structures if the precursor Lc3 is available. In contrast, adenocarcinoma tissues showed significant native immunofluorescence with the antibody. These data suggest that an accumulation of both type 1 and 2 chain lacto series glycolipids with alpha 1----3- or alpha 1----4fucosyl substitution in human adenocarcinoma is due to enhanced beta 1----3N-acetylglucosaminyltransferase rather than enhancement of other enzymes. This enzyme may play a key role in regulating the level of various types of lacto series tumor-associated antigens with the lacto type 1 or 2 chain.

Synthetic Antigens as Immunogens: Part I The Combining Site Specificities of Antibodies Formed in Rabbits to Synthetic Disaccharide α-L-Fuc-(1→3)-D-Gal

Antibody to chemically synthesized Fuc alpha 1----3 Gal-1----OC6H4N = N bovine serum albumin was developed. Specificity analysis using related synthetic saccharides was performed. The antisera were shown to be specific for both sugars in the disaccharide, to the 1----3 positional linkage and to the alpha anomeric configuration. Immunohistochemical staining was performed on benign disease and adenocarcinoma tissue of the colon. 5/8 of the benign colon polyp tissue and 4/6 of the colonic adenocarcinoma tissue showed strong granular cytoplasmic staining of the epithelia cells lining the ducts. None of the six normal colon tissues tested showed any reactivity. Immunohistochemical staining was also performed on tissue from benign disease and from carcinoma of the mammary gland. 2/5 of the tissue from benign disease of the breast and 3/6 of the tissue from breast carcinoma showed strong granular cytoplasmic staining of the ductal epithelial cells.

Dynamics of glycoprotein metabolism in familial polyposis coli-derived colonic epithelial tissue.

Familial polyposis coli is a disease state that offers an opportunity to obtain colon tissue in a predictable manner in terms of identifying putative premalignant markers. Our results support the hypothesis that a sequential series of alterations occur in the dynamics of glycoprotein metabolism as normal colonic mucosa progresses to colon adenocarcinoma. Specifically, both nonmalignant polyps and colon adenocarcinoma tissue expressed diminished glycosylation of proteins, whereas an elevated level of activity for membrane-associated glycosyltransferases does not appear until colon cancer itself develops.

Glycosaminoglycans of human colonic mucosa and colonic adenocarcinoma.

The mucosal scrapings of the normal human colons and the colonic adenocarcinoma tissues were digested with pronase, and the glycosaminoglycan fractions were prepared by fractionation with cetylpyridinium chloride after treatment with deoxyribonuclease. Chondroitin sulfate A/C, heparan sulfate, and hyaluronic acid were contained in the glycosaminoglycan fraction of the normal tissue, but dermatan sulfate was undetectable. In contrast, the glycosaminoglycan fractions derived from the tumors contained substantial amounts of dermatan sulfate together with the foregoing three glycosaminoglycans. The total content of glycosaminoglycans per wet tissue weight was elevated in the tumor tissues, which was consistent with histochemical findings.

Cells from normal and malignant human colon mucosa differentially inhibit embryonic cell aggregation.

A model system for studying some aspects of the interaction of cancer cells in tumors and their surrounding nonmalignant tissue is the co-culture of cancer cells and embryonic chick neural retinal cells in gyratory shakers. Neural retina cell aggregation, under these conditions, has been shown to be differentially inhibited by small numbers of cultured mouse and human cancer cells. We extend here these observations to co-cultures of retinal cells with small numbers (60:1 ratio) of human cells isolated from normal colon mucosa or colonic adenocarcinoma tissue. The cells from the malignant tissue samples inhibited aggregation to a significantly greater extent than the cells from normal mucosa, even when both were from the same individual. Cells derived from nonmalignant tumors were more inhibitory than those from normal individuals, which is consistent with described differences in this 'transitional' region. Thus, the aggregation inhibition assay appears applicable to freshly isolated human tissues.