Biomedical subjects
Publications and source records attributed to S H Itzkowitz.
The Mount Sinai Division of Gastroenterology has an international reputation for outstanding contributions to the study of digestive diseases, especially inflammatory bowel disease. A discussion of the current structure of the gastroenterology (GI) fellowship training program is provided, along with an overview of the GI Division at the turn of the 21st century.
The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc alpha2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases.
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BACKGROUND: Malignant cells exhibit increased glycolytic metabolism, and in many cases increased glucose transporter gene expression. The authors hypothesized that GLUT1 glucose transporter expression is increased in colorectal carcinoma, and that the degree of expression might have prognostic significance. METHODS: GLUT1 glucose transporter immunostaining was studied in normal colon and benign colon adenomas and in 112 colorectal carcinomas from patients for whom long term clinical outcome was known. RESULTS: GLUT1 immunostaining was absent in normal colorectal epithelium and tubular adenomas, and absent or only weakly apparent in tubulovillous adenomas. The majority of carcinomas (101 of 112; 90%) had GLUT1 immunostaining. Tumors from 92 patients had low GLUT1 expression (< 50% of cells were GLUT1 positive) and 19 of these patients (21%) died of disease during follow-up. In contrast, tumors from 20 patients had high GLUT1 expression (> 50% of cells were GLUT1 positive) and 9 of these patients (45%) died of disease during follow-up. Disease specific mortality was greater in patients with high GLUT1 tumors (relative risk of 2.4; P=0.02). In a multivariate analysis to assess whether high GLUT1 staining correlated with increased mortality independently of Dukes stage, the risk of death from colon carcinoma in the group with high GLUT1 staining was 2.3 times that in the group with low GLUT1 staining, a difference that approached statistical significance (P=0.07). CONCLUSIONS: GLUT1 glucose transporter expression is associated strongly with neoplastic progression in the colon, and assessment of the extent of GLUT1 immunostaining in colorectal carcinoma identifies patients with a poorer prognosis.
Mucin production by human colon cancer cells correlates with liver metastasis in animal models, but it is not known which steps in metastasis depend on specific alterations in mucin synthesis. Clonal variants of cell line LS174T selected for differences in mucin core carbohydrate expression have been further characterized biochemically, and tested for their ability to participate in metastasis-related events. LS-C mucin contains truncated carbohydrates enriched for sialyl Tn and these cells bind to basement membrane matrix to a greater extent than LS-B cells. This binding is partially inhibitable by antibody to sialyl Tn. LS-B produces more fully glycosylated mucin and preferentially binds to hepatic sinusoidal endothelial cells and E-selectin through sialylated peripheral mucin-associated carbohydrate structures. Adhesion of LS-B to endothelial cells is inhibited by neutralizing antibody to E-selectin, and inhibition of glycosylation or desialylation of LS-B mucin abrogates binding to E-selectin in vitro. LS-B cells spontaneously metastasized from cecum to liver and colonized the liver of athymic mice after splenic-portal injection to a significantly greater extent than LS-C, suggesting that expression of peripheral mucin carbohydrate structures is most important for metastasis of human colon cancer cells.
Monoclonal antibodies TKH2 and B72.3, which react with the mucin-associated sialyl-Tn(STn) antigen, preferentially bind to cancerous but not normal colonic tissues. If O-acetyl groups are removed by saponification of tissues, MAb TKH2 will react with normal colonocytes, whereas MAb B72.3 remains non-reactive. To explain this difference in binding specificity, we tested both MAbs against synthetic constructs of single (monomeric) or clustered (trimeric) STn epitopes by enzyme immunoassay. Both MAb TKH2 and MAb B72.3 reacted with trimeric STn, but MAb TKH2 demonstrated greater binding than MAb B72.3 to monomeric STn. This suggests that normal colonic mucosa expresses monomeric STn epitopes, but that with transformation to malignancy, clustered STn epitopes appear. The appearance of clustered STn epitopes during colonic carcinogenesis represents a novel pattern of carbohydrate antigen expression and implicates alterations at the level of apomucins and/or glycosyltransferases responsible for cluster epitope formation.
Sialyl-Tn antigen (SAalpha2-6 GalNAc alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc alpha-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal beta1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R beta3-Gal-transferase (core 1 beta3-Gal-transferase) was lacking. Core 1 beta3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc beta1-6 (Gal beta1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 beta3-Gal-transferase activity. The lack of core 1 beta3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 beta3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.
BACKGROUND: In human colon, binding of the lectin Amaranthus caudatus has been considered to be a marker of cellular proliferation and malignant progression. We studied regional amaranthin binding in rat colon and correlated this with physiologic manipulations of proliferation. METHODS: Binding of amaranthin in segments of proximal and distal colon was studied in starved, refed, and control Wistar rats and was compared to tritiated thymidine labeling and proliferating cell nuclear-antigen expression. RESULTS: Amaranthin bound mainly to cells in the lower crypt of distal colon and midcrypt of proximal colon, paralleling the distribution of proliferative markers. Binding occurred in the supranuclear region in distal colon and the pericellular membrane in proximal colon. Starvation/refeeding was associated with a change in amaranthin binding intensity in distal colon, but not in proximal colon. CONCLUSION: The pattern of amaranthin binding during starvation/refeeding seems to reflect physiologic changes in several areas of the colon.
Recent developments in mucin histochemistry and immunohistochemistry have made reliable determination of the gastric and intestinal phenotypes of gastric carcinoma cells possible. Phenotypic expression changes from gastric epithelial cell type to intestinal epithelial cell type with the growth of gastric tumours in experimental animals. We studied cell differentiation in gastric signet ring cell carcinomas with progression in 203 surgically obtained specimens. The results showed that the proportion of gastric phenotype carcinomas, in which over 90% of the tissue consists of gastric epithelial cell type cells, decreases with the depth of invasion. The proportion of mixed phenotype carcinomas (between 10% and 90% of the tissue made up of gastric and/or intestinal epithelial cell type cells) increases. The intestinal phenotype (over 90% intestinal epithelial cell type carcinoma cells) was found in four carcinomas (about 2%) involving the serosa. No clear relationship was evident between phenotypic expression of carcinoma cells and the degree of intestinal metaplasia of the surrounding mucosa. Progression of gastric signet ring cell carcinomas is associated with a phenotypic shift from gastric to intestinal type expression.
Colorectal cancer is a significant clinical problem for patients with long-standing ulcerative colitis and Crohn's disease. Traditional risk factors include long disease duration and greater extent of colonic disease, but newer factors such as associated primary sclerosing cholangitis, folate deficiency, and family history of colon cancer may help to refine risk stratification. Molecular pathogenesis of colitis-associated colon cancer shares some of the same genetic abnormalities as sporadic colon cancer, but the timing of certain alterations suggests different carcinogenic pathways. Some molecular alterations show promise as complementary markers to dysplasia. Clinical studies of colonoscopic surveillance indicate that colon cancers can be detected early and that mortality may therefore be improved. A suggested surveillance strategy is proposed.
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BACKGROUND & AIMS: Expression of the carbohydrate-associated sialosyl-Tn (STn) antigen has been correlated with carcinogenesis in sporadic colon cancer. This retrospective study analyzed surveillance colonoscopy biopsy specimens to determine whether STn antigen could serve as a marker for neoplasia risk in patients with long-standing ulcerative colitis. METHODS: Eleven patients who developed dysplasia or cancer and who had undergone at least three surveillance colonoscopies were matched with 11 controls who had not developed neoplasia. Sections from 969 available surveillance biopsy specimens were stained immunohistochemically with monoclonal antibody TKH2 and were interpreted blindly. RESULTS: Compared with controls, patients expressed STn antigen more frequently in their biopsy specimens (44% patient biopsy specimens vs. 11% control biopsy specimens). Patients were also more likely to undergo colonoscopies with two or more colonic segments expressing STn antigen (73% vs. 22%), repeated STn antigen expression in two or more consecutive colonoscopies (91% vs. 27%), and STn antigen expression proximal to the hepatic flexure (64% vs. 0%). STn antigen appearance preceded the detection of neoplasia by 7 or fewer years, and its preferential expression in patients was not confounded by the degree of active inflammation. CONCLUSIONS: Repeated STn antigen expression in nondysplastic colonic mucosa may presage the development of neoplasia in long-standing ulcerative colitis and may be a useful adjunct to dysplasia during colonoscopic surveillance.
MUC2 and MUC3 are prominent mucin genes expressed in the human intestine. Using in situ hybridization with RNA probes, we examined the cellular distribution of MUC2 and MUC3 mRNA in normal, malignant, and inflammatory human intestinal tissues. In normal small intestine and colon, MUC2 mRNA was expressed exclusively in goblet cells and occurred throughout the entire height of the mucosa. MUC3 mRNA was expressed by goblet and columnar cells but was restricted to the villous compartment of the small intestine and the surface epithelium of the colon. Expression of MUC2 and MUC3 mRNA were both markedly decreased in poorly, moderately, and well-differentiated colon cancers but were preserved in mucinous colon cancers. In ulcerative colitis and Crohn's colitis tissues, MUC2 and MUC3 mRNA expression displayed a normal pattern regardless of whether the mucosa manifested active or quiescent inflammation. These findings indicate that MUC2 is goblet cell-specific, whereas MUC3 is related to maturation of intestinal epithelial cells. In colon cancers, the genetic regulation of MUC2 and MUC3 is different depending on the histological type of tumor. The constitutive expression of MUC2 and MUC3 mRNA in inflammatory bowel diseases suggests that these genes may be necessary for maintenance of normal epithelial cell function during inflammation.
Adenomatous polyps of the gastrointestinal tract are dysplastic precursor lesions of adenocarcinoma. The features of an adenoma that are associated with a greater tendency to progress to carcinoma include larger polyp size, high grade dysplasia, and increased villous glandular architecture. Alterations in particular oncogenes and tumor-suppressor genes have been correlated with various stages of colonic adenoma formation, thereby facilitating our knowledge of adenoma pathogenesis. Heredity and environment contribute to the risk of developing adenomatous polyps of the colon. The incidence of colorectal cancer can be decreased by the endoscopic removal of adenomas, thereby providing good rationale for screening and surveillance programs.
Sialyl Tn (sTn) is a mucin-associated carbohydrate antigen expressed in most types of human adenocarcinoma. Defining the configuration of tumor cell surface sTn recognized by antibodies is important for understanding the basis for the cancer cell specificity of sTn-reactive mAbs, for the development of more effective mAbs, and for designing cancer vaccines against sTn. In this study, we compared the immunogenicity of synthetic single sTn disaccharide epitopes and clusters [sTn(C)] of 3 sTn epitopes covalently linked via serine to keyhole limpet hemocyanin [KLH; sTn-KLH and sTn(C)-KLH, respectively]. The cell surface sTn configurations were analyzed with the use of sera from mice immunized with these neoglycoproteins and a panel of sTn-reactive mAb. Sera from mice immunized with sTn-KLH reacted in direct and inhibition assays with sTn-human serum albumin (HSA) but only weakly with sTn(C)-HSA, whereas sera from mice immunized with sTn(C)-KLH reacted with sTn(C)-HSA but not with sTn-HSA. Both anti-sTn and anti-sTn(C) sera reacted with ovine submaxillary mucin (a natural source of sTn) and with sTn-positive human tumor cell line LS-C but not with sTn-negative LS-B cells. With regard to the sTn-reactive mAbs, B72.3 reacted exclusively with clustered sTn, whereas mAb B195.3R11 reacted preferentially with unclustered sTn. Results with mAbs TKH2, B239.1, and CC49 were less clear, although all reacted more strongly with clustered sTn than with unclustered sTn. These results suggest that sTn is recognized at the tumor cell surface in at least two quite distinct configurations, as clustered and nonclustered arrays.
Immunohistochemical studies have indicated that sialylated carbohydrate antigens such as sialyl-Tn, sialyl-Le(a), and sialyl-Le(x) are expressed in a tumor-associated fashion in human colon. Since sialic acid residues are O-acetylated more extensively in normal colonic epithelium than in colon cancer cells, we examined whether deacetylation of colonic tissues might enable monoclonal antibodies to recognize these tumor-associated sialylated antigens. In normal colon, deacetylation turned most cases (82%) positive with anti-sialyl-Tn mAb TKH2; and in colon cancers, it increased the number of TKH2-positive cells. Sialyl-Le(a) and sialyl-Le(x) detection was also increased after deacetylation of normal and malignant colonic tissues so that the frequency of positive cases in normal tissues was similar to that in the cancers. However, in the stomach and pancreas, the same treatment rarely increased the detection of the sialylated epitopes in normal or cancerous tissues. Thus, the same sialylated epitopes can be expressed in a tumor-associated fashion by different mechanisms in different gastrointestinal organs; in the colon, these antigens are constitutively expressed and O-acetylated, whereas in the upper gastrointestinal tract, they are rarely O-acetylated, suggesting that other mechanisms such as differences in glycosylation account for the cancer-associated expression.