Altered expression of sialylated carbohydrate antigens in HT29 colonic carcinoma cells David R. Mack 1 , Pi-Wan Cheng 2,3 , Fulvio Perini 3 , Shu Wei 1 , and Michael A. Hollingsworth 3 Combined Section of Pediatric Gastroenterology, 1 Department of Pediatrics, 2 Biochemistry and Molecular Biology, and 3 Eppley Institute for Research in Cancer and Allied Sciences, University of Nebraska Medical Center, and Center for Human Nutrition, Omaha*, Nebraska, USA 69198 Keywords: glycoproteins, mucins, Lewis antigens Abbreviations: Le a - Lewis A, S Le c - sialyl Lewis C (NeuAca2,3Gal-b1,3-GlcNAc), Le x - Lewis X, S Le x - sialyl Lewis X, S Le a - sialyl Lewis A, Le y - Lewis Y, S Le y - sialyl Lewis Y, STn - sialyl Tn, HPLC - high pressure liquid chromatography, PBS - phosphate buffered saline To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose con- taining media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates. Introduction Alteration of cell surface carbohydrates during malignant transformation is a recognized phenomenon in a variety of tumors [1,2]. There has been interest in the specific role that carbohydrate antigens might play in tumor invasion and metastatic potential. For instance, a poor prognosis in patients with colorectal carcinoma was found to corre- late with expression of the carbohydrate antigen sialosyl- Tn on this tumor and this correlation was independent of tumor grade and stage [3]. Differences in metastatic potential are attributable to various cellular properties [4,5] including sialylated complex carbohydrates [6,7]. The biological factors that cause increases in sialylated carbo- hydrate antigens in the metastases remain unknown. It has been postulated that biosynthesis of sialylated mucin core and peripheral carbohydrate structures might be affected by factors at the site of metastases or selective metastases of subpopulations of cancer cells producing these antigens [7–9]. Thus, the microenvironment of the primary tumor may be responsible for enhanced expression of sialylated antigens with metastases in part resulting from prefer- ential colonization of differentially glycosylated tumor cells. In the present study, we examined immunoreactivity of antibodies to core and peripheral mucin carbohydrate structures and glycosyltransferases responsible for the syn- thesis of these carbohydrate structures on HT29 colonic carcinoma cell lines that were grown in glucose- and galac- tose containing media. These different culture conditions have previously been associated with alterations in growth rate, morphological appearance, disaccharidase activity and mucin gene expression [10,11]. Differences in the anti- body reactivity with cell surface and secreted products from the cells and sialyltransferase activities were observed when the growth conditions in which the cells were grown were altered. Glycoconjugate Journal 15, 1155–1163 (1998) © 1998 Kluwer Academic Publishers. Manufactured in The Netherlands *To whom correspondence should be addressed, Tel: (402) 559-7390; Fax: (402) 559-5763; E-mail: drmack@unmc.edu