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Expression and Characterization of Mucins Associated with the Resistance to Methotrexate of Human Colonic Adenocarcinoma Cell Line HT29¹

Gastrointestinal Research Laboratory, VA Medical Center and University of California, San Francisco, San Francisco, California 94121 [R. D., K-S. K., J. C. B., Y. S. K.], and Unite de Recherches sur la Differenciation Cellulaire Intestinale (INSERM U 178), 16, Avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France [T. L., A. B., A. Z.]

A series of experiments were conducted to study synthesis and secretion of mucin in mucus-secreting subpopulations of HT29 human colonic adenocarcinoma cells selected by resistance to methotrexate (MTX). Mucin was quantitated by [³H]glucosamine labeling and chromatography on Sepharose CL-4B. The mucinous nature of the labeled high molecular weight glycoprotein was verified by alkaline borohydride treatment, cesium chloride density gradient ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these experiments demonstrated that MTX-treated cells have increased amounts of mucin in medium, cytosol, and membrane fractions. This was associated with the increase in the activities of polypeptidyl-N-acetylgalactosaminyltransferase and ß-1,3-galactosyltransferase compared to control cells. DEAE-Sephacel chromatography of [³H]glucosamine-labeled high molecular weight glycoproteins suggest that MTX-treated cells are less acidic compared to controls. Using complementary DNA probes for two distinct human intestinal mucins (MUC2 and MUC3) and one mammary mucin (MUC1), it was found that MTX-treated cells expressed more mucin messages compared to untreated cells. These results were consistent with immunoblots using anti-MRP (MUC2 repeat peptide), anti-M3P (MUC3 repeat peptide), 139H2 (MUC1 peptide), anti-T (peanut lectin), anti-Tn (91S8), and anti-sialosyl Tn (JT10e) antibodies. These data indicate that MTX-resistant HT29 cells show enhanced secretion and synthesis of mucin as well as expression of MUC1-, MUC2-, and MUC3-related mucin polypeptide epitopes.

¹ This work was supported by the Department of Veterans Affairs Medical Research Service, National Cancer Institute Grant CA45967 (R. D., J. C. B., Y. S. K.).

² To whom requests for reprints should be addressed, at Department of Urology (112F), VA Medical Center, 4150 Clement St., San Francisco, CA 94121.

Received 1/17/92. Accepted 6/18/92.

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