Volume 123, Issue 3, Pages 817-826 (September 2002) Expression of human intestinal mucin is modulated by the β-galactoside binding protein galectin-3 in colon cancer  Steven P. Dudas, Christopher K. Yunker, Lawrence R. Sternberg, James C. Byrd, Robert S. Bresalier  Gastroenterology  Volume 123, Issue 3, Pages 817-826 (September 2002) DOI: 10.1053/gast.2002.35395 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of galectin-3 and MUC2 in human colon cancer line HM7. Cell lysates were subject to Western analysis using galectin-3–specific mAb TIB-166 and MUC2-specific mAb CCP58. Equal amounts of homogenate protein (30 μg) were added to each lane, and proteins were separated by electrophoresis on 7.5% sodium dodecyl sulfate/polyacrylamide gels with 3% stacking gels. MUC2 has a reported Mr of >600 kilodaltons and therefore migrates much more slowly than the slowest migrating standard. Arrow indicates interface between stacking gel and running gel. Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Mucin secretion in human colon cancer cell lines. (A) Western analysis with antibody MUC2N3 of lysates and secreted high-density and low-density glycoproteins from colon cancer cell lines HM7-E3 and HM7-E8. This antibody recognizes an epitope that is unaffected by glycosylation of the apoprotein. Aliquots of 30 μg of protein per lane were analyzed on 3%–8% NuPAGE gels under reducing conditions. Arrow indicates top of gel. (B) Analysis of synthesis and secretion of total mucin in HM7-E3 and HM7-E8. Cells were labeled for 21 hours with tritiated glucosamine as described in Materials and Methods, and the “cytosol” and dialyzed medium (secreted mucin) were analyzed by gel filtration on Superose 6. Results show mean and SD of 3 analyses. Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of stable introduction of sense or antisense constructs of galectin-3 on galectin-3 and MUC2 protein and RNA expression. (A) Relative abundance of galectin-3 and MUC2 protein in cell lines LS174T, LSLim6, HM7 controls, and derivatives transformed with galectin-3 sense and antisense constructs. Western analysis was performed on 3%–8% NuPAGE gels under reducing conditions using antibodies TIB-166 and CCP58. (B) Northern analysis. Total RNA was electrophoresed on 1.2% agarose formaldehyde gels, transferred to nylon membranes, and serially probed for the relative abundance of galectin-3, MUC2, and β-actin messenger RNA. Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of induction of galectin-3 antisense on expression of galectin-3 and MUC2 protein. Clonal derivatives of human colon cancer cell line HM7 (AG1, AG4, AG11) containing galectin-3 antisense under control of a tetracycline-inducible promoter were grown in the absence (−) and presence (+) of 2 μg/mL tetracycline. (A) Total cellular galectin-3 and MUC2. Equal amounts of homogenate protein were electrophoresed and immunoblotted for detection of galectin-3, MUC2, and β-actin as described in Materials and Methods and Figure 3. (B) Cell surface galectin-3. Single cell suspensions of colon cancer cells were exposed to anti–galectin-3 mAb TIB-166 and submitted to flow analysis as described in Materials and Methods. Induction of galectin-3 antisense (plus tetracycline) resulted in reduction in cell surface galectin-3 in all cell lines. Solid areas depict controls (second antibody only). Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of induction of galectin-3 antisense on mucin synthesis and secretion. (A) Analysis of synthesis and secretion of total mucin in cell line AG4 with and without tetracycline treatment to induce antisense galectin-3. Cells were labeled for 21 hours with tritiated glucosamine as described in Materials and Methods, and the “cytosol” and dialyzed medium (secreted mucin) were analyzed by gel filtration on Superose 6. Results show mean and SD of 3 analyses. (B) Western analysis using biotinylated jacalin (Artocarpus integrifolia) agglutinin (jacalin). Aliquots of 30 μg protein from lysates of AG4 cells treated with or without tetracycline were subjected to electrophoresis under reducing conditions on 3%–8% NuPAGE gels. Blots were incubated with 0.5 μg/mL biotinyl jacalin to detect mucins containing the Gal-β-1,3-GalNAc core structure with or without sialylation. The identity of the low Mr bands is unknown but quantitatively unchanged by tetracycline. Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 The effect of induction of galectin-3 antisense on galectin-3 and MUC2 is reversible. Cells lines AG1 and AG4 were grown for 2 weeks in the presence (+) of 2 μg/mL tetracycline to induce antisense galectin-3 or in the absence of tetracycline as control (−). Cell cultures were subsequently washed to remove tetracycline and grown for an additional 2 weeks in fresh media minus tetracycline (R). Cell homogenates were subjected to Western analysis as described in Figure 3. Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of induction of galectin-3 antisense on galectin-3 and MUC2 expression in vivo. Athymic nude mice were injected in the cecal wall with colon cancer cell line AG4 containing antisense to galectin-3 under control of a tetracycline-inducible promoter. Animals were treated with 1 μg/mL tetracycline in the drinking water for 1 week before injection and continuously for 6 weeks thereafter to induce galectin-3 antisense (right column) or with water minus tetracycline (left column). Cecal xenografts were removed and stained for galectin-3 and MUC2 and with periodic acid–Schiff or high iron diamine/alcian blue as described in Materials and Methods. Gastroenterology 2002 123, 817-826DOI: (10.1053/gast.2002.35395) Copyright © 2002 American Gastroenterological Association Terms and Conditions