Immunohistochemical detection of SERCA3 protein in normal adult and fetal colonic epithelium and in colon tumors. A: Internal positive controls for PLIM430 monoclonal antibody labeling. Endothelial cells (1) and lymphocytes (2) exhibit a strong cytoplasmic staining. B: PLIM430 labeling of normal adult colonic mucosa. At low magnification (1), epithelial cellsexhibit a strong labeling with a progressive increase along the crypt toward the upper part (2) from the deeper part (3). C: PLIM430 labeling of normal fetal colonic mucosa. At low magnification, staining of epithelial cells is strong (1) and located at the basal area of cells (3). The gradient of labeling along the crypts is less pronounced than in normal adult colonic mucosa (2). D: PLIM430 labeling in hyperplastic polyps. At low magnification, crypts are elongated, tortuous, and covered by strongly labeled epithelial cells (1). The gradient of labeling along the crypts is more pronounced than in normal adult colonic mucosa, toward the upper part (2) from the deeper part (3). E: PLIM430 labeling of adenomas. At low magnification (1 and 4), the intensity of adenoma staining is globally lower than in normal colonic mucosa. Between different fields, labeling is heterogeneous (2, 3, 5, and 6). In adenomatous structures with low grade dysplasia, epithelial staining is more preserved than in adenomatous structures with higher grade dysplasia (arrows). These regions are generally located in the upper part of adenomas (5). F: PLIM430 labeling of adenocarcinomas. Whereas adjacent normal epithelium is strongly labeled, the staining of adenocarcinomatous tissue is very weak or null (1 to 3). The weak variations of labeling of the neoplastic cells are related to the differentiation of the tumor. In well-differentiated adenocarcinoma, cell are weakly labeled (4 and 5), whereas in poorly differentiated adenocarcinoma, tumor cells do not show any staining (9). In moderately differentiated cases, labeling is more heterogeneous (6) with alternating areas of weak (7) and null (8) staining according to local differentiation of tumor cells.

Induction of SERCA3 expression in colon cancer cells by dominant-negative TCF4. Ls174T cells transfected with repressor only (LsTR4 cells) or a doxicycline-inducible vector coding for dominant-negative TCF4 (LsL8 cells) were grown in the absence or presence of 2 μmol/L doxicycline for 3 days. After loading equal amounts of total cellular protein on SDS-polyacrylamide gels, electrophoresis, and transfer, SERCA3 expression was determined by Western immunoblotting using the SERCA3-specific PLIM430 monoclonal antibody. In parallel, as positive controls for SERCA3 induction, LsL8 cells and KATO-III cells were treated with 2 mmol/L butyrate for the same period. Expression of dominant-negative TCF4 leads to SERCA3 expression.

Inhibition of SERCA3 expression by mithramycin-A. A: Inhibition of valerate-induced SERCA3 expression by various concentrations of mithramycin-A: DLD-1 colon cancer cells were treated with valerate in the absence or the presence of various concentrations of mithramycin-A for 2 days, and SERCA3 protein was quantitated by Western blotting. Untreated cells expressed undetectable levels of SERCA3 protein. Induction of SERCA3 expression was obtained by treatment with valerate, and valerate-induced SERCA3 expression was inhibited by mithramycin-A in a dose-dependent fashion in the high nanomolar concentration range. SERCA2 expression was not significantly affected by the treatments. B: Inhibition by mithramycin-A of SERCA3 expression induced by various concentrations of butyrate: KATO-III cells were treated for 2 days with various concentrations of butyrate in the absence or in the presence of 10 μmol/L mithramycin-A, and SERCA2 as well as SERCA3 protein levels were determined. Butyrate-induced SERCA3 expression was abolished by mithramycin-A. C: Inhibition of constitutive SERCA3 expression in COLO-205 cells by various concentrations of mithramycin-A: COLO-205 colon carcinoma cells were treated for 3 days with vehicle or various concentrations of mithramycin-A, and SERCA2 as well as SERCA3 expression was determined. Although mithramycin-A did not affect SERCA2 expression, SERCA3 expression was inhibited by the drug in a concentration-dependent manner.

Induction of carcinoembryonic antigen expression in Caco-2 cells by SERCA inhibitors. Caco-2 cells at day 1 postconfluence were treated for 7 days with vehicle or with 10 μmol/L tBHQ, 6 μmol/L cyclopiazonic acid (CPA), or 0.125 nmol/L thapsigargin (Tg), and cellular carcinoembryonic antigen was detected by Western blotting. Carcinoembryonic antigen levels were increased by all three structurally unrelated SERCA inhibitors when compared with untreated cells.