Volume 133, Issue 5, Pages 1569-1578 (November 2007) Functional Interaction of DNA Topoisomerase IIα With the β-Catenin and T-Cell Factor-4 Complex  Lin Huang, Miki Shitashige, Reiko Satow, Kazufumi Honda, Masaya Ono, Jisoo Yun, Akihiro Tomida, Takashi Tsuruo, Setsuo Hirohashi, Tesshi Yamada  Gastroenterology  Volume 133, Issue 5, Pages 1569-1578 (November 2007) DOI: 10.1053/j.gastro.2007.08.011 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Interaction of Topo IIα and the β-catenin/TCF-4 complex. (A) A nuclear extract of DLD-1 cells was immunoprecipitated with anti–Topo IIα goat polyclonal antibody (topo), normal goat IgG, anti–β-catenin monoclonal antibody (β-cat), anti–TCF-4 monoclonal antibody (tcf), or normal mouse IgG. The nuclear extract (Input) and immunoprecipitates (ip) were blotted with the indicated antibodies. (B) Immunofluorescence microscopy showing the subcellular localization of the β-catenin (green) and Topo IIα (red) proteins in DLD-1 cells. The images in the bottom panels are enlargements of the framed areas in the corresponding top panels. (C) Representation of Topo IIα mutants examined on binding to β-catenin by 2-hybrid assay. Numbers indicate the amino acids included in each (SwissProt accession number P11388). wt, wild type; lz, leucine zipper; nls, nuclear localization signal. (D) Determination of the region of Topo IIα necessary for binding to β-catenin by 2-hybrid assay. HeLa cells were co-transfected with the pBIND vector carrying the entire coding sequence of β-catenin complementary DNA (pBIND-β-catenin, ■) or empty vector (pBIND-control, □), pACT plasmid carrying one of the truncated forms of Topo IIα indicated in C or empty vector (Control), and pG5luc plasmid. Forty-eight hours after the transfection, luciferase activity was measured by using Renilla reniformis luciferase activity as an internal control. Bars, SD. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Effect of overexpression of Topo IIα on TCF/LEF transcriptional activity. (A) HeLa cells were co-transfected in triplicate with TOP-FLASH (■) or FOP-FLASH (□) TCF/LEF luciferase reporter plasmid, FLAG-β-catenin ΔN134 or control plasmid (0.06 μg), and FLAG-Topo IIα or control plasmid (0.18 μg). Twenty-four hours after the transfection the luciferase activities were measured. Bars, SD. (B) The indicated amounts (in μg) of FLAG–Topo IIα plasmid were co-transfected in triplicate into HeLa cells along with FLAG–β-catenin ΔN134 or control plasmid (0.06 μg) and one of the reporter plasmids. Twenty-four hours after the transfection the luciferase activities were measured. Bars, SD. (C) DLD-1 cells were co-transfected in triplicate with FLAG–Topo IIα or control plasmid (0.2 μg) and one of the reporter plasmids. Twenty-four hours after the transfection the luciferase activities were measured. Bars, SD. (D) The indicated amounts (in μg) of FLAG–Topo IIα plasmid were co-transfected in triplicate into DLD-1 cells along with one of the reporter plasmids. Twenty-four hours after the transfection the luciferase activities were measured. Bars, SD. (E) DLD-1 cells were transfected with FLAG–Topo IIα or control plasmid. The expression levels of the indicated proteins were determined by immunoblotting (left). The relative expression levels of the MMP7, MYC, and AXIN2 genes were quantified by real-time reverse-transcription polymerase chain reaction (right). Bars, SD. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Effect of Topo IIα knockdown on TCF/LEF transcriptional activity. (A) DLD-1 cells were transfected with Topo IIα–interfering RNA (1 and 3) or control RNA (XII and 1), treated with Lipofectamine 2000 alone (without RNA) (la), or untreated (u). The levels of expression of Topo IIα, β-catenin, and β-actin (loading control) proteins were analyzed by immunoblotting 48 hours later. (B) DLD-1 cells were co-transfected with TOP-FLASH (■) or FOP-FLASH (□) TCF/LEF luciferase reporter plasmid and no RNA (la), Topo IIα–interfering RNA (1 and 3), or control RNA (XII and 1). Luciferase activities were measured 48 hours later. Bars, SD. (C) HCT116 cells were transfected with Topo IIα–interfering RNA (1 and 3) or control RNA (XII and 1), treated with Lipofectamine 2000 alone (without RNA) (la), or untreated (u). The levels of expression of Topo IIα, β-catenin, and β-actin (loading control) proteins were analyzed by immunoblotting 48 hours later. (D) HCT116 cells were co-transfected with TOP-FLASH (■) or FOP-FLASH (□) TCF/LEF luciferase reporter plasmid and no RNA (la), Topo IIα–interfering RNA (1 and 3), or control RNA (XII and 1). Luciferase activities were measured 48 hours later. Bars, SD. (E) DLD-1 cells were transfected with Topo IIα–interfering RNA (1 and 3) or control RNA (XII and 1). The relative expression levels of the TOP2A, MMP7, MYC, and AXIN2 genes were quantified by real-time reverse-transcription polymerase chain reaction. Bars, SD. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Suppression of TCF/LEF transcriptional activity by Topo II inhibitors. (A) HeLa cells were co-transfected in triplicate with TOP-FLASH (■) or FOP-FLASH (□) TCF/LEF luciferase reporter plasmid, FLAG-β-catenin ΔN134 or control plasmid (0.06 μg), and FLAG-Topo IIα or control plasmid (0.18 μg). The indicated amounts of merbarone (in μmol/L) were added to the cells 4 hours later. Twenty-four hours after the transfection the luciferase activities were measured. Bars, SD. (B) DLD-1 cells were co-transfected in triplicate with one of the reporter plasmids and FLAG–Topo IIα or control plasmid (0.2 μg). The indicated amounts of merbarone (in μmol/L) were added to the cells 4 hours later. (C) HCT116 cells were co-transfected in triplicate with one of the reporter plasmids and FLAG–Topo IIα or control plasmid (0.2 μg). The indicated amounts of merbarone (in μmol/L) were added to the cells 4 hours later. (D) HeLa cells were co-transfected in triplicate with one of the reporter plasmids, FLAG–β-catenin ΔN134 or control plasmid (0.06 μg), and FLAG–Topo IIα or control plasmid (0.18 μg). The indicated amounts of etoposide (in μmol/L) were added to the cells 4 hours later. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Augmentation of Topo II activity by β-catenin. (A and B) Twenty-four hours after transfection with FLAG–β-catenin ΔN134 (β) or control (c) plasmid, human embryonic kidney (HEK) 293 cells were treated without or with etoposide for 2 hours. (A) The expression of β-catenin, Topo IIα, and histone H4 (loading control) was analyzed by immunoblotting. (B) The relative amounts of decatenated kDNA were quantified by scanning the bands in the gel. wt, wild type; cat, catenated; decat, decatenated. (C and D) Twenty-four hours after transfection with FLAG–β-catenin ΔN134 (β) or control (c) plasmid, HeLa cells were treated without or with etoposide (e) or merbarone (m) for 2 hours. (C) The expression of β-catenin, Topo IIα, and histone H4 (loading control) was analyzed by immunoblotting. (D) The relative amounts of decatenated kDNA were quantified by scanning the bands in the gel. wt, wild type; cat, catenated; decat, decatenated. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Expression of Topo IIα and β-catenin proteins in normal and tumor tissues of the colon and rectum. (A–G) Immunohistochemical analysis of (A–D, F, and G) Topo IIα and (E) β-catenin proteins in the morphologically (A–C) normal large intestine, (D and E) adenoma, and (F and G) adenocarcinoma of familial adenomatous polyposis patients. C is an enlarged image of the framed area in B. a, adenoma; c, carcinoma; n, normal. (H–K) Immunohistochemical analysis of (H and I) Topo IIα and (J and K) β-catenin in serial sections of sporadic colorectal adenocarcinoma. c, carcinoma; n, normal. Original magnification: (D–F, H, and J) 4×, (A and B) 10×, and (C, G, I, and K) 40×. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Co-expression of Topo IIα and β-catenin proteins in colorectal cancer. Immunofluorescence microscopy images showing the localization of the (A, D, and G and green in C, F, and I) β-catenin and (B, E, and H and red in C, F, and I) Topo IIα proteins in (A–C) morphologically normal large intestine mucosa and (D–I) adenocarcinomas of sporadic colorectal cancer patients. β-catenin accumulates in the nucleus, cytoplasm, and cell membrane of cancer cells (D and G and green in F and I). Yellow nuclei in F and I indicate colocalization of the 2 proteins. Original magnification: (A–F) 40× and (G–I) 100×. Gastroenterology 2007 133, 1569-1578DOI: (10.1053/j.gastro.2007.08.011) Copyright © 2007 AGA Institute Terms and Conditions