1. Volume 128, Issue 7, Pages 1919-1936 (June 2005)
Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β- Catenin Complex 
Masashi Idogawa, Tesshi Yamada, Kazufumi Honda, Satoshi Sato, Kohzoh Imai, Setsuo Hirohashi 
Gastroenterology 
Volume 128, Issue 7, Pages (June 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Identification of an interaction between PARP-1 and TCF-4. (A) SDS-PAGE analysis of the immunoprecipitates from HEK293 cells transfected with FLAG-TCF-4 or control FLAG-MOCK. Twenty-four hours after transfection, total cell lysates (left) or nuclear extracts (right) were immunoprecipitated with anti-FLAG affinity gel and analyzed by SDS-PAGE. The open arrowhead is pointing to FLAG-TCF-4 and the closed arrowhead to the 112-kilodalton protein. (B) Mass spectrum of the 112-kilodalton protein digested with modified trypsin. (C) Amino acid sequence of PARP-1. Underlining indicates peptides that correspond to peaks identified by mass spectrometry. (D) Western blot analysis of the immunoprecipitates of HEK293 cells transfected with FLAG-TCF-4 (+) or control FLAG-MOCK (−). The input cell lysates (total) and immunoprecipitates with anti-FLAG antibody (IP: FLAG) were blotted with anti-FLAG and anti-PARP-1 antibodies. (E) (Top) Lysates of HEK293 cells transfected with FLAG-TCF-4 or untransfected HEK293 cells (parent) were blotted with anti-FLAG or anti-PARP-1 antibody. (Bottom) Lysates of HEK293 cells transfected with FLAG-TCF-4 were immunoprecipitated with anti-PARP-1 antibody or normal mouse IgG and analyzed by blotting with anti-FLAG and anti-PARP-1 antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 1
Identification of an interaction between PARP-1 and TCF-4. (A) SDS-PAGE analysis of the immunoprecipitates from HEK293 cells transfected with FLAG-TCF-4 or control FLAG-MOCK. Twenty-four hours after transfection, total cell lysates (left) or nuclear extracts (right) were immunoprecipitated with anti-FLAG affinity gel and analyzed by SDS-PAGE. The open arrowhead is pointing to FLAG-TCF-4 and the closed arrowhead to the 112-kilodalton protein. (B) Mass spectrum of the 112-kilodalton protein digested with modified trypsin. (C) Amino acid sequence of PARP-1. Underlining indicates peptides that correspond to peaks identified by mass spectrometry. (D) Western blot analysis of the immunoprecipitates of HEK293 cells transfected with FLAG-TCF-4 (+) or control FLAG-MOCK (−). The input cell lysates (total) and immunoprecipitates with anti-FLAG antibody (IP: FLAG) were blotted with anti-FLAG and anti-PARP-1 antibodies. (E) (Top) Lysates of HEK293 cells transfected with FLAG-TCF-4 or untransfected HEK293 cells (parent) were blotted with anti-FLAG or anti-PARP-1 antibody. (Bottom) Lysates of HEK293 cells transfected with FLAG-TCF-4 were immunoprecipitated with anti-PARP-1 antibody or normal mouse IgG and analyzed by blotting with anti-FLAG and anti-PARP-1 antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 2
Inclusion of PARP-1 in the native TCF-4/β-catenin complex. (A) Nuclear extracts of SW480 (Total) were immunoprecipitated with anti-PARP-1 antibody (IP: PARP-1) or normal mouse IgG (IP: IgG) and blotted with anti-TCF-3/4 (TCF-4) and anti-PARP-1 antibodies. (B) SW480 cell lysates were immunoprecipitated with anti-PARP-1 antibody (IP: PARP-1) or normal mouse IgG (IP: IgG) and blotted with anti-β-catenin and anti-PARP-1 antibodies. (C) Nuclear extracts of SW480 (Nuclear) were immunoprecipitated with anti-TCF-4 antibody (IP: TCF-4), anti-β-catenin antibody (IP: β-catenin), or normal mouse IgG (IP: IgG) and blotted with anti-PARP-1, anti-β-catenin, and anti-TCF-3/4 (TCF-4) antibodies. (D) Immunoprecipitation of lysates of HEK293 cells cotransfected with pCR3.1-β-cateninΔN134 (β-cateninΔN) and/or FLAG-TCF-4. The input cell lysates (Total) and immunoprecipitates with anti-PARP-1 antibody (IP: PARP-1) were blotted with anti-FLAG, anti-β-catenin, and anti-PARP-1 antibodies. (E) HEK293 cells were cotransfected with FLAG-TCF-4, pCR3.1-β-cateninΔN134 (β-cateninΔN), or their corresponding control plasmids (−). Twenty-four hours after transfection, the input cell lysates (Total) and the immunoprecipitates with anti-FLAG affinity gel (IP: FLAG) were analyzed by blotting with anti-FLAG, anti-PARP-1, and anti-β-catenin antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 2
Inclusion of PARP-1 in the native TCF-4/β-catenin complex. (A) Nuclear extracts of SW480 (Total) were immunoprecipitated with anti-PARP-1 antibody (IP: PARP-1) or normal mouse IgG (IP: IgG) and blotted with anti-TCF-3/4 (TCF-4) and anti-PARP-1 antibodies. (B) SW480 cell lysates were immunoprecipitated with anti-PARP-1 antibody (IP: PARP-1) or normal mouse IgG (IP: IgG) and blotted with anti-β-catenin and anti-PARP-1 antibodies. (C) Nuclear extracts of SW480 (Nuclear) were immunoprecipitated with anti-TCF-4 antibody (IP: TCF-4), anti-β-catenin antibody (IP: β-catenin), or normal mouse IgG (IP: IgG) and blotted with anti-PARP-1, anti-β-catenin, and anti-TCF-3/4 (TCF-4) antibodies. (D) Immunoprecipitation of lysates of HEK293 cells cotransfected with pCR3.1-β-cateninΔN134 (β-cateninΔN) and/or FLAG-TCF-4. The input cell lysates (Total) and immunoprecipitates with anti-PARP-1 antibody (IP: PARP-1) were blotted with anti-FLAG, anti-β-catenin, and anti-PARP-1 antibodies. (E) HEK293 cells were cotransfected with FLAG-TCF-4, pCR3.1-β-cateninΔN134 (β-cateninΔN), or their corresponding control plasmids (−). Twenty-four hours after transfection, the input cell lysates (Total) and the immunoprecipitates with anti-FLAG affinity gel (IP: FLAG) were analyzed by blotting with anti-FLAG, anti-PARP-1, and anti-β-catenin antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 3
Identification of the regions required for interaction between TCF-4 and PARP-1. (A–C) Full-length or truncated forms of FLAG-TCF-4 purified on anti-FLAG affinity gel were incubated with the biotinylated PARP-1 protein prepared by in vitro translation. The complexes were thoroughly washed and analyzed by blotting with anti-FLAG antibody (FLAG) and avidin/horseradish peroxidase (Avidin). Full-length and truncated forms of TCF-4 are schematically represented at the bottom. Asterisks indicate the TCF-4 constructs that bound to the PARP-1 protein. (D) Full-length and truncated forms of biotinylated PARP-1 were prepared by in vitro translation, and their expression was confirmed by blotting with avidin/horseradish peroxidase (Avidin, input). The full-length FLAG-TCF-4 protein was purified with anti-FLAG affinity gel and incubated with each form of PARP-1 for 12 hours at 4°C. The complexes were thoroughly washed, eluted from the gels, and analyzed by blotting with avidin-HRP (Avidin, output) or anti-FLAG antibody (FLAG). The full-length and truncated forms of PARP-1 are illustrated at the bottom (WT, wild-type). Asterisks indicate the PARP-1 constructs that bound to TCF-4.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 3
Identification of the regions required for interaction between TCF-4 and PARP-1. (A–C) Full-length or truncated forms of FLAG-TCF-4 purified on anti-FLAG affinity gel were incubated with the biotinylated PARP-1 protein prepared by in vitro translation. The complexes were thoroughly washed and analyzed by blotting with anti-FLAG antibody (FLAG) and avidin/horseradish peroxidase (Avidin). Full-length and truncated forms of TCF-4 are schematically represented at the bottom. Asterisks indicate the TCF-4 constructs that bound to the PARP-1 protein. (D) Full-length and truncated forms of biotinylated PARP-1 were prepared by in vitro translation, and their expression was confirmed by blotting with avidin/horseradish peroxidase (Avidin, input). The full-length FLAG-TCF-4 protein was purified with anti-FLAG affinity gel and incubated with each form of PARP-1 for 12 hours at 4°C. The complexes were thoroughly washed, eluted from the gels, and analyzed by blotting with avidin-HRP (Avidin, output) or anti-FLAG antibody (FLAG). The full-length and truncated forms of PARP-1 are illustrated at the bottom (WT, wild-type). Asterisks indicate the PARP-1 constructs that bound to TCF-4.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 4
PARP-1 augments the β-catenin-evoked transcriptional activity of TCF/LEF. (A) HEK293 cells were cotransfected with 0.4 μg of PARP-1-myc (PARP-1 [+]) or its relevant mock (PARP-1 [−]) plasmid, 0.4 μg of FLAG-β-cateninΔN134 (β-catΔN [+]) or its corresponding mock (β-catΔN [−]) plasmid, and 0.2 μg of canonical (TOP-FLASH) or mutant (FOP-FLASH) TCF/LEF luciferase reporter. After culture for 24 hours without (−) or with the indicated concentration of 3-AB, the luciferase activity of TOP-FLASH (solid bars) and FOP-FLASH (gray bars) was measured. Activity was adjusted to the TOP-FLASH activity of the mock transfectant (PARP-1 [−] and β-catΔN [−]) and expressed as fold increase. (B) Expression of endogenous and transfected β-catenin and PARP-1 proteins. The lysates of cells prepared in A were analyzed by blotting with anti-c-myc, anti-PARP-1, and anti-β-catenin antibodies. (C) The indicated amounts of pcDNA3.1-PARP-1-myc plasmids (0-0.6 μg) were cotransfected into HEK293 cells along with 0.2 μg of FLAG-β-cateninΔN134 and 0.2 μg of luciferase reporter construct; 24 hours after transfection, the luciferase activity of TOP-FLASH (solid bars) and FOP-FLASH (gray bars) was measured. The total amount of plasmid DNA was kept constant by adding empty pcDNA3.1/myc-His DNA. The luciferase activity was adjusted to that of the control transfectant (TOP-FLASH of PARP-1 [−] and β-catΔN [−]) and expressed as fold increase. (D) Enhancement of TCF/β-catenin target gene expression by PARP-1. HEK293 cells were transiently transfected with FLAG-β-cateninΔN134 (β-catΔN), PARP-1-myc (PARP-1), and/or relevant mock plasmids, as indicated at the bottom. The expression levels of cyclin D1, c-myc, matrilysin, and GAPDH were analyzed by reverse-transcription PCR, adjusted to those of GAPDH, and expressed as the fold increase compared with the control transfectant (PARP-1 [−], β-catΔN [−]). (E) The full-length (WT, wild-type) or truncated (DBD + AD and ΔN217) forms of PARP-1 plasmid were cotransfected into HEK293 cells with the same amount of FLAG-β-cateninΔN134 (β-catΔN [+]) or its mock (β-catΔN [−]) plasmid along with luciferase reporters. The activity was adjusted to that of the TOP-FLASH of the transfectant alone with β-cateninΔN134 (β-catΔN [+] and MOCK) and expressed as fold increase. The amounts of endogenous and transfected PARP-1 and β-catenin proteins were confirmed by Western blotting with anti-PARP-1 and anti-β-catenin antibodies. Each column displays the mean ± SD of data from 3 separate experiments in A, C, and D.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 5
Knockdown of PARP-1 by RNA interference. (A) Western blot analysis showing the protein level of PARP-1 (top) and β-actin (loading control, bottom) of HCT116 cells transfected with pSUPER-PARP-1 (A) or (B) or pSUPER-control. (B) TCF/LEF transcriptional activity of HCT116 cells transfected with pSUPER-PARP-1 (A) or (B) or pSUPER-control. Each column displays the mean ± SD of data from 3 separate experiments. (C) Colony formation of HCT116 cells transfected with pSUPER-PARP-1 (A) or (B) or pSUPER-control. Transfectants were selected with geneticin for 7 days. (D) Cell cycle analysis of HCT116 cells transfected with pSUPER-PARP-1 (A) or (B) and pSUPER-control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

10. Figure 6
Effect of poly-ADP-ribosylation on the interaction between TCF-4 and PARP-1. (A) Auto-polyADP-ribosylation of the PARP-1 protein in vitro. The recombinant PARP-1 protein was auto-polyADP-ribosylated in the presence of biotinylated NAD+. The polyADP-ribosylated PARP-1 protein was detected by blotting with avidin/horseradish peroxidase. (B) Interaction between TCF-4 and polyADP-ribosylated PARP-1. The FLAG-TCF-4 protein immobilized on anti-FLAG affinity gel was incubated with the polyADP-ribosylated (pADP-rPARP-1) or unmodified (rPARP-1) PARP-1 protein. The complexes were thoroughly washed, eluted, and analyzed by blotting with anti-PARP-1 and anti-FLAG antibodies. (C) Interaction between endogenous TCF-4 and polyADP-ribosylated PARP-1. SW480 cells were incubated with or without bleomycin (50 μg/mL) for 12 hours. Each nuclear extract (Nuclear) was immunoprecipitated with anti-TCF-4 antibody (IP: TCF-4) and blotted with anti-PARP-1 (PARP-1), anti-TCF-3/4 (TCF-4), and anti-polyADP-ribose (pADPr) antibodies. (D) Absence of polyADP-ribosylation of TCF-4 and β-catenin. The polyADP-ribosylation reaction was performed in vitro on the FLAG-TCF-4 or FLAG-β-catenin protein in the presence of biotinylated NAD+. Histone was included in each sample as a positive control. Samples were analyzed by blotting with avidin/horseradish peroxidase (for detection of polyADP-ribosylated proteins), anti-PARP-1, and anti-FLAG antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

11. Figure 7
Immunohistochemistry of the normal intestine and tumor tissues of patients with FAP and Min mice. Expression of the (A–C, E, G, J, and K) PARP-1 and (D, F, and H) β-catenin proteins in the (A and B) normal large intestine, (C–J) adenoma, and (K) adenocarcinoma tissues of patients with FAP. Expression of the PARP-1 protein in the (L–N and R) normal and (P, Q, and S) adenoma tissues of the (L–Q) small and (R and S) large intestine of Min mice. (A and B) Expression of PARP-1 in normal large intestine of a patient with FAP. An area of A containing the bottom portions of normal crypts is shown enlarged in B. (C–H) Concurrent expression of PARP-1 and β-catenin in adenoma of patients with FAP. C and D, E and F, and G and H are the same fields of serial sections stained with (C, E, and G) anti-PARP-1 and (D, F, and H) anti-β-catenin antibodies. (J) Nuclear expression of PARP-1 in adenoma cells of a patient with FAP. (K) Expression of PARP-1 in colorectal carcinoma of a patient with FAP. (L–N) Expression of PARP-1 in normal small intestine of a Min mouse. Two areas in the boxes of L are shown enlarged in M (bottom of a crypt) and N (tip of a villus). (P and Q) Expression of PARP-1 in adenoma of a Min mouse. An area of P is shown enlarged in Q. Note that tumor cells (t) express a higher amount of nuclear PARP-1 protein than the covering normal epithelial cells (n). (R and S) Expression of PARP-1 in (R) normal mucosa and (S) adenoma of the large intestine of Min mice. t, tumor cells; n, normal epithelial cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

12. Figure 7
Immunohistochemistry of the normal intestine and tumor tissues of patients with FAP and Min mice. Expression of the (A–C, E, G, J, and K) PARP-1 and (D, F, and H) β-catenin proteins in the (A and B) normal large intestine, (C–J) adenoma, and (K) adenocarcinoma tissues of patients with FAP. Expression of the PARP-1 protein in the (L–N and R) normal and (P, Q, and S) adenoma tissues of the (L–Q) small and (R and S) large intestine of Min mice. (A and B) Expression of PARP-1 in normal large intestine of a patient with FAP. An area of A containing the bottom portions of normal crypts is shown enlarged in B. (C–H) Concurrent expression of PARP-1 and β-catenin in adenoma of patients with FAP. C and D, E and F, and G and H are the same fields of serial sections stained with (C, E, and G) anti-PARP-1 and (D, F, and H) anti-β-catenin antibodies. (J) Nuclear expression of PARP-1 in adenoma cells of a patient with FAP. (K) Expression of PARP-1 in colorectal carcinoma of a patient with FAP. (L–N) Expression of PARP-1 in normal small intestine of a Min mouse. Two areas in the boxes of L are shown enlarged in M (bottom of a crypt) and N (tip of a villus). (P and Q) Expression of PARP-1 in adenoma of a Min mouse. An area of P is shown enlarged in Q. Note that tumor cells (t) express a higher amount of nuclear PARP-1 protein than the covering normal epithelial cells (n). (R and S) Expression of PARP-1 in (R) normal mucosa and (S) adenoma of the large intestine of Min mice. t, tumor cells; n, normal epithelial cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

13. Figure 8
Transcriptional regulation of the PARP-1 gene by ETSs. (A) Expression of PARP-1 and GAPDH mRNA in samples of normal intestine (n) and cancer (t) tissue from 5 patients with sporadic colorectal cancer. (B) Expression of PARP-1 in normal (n) mucosa and adenocarcinoma (t) of the large intestine. (C) Membrane/cytoplasmic and focal nuclear (arrows) expression of PARP-1 in adenocarcinoma of the large intestine. Note the nuclear expression of PARP-1 in leucocytes (leu) infiltrating the stroma. (D) Expression of Parp-1, Ets1, Ets2, and Gapdh mRNA in samples of the normal intestine (n) and polyp (t) tissues from Min mice. (E and F) Transactivation of the PARP-1 gene promoter by ETSs. pcDNA3.1-ETS1-myc, pcDNA3.1-ETS2-myc, or empty pcDNA3.1-myc (MOCK) and pGL3-basic-PARP-1-prom were transiently transfected into HEK293 cells (E) without or (F) with dominant-negative ETS2. The activity was adjusted to that of the mock transfectant and expressed as fold increase. Western blot analysis using anti-myc antibody revealed equivalent expression of dominant-negative ETS2 (F). Each column displays the mean ± SD of data from 3 separate experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

14. Figure 8
Transcriptional regulation of the PARP-1 gene by ETSs. (A) Expression of PARP-1 and GAPDH mRNA in samples of normal intestine (n) and cancer (t) tissue from 5 patients with sporadic colorectal cancer. (B) Expression of PARP-1 in normal (n) mucosa and adenocarcinoma (t) of the large intestine. (C) Membrane/cytoplasmic and focal nuclear (arrows) expression of PARP-1 in adenocarcinoma of the large intestine. Note the nuclear expression of PARP-1 in leucocytes (leu) infiltrating the stroma. (D) Expression of Parp-1, Ets1, Ets2, and Gapdh mRNA in samples of the normal intestine (n) and polyp (t) tissues from Min mice. (E and F) Transactivation of the PARP-1 gene promoter by ETSs. pcDNA3.1-ETS1-myc, pcDNA3.1-ETS2-myc, or empty pcDNA3.1-myc (MOCK) and pGL3-basic-PARP-1-prom were transiently transfected into HEK293 cells (E) without or (F) with dominant-negative ETS2. The activity was adjusted to that of the mock transfectant and expressed as fold increase. Western blot analysis using anti-myc antibody revealed equivalent expression of dominant-negative ETS2 (F). Each column displays the mean ± SD of data from 3 separate experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions