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Signaling and Regulation

The Interaction of Specific Peptide Aptamers With the DNA Binding Domain and the Dimerization Domain of the Transcription Factor Stat3 Inhibits Transactivation and Induces Apoptosis in Tumor Cells11Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung für Krebsforschung, Bonn (10-1626-Gr2), and Novartis Stiftung für Therapeutische Forschung.

Kerstin Nagel-Wolfrum, Claudia Buerger, Ilka Wittig, Karin Butz, Felix Hoppe-Seyler and Bernd Groner
Kerstin Nagel-Wolfrum
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Claudia Buerger
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Ilka Wittig
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Karin Butz
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Felix Hoppe-Seyler
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Bernd Groner
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DOI:  Published March 2004
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  • FIGURE 1.
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    FIGURE 1.

    Interaction of isolated peptide aptamers with Stat3. A. Functional domains of the Stat3 molecule, the cooperative binding domain, the DNA binding domain, and the transactivation domain are indicated. Phosphorylation of the tyrosine 705 in the dimerization region mediates interaction with the SH2 domain of another monomer and causes Stat3 dimer formation. Tyrosine 705 and serine 727 are key phosphorylation sites. Domains used as bait constructs in the yeast two-hybrid screens are indicated. B. Isolated peptide aptamers interact specifically with corresponding bait constructs. Empty bait vector (pPC97) or bait constructs (pPC97-Stat5-DD, pPC97-Stat3-DD, and pPC97-Stat3-DBD) were transformed in haploid yeast strain PJ69α and mated with haploid yeast strain PJ69a expressing indicated peptide aptamers. Interaction of bait and prey was monitored under selection conditions. C. In vitro interaction of selected peptide aptamers with full-length Stat3. Whole cell extracts of RPMI 8226 cells were preincubated with recombinant expressed peptide aptamers prior to incubation with anti-Stat3 antibodies and Sepharose A beads. After removing unbound peptide aptamers, samples were subjected to SDS-PAGE, and retained peptide aptamers in the complex were visualized by Western blotting with an anti-Trx antibody.

  • FIGURE 2.
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    FIGURE 2.

    Peptide aptamers inhibit Stat3 signaling in Herc cells. A. Peptide aptamers inhibit Stat3 transactivation in transiently transfected Herc cells. Herc cells were transiently transfected with increasing amounts of pRc/CMV-VP22 peptide aptamer vectors (0.5, 1, or 1.5 μg or 1.5 μg pRc/CMV-VP22-Trx), Stat3 luciferase reporter construct (500 ng), and β-galactosidase expression plasmid (30 ng). Twenty-four hours after transfection, cells were starved in serum-free medium for 10 h followed by EGF treatment (50 ng/ml) overnight. Luciferase activity was measured and normalized for transfection efficiency using β-galactosidase activity as an internal control. Fold induction was calculated as the ratio of light units from stimulated to unstimulated luciferase activity. Fold induction of wild-type cells was set to 100%. Peptide aptamers significantly decreased Stat3 transactivation compared with control and Trx-transfected cells (P ≤ 0.05). Columns, means of three independent experiments; bars, SD. B. DNA binding activity of Stat3 is reduced in Herc cells stably transfected with peptide aptamers. Stat3 DNA binding activity was determined via EMSA using whole cell extracts (4 μg) and a 32P-labeled oligonucleotide probe containing a consensus binding motif for Stat3 (high-affinity mutant of the sis-inducible element). For supershift analysis, whole cell extracts were preincubated with specific antibody for Stat3 (Santa Cruz Biotechnology). Bottom, expression levels of the peptide aptamers. C1, C4, and C10 are single clones of transfected cells. Arrow, supershift; asterisk, DNA binding.

  • FIGURE 3.
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    FIGURE 3.

    DD peptide aptamers specifically interfere with Stat3 phosphorylation. A. EGF-induced phosphorylation of Stat3 is reduced in Herc cells in the presence of DD peptide aptamers. Stably transfected Herc cells were starved overnight and induced with EGF (50 ng/ml) for 20 min and harvested. Cell lysates (50 μg) were subjected to Western blot analysis using a phospho-Stat3-specific antibody. After stripping, the membrane was reprobed with a total Stat3-specific antibody to ensure equal loading. B and C. Activation of EGFR and the downstream target MAPK p42/p44 is not affected in the presence of peptide aptamers. Stably transfected Herc cells were starved overnight and induced with EGF (50 ng/ml) for 20 min and harvested. B. EGFR was precipitated with an EGFR-specific antibody. Complexes were subjected to Western blotting, where the phosphorylation status of the receptor was detected using an anti-phosphotyrosine-specific antibody. Reprobing the blot with an anti-EGFR-specific antibody revealed equal protein amounts loaded on the gel. C. Total lysates were subjected to Western blot analysis using specific anti-phospho-MAPK p42/p44 antibodies. To monitor equal loading, the membrane was stripped and reprobed with an antibody recognizing total p42/p44 MAPK.

  • FIGURE 4.
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    FIGURE 4.

    Peptide aptamer DBD-1 reduces Stat3-mediated transcriptional activity and decreases viability in B16 cells. A. B16 cells were transiently transfected with indicated pRc/CMV-VP22 peptide aptamer vectors (1.5 μg), a Stat3 luciferase reporter construct (500 ng), and a β-galactosidase expression plasmid (30 ng). Thirty-six hours after transfection, luciferase activity was measured and normalized for transfection efficiency using β-galactosidase activity as an internal control. Luciferase activity of wild-type cells was designated to 100%. Peptide aptamer DBD-1 significantly inhibits Stat3 activity (P ≤ 0.05). Columns, means of three independent experiments; bars, SD. B. B16 cells were transiently transfected with indicated pRc/CMV-VP22 peptide aptamer vectors (2 μg) and seeded in triplicates in 96-well plates. Every 24 h, relative number of viable cells was assessed using the XTT-based proliferation kit (Roche Molecular Biochemicals). Peptide aptamer DBD-1 significantly reduced relative numbers of viable cells (P ≤ 0.05). Columns, means of three independent experiments; bars, SD. Embedded Image, Control; Embedded Image, Trx; Embedded Image, DD-1; Embedded Image, DD-2; Embedded Image, DBD-1.

  • FIGURE 5.
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    FIGURE 5.

    DBD-1 induces apoptosis in B16 cells. Murine melanoma cells (B16) were transiently transfected with indicated pRc/CMV-VP22 peptide aptamer vectors (2 μg). Forty-eight hours after transfection, cells were stained for DNA fragmentation (TUNEL; red) followed by indirect immunostaining for peptide aptamer expression (anti-Trx antibody; green). Nuclei were stained with 4′,6-diamino-2-phenylindole (blue). Asterisks, TUNEL-positive and transfected B16 cells; arrow, TUNEL-positive but untransfected cell.

  • FIGURE 6.
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    FIGURE 6.

    Dose-dependent growth inhibition of tumor cells transduced with peptide aptamer DBD-1-9R. A. B16 cells (103 cells/well) were seeded and transduced every 12 h with indicated concentration of peptide aptamer DBD-1-9R or Trx-9R. As control, cells were treated with dialysis buffer. Every 24 h, relative number of viable cells was assessed in triplicates using the XTT-based proliferation kit (Roche Molecular Biochemicals) in 96-well plates. Control-treated cells were assigned to 100%. DBD-1-9R significantly reduced relative numbers of viable cells compared with control and Trx-9R-treated cells (P ≤ 0.05). Columns, means of three independent experiments; bars, SD. B and C. Dose-dependent growth inhibition in human myeloma cells transduced with DBD peptide aptamer. U266 cells (2 × 105 cells/ml) were seeded and transduced every 12 h with indicated concentration of peptide aptamer DBD-1-9R or Trx-9R. As control, cells were treated with dialysis buffer. Embedded Image, Control; Embedded Image, Trx (180 nm); Embedded Image, Trx (360 nm); Embedded Image, DBD-1 (180 nm); Embedded Image, DBD-1 (360 nm). B. Cell numbers were determined in 24 h intervals—, Control; Embedded Image, Trx; Embedded Image, DBD-1 (90 nm); Embedded Image, DBD-1 (180 nm); Embedded Image, DBD-1 (270 nm); Embedded Image, DBD-1 (360 nm); Embedded Image, DBD-1 (450 nm). C. Every 24 h, relative number of viable cells was assessed using the XTT-based proliferation kit (Roche Molecular Biochemicals). Control-treated cells were assigned to 100%. DBD-1-9R significantly reduced relative numbers of viable cells compared with control and Trx-9R-treated cells (P ≤ 0.05). Embedded Image, Control; Embedded Image, Trx (180 nm); Embedded Image, Trx (360 nm); Embedded Image, DBD-1 (180 nm); Embedded Image, DBD-1 (360 nm). Columns, means of three independent experiments; bars, SD. D. RPMI 8226 cells (2 × 105 cells/ml) were seeded and transduced every 12 h with indicated concentration of peptide aptamer DBD-1-9R or Trx-9R. As control, cells were treated with dialysis buffer. Every 24 h, relative number of viable cells was assessed using the XTT-based proliferation kit (Roche Molecular Biochemicals). Control-treated cells were assigned to 100%. DBD-1-9R slightly reduces relative number of viable cells compared with Trx-9R and control treated cells. Embedded Image, Control; Embedded Image, Trx (180 nm); Embedded Image, Trx (360 nm); Embedded Image, DBD-1 (180 nm); Embedded Image, DBD-1 (360 nm).

  • FIGURE 7.
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    FIGURE 7.

    Peptide aptamer DBD-1 and DD-2 induce proapoptotic markers in U266 cells. A. Bcl-xL expression is down-regulated in cells transduced with peptide aptamers. U266 cells were transduced with indicated peptide aptamers (360 nm) every 12 h for 2.5 days. Western blot analyses were performed using whole cell extracts (80 μg) and probed with Bcl-xL, Stat3, and actin antibodies. Buffer-treated and Trx-9R-treated cells served as negative controls. Quantification of the Bcl-xL levels was normalized to the actin levels with the TINA software. B. Cleavage of caspase-3 and PARP in DD-2- and DBD-1-transduced cells. Cells were treated with DBD-1-9R (360 nm), DD-2-9R (1 μm), or Trx-9R (1 μm) to study caspase-3 and PARP cleavage. RIPA extracts (100 μg) were subjected to immunoblot analysis using a caspase-3 antibody, which recognizes the cleaved fragments (17 and 19 kDa) and an anti-PARP antibody that recognizes full-length PARP and the cleaved 89 kDa fragment. Membrane was reprobed with anti-Stat3 antibodies to ensure equal loading.

  • FIGURE 8.
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    FIGURE 8.

    Model for the inhibition of Stat3 signaling by peptide aptamers. Peptide aptamers selected for interaction with the Stat3-DD associate with monomeric Stat molecules form a complex and as a consequence prevent phosphorylation of Stat3 molecules (I). Peptide aptamer DBD-1, which was selected for interaction with Stat3-DBD, does not interfere with the phosphorylation of Stat3 (II) but block the binding of Stat3 molecules to the DNA (III). As a result of inhibiting either the phosphorylation or the DNA binding of Stat3 molecules, Stat3 signaling is blocked and the transcription of target genes is reduced (IV). As a result of the inhibition of Stat3 activation, phenotypical changes including growth inhibition and/or induction of apoptosis occur in Stat3-dependent tumor cells.

Tables

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  • TABLE 1.

    Quantification of TUNEL-Positive B16 cells Following Transfection of Peptide Aptamers

    Peptide AptamerTUNEL+ (% of All Cells)Trx+/TUNEL+ (% of All Cells)Trx−/TUNEL+ (% of All Cells)
    Co2.02.00.0
    Trx2.92.70.2
    DD-12.32.00.3
    DD-22.11.90.2
    DBD51.140.011.1
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Molecular Cancer Research: 2 (3)
March 2004
Volume 2, Issue 3
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The Interaction of Specific Peptide Aptamers With the DNA Binding Domain and the Dimerization Domain of the Transcription Factor Stat3 Inhibits Transactivation and Induces Apoptosis in Tumor Cells11Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung für Kre…
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The Interaction of Specific Peptide Aptamers With the DNA Binding Domain and the Dimerization Domain of the Transcription Factor Stat3 Inhibits Transactivation and Induces Apoptosis in Tumor Cells11Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung für Krebsforschung, Bonn (10-1626-Gr2), and Novartis Stiftung für Therapeutische Forschung.
Kerstin Nagel-Wolfrum, Claudia Buerger, Ilka Wittig, Karin Butz, Felix Hoppe-Seyler and Bernd Groner
Mol Cancer Res March 1 2004 (2) (3) 170-182;

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The Interaction of Specific Peptide Aptamers With the DNA Binding Domain and the Dimerization Domain of the Transcription Factor Stat3 Inhibits Transactivation and Induces Apoptosis in Tumor Cells11Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung für Krebsforschung, Bonn (10-1626-Gr2), and Novartis Stiftung für Therapeutische Forschung.
Kerstin Nagel-Wolfrum, Claudia Buerger, Ilka Wittig, Karin Butz, Felix Hoppe-Seyler and Bernd Groner
Mol Cancer Res March 1 2004 (2) (3) 170-182;
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