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MET process by pyrrole–imidazole polyamide targeting human transforming growth factor-β1
Accepted Manuscript Title: Modulation of the EMT/MET process by pyrrole-imidazole polyamide targeting human transforming growth factor-1 Author: Kosuke Saito Noboru Fukuda Ken-ichi Shinohara Yoshikazu Masuhiro Shigemasa Hanazawa Hiroyuki Matsuda Kyoko Fujiwara Takahiro Ueno Masayoshi Soma PII: DOI: Reference:
S1357-2725(15)00196-X http://dx.doi.org/doi:10.1016/j.biocel.2015.07.011 BC 4667
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
6-3-2015 19-6-2015 23-7-2015
Please cite this article as: Saito, K., Fukuda, N., Shinohara, K.-i., Masuhiro, Y., Hanazawa, S., Matsuda, H., Fujiwara, K., Ueno, T., and Soma, M.,Modulation of the EMT/MET process by pyrrole-imidazole polyamide targeting human transforming growth factor-rmbeta1, International Journal of Biochemistry and Cell Biology (2015), http://dx.doi.org/10.1016/j.biocel.2015.07.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Modulation of the EMT/MET process by pyrrole-imidazole polyamide targeting human transforming growth factor-β1 Short title: Modulation of EMT/MET with PI polyamide to TGF-β1
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Kosuke Saito a, Noboru Fukuda b,c,*, Ken-ichi Shinohara d, Yoshikazu Masuhiro e, Shigemasa Hanazawa e, Hiroyuki Matsuda a, Kyoko Fujiwara a, Takahiro Ueno c,
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Masayoshi Soma a
Department of General Medicine, Nihon University School of Medicine, Tokyo, Japan
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Division of Life Science, Advanced Research Institute for the Sciences and Humanities,
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Nihon University Graduate School, Tokyo, Japan
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Division of Nephrology Hypertension and Endocrinology, Department of Medicine,
Nihon University School of Medicine, Tokyo, Japan
Division of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba, Japan
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Department of Applied Biological Sciences, College of Bioresource Sciences, Nihon
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University, Kanagawa, Japan
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* Corresponding author: Noboru Fukuda, Department of General Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30-1, Itabashi-ku, Tokyo 173-8610, Japan.
Phone: 81-3-3972-8111; Fax: 81-3-3972-8666, E-mail: [email protected] Word Count: 4,902 words
Abstract Length: 177 words
Number of Figures: 6 Figures, 1 Supplemental table, 3 Supplemental figures
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Highlights PI polyamide targeting human TGF-β1 gene promoter region regulates the EMT/MET process in MCF10A cells.
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PI polyamide significantly increased reprogramming efficiency via enhanced MET in iPSC induction of human fibroblasts.
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PI polyamide to human TGF-β is a novel compound that can control EMT/MET of
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human epithelial cells.
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ABSTRACT Transforming growth factor-β1 (TGF-β1) is a potent induction factor for epithelial-mesenchymal transition (EMT). Mesenchymal-epithelial transition (MET), as
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the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole-imidazole (PI)
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polyamide, a novel gene regulator that targets human TGF-β1, and investigated its effects
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on the EMT/MET process. PI polyamide targeted to TGF-β1 significantly inhibited the mRNA expression of TGF-β1 and SNAI1 as an EMT marker and increased mRNA and
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protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-β1 was applied to human
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fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline
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phosphatase-positive colonies. The expression of undifferentiated markers was also
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observed in these colonies. These results suggest that PI polyamide targeted to human TGF-β is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.
Keywords: epithelial-mesenchymal transition, mesenchymal-epithelial transition, pyrrole-imidazole polyamide, transforming growth factor- 1.
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1. Introduction Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell adhesion and gain migratory and invasive properties to transform into the
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mesenchymal phenotype (Boyer et al., 2000). In the EMT process, epithelial cells lose their rigid cell-cell junction capability and apicobasal polarity, and their morphology is
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altered. The epithelial cells become spindle-shaped and obtain the properties of cell
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migration and invasion. Mesenchymal-epithelial transition (MET), the inverse process of EMT, occurs in the reprogramming process of cells (Yang and Weinberg, 2008). The
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progression of MET contributes to embryonic development and cancer metastasis. Transforming growth factor-β (TGF-β) is known to be a potent EMT induction factor
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associated with malignant transformation (Zavadil and Böttinger, 2005). In tumor progression and metastasis, the EMT process is initiated to obtain mesenchymal
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phenotypes that show decreased expression of adhesion molecule E-cadherin, as an
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epithelial marker, and increased expression of fibronectin, vimentin, and α-smooth muscle actin, as mesenchymal markers, via the activating receptors of TGF-β1 and TGF-β2 to induce Smad signaling. They are also involved in increasing the expression of the EMT transcription factor of snail family zinc finger 1 (SNAI1), ZEB, the Six family of homeobox genes, and Forkhead and High Mobility Group proteins (Imamura et al., 2012; Wendt et al., 2010; Morrison et al., 2013). The MET processes are also characterized by decreases in the expression of mesenchymal markers accompanied by increases in epithelial markers (Yao et al., 2011). The progression of MET is induced by cytokines of the TGF-β superfamily and bone morphogenetic proteins (BMPs) (Morrison et al., 2013). BMP-7 counteracts TGF-β1-induced EMT through the enhanced expression
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of E-cadherin by activating Smad 1, 5, or 8 internal binding to Smad 4 in cancer cells (Na et al., 2009; Zeisberg et al., 2003). It was recently reported that the MET process is associated with the progression of
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reprogramming in induced pluripotent stem cells (iPSCs) derived from fibroblasts (Lin et al., 2009; Li et al., 2010). iPSCs are generated by inducing reprogramming transcription
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factor genes such as Sox2, Pou5f1 (Oct4), Klf4, and c-Myc in somatic cells (Takahashi
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and Yamanaka, 2006). During the reprogramming process, these reprogramming factors activate an epithelial program and shut down key mesenchymal genes to overcome the
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EMT epigenetic barrier of fibroblasts and allow their successful reprogramming into iPSCs (Li et al., 2010).
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Pyrrole-imidazole (PI) polyamides are potent gene regulating compounds that can inhibit DNA-protein interaction by binding to the minor groove of double-helical DNA
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with high affinity and sequence specificity (Trauger et al., 1996; Gottesfeld et al., 1997).
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PI polyamides are small synthetic molecules composed of the aromatic rings of the N-methylpyrrole and N-methylimidazole amino acids. Various types of sequence-specific PI polyamides have been developed to control gene expression. DNA recognition depends on a code of side-by-side pairing of pyrrole and imidazole in the minor groove. The pairing of imidazole opposite pyrrole targets the guanine (G)-cytosine (C) base pair. PI targets the C-G base pair, and the pyrrole-pyrrole pairing degenerately targets the thymine (T)-adenine (A) and A-T base pairs. PI polyamides do not require vector-assisted delivery systems because of their cell permeability and may easily enter nuclei. Interfering with DNA-protein interfaces using PI polyamides may inhibit the initiation of gene transcription and modulate target gene induction. PI polyamides may
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therefore have potential use as novel transcriptional gene regulating agents for the treatment of diseases. We previously reported that PI polyamide targeting human TGF-β1 promoter
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significantly inhibits both TGF-β1 promoter activity and the expression of TGF-β1 mRNA and protein in human cells (Lai et al., 2005; Matsuda et al., 2006). In the present
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study, we investigated the effects of PI polyamide targeting human TGF-β1 applied to the
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regulation of EMT/MET processes by which the induction of iPSCs are potentiated.
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2. Experimental
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2.1. Design and synthesis of PI polyamide targeted to human TGF-β1 promoter region
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PI polyamides were synthesized according to previously described methods (Bando
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et al., 2002). We designed both a PI polyamide that binds to the DNA sequence of the near FSE2 site (-1383 to -1376 nucleotides upstream of the transcription initiation point) of the promoter region of the human TGF-β1 gene (Suppl. Fig. 1A) and a Mismatch PI polyamide that does not bind to the FSE2-targeted PI polyamide binding site. These polyamides were described in a previous report (Lai et al., 2005).
2.2. Cell culture
MCF10A cells (American Type Cell Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium-F12 (DMEM/F-12) (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA) containing 5% horse serum (Life Technologies), 500
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ng/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 10 ng/ml recombinant human epidermal growth factor (Peprotech, Rocky Hill, NJ), 5 μg/ml insulin (Sigma-Aldrich), and 100 ng/ml cholera toxin (Wako Pure Chemical Industries, Osaka, Japan) at 37°C in
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a humidified atmosphere of 95% air/5% CO2. Human dermal fibroblast (HDF) cells from facial dermis (Cell Applications, San
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Diego, CA), were maintained in fibroblast growth medium (Cell Applications) or
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DMEM supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan).
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Generated human iPSCs were maintained on mitomycin C-treated mouse embryo fibroblast (MEF) feeder cells (Repro CELL, Kanagawa, Japan) on 0.1% gelatin
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(Sigma-Aldrich)-coated 6-well plate dishes (Corning, Corning, NY) in human embryonic stem (ES)-induced medium. DMEM/F-12 was supplemented with 20%
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KnockOutTM serum replacement (Life Technologies), 10 mM MEM-NEAA solution
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(Life Technologies), 55 mM 2-Mercaptoethanol (Life Technologies), and 4 ng/ml basic fibroblast growth factor (bFGF) (Wako) at 37°C in a humidified atmosphere of 95% air/5% CO2.
2.3. Gel shift assay
Fluorescein-labeled oligonucleotides, which included the FSE2 binding sequence,
were synthesized for a gel mobility shift assay (Eurofins Genomics, Tokyo, Japan). One millimole of the fluorescein-labeled sense oligonucleotides and the anti-sense oligonucleotides were dissolved in annealing buffer (250 mM Tris-HCl, 87.5 mM EDTA, 200 mM NaCl) and incubated for 5 min at 95°C. The solution of
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double-stranded oligonucleotides was mixed with 1 μM PI polyamide and gel shift binding buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 25 mM dithiothreitol, 250 mM NaCl, 20% glycerol) and incubated for one hour at room temperature. The
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resulting complexes were separated by electrophoresis in a 20% Tris-buffered EDTA acrylamide (Life Technologies) and visualized with an LAS-4000 luminescent image
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analyzer (Fujifilm, Tokyo, Japan).
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2.4. Distribution of PI polyamide
MCF10A cells were cultured on 6-well plate dishes at a density of 1.0 × 105
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cells/well. Cells were incubated with 1 μM fluorescein isothiocyanate (FITC)-conjugated PI polyamide in MCF10A medium for 72 hours and fixed for 10 min
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with ice-cold methanol (Wako) and 1 min with ice-cold acetone (Wako). Nuclear
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staining was achieved with 1 μg/ml 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and visualized with an Axiovert 200 (Carl Zeiss, Oberkochen, Germany) using appropriate filters.
2.5. Cell proliferation assay
Cell proliferation was assessed by cell count reagent SF (Nacalai Tesque, Kyoto,
Japan). Cells were seeded at a density of 3500 cells/well in 96-well plate dishes (Corning) and cultured with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 24 hours and next, at the concentration of 0.01, 0.1, 1, 3 μM with PI polyamide for 48 hours. After the treatment, 10 μl of cell count reagent SF was
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applied to the cultured cells, and the cells were further incubated for one hour. Next, absorbance was measured at 450 nm using an ARVO MX microplate reader (Perkin
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Elmer, Waltham, MA).
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2.6. Induction of iPSCs with PI polyamide
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Induction of human iPSCs from HDF cells was performed using Sox2, Oct4, Klf4, and c-Myc factors containing the non-integrating CytoTuneTM-Sendai viral vector kit
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(DNAVEC, Ibaraki, Japan). HDF cells were seeded at a density of 3 × 105 cells per well on tissue culture-treated 6-well plate dishes with DMEM containing 10% FBS. The next
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day, plating cells were infected with CytoTuneTM-Sendai viral vector at multiplicity of infection of 3, and PI or Mismatch PI polyamide was applied on 6-well plate dishes and
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cultured in DMEM containing 10% FBS. After 3 days, the cells were reseeded at a
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density of 3 × 104 cells per well into mitomycin C-treated MEF feeder cells on 0.1% gelatin-coated 6-well plate dishes, and then PI polyamide or Mismatch PI polyamide was applied. Thereafter, the culture primate ES medium with bFGF was changed every day, whereas that with the PI polyamide was changed every 3 days. When ES-like colonies appeared, the medium was changed to primate ES medium with bFGF, and the growing colonies were harvested and then plated on MEF feeder cells to avoid spontaneous differentiation.
2.7. Determining reprogramming efficiency
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Reprogramming efficiency was calculated as the number of alkaline phosphatase (ALP)-positive colonies formed per the number of infected cells seeded. iPSCs colonies were identified based on ES-like morphology and ALP staining. ALP staining, which was
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performed after fixing with ALP substrate (Sigma-Aldrich), was used to facilitate the
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identification of iPSCs colonies.
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2.8. Scratch wound healing assay
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The MCF10A cells on the 6-well plate dishes were allowed to grow to full confluence. Growth was then arrested by treatment with 4 μg/ml mitomycin C (Kyowa
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Hakko Kirin, Tokyo, Japan). After 2 hours incubation and two washings in phosphate buffered saline (PBS) (Nacalai Tesque), the cells were cultured in MCF10A medium with
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100 nM PMA and/or PI polyamide for 48 hours. We used PMA as an inducer of EMT in
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the MCF10A cells because PMA has been reported to induce EMT in cancer and progression of tumor phenotype (cell invasion in 3-dimensional epithelial cells via the activation of TGF-β1 signaling-mediated protein kinase C) (He et al., 2010; Klos et al., 2014). The cells were then scratched by a sterile pipette tip, and images were captured under a light microscope (Leica Microsystems, Wetzlar, Germany).
2.9. RNA isolation, reverse transcription (RT) and real-time PCR
Total RNA was isolated from the cells using an RNeasy Mini Kit (Qiagen, Valencia, CA). Aliquots of total RNA were reverse transcribed into single-stranded cDNA using a PrimeScript™ RT-PCR Kit (Takara Bio, Shiga, Japan). Human sequence-specific
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primers for the SYBR Green assay were designed with Primer 3.0. Real-time PCR was performed in triplicate on a Thermal Cycler Dice® Real Time System (Takara Bio). Relative gene expression was analyzed by the comparative Ct method with GAPDH
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RNA as the endogenous control after confirming that the efficiencies of the target and the endogenous control amplifications were approximately equal. Results are presented as
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target gene expression normalized to GAPDH RNA.
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2.10. PrimerArray® assay for marker gene of ES cells in iPSCs
Total RNA was extracted from the induced iPSC colonies at 14 days with or without
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PI polyamide using an RNeasy Mini Kit (Qiagen). The aliquots of total RNA were reverse transcribed into single-stranded cDNA using a PrimeScript™ RT-PCR Kit
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(Takara Bio), and ES cells, germ layer/trophoblast, and ES cell differentiation marker
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gene mRNA levels were assessed by PrimerArray® Embryonic Stem Cells (Human) (Takara Bio), according to the manufacturer’s instructions (n=3). Real-time PCR was performed in triplicate on a Thermal Cycler Dice® Real Time System (Takara Bio). Results are presented as target gene expression normalized to GAPDH RNA.
2.11. Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and washed twice with ice-cold PBS, permeabilized for 10 min with PBS and 0.25% Triton-X 100 at room temperature and washed three times for 5 min with Tris Buffered Saline (TBS) (Sigma-Aldrich), blocked with 1% goat serum and 1% bovine serum albumin
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(BSA) in TBS containing 0.1% Tween 20 (TBS-T), and incubated overnight at 4oC with primary antibody, including rabbit anti-E-cadherin (1:50, Abcam, Cambridge, UK) and rabbit anti-Nanog (1:800), anti-Sox2 (1:800), anti-Oct4 (1:800), and mouse anti-SSEA4
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(1:500; all from Cell Signaling Technology, Danvers, MA). These primary antibodies were diluted in PBS containing 1% BSA and 0.25% Triton-X 100. After the samples were
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washed three times for 10 min with PBS, they were incubated with secondary antibody,
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Alexa 488 (or 594) goat anti-rabbit (or mouse) IgG antibody (1:1000, Life Technologies), in PBS for one hour at room temperature. After immunofluorescence staining, the cells
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were washed in PBS, and the nuclei were stained using 1 μg/ml DAPI in PBS for 10 min at room temperature. The cells were mounted in Fluoromount-G (Southern
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Biotechnology, Birmingham, AL), and coverslips were placed over the cells. Staining was visualized and photographed with an Axiovert 200 (Carl Zeiss) and FluoView
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FV10i confocal microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).
2.12. Western blotting
Cultured MCF10A cells were incubated with 100 nM PMA for 24 hours. Next, 1 M
TGF-β1 receptor inhibitor, SB431542 (Sigma-Aldrich), or 1
M PI polyamide was
applied for 48 hours. Cells were washed twice in ice-cold PBS. The cells were solubilized by RIPA Buffer (Nacalai Tesque), and cell lysates were homogenized by sonication using a Bioruptor UCD-250 (Cosmo Bio, Tokyo, Japan). The resultant supernatant was transferred to a fresh microcentrifuge tube. Protein content was assayed using a Pierce BSA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. After boiling at 70°C for 10 min, 25-µg protein samples
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were electrophoretically separated in NuPAGE® Novex 4-12% Bis-Tris gel (Life Technologies) and transferred to iBlot Gel Transfer Stacks PVDF Regular (Life Technologies). After transfer, blots were blocked in Blocking One (Nacalai Tesque) for
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one hour at room temperature. The primary antibodies, rabbit anti-E-cadherin (1:500, Abcam), anti-GAPDH (1:2500, Abcam), and mouse anti-Vimentin (1:200, Abcam), were
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diluted in Can Get Signal® immunoreactions Enhancer Solution 1 (Toyobo Life Science,
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Osaka, Japan), and blots were incubated in the solutions overnight at 4°C. After washing the membranes with TBS-T, they were incubated at room temperature for 1 h with
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secondary antibody, donkey anti-rabbit (or mouse) IgG, and horseradish peroxidase-linked species-specific whole antibody (1:10000, GE Healthcare, Little
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Chalfont, UK) diluted in TBS-T. The signals were detected with ECL Prime Western Blotting Detection Reagent (GE Healthcare) using an LAS-4000 Luminescent Image
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Analyzer (Fujifilm). The bands of gel images were quantified using TotalLab Quant
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software v13.2 (Nonlinear Dynamics Ltd., Newcastle, UK).
2.13. Statistical analysis
Values are reported as mean ± standard error of the mean (SEM). Statistical analysis
was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis to compare multiple conditions. P < 0.05 was considered statistically significant.
3. Results
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3.1. PI polyamide binding to double-stranded oligonucleotides and incorporation into nuclei in MCF10A cells
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We designed and synthesized a PI polyamide with a target of binding to fat-specific element 2 (FSE2) of the promoter region of the human TGF-β1 gene (Suppl. Fig. 1A).
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The chemical structure of the PI polyamide is shown in Supplemental Figure 1B. To
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determine whether the PI polyamides could bind to the target sequence DNA, the gel shift assay allowed the determination of binding affinity and specificity of the polyamides for
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double-stranded DNA. The PI polyamide designed specifically for the TGF-β1 promoter region bound to the FSE2 target double-stranded oligonucleotide, whereas the PI
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polyamide did not bind to non-target double-stranded DNA (Fig. 1A). Therefore, these results suggested that the PI polyamide can bind to the target sequence DNA.
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Next, we observed the distribution of the PI polyamide in the cells using
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FITC-labelled PI polyamide. When MCF10A cells were incubated with 1 μM FITC-labelled PI polyamide targeted to TGF-β1 for 72 hours, strong fluorescent signals were detected in the nuclei of most MCF10A cells (Fig. 1B). These findings indicated that the PI polyamides could bind the target DNA in the nuclei.
3.2. Effect of PI polyamide on expression of TGF-β1, E-cadherin, SNAI1, and fibronectin mRNA in MCF10A cells stimulated with PMA
MCF10A cells were incubated with PMA to induce EMT through TGF-β activation. The abundance of TGF-β1 mRNA was increased in MCF10A cells (Suppl. Fig. 2A), and the morphology of the cells changed from the epithelial form to the mesenchymal-like
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form (Suppl. Fig. 2A and B). A concentration of 1 μM PI polyamide decreased the PMA-increased abundance of TGF-β1 mRNA (Fig. 2A). A concentration of 100 nM PI polyamide decreased the PMA-increased abundance of SNAI1 mRNA (Fig. 2B).
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Concentrations of 10 nM to 1 μM PI polyamide increased the PMA-inhibited abundance of E-cadherin mRNA (Fig. 2C). Concentrations of 10 nM to 1 μM PI polyamide did not
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decrease the PMA-increased abundance of fibronectin mRNA (Fig. 2D). These findings
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suggested that the PI polyamide targeting TGF- 1 exerts inhibitory effects on the EMT
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process.
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3.3. Effects of PI polyamide on expression of E-cadherin and vimentin protein
To further confirm the effects of the PI polyamide targeting TGF- 1 on the
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expression of E-cadherin in MCF10A cells stimulated with PMA, immunocytochemistry
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was performed in MCF10A cells stimulated with 100 nM PMA in the presence or absence of PI polyamide for 48 hours. PMA obviously suppressed the staining of E-cadherin in MCF10A cells, whereas PI polyamide recovered the suppressed staining of E-cadherin in MCF10A cells (Fig. 3A). We performed western blot analysis for the expression of E-cadherin and vimentin proteins in cultured MCF10A cells incubated with PMA for 24 hours and subsequently incubated with SB431542 or PI polyamide for 48 hours (Fig. 3B). PMA completely suppressed the abundance of E-cadherin protein, whereas PI polyamide recovered the abundance of E-cadherin protein suppressed by PMA (Fig. 3B and C), and PI polyamide inhibited the PMA-increased abundance of vimentin protein (not statistically significant) (Fig. 3B and D).
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3.4. Effect of PI polyamide on cell proliferation and migration of MCF10A cells
To evaluate the effect of PI polyamide on the cell proliferation of MCF10A cells, cell
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proliferation activity was analyzed by WST-8 assay. PI polyamide had no significant effect on cell proliferation in MCF10A cells after 72 hours (Suppl. Fig. 3).
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The effect of PI polyamide on the EMT process was also assessed by cell migration
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with PMA. We performed a Scratch wound healing assay in cultured MCF10A cells stimulated with 100 nM PMA in the presence or absence of PI polyamide for 48 hours.
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The PI polyamide abolished cell migration with PMA in the MCF 10A cells (Fig. 4). These findings suggested that the PI polyamide did not affect cell proliferation but did
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inhibit the cell migration associated with the EMT process.
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3.5. Effect of PI polyamide on induction of iPSCs
Concerning the effects of PI polyamide targeting human TGF- 1 on the induction
efficiency of human iPSCs, at 14 days after transfection of Sox2, Oct4, Klf4, and c-Myc genes with Sendai virus into HDF cells, the number of ALP-positive colonies was increased in comparison to the non-treated group. PI polyamide increased the number of iPS colonies by 180%. Mismatch PI polyamide did not affect the number of iPS colonies (Fig. 5A and B). The human iPS-like morphology of the colonies was found to be Nanog positive by immunostaining (Fig. 5C). To assess the characteristics of the iPS colonies, we performed PrimerArray® assay for germ layer/trophoblast and ES cell differentiation markers on the iPS colonies with or without PI polyamide. Data obtained from the gene list are shown in Supplemental Table 1. Up-regulated genes (fold change > 2) were the
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CCL2 (chemokine [C-C motif] ligand 2) gene, with a fold change = 2.66, and the CCR7 (chemokine [C-C motif] receptor 7) gene, with a fold change = 2.07. The down-regulated gene (fold changes < 0.5) was NPPA (natriuretic peptide A), with a fold change = 0.29.
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iPS colonies both with and without PI by mRNA expression profiling presented similar gene expression patterns (Fig. 5D).
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We performed immunofluorescence staining of undifferentiated markers generating
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human iPSC-like colonies treated with PI polyamide targeting human TGF- 1. In 2 passages of these colonies after treatment with PI polyamide, immunostaining showed
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staining of undifferentiated markers Nanog, SSEA4, Sox2, and Oct4 proteins (Fig. 6). These results indicated that the PI polyamide increased the efficiency of iPSC generation
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4. Discussion
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iPSCs.
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with no adverse effects on the molecules involved in the process of reprogramming
Synthetic PI polyamides targeting gene promoters will have potential use as practical
medicines for the regulation of gene transcription because the polyamides have biochemical characteristics such as cell permeability and specific suppression of expression of the target gene to bind into the DNA sequence. We have already demonstrated that PI polyamide targeting TGF-β1 effectively attenuates progressive renal disease (Matsuda et al., 2011), stenosis of the carotid artery after angioplasty (Yao et al., 2009), alkali burn of the cornea (Chen et al., 2010), and hypertrophic scars (Washio et al., 2011). PI polyamides targeted to TGF-β1 may thus be a potent gene silencer at the transcription level. We also demonstrated that a PI polyamide targeted to the FSE2 binding site of human TGF-β1 promoter inhibited the PMA-stimulated expression of 17 Page 17 of 36
TGF-β1 mRNA in vascular smooth muscle cells (Lai et al., 2005). The EMT process is an essential part of pathogenesis in tumor progression and organ fibrotic diseases including liver cirrhosis and renal fibrosis (Zeisberg et al., 2003; Bates and Mercurio, 2005; Choi
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and Diehl, 2009). The EMT process has been reported to be mainly mediated by the induction of TGF-β1 accompanied by increases in SNAI1 and decreases in E-cadherin
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(Lamouille et al., 2014). Thus, it is thought that PI polyamides targeting the human
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TGF-β1 promoter modulate the EMT process by decreasing SNAI1 expression and increasing E-cadherin expression.
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In the present experiments, PI polyamide targeting human TGF-β1 significantly decreased the expression of SNAI1 mRNAs and inhibited vimentin protein as an EMT
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marker gene and significantly increased the expression of E-cadherin mRNA and protein. SNAI1 is a basic helix-loop-helix transcription factor that binds to specific cognate
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sequences, termed E-boxes, and represses the transcription of E-cadherin and other key
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epithelial regulators (Cano et al., 2000). E-cadherin is a transmembrane constituent of the intercellular adherens junctions responsible for maintaining epithelial cohesion and has also been linked to the control of the EMT and ES cell pluripotency (Cavallaro and Christofori, 2004; Soncin et al., 2009). Moreover, cell migration is the main characteristic of EMT obtained through the decreased activity of E-cadherin. (Boyer et al., 2000). We investigated the effects of PI polyamide targeting human TGF-β1 on the
induction of iPSCs through suppression of the EMT and enhancement of the MET processes. iPSCs are generated through multiple-steps mediated by reprogramming transcription factors, which progressively induce the expression of ES cell-like genes and suppress the somatic cell genetic program (Takahashi and Yamanaka, 2006; Brambrink et al., 2008). The mechanisms underlying the induction of iPSCs with Yamanaka
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transcription factors have not been identified. Recently, Wang et al. (2010) investigated the transcriptional roadmap to iPSCs in somatic cells by microarray analysis and found that MET is a pivotal process in the induction of iPSCs. In addition, Li et al. (2010)
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demonstrated that the MET process is required to generate iPSCs from mouse fibroblasts, which is organized by suppressing molecules related to EMT. PI polyamide targeted to
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TGF-β1 increased the number of ALP-positive colonies by 180%. We confirmed the
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expression of undifferentiated markers in these colonies. Previous studies have
demonstrated the greater efficiency of iPSC generation using chemical compounds or
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micro RNAs that inhibit TGF-β signaling (Hou et al., 2013; Li and Rana, 2012; Li et al., 2009; Li et al., 2011). Keratinocytes can be reprogrammed with higher efficiency because
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of it reside within epithelium properties (Maherali et al., 2008), and E-cadherin improves reprogramming efficiency. Moreover, it has been established that the process of
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reprogramming efficiency of ES cells is associated with the higher expression of
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E-cadherin (Li et al., 2010; Huangfu et al., 2008; Shi et al., 2008). We found that PI polyamide targeted to TGF-β1 efficiently enhanced the induction of iPSCs by increasing the endogenous E-cadherin through the activity of MET in the present experiments. The PrimerArray® assay showed that two molecules, chemokine the CCL2 and
CCR7 genes, were up-regulated (respective fold changes = 2.66, 2.07) in iPSC colonies with PI polyamide compared to those without PI polyamide. Hasegawa et al. (2011) reported that CCL2 enhanced pluripotency in the induction of iPSCs via mediation by activating the Stat3 pathway and Klf4 up-regulation. Our results demonstrated that PI polyamide targeted to TGF-β1 enhanced efficiency of generated iPSCs, and it is thought that one of the reasons the PI polyamide increase expression of CCL2 and CCR7 in process of induced iPSCs.
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In conclusion, PI polyamide targeting the human TGF-β1 gene promoter region was found to regulate the EMT/MET process in MCF10A cells and increase the efficiency of iPSC induction in human fibroblasts. The results suggest that PI polyamide targeted to
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epithelial cells and enhance the iPSC induction of human fibroblasts.
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human TGF-β is a novel compound that can control the EMT/MET process of human
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Acknowledgements
We acknowledge support of this study by a financial grant from the “Strategic
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Research Base Development” Program for Private Universities subsidized by MEXT (S1101018), a Nihon University Multidisciplinary Research Grant in 2014 (14-024), and
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a Grant-in-Aid for Scientific Research (KAKENHI) in 2014 (26830134).
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REFERENCES Bando T, Narita A, Saito I, Sugiyama H. Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation. Chemistry 2002;8:4781–90.
cancer progression. Cancer Biol Ther 2005;4:365–70.
ip t
Bates RC, Mercurio AM. The epithelial-mesenchymal transition (EMT) and colorectal
cr
Boyer B, Vallés AM, Edme N. Induction and regulation of epithelial-mesenchymal
us
transitions. Biochem Pharmacol 2000;60:1091–9.
Brambrink T, Foreman R, Welstead GG, Lengner CJ, Wernig M, Suh H, et al. Sequential
an
expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2008;2:151–9.
M
Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing
d
E-cadherin expression. Nat Cell Biol 2000;2:76–83.
Ac ce pt e
Cavallaro U, Christofori G. Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 2004;4:118–32.
Chen M, Matsuda H, Wang L, Watanabe T, Kimura MT, Igarashi J, et al. Pretranscriptional regulation of TGF-β1 by PI polyamide prevents scarring and accelerates wound healing of the cornea after exposure to alkali. Mol Ther 2010;18:519–27.
Choi SS, Diehl AM. Epithelial-to-mesenchymal transitions in the liver. Hepatology 2009;50:2007–13. Gottesfeld JM, Neely L, Trauger JW, Baird EE, Dervan PB. Regulation of gene expression by small molecules. Nature 1997;387:202–5.
21 Page 21 of 36
Hasegawa Y, Takahashi N, Forrest AR, Shin JW, Kinoshita Y, Suzuki H, et al. CC chemokine ligand 2 and leukemia inhibitory factor cooperatively promote pluripotency in mouse induced pluripotent cells. Stem Cells 2011;29:1196–205.
ip t
He H, Davidson AJ, Wu D, Marshall FF, Chung LW, Zhau HE, et al. Phorbol ester phorbol-12-myristate-13-acetate induces epithelial to mesenchymal transition in
cr
human prostate cancer ARCaPE cells. Prostate 2010;70:1119–26.
us
Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 2013;341:651–4.
an
Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule
M
compounds. Nat Biotechnol 2008;26:795–7.
Imamura T, Hikita A, Inoue Y. The roles of TGF-β signaling in carcinogenesis and breast
d
cancer metastasis. Breast Cancer 2012;19:118–24.
Ac ce pt e
Klos KS, Warmka JK, Drachenberg DM, Chang L, Luxton GW, Leung CT, et al. Building bridges toward invasion: tumor promoter treatment induces a novel protein kinase C-dependent phenotype in MCF10A mammary cell acini. PLoS One 2014;9:e90722.
Lai YM, Fukuda N, Ueno T, Matsuda H, Saito S, Matsumoto K, et al. Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-β1 gene. J Pharmacol Exp Ther 2005;315:571–5.
Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial–mesenchymal transition. Nature Rev Mol Cell Biol 2014;15:178–96.
22 Page 22 of 36
Li R, Liang J, Ni S, Zhou T, Qing X, Li H, et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 2010;7:51–63.
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Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, et al. A chemical platform for improved induction of human iPSCs. Nat Methods 2009;6:805–8.
cr
Li W, Wei W, Zhu S, Zhu J, Shi Y, Lin T, et al. Generation of rat and human induced
us
pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 2009;4:16–9.
an
Li Z, Rana TM. A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation. Nat Commun 2012;3:1085.
M
Li Z, Yang CS, Nakashima K, Rana TM. Small RNA-mediated regulation of iPS cell generation. EMBO J 2011;30:823–34.
d
Maherali N, Ahfeldt T, Rigamonti A, Utikal J, Cowan C, Hochedlinger K. A
Ac ce pt e
high-efficiency system for the generation and study of human induced pluripotent
stem cells. Cell Stem Cell 2008;3:340–5.
Matsuda H, Fukuda N, Ueno T, Katakawa M, Wang X, Watanabe T, et al. Transcriptional inhibition of progressive renal disease by gene silencing pyrrole-imidazole polyamide targeting of the transforming growth factor-β1 promoter. Kidney Int 2011;79:46–56.
Matsuda H, Fukuda N, Ueno T, Tahira Y, Ayame H, Zhang W, et al. Development of gene silencing pyrrole-imidazole polyamide targeting the TGF-β1 promoter for treatment of progressive renal diseases. J Am Soc Nephrol 2006;17: 422–32. Morrison CD, Parvani JG, Schiemann WP. The relevance of the TGF-β Paradox to EMT-MET programs. Cancer Lett 2013;341:30–40.
23 Page 23 of 36
Na YR, Seok SH, Kim DJ, Han JH, Kim TH, Jung H, et al. Bone morphogenetic protein 7 induces mesenchymal-to-epithelial transition in melanoma cells, leading to inhibition of metastasis. Cancer Sci 2009;100:2218–25.
ip t
Shi Y, Desponts C, Do JT, Hahm HS, Schöler HR, Ding S. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule
cr
compounds. Cell Stem Cell 2008;3:568–74.
us
Soncin F, Mohamet L, Eckardt D, Ritson S, Eastham AM, Bobola N, et al. Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in
an
reversible LIF-independent self-renewal. Stem Cells 2009;27:2069–80. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic
M
and adult fibroblast cultures by defined factors. Cell 2006;126:663–76. Trauger JW, Baird EE, Dervan PB. Recognition of DNA by designed ligands at
d
subnanomolar concentrations. Nature 1996;382:559–61.
Ac ce pt e
Wang Y, Mah N, Prigione A, Wolfrum K, Andrade-Navarro MA, Adjaye J. A transcriptional roadmap to the induction of pluripotency in somatic cells. Stem Cell Rev 2010;6:282–96.
Washio H, Fukuda N, Matsuda H, Nagase H, Watanabe T, Matsumoto Y, et al. Transcriptional inhibition of hypertrophic scars by a gene silencer, pyrrole-imidazole polyamide, targeting the TGF-β1 promoter. J Invest Dermatol 2011;131: 1987–95.
Wendt MK, Smith JA, Schiemann WP. Transforming growth factor-β-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression. Oncogene 2010;29:6485–98. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 2008;14:818–29.
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Yao D, Dai C, Peng S. Mechanism of the mesenchymal-epithelial transition and its relationship with metastatic tumor formation. Mol Cancer Res 2011;9:1608–20. Yao EH, Fukuda N, Ueno T, Matsuda H, Nagase H, Matsumoto Y, et al. A
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pyrrole-imidazole polyamide targeting transforming growth factor-beta1 inhibits restenosis and preserves endothelialization in the injured artery. Cardiovasc Res
cr
2009;81:797–804.
us
Zavadil J, Böttinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005;24:5764–74.
an
Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, et al. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses
Ac ce pt e
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chronic renal injury. Nat Med 2003;9:964–8.
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Figure legends
Fig 1. Binding of pyrrole-imidazole (PI) polyamide to target DNA and distribution of PI
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polyamide in cells. (A) Binding of PI polyamide to the target sequence assessed by gel
transforming growth factor (TGF)-β1 promoter region DNA
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shift assay. Lane 1: Fluorescein-labeled FSE2 target double-stranded DNA on human
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(5’-GCAATTCTTACA-3’/5’-TGTAAGAATTGC-3’). Lane 2: Fluorescein-labeled FSE2 target double-stranded DNA on human TGF-β1 promoter region plus PI
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polyamide. Lane 3: Fluorescein-labeled non-target double-stranded DNA on human TGF-β1 promoter region number 1
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(5’-CTGACTCTCCTT-3’/5’-AAGGAGAGTCAG-3’). Lane 4: Fluorescein-labeled non-target double-stranded DNA on human TGF-β1 promoter region number 1 plus PI
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polyamide. Lane 5: Fluorescein-labeled non-target double-stranded DNA on TGF-β1
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promoter region number 2 (5’-CCTGTGTCTCAT-3’/5’-ATGAGACACAGG-3’). Lane 6: Fluorescein-labeled non-target double-stranded DNA on human TGF-β1 promoter region number 2 plus PI polyamide. (B) Distribution of FITC-labelled PI polyamide in MCF10A cells, which were incubated with 1 μM FITC-labelled PI polyamide for 72 hours. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI). Scale bars represent 50 μm.
Fig 2. Effects of pyrrole-imidazole (PI) polyamide targeted to human transforming growth factor (TGF)-β1 on the expression of TGF-β1, SNAI1, and E-cadherin mRNAs in MCF10A cells stimulated with PMA. (A) Expression levels of the mRNA encoding human TGF-β1 in MCF10A cells cultured with 100 nM PMA for 24 hours (white bar)
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and at the indicated concentrations with PI polyamide for 24 hours (black bars). (B) Expression levels of the mRNA encoding SNAI1, (C) E-cadherin, and (D) fibronectin in MCF10A cultured with 100 nM PMA for 24 hours (white bar in each panel) and at the
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indicated concentrations with PI polyamide for 48 hours (black bars in each panel). GAPDH mRNA was used to normalize the variability in template loading. Data are mean
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± SEM (n=3). *P < 0.05, **P < 0.01 in the indicated columns. PI: PI polyamide targeted
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to TGF- 1.
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Fig 3. Effects of PMA on expression of E-cadherin and vimentin protein. (A) Immunofluorescence staining of MCF10A cells stimulated with 100 nM PMA in the
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presence or absence of pyrrole-imidazole (PI) polyamide targeted to human transforming growth factor (TGF)-β1 for 48 hours. Nuclei were stained with DAPI. Scale bars
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represent 50 μm. (B) Western blotting analysis of E-cadherin and vimentin protein on
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cultured MCF10A cells stimulated with 100 nM PMA for 24 hours and next with PI polyamide for 48 hours. SB431542 was used as a positive control. GAPDH protein level was used to normalize the variability in template loading. These experiments were performed using anti-E-cadherin, GAPDH, and vimentin primary antibody. (C) Quantification of protein levels of E-cadherin, and (D) Vimentin. Data are mean ± SEM (n=3). *P < 0.05.
Fig 4. Effects of pyrrole-imidazole (PI) polyamide targeted to human transforming growth factor (TGF)- 1 on cell migration. MCF10A cells were incubated with 100 nM PMA in the presence or absence of PI polyamide targeting TGF- 1 for 48 hours.
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Control: Non-treated control group. Polyamide: PI polyamide targeting TGF- 1. Scale bars represent 200 μm.
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Fig 5. Effects of pyrrole-imidazole (PI) polyamide targeted to transforming growth factor (TGF)- 1 on the induction of induced pluripotent stem cells (iPSCs). (A) Representative
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images. (B) Evaluation of the number of ALP-positive colonies. (C)
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Immunofluorescence staining of the human iPS-like morphology of colonies using anti-Nanog primary antibody. (D) mRNA expression profiles of iPSCs colonies with or
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without PI by PrimerArray® assay for embryonic stem cells, germ layer/trophoblast, and embryonic stem cell differentiation marker gene expression. Control: Non-treated control
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group. PI: PI polyamide; Mismatch PI: Mismatch PI polyamide. Data are mean ± SEM
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(n=3). **P < 0.01. Scale bars represent 125 μm.
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Fig 6. Immunofluorescence staining of undifferentiated markers generating colonies with human iPS-like morphology after treatment with pyrrole-imidazole (PI) polyamide targeted to transforming growth factor (TGF)- 1. After 2 passages from generated colonies with human iPS-like morphology after treatment with PI polyamide, it was performed immunofluorescence staining using anti-Nanog, SSEA4, Sox2, and Oct4 primary antibody. Nuclei were stained with DAPI. Scale bars represent 50 μm.
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S1357-2725(15)00196-X http://dx.doi.org/doi:10.1016/j.biocel.2015.07.011 BC 4667
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
6-3-2015 19-6-2015 23-7-2015
Please cite this article as: Saito, K., Fukuda, N., Shinohara, K.-i., Masuhiro, Y., Hanazawa, S., Matsuda, H., Fujiwara, K., Ueno, T., and Soma, M.,Modulation of the EMT/MET process by pyrrole-imidazole polyamide targeting human transforming growth factor-rmbeta1, International Journal of Biochemistry and Cell Biology (2015), http://dx.doi.org/10.1016/j.biocel.2015.07.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Modulation of the EMT/MET process by pyrrole-imidazole polyamide targeting human transforming growth factor-β1 Short title: Modulation of EMT/MET with PI polyamide to TGF-β1
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Kosuke Saito a, Noboru Fukuda b,c,*, Ken-ichi Shinohara d, Yoshikazu Masuhiro e, Shigemasa Hanazawa e, Hiroyuki Matsuda a, Kyoko Fujiwara a, Takahiro Ueno c,
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Masayoshi Soma a
Department of General Medicine, Nihon University School of Medicine, Tokyo, Japan
b
Division of Life Science, Advanced Research Institute for the Sciences and Humanities,
c
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Nihon University Graduate School, Tokyo, Japan
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a
Division of Nephrology Hypertension and Endocrinology, Department of Medicine,
Nihon University School of Medicine, Tokyo, Japan
Division of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba, Japan
e
Department of Applied Biological Sciences, College of Bioresource Sciences, Nihon
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University, Kanagawa, Japan
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* Corresponding author: Noboru Fukuda, Department of General Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30-1, Itabashi-ku, Tokyo 173-8610, Japan.
Phone: 81-3-3972-8111; Fax: 81-3-3972-8666, E-mail: [email protected] Word Count: 4,902 words
Abstract Length: 177 words
Number of Figures: 6 Figures, 1 Supplemental table, 3 Supplemental figures
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Highlights PI polyamide targeting human TGF-β1 gene promoter region regulates the EMT/MET process in MCF10A cells.
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PI polyamide significantly increased reprogramming efficiency via enhanced MET in iPSC induction of human fibroblasts.
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PI polyamide to human TGF-β is a novel compound that can control EMT/MET of
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human epithelial cells.
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ABSTRACT Transforming growth factor-β1 (TGF-β1) is a potent induction factor for epithelial-mesenchymal transition (EMT). Mesenchymal-epithelial transition (MET), as
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the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole-imidazole (PI)
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polyamide, a novel gene regulator that targets human TGF-β1, and investigated its effects
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on the EMT/MET process. PI polyamide targeted to TGF-β1 significantly inhibited the mRNA expression of TGF-β1 and SNAI1 as an EMT marker and increased mRNA and
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protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-β1 was applied to human
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fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline
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phosphatase-positive colonies. The expression of undifferentiated markers was also
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observed in these colonies. These results suggest that PI polyamide targeted to human TGF-β is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.
Keywords: epithelial-mesenchymal transition, mesenchymal-epithelial transition, pyrrole-imidazole polyamide, transforming growth factor- 1.
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1. Introduction Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell adhesion and gain migratory and invasive properties to transform into the
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mesenchymal phenotype (Boyer et al., 2000). In the EMT process, epithelial cells lose their rigid cell-cell junction capability and apicobasal polarity, and their morphology is
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altered. The epithelial cells become spindle-shaped and obtain the properties of cell
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migration and invasion. Mesenchymal-epithelial transition (MET), the inverse process of EMT, occurs in the reprogramming process of cells (Yang and Weinberg, 2008). The
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progression of MET contributes to embryonic development and cancer metastasis. Transforming growth factor-β (TGF-β) is known to be a potent EMT induction factor
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associated with malignant transformation (Zavadil and Böttinger, 2005). In tumor progression and metastasis, the EMT process is initiated to obtain mesenchymal
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phenotypes that show decreased expression of adhesion molecule E-cadherin, as an
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epithelial marker, and increased expression of fibronectin, vimentin, and α-smooth muscle actin, as mesenchymal markers, via the activating receptors of TGF-β1 and TGF-β2 to induce Smad signaling. They are also involved in increasing the expression of the EMT transcription factor of snail family zinc finger 1 (SNAI1), ZEB, the Six family of homeobox genes, and Forkhead and High Mobility Group proteins (Imamura et al., 2012; Wendt et al., 2010; Morrison et al., 2013). The MET processes are also characterized by decreases in the expression of mesenchymal markers accompanied by increases in epithelial markers (Yao et al., 2011). The progression of MET is induced by cytokines of the TGF-β superfamily and bone morphogenetic proteins (BMPs) (Morrison et al., 2013). BMP-7 counteracts TGF-β1-induced EMT through the enhanced expression
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of E-cadherin by activating Smad 1, 5, or 8 internal binding to Smad 4 in cancer cells (Na et al., 2009; Zeisberg et al., 2003). It was recently reported that the MET process is associated with the progression of
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reprogramming in induced pluripotent stem cells (iPSCs) derived from fibroblasts (Lin et al., 2009; Li et al., 2010). iPSCs are generated by inducing reprogramming transcription
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factor genes such as Sox2, Pou5f1 (Oct4), Klf4, and c-Myc in somatic cells (Takahashi
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and Yamanaka, 2006). During the reprogramming process, these reprogramming factors activate an epithelial program and shut down key mesenchymal genes to overcome the
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EMT epigenetic barrier of fibroblasts and allow their successful reprogramming into iPSCs (Li et al., 2010).
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Pyrrole-imidazole (PI) polyamides are potent gene regulating compounds that can inhibit DNA-protein interaction by binding to the minor groove of double-helical DNA
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with high affinity and sequence specificity (Trauger et al., 1996; Gottesfeld et al., 1997).
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PI polyamides are small synthetic molecules composed of the aromatic rings of the N-methylpyrrole and N-methylimidazole amino acids. Various types of sequence-specific PI polyamides have been developed to control gene expression. DNA recognition depends on a code of side-by-side pairing of pyrrole and imidazole in the minor groove. The pairing of imidazole opposite pyrrole targets the guanine (G)-cytosine (C) base pair. PI targets the C-G base pair, and the pyrrole-pyrrole pairing degenerately targets the thymine (T)-adenine (A) and A-T base pairs. PI polyamides do not require vector-assisted delivery systems because of their cell permeability and may easily enter nuclei. Interfering with DNA-protein interfaces using PI polyamides may inhibit the initiation of gene transcription and modulate target gene induction. PI polyamides may
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therefore have potential use as novel transcriptional gene regulating agents for the treatment of diseases. We previously reported that PI polyamide targeting human TGF-β1 promoter
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significantly inhibits both TGF-β1 promoter activity and the expression of TGF-β1 mRNA and protein in human cells (Lai et al., 2005; Matsuda et al., 2006). In the present
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study, we investigated the effects of PI polyamide targeting human TGF-β1 applied to the
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regulation of EMT/MET processes by which the induction of iPSCs are potentiated.
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2. Experimental
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2.1. Design and synthesis of PI polyamide targeted to human TGF-β1 promoter region
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PI polyamides were synthesized according to previously described methods (Bando
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et al., 2002). We designed both a PI polyamide that binds to the DNA sequence of the near FSE2 site (-1383 to -1376 nucleotides upstream of the transcription initiation point) of the promoter region of the human TGF-β1 gene (Suppl. Fig. 1A) and a Mismatch PI polyamide that does not bind to the FSE2-targeted PI polyamide binding site. These polyamides were described in a previous report (Lai et al., 2005).
2.2. Cell culture
MCF10A cells (American Type Cell Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium-F12 (DMEM/F-12) (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA) containing 5% horse serum (Life Technologies), 500
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ng/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 10 ng/ml recombinant human epidermal growth factor (Peprotech, Rocky Hill, NJ), 5 μg/ml insulin (Sigma-Aldrich), and 100 ng/ml cholera toxin (Wako Pure Chemical Industries, Osaka, Japan) at 37°C in
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a humidified atmosphere of 95% air/5% CO2. Human dermal fibroblast (HDF) cells from facial dermis (Cell Applications, San
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Diego, CA), were maintained in fibroblast growth medium (Cell Applications) or
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DMEM supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan).
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Generated human iPSCs were maintained on mitomycin C-treated mouse embryo fibroblast (MEF) feeder cells (Repro CELL, Kanagawa, Japan) on 0.1% gelatin
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(Sigma-Aldrich)-coated 6-well plate dishes (Corning, Corning, NY) in human embryonic stem (ES)-induced medium. DMEM/F-12 was supplemented with 20%
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KnockOutTM serum replacement (Life Technologies), 10 mM MEM-NEAA solution
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(Life Technologies), 55 mM 2-Mercaptoethanol (Life Technologies), and 4 ng/ml basic fibroblast growth factor (bFGF) (Wako) at 37°C in a humidified atmosphere of 95% air/5% CO2.
2.3. Gel shift assay
Fluorescein-labeled oligonucleotides, which included the FSE2 binding sequence,
were synthesized for a gel mobility shift assay (Eurofins Genomics, Tokyo, Japan). One millimole of the fluorescein-labeled sense oligonucleotides and the anti-sense oligonucleotides were dissolved in annealing buffer (250 mM Tris-HCl, 87.5 mM EDTA, 200 mM NaCl) and incubated for 5 min at 95°C. The solution of
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double-stranded oligonucleotides was mixed with 1 μM PI polyamide and gel shift binding buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 25 mM dithiothreitol, 250 mM NaCl, 20% glycerol) and incubated for one hour at room temperature. The
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resulting complexes were separated by electrophoresis in a 20% Tris-buffered EDTA acrylamide (Life Technologies) and visualized with an LAS-4000 luminescent image
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analyzer (Fujifilm, Tokyo, Japan).
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2.4. Distribution of PI polyamide
MCF10A cells were cultured on 6-well plate dishes at a density of 1.0 × 105
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cells/well. Cells were incubated with 1 μM fluorescein isothiocyanate (FITC)-conjugated PI polyamide in MCF10A medium for 72 hours and fixed for 10 min
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with ice-cold methanol (Wako) and 1 min with ice-cold acetone (Wako). Nuclear
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staining was achieved with 1 μg/ml 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and visualized with an Axiovert 200 (Carl Zeiss, Oberkochen, Germany) using appropriate filters.
2.5. Cell proliferation assay
Cell proliferation was assessed by cell count reagent SF (Nacalai Tesque, Kyoto,
Japan). Cells were seeded at a density of 3500 cells/well in 96-well plate dishes (Corning) and cultured with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 24 hours and next, at the concentration of 0.01, 0.1, 1, 3 μM with PI polyamide for 48 hours. After the treatment, 10 μl of cell count reagent SF was
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applied to the cultured cells, and the cells were further incubated for one hour. Next, absorbance was measured at 450 nm using an ARVO MX microplate reader (Perkin
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Elmer, Waltham, MA).
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2.6. Induction of iPSCs with PI polyamide
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Induction of human iPSCs from HDF cells was performed using Sox2, Oct4, Klf4, and c-Myc factors containing the non-integrating CytoTuneTM-Sendai viral vector kit
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(DNAVEC, Ibaraki, Japan). HDF cells were seeded at a density of 3 × 105 cells per well on tissue culture-treated 6-well plate dishes with DMEM containing 10% FBS. The next
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day, plating cells were infected with CytoTuneTM-Sendai viral vector at multiplicity of infection of 3, and PI or Mismatch PI polyamide was applied on 6-well plate dishes and
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cultured in DMEM containing 10% FBS. After 3 days, the cells were reseeded at a
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density of 3 × 104 cells per well into mitomycin C-treated MEF feeder cells on 0.1% gelatin-coated 6-well plate dishes, and then PI polyamide or Mismatch PI polyamide was applied. Thereafter, the culture primate ES medium with bFGF was changed every day, whereas that with the PI polyamide was changed every 3 days. When ES-like colonies appeared, the medium was changed to primate ES medium with bFGF, and the growing colonies were harvested and then plated on MEF feeder cells to avoid spontaneous differentiation.
2.7. Determining reprogramming efficiency
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Reprogramming efficiency was calculated as the number of alkaline phosphatase (ALP)-positive colonies formed per the number of infected cells seeded. iPSCs colonies were identified based on ES-like morphology and ALP staining. ALP staining, which was
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performed after fixing with ALP substrate (Sigma-Aldrich), was used to facilitate the
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identification of iPSCs colonies.
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2.8. Scratch wound healing assay
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The MCF10A cells on the 6-well plate dishes were allowed to grow to full confluence. Growth was then arrested by treatment with 4 μg/ml mitomycin C (Kyowa
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Hakko Kirin, Tokyo, Japan). After 2 hours incubation and two washings in phosphate buffered saline (PBS) (Nacalai Tesque), the cells were cultured in MCF10A medium with
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100 nM PMA and/or PI polyamide for 48 hours. We used PMA as an inducer of EMT in
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the MCF10A cells because PMA has been reported to induce EMT in cancer and progression of tumor phenotype (cell invasion in 3-dimensional epithelial cells via the activation of TGF-β1 signaling-mediated protein kinase C) (He et al., 2010; Klos et al., 2014). The cells were then scratched by a sterile pipette tip, and images were captured under a light microscope (Leica Microsystems, Wetzlar, Germany).
2.9. RNA isolation, reverse transcription (RT) and real-time PCR
Total RNA was isolated from the cells using an RNeasy Mini Kit (Qiagen, Valencia, CA). Aliquots of total RNA were reverse transcribed into single-stranded cDNA using a PrimeScript™ RT-PCR Kit (Takara Bio, Shiga, Japan). Human sequence-specific
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primers for the SYBR Green assay were designed with Primer 3.0. Real-time PCR was performed in triplicate on a Thermal Cycler Dice® Real Time System (Takara Bio). Relative gene expression was analyzed by the comparative Ct method with GAPDH
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RNA as the endogenous control after confirming that the efficiencies of the target and the endogenous control amplifications were approximately equal. Results are presented as
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target gene expression normalized to GAPDH RNA.
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2.10. PrimerArray® assay for marker gene of ES cells in iPSCs
Total RNA was extracted from the induced iPSC colonies at 14 days with or without
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PI polyamide using an RNeasy Mini Kit (Qiagen). The aliquots of total RNA were reverse transcribed into single-stranded cDNA using a PrimeScript™ RT-PCR Kit
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(Takara Bio), and ES cells, germ layer/trophoblast, and ES cell differentiation marker
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gene mRNA levels were assessed by PrimerArray® Embryonic Stem Cells (Human) (Takara Bio), according to the manufacturer’s instructions (n=3). Real-time PCR was performed in triplicate on a Thermal Cycler Dice® Real Time System (Takara Bio). Results are presented as target gene expression normalized to GAPDH RNA.
2.11. Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and washed twice with ice-cold PBS, permeabilized for 10 min with PBS and 0.25% Triton-X 100 at room temperature and washed three times for 5 min with Tris Buffered Saline (TBS) (Sigma-Aldrich), blocked with 1% goat serum and 1% bovine serum albumin
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(BSA) in TBS containing 0.1% Tween 20 (TBS-T), and incubated overnight at 4oC with primary antibody, including rabbit anti-E-cadherin (1:50, Abcam, Cambridge, UK) and rabbit anti-Nanog (1:800), anti-Sox2 (1:800), anti-Oct4 (1:800), and mouse anti-SSEA4
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(1:500; all from Cell Signaling Technology, Danvers, MA). These primary antibodies were diluted in PBS containing 1% BSA and 0.25% Triton-X 100. After the samples were
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washed three times for 10 min with PBS, they were incubated with secondary antibody,
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Alexa 488 (or 594) goat anti-rabbit (or mouse) IgG antibody (1:1000, Life Technologies), in PBS for one hour at room temperature. After immunofluorescence staining, the cells
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were washed in PBS, and the nuclei were stained using 1 μg/ml DAPI in PBS for 10 min at room temperature. The cells were mounted in Fluoromount-G (Southern
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Biotechnology, Birmingham, AL), and coverslips were placed over the cells. Staining was visualized and photographed with an Axiovert 200 (Carl Zeiss) and FluoView
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FV10i confocal microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).
2.12. Western blotting
Cultured MCF10A cells were incubated with 100 nM PMA for 24 hours. Next, 1 M
TGF-β1 receptor inhibitor, SB431542 (Sigma-Aldrich), or 1
M PI polyamide was
applied for 48 hours. Cells were washed twice in ice-cold PBS. The cells were solubilized by RIPA Buffer (Nacalai Tesque), and cell lysates were homogenized by sonication using a Bioruptor UCD-250 (Cosmo Bio, Tokyo, Japan). The resultant supernatant was transferred to a fresh microcentrifuge tube. Protein content was assayed using a Pierce BSA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. After boiling at 70°C for 10 min, 25-µg protein samples
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were electrophoretically separated in NuPAGE® Novex 4-12% Bis-Tris gel (Life Technologies) and transferred to iBlot Gel Transfer Stacks PVDF Regular (Life Technologies). After transfer, blots were blocked in Blocking One (Nacalai Tesque) for
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one hour at room temperature. The primary antibodies, rabbit anti-E-cadherin (1:500, Abcam), anti-GAPDH (1:2500, Abcam), and mouse anti-Vimentin (1:200, Abcam), were
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diluted in Can Get Signal® immunoreactions Enhancer Solution 1 (Toyobo Life Science,
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Osaka, Japan), and blots were incubated in the solutions overnight at 4°C. After washing the membranes with TBS-T, they were incubated at room temperature for 1 h with
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secondary antibody, donkey anti-rabbit (or mouse) IgG, and horseradish peroxidase-linked species-specific whole antibody (1:10000, GE Healthcare, Little
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Chalfont, UK) diluted in TBS-T. The signals were detected with ECL Prime Western Blotting Detection Reagent (GE Healthcare) using an LAS-4000 Luminescent Image
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Analyzer (Fujifilm). The bands of gel images were quantified using TotalLab Quant
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software v13.2 (Nonlinear Dynamics Ltd., Newcastle, UK).
2.13. Statistical analysis
Values are reported as mean ± standard error of the mean (SEM). Statistical analysis
was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis to compare multiple conditions. P < 0.05 was considered statistically significant.
3. Results
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3.1. PI polyamide binding to double-stranded oligonucleotides and incorporation into nuclei in MCF10A cells
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We designed and synthesized a PI polyamide with a target of binding to fat-specific element 2 (FSE2) of the promoter region of the human TGF-β1 gene (Suppl. Fig. 1A).
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The chemical structure of the PI polyamide is shown in Supplemental Figure 1B. To
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determine whether the PI polyamides could bind to the target sequence DNA, the gel shift assay allowed the determination of binding affinity and specificity of the polyamides for
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double-stranded DNA. The PI polyamide designed specifically for the TGF-β1 promoter region bound to the FSE2 target double-stranded oligonucleotide, whereas the PI
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polyamide did not bind to non-target double-stranded DNA (Fig. 1A). Therefore, these results suggested that the PI polyamide can bind to the target sequence DNA.
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Next, we observed the distribution of the PI polyamide in the cells using
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FITC-labelled PI polyamide. When MCF10A cells were incubated with 1 μM FITC-labelled PI polyamide targeted to TGF-β1 for 72 hours, strong fluorescent signals were detected in the nuclei of most MCF10A cells (Fig. 1B). These findings indicated that the PI polyamides could bind the target DNA in the nuclei.
3.2. Effect of PI polyamide on expression of TGF-β1, E-cadherin, SNAI1, and fibronectin mRNA in MCF10A cells stimulated with PMA
MCF10A cells were incubated with PMA to induce EMT through TGF-β activation. The abundance of TGF-β1 mRNA was increased in MCF10A cells (Suppl. Fig. 2A), and the morphology of the cells changed from the epithelial form to the mesenchymal-like
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form (Suppl. Fig. 2A and B). A concentration of 1 μM PI polyamide decreased the PMA-increased abundance of TGF-β1 mRNA (Fig. 2A). A concentration of 100 nM PI polyamide decreased the PMA-increased abundance of SNAI1 mRNA (Fig. 2B).
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Concentrations of 10 nM to 1 μM PI polyamide increased the PMA-inhibited abundance of E-cadherin mRNA (Fig. 2C). Concentrations of 10 nM to 1 μM PI polyamide did not
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decrease the PMA-increased abundance of fibronectin mRNA (Fig. 2D). These findings
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suggested that the PI polyamide targeting TGF- 1 exerts inhibitory effects on the EMT
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process.
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3.3. Effects of PI polyamide on expression of E-cadherin and vimentin protein
To further confirm the effects of the PI polyamide targeting TGF- 1 on the
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expression of E-cadherin in MCF10A cells stimulated with PMA, immunocytochemistry
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was performed in MCF10A cells stimulated with 100 nM PMA in the presence or absence of PI polyamide for 48 hours. PMA obviously suppressed the staining of E-cadherin in MCF10A cells, whereas PI polyamide recovered the suppressed staining of E-cadherin in MCF10A cells (Fig. 3A). We performed western blot analysis for the expression of E-cadherin and vimentin proteins in cultured MCF10A cells incubated with PMA for 24 hours and subsequently incubated with SB431542 or PI polyamide for 48 hours (Fig. 3B). PMA completely suppressed the abundance of E-cadherin protein, whereas PI polyamide recovered the abundance of E-cadherin protein suppressed by PMA (Fig. 3B and C), and PI polyamide inhibited the PMA-increased abundance of vimentin protein (not statistically significant) (Fig. 3B and D).
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3.4. Effect of PI polyamide on cell proliferation and migration of MCF10A cells
To evaluate the effect of PI polyamide on the cell proliferation of MCF10A cells, cell
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proliferation activity was analyzed by WST-8 assay. PI polyamide had no significant effect on cell proliferation in MCF10A cells after 72 hours (Suppl. Fig. 3).
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The effect of PI polyamide on the EMT process was also assessed by cell migration
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with PMA. We performed a Scratch wound healing assay in cultured MCF10A cells stimulated with 100 nM PMA in the presence or absence of PI polyamide for 48 hours.
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The PI polyamide abolished cell migration with PMA in the MCF 10A cells (Fig. 4). These findings suggested that the PI polyamide did not affect cell proliferation but did
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inhibit the cell migration associated with the EMT process.
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3.5. Effect of PI polyamide on induction of iPSCs
Concerning the effects of PI polyamide targeting human TGF- 1 on the induction
efficiency of human iPSCs, at 14 days after transfection of Sox2, Oct4, Klf4, and c-Myc genes with Sendai virus into HDF cells, the number of ALP-positive colonies was increased in comparison to the non-treated group. PI polyamide increased the number of iPS colonies by 180%. Mismatch PI polyamide did not affect the number of iPS colonies (Fig. 5A and B). The human iPS-like morphology of the colonies was found to be Nanog positive by immunostaining (Fig. 5C). To assess the characteristics of the iPS colonies, we performed PrimerArray® assay for germ layer/trophoblast and ES cell differentiation markers on the iPS colonies with or without PI polyamide. Data obtained from the gene list are shown in Supplemental Table 1. Up-regulated genes (fold change > 2) were the
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CCL2 (chemokine [C-C motif] ligand 2) gene, with a fold change = 2.66, and the CCR7 (chemokine [C-C motif] receptor 7) gene, with a fold change = 2.07. The down-regulated gene (fold changes < 0.5) was NPPA (natriuretic peptide A), with a fold change = 0.29.
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iPS colonies both with and without PI by mRNA expression profiling presented similar gene expression patterns (Fig. 5D).
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We performed immunofluorescence staining of undifferentiated markers generating
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human iPSC-like colonies treated with PI polyamide targeting human TGF- 1. In 2 passages of these colonies after treatment with PI polyamide, immunostaining showed
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staining of undifferentiated markers Nanog, SSEA4, Sox2, and Oct4 proteins (Fig. 6). These results indicated that the PI polyamide increased the efficiency of iPSC generation
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4. Discussion
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iPSCs.
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with no adverse effects on the molecules involved in the process of reprogramming
Synthetic PI polyamides targeting gene promoters will have potential use as practical
medicines for the regulation of gene transcription because the polyamides have biochemical characteristics such as cell permeability and specific suppression of expression of the target gene to bind into the DNA sequence. We have already demonstrated that PI polyamide targeting TGF-β1 effectively attenuates progressive renal disease (Matsuda et al., 2011), stenosis of the carotid artery after angioplasty (Yao et al., 2009), alkali burn of the cornea (Chen et al., 2010), and hypertrophic scars (Washio et al., 2011). PI polyamides targeted to TGF-β1 may thus be a potent gene silencer at the transcription level. We also demonstrated that a PI polyamide targeted to the FSE2 binding site of human TGF-β1 promoter inhibited the PMA-stimulated expression of 17 Page 17 of 36
TGF-β1 mRNA in vascular smooth muscle cells (Lai et al., 2005). The EMT process is an essential part of pathogenesis in tumor progression and organ fibrotic diseases including liver cirrhosis and renal fibrosis (Zeisberg et al., 2003; Bates and Mercurio, 2005; Choi
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and Diehl, 2009). The EMT process has been reported to be mainly mediated by the induction of TGF-β1 accompanied by increases in SNAI1 and decreases in E-cadherin
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(Lamouille et al., 2014). Thus, it is thought that PI polyamides targeting the human
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TGF-β1 promoter modulate the EMT process by decreasing SNAI1 expression and increasing E-cadherin expression.
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In the present experiments, PI polyamide targeting human TGF-β1 significantly decreased the expression of SNAI1 mRNAs and inhibited vimentin protein as an EMT
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marker gene and significantly increased the expression of E-cadherin mRNA and protein. SNAI1 is a basic helix-loop-helix transcription factor that binds to specific cognate
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sequences, termed E-boxes, and represses the transcription of E-cadherin and other key
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epithelial regulators (Cano et al., 2000). E-cadherin is a transmembrane constituent of the intercellular adherens junctions responsible for maintaining epithelial cohesion and has also been linked to the control of the EMT and ES cell pluripotency (Cavallaro and Christofori, 2004; Soncin et al., 2009). Moreover, cell migration is the main characteristic of EMT obtained through the decreased activity of E-cadherin. (Boyer et al., 2000). We investigated the effects of PI polyamide targeting human TGF-β1 on the
induction of iPSCs through suppression of the EMT and enhancement of the MET processes. iPSCs are generated through multiple-steps mediated by reprogramming transcription factors, which progressively induce the expression of ES cell-like genes and suppress the somatic cell genetic program (Takahashi and Yamanaka, 2006; Brambrink et al., 2008). The mechanisms underlying the induction of iPSCs with Yamanaka
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transcription factors have not been identified. Recently, Wang et al. (2010) investigated the transcriptional roadmap to iPSCs in somatic cells by microarray analysis and found that MET is a pivotal process in the induction of iPSCs. In addition, Li et al. (2010)
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demonstrated that the MET process is required to generate iPSCs from mouse fibroblasts, which is organized by suppressing molecules related to EMT. PI polyamide targeted to
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TGF-β1 increased the number of ALP-positive colonies by 180%. We confirmed the
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expression of undifferentiated markers in these colonies. Previous studies have
demonstrated the greater efficiency of iPSC generation using chemical compounds or
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micro RNAs that inhibit TGF-β signaling (Hou et al., 2013; Li and Rana, 2012; Li et al., 2009; Li et al., 2011). Keratinocytes can be reprogrammed with higher efficiency because
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of it reside within epithelium properties (Maherali et al., 2008), and E-cadherin improves reprogramming efficiency. Moreover, it has been established that the process of
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reprogramming efficiency of ES cells is associated with the higher expression of
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E-cadherin (Li et al., 2010; Huangfu et al., 2008; Shi et al., 2008). We found that PI polyamide targeted to TGF-β1 efficiently enhanced the induction of iPSCs by increasing the endogenous E-cadherin through the activity of MET in the present experiments. The PrimerArray® assay showed that two molecules, chemokine the CCL2 and
CCR7 genes, were up-regulated (respective fold changes = 2.66, 2.07) in iPSC colonies with PI polyamide compared to those without PI polyamide. Hasegawa et al. (2011) reported that CCL2 enhanced pluripotency in the induction of iPSCs via mediation by activating the Stat3 pathway and Klf4 up-regulation. Our results demonstrated that PI polyamide targeted to TGF-β1 enhanced efficiency of generated iPSCs, and it is thought that one of the reasons the PI polyamide increase expression of CCL2 and CCR7 in process of induced iPSCs.
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In conclusion, PI polyamide targeting the human TGF-β1 gene promoter region was found to regulate the EMT/MET process in MCF10A cells and increase the efficiency of iPSC induction in human fibroblasts. The results suggest that PI polyamide targeted to
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epithelial cells and enhance the iPSC induction of human fibroblasts.
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human TGF-β is a novel compound that can control the EMT/MET process of human
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Acknowledgements
We acknowledge support of this study by a financial grant from the “Strategic
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Research Base Development” Program for Private Universities subsidized by MEXT (S1101018), a Nihon University Multidisciplinary Research Grant in 2014 (14-024), and
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a Grant-in-Aid for Scientific Research (KAKENHI) in 2014 (26830134).
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REFERENCES Bando T, Narita A, Saito I, Sugiyama H. Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation. Chemistry 2002;8:4781–90.
cancer progression. Cancer Biol Ther 2005;4:365–70.
ip t
Bates RC, Mercurio AM. The epithelial-mesenchymal transition (EMT) and colorectal
cr
Boyer B, Vallés AM, Edme N. Induction and regulation of epithelial-mesenchymal
us
transitions. Biochem Pharmacol 2000;60:1091–9.
Brambrink T, Foreman R, Welstead GG, Lengner CJ, Wernig M, Suh H, et al. Sequential
an
expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2008;2:151–9.
M
Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing
d
E-cadherin expression. Nat Cell Biol 2000;2:76–83.
Ac ce pt e
Cavallaro U, Christofori G. Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 2004;4:118–32.
Chen M, Matsuda H, Wang L, Watanabe T, Kimura MT, Igarashi J, et al. Pretranscriptional regulation of TGF-β1 by PI polyamide prevents scarring and accelerates wound healing of the cornea after exposure to alkali. Mol Ther 2010;18:519–27.
Choi SS, Diehl AM. Epithelial-to-mesenchymal transitions in the liver. Hepatology 2009;50:2007–13. Gottesfeld JM, Neely L, Trauger JW, Baird EE, Dervan PB. Regulation of gene expression by small molecules. Nature 1997;387:202–5.
21 Page 21 of 36
Hasegawa Y, Takahashi N, Forrest AR, Shin JW, Kinoshita Y, Suzuki H, et al. CC chemokine ligand 2 and leukemia inhibitory factor cooperatively promote pluripotency in mouse induced pluripotent cells. Stem Cells 2011;29:1196–205.
ip t
He H, Davidson AJ, Wu D, Marshall FF, Chung LW, Zhau HE, et al. Phorbol ester phorbol-12-myristate-13-acetate induces epithelial to mesenchymal transition in
cr
human prostate cancer ARCaPE cells. Prostate 2010;70:1119–26.
us
Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 2013;341:651–4.
an
Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule
M
compounds. Nat Biotechnol 2008;26:795–7.
Imamura T, Hikita A, Inoue Y. The roles of TGF-β signaling in carcinogenesis and breast
d
cancer metastasis. Breast Cancer 2012;19:118–24.
Ac ce pt e
Klos KS, Warmka JK, Drachenberg DM, Chang L, Luxton GW, Leung CT, et al. Building bridges toward invasion: tumor promoter treatment induces a novel protein kinase C-dependent phenotype in MCF10A mammary cell acini. PLoS One 2014;9:e90722.
Lai YM, Fukuda N, Ueno T, Matsuda H, Saito S, Matsumoto K, et al. Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-β1 gene. J Pharmacol Exp Ther 2005;315:571–5.
Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial–mesenchymal transition. Nature Rev Mol Cell Biol 2014;15:178–96.
22 Page 22 of 36
Li R, Liang J, Ni S, Zhou T, Qing X, Li H, et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 2010;7:51–63.
ip t
Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, et al. A chemical platform for improved induction of human iPSCs. Nat Methods 2009;6:805–8.
cr
Li W, Wei W, Zhu S, Zhu J, Shi Y, Lin T, et al. Generation of rat and human induced
us
pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 2009;4:16–9.
an
Li Z, Rana TM. A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation. Nat Commun 2012;3:1085.
M
Li Z, Yang CS, Nakashima K, Rana TM. Small RNA-mediated regulation of iPS cell generation. EMBO J 2011;30:823–34.
d
Maherali N, Ahfeldt T, Rigamonti A, Utikal J, Cowan C, Hochedlinger K. A
Ac ce pt e
high-efficiency system for the generation and study of human induced pluripotent
stem cells. Cell Stem Cell 2008;3:340–5.
Matsuda H, Fukuda N, Ueno T, Katakawa M, Wang X, Watanabe T, et al. Transcriptional inhibition of progressive renal disease by gene silencing pyrrole-imidazole polyamide targeting of the transforming growth factor-β1 promoter. Kidney Int 2011;79:46–56.
Matsuda H, Fukuda N, Ueno T, Tahira Y, Ayame H, Zhang W, et al. Development of gene silencing pyrrole-imidazole polyamide targeting the TGF-β1 promoter for treatment of progressive renal diseases. J Am Soc Nephrol 2006;17: 422–32. Morrison CD, Parvani JG, Schiemann WP. The relevance of the TGF-β Paradox to EMT-MET programs. Cancer Lett 2013;341:30–40.
23 Page 23 of 36
Na YR, Seok SH, Kim DJ, Han JH, Kim TH, Jung H, et al. Bone morphogenetic protein 7 induces mesenchymal-to-epithelial transition in melanoma cells, leading to inhibition of metastasis. Cancer Sci 2009;100:2218–25.
ip t
Shi Y, Desponts C, Do JT, Hahm HS, Schöler HR, Ding S. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule
cr
compounds. Cell Stem Cell 2008;3:568–74.
us
Soncin F, Mohamet L, Eckardt D, Ritson S, Eastham AM, Bobola N, et al. Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in
an
reversible LIF-independent self-renewal. Stem Cells 2009;27:2069–80. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic
M
and adult fibroblast cultures by defined factors. Cell 2006;126:663–76. Trauger JW, Baird EE, Dervan PB. Recognition of DNA by designed ligands at
d
subnanomolar concentrations. Nature 1996;382:559–61.
Ac ce pt e
Wang Y, Mah N, Prigione A, Wolfrum K, Andrade-Navarro MA, Adjaye J. A transcriptional roadmap to the induction of pluripotency in somatic cells. Stem Cell Rev 2010;6:282–96.
Washio H, Fukuda N, Matsuda H, Nagase H, Watanabe T, Matsumoto Y, et al. Transcriptional inhibition of hypertrophic scars by a gene silencer, pyrrole-imidazole polyamide, targeting the TGF-β1 promoter. J Invest Dermatol 2011;131: 1987–95.
Wendt MK, Smith JA, Schiemann WP. Transforming growth factor-β-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression. Oncogene 2010;29:6485–98. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 2008;14:818–29.
24 Page 24 of 36
Yao D, Dai C, Peng S. Mechanism of the mesenchymal-epithelial transition and its relationship with metastatic tumor formation. Mol Cancer Res 2011;9:1608–20. Yao EH, Fukuda N, Ueno T, Matsuda H, Nagase H, Matsumoto Y, et al. A
ip t
pyrrole-imidazole polyamide targeting transforming growth factor-beta1 inhibits restenosis and preserves endothelialization in the injured artery. Cardiovasc Res
cr
2009;81:797–804.
us
Zavadil J, Böttinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005;24:5764–74.
an
Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, et al. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses
Ac ce pt e
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chronic renal injury. Nat Med 2003;9:964–8.
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Figure legends
Fig 1. Binding of pyrrole-imidazole (PI) polyamide to target DNA and distribution of PI
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polyamide in cells. (A) Binding of PI polyamide to the target sequence assessed by gel
transforming growth factor (TGF)-β1 promoter region DNA
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shift assay. Lane 1: Fluorescein-labeled FSE2 target double-stranded DNA on human
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(5’-GCAATTCTTACA-3’/5’-TGTAAGAATTGC-3’). Lane 2: Fluorescein-labeled FSE2 target double-stranded DNA on human TGF-β1 promoter region plus PI
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polyamide. Lane 3: Fluorescein-labeled non-target double-stranded DNA on human TGF-β1 promoter region number 1
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(5’-CTGACTCTCCTT-3’/5’-AAGGAGAGTCAG-3’). Lane 4: Fluorescein-labeled non-target double-stranded DNA on human TGF-β1 promoter region number 1 plus PI
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polyamide. Lane 5: Fluorescein-labeled non-target double-stranded DNA on TGF-β1
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promoter region number 2 (5’-CCTGTGTCTCAT-3’/5’-ATGAGACACAGG-3’). Lane 6: Fluorescein-labeled non-target double-stranded DNA on human TGF-β1 promoter region number 2 plus PI polyamide. (B) Distribution of FITC-labelled PI polyamide in MCF10A cells, which were incubated with 1 μM FITC-labelled PI polyamide for 72 hours. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI). Scale bars represent 50 μm.
Fig 2. Effects of pyrrole-imidazole (PI) polyamide targeted to human transforming growth factor (TGF)-β1 on the expression of TGF-β1, SNAI1, and E-cadherin mRNAs in MCF10A cells stimulated with PMA. (A) Expression levels of the mRNA encoding human TGF-β1 in MCF10A cells cultured with 100 nM PMA for 24 hours (white bar)
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and at the indicated concentrations with PI polyamide for 24 hours (black bars). (B) Expression levels of the mRNA encoding SNAI1, (C) E-cadherin, and (D) fibronectin in MCF10A cultured with 100 nM PMA for 24 hours (white bar in each panel) and at the
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indicated concentrations with PI polyamide for 48 hours (black bars in each panel). GAPDH mRNA was used to normalize the variability in template loading. Data are mean
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± SEM (n=3). *P < 0.05, **P < 0.01 in the indicated columns. PI: PI polyamide targeted
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to TGF- 1.
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Fig 3. Effects of PMA on expression of E-cadherin and vimentin protein. (A) Immunofluorescence staining of MCF10A cells stimulated with 100 nM PMA in the
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presence or absence of pyrrole-imidazole (PI) polyamide targeted to human transforming growth factor (TGF)-β1 for 48 hours. Nuclei were stained with DAPI. Scale bars
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represent 50 μm. (B) Western blotting analysis of E-cadherin and vimentin protein on
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cultured MCF10A cells stimulated with 100 nM PMA for 24 hours and next with PI polyamide for 48 hours. SB431542 was used as a positive control. GAPDH protein level was used to normalize the variability in template loading. These experiments were performed using anti-E-cadherin, GAPDH, and vimentin primary antibody. (C) Quantification of protein levels of E-cadherin, and (D) Vimentin. Data are mean ± SEM (n=3). *P < 0.05.
Fig 4. Effects of pyrrole-imidazole (PI) polyamide targeted to human transforming growth factor (TGF)- 1 on cell migration. MCF10A cells were incubated with 100 nM PMA in the presence or absence of PI polyamide targeting TGF- 1 for 48 hours.
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Control: Non-treated control group. Polyamide: PI polyamide targeting TGF- 1. Scale bars represent 200 μm.
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Fig 5. Effects of pyrrole-imidazole (PI) polyamide targeted to transforming growth factor (TGF)- 1 on the induction of induced pluripotent stem cells (iPSCs). (A) Representative
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images. (B) Evaluation of the number of ALP-positive colonies. (C)
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Immunofluorescence staining of the human iPS-like morphology of colonies using anti-Nanog primary antibody. (D) mRNA expression profiles of iPSCs colonies with or
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without PI by PrimerArray® assay for embryonic stem cells, germ layer/trophoblast, and embryonic stem cell differentiation marker gene expression. Control: Non-treated control
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group. PI: PI polyamide; Mismatch PI: Mismatch PI polyamide. Data are mean ± SEM
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(n=3). **P < 0.01. Scale bars represent 125 μm.
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Fig 6. Immunofluorescence staining of undifferentiated markers generating colonies with human iPS-like morphology after treatment with pyrrole-imidazole (PI) polyamide targeted to transforming growth factor (TGF)- 1. After 2 passages from generated colonies with human iPS-like morphology after treatment with PI polyamide, it was performed immunofluorescence staining using anti-Nanog, SSEA4, Sox2, and Oct4 primary antibody. Nuclei were stained with DAPI. Scale bars represent 50 μm.
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Fig. 3A
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Fig. 3B, 3C and 3D
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Fig. 4
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Fig. 5A, 5B and 5C
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Fig. 5D
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Fig. 6
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