Human fibroblasts-derived BWS6 and BWS9 cells, which have been established from Beckwith-Wiedemann syndrome patients, were kindly provided by Dr M.J. Higgins (Roswell Park Cancer Institute), and Human Wilms’ tumor G401 cells were purchased from Human Science (Kyoto, Japan). Both BWS6 and BWS9 cells were maintained in DMEM (Nakarai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Nichirei Bioscience, Tokyo, Japan) and 100 IU/ml of penicillin/streptomycin/glutamine (PSG) (Life Technologies, Carlsbad, CA, USA). G401 cells were cultured in McCoy’s 5A (Life Technologies) supplemented with 10% heat-inactivated FBS and 100 IU/ml of PSG. Cells were grown at 37°C in a CO₂ incubator with a humidified atmosphere containing 5% CO₂ and 95% air.

PI polyamides used in this study were synthesized by a machine-assisted automatic synthesis system, PSSM-8 peptide synthesizer (Shimadzu, Kyoto, Japan) as previously described (23,24). Two PI polyamides termed PI-a and PI-b were designed to span the boundary of 2 of 4 CCAAT boxes within human KCNQ1OT1 promoter region (Fig. 1). Mismatch PI polyamide, which does not recognize CCAAT sequence, was also designed and produced. After synthesis and purification, these PI polyamides were dissolved in ultra pure water at a final concentration of 1 mM as stock solutions.

FITC-labeled oligonucleotides containing target sequences of PI polyamides were synthesized by Eurofin Genomics (Tokyo, Japan). Sequences of the oligonucleotides are as follows: the oligonucleotide containing PI-a target sequence, 5′-GAGTCTAACCGGGTCGCCCTTTTGGGCGAC CCGGTTAGACTC-3′ and the oligonucleotide containing PI-b target sequence, 5′-GGTGGTCGAACTAA CCCGCTTTTGCGGGTTAGTCGACCACC-3′. To generate hairpin structure of double-strand DNA, 1 μM of oligonucleotides dissolved in an annealing buffer (5 M NaCl, 500 mM EDTA, 1 M Tris-HCl pH 8.0) were boiled at 100°C for 10 min, and then left at room temperature to gradually cool them down. The resultant FITC-labeled hairpin oligonucleotides were incubated in the presence or absence of 1 or 3 μM of PI polyamides for 1 h at room temperature. The reaction mixtures were separated by electrophoresis through 20% polyacrylamide gel in Tris-borate-EDTA buffer and then visualized with the ImageQant LAS4000 (Fujifilm, Tokyo, Japan).

BWS6 and G401 cells were maintained in the presence or absence of FITC-labeled PI polyamides for 24 h. After washing in ice-cold phosphate-buffered saline (PBS, Nakarai Tesque), cells were fixed in 4% formaldehyde solution (Wako Pure Chemical Industries, Osaka, Japan) for 30 min, and then stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, St. Louis, MO, USA). Finally, cells were observed under fluorescence microscopy Axiovert 2000 (Carl Zeiss Microscopy, Oberkochen, Germany).

Seventy-two hours after incubation in the presence or absence of PI polyamides, cells were washed in ice-cold PBS and lysed in TRIzol reagent (Life Technologies). Then, total RNA was prepared according to the manufacturer’s instructions, and 500 ng of total RNA was treated with DNaseI (Qiagen, Hilden, Germany), followed by reverse transcription using Prime Script (Takara). The resultant cDNA was subjected to quantitative real-time RT-PCR analysis using Dimer Eraser (Takara). The primers used in this analysis are as follows: KCNQ1OT1 (forward: 5′-AATGGG GATGTGAGGATCAGG-3′, reverse: 5′-TGACCCCAGTG GAATATGTGC-3′); GAPDH (forward: 5′-GCACCGTCAA GGCTGAGAAC-3′, reverse: 5′-TGGTGAAGACGCCAGT GGA-3′). GAPDH was used as an internal control.

G401 cells were seeded at 2×10³ cells/96-well microplates. Then cells were exposed to the indicated concentrations of PI polyamides or left untreated. At the indicated time points after treatment, cell viability was measured by standard WST-8 assay (Nacalai tesque).

G401 cells were seeded at 5×10⁴ cells/2-cm diameter dishes. Then, cells were treated with or without the indicated PI polyamides. Seventy-two hours after treatment, cells were washed in ice-cold PBS, treated with trypsin/EDTA, and 2×10⁵ cells were subsequently stained with Annexin V-FITC plus propidium iodide using Annexin V-FITC Apoptosis Detection kit (Abcam, Bristol, UK) according to the manufacturer’s protocols. Finally, cells were sorted by FACSCalibur flow cytometer with FL-1 (Annexin V channels) and FL-2 (PI channels), and then analyzed by CellQuest (BD Biosciences, Rockville, MD, USA). As a positive control, cells were treated with 10 μM of H₂O₂ for 9 h to induce cell death.

Statistical significance was calculated using the Student’s t-test. Results were considered as significant with P-value of <0.05.

To verify whether the synthesized PI polyamides (termed PI-a and PI-b) could bind to their target DNA sequences, gel mobility shift assays were performed. For this purpose, the oligonucleotide containing target sequence of PI-a was incubated with DW, PI-a or non-specific PI polyamide (termed mismatch), and then reaction mixtures were analyzed by poly-acrylamide gel electrophoresis. As shown in Fig. 2A, a clearly retarded band was observed in the presence of PI-a but not of mismatch PI polyamide. Similarly, an oligonucleotide/PI-b complex was detectable in the reaction mixture containing PI-b and its target DNA sequence, whereas mismatch PI polyamide failed to bind to oligonucleotide containing the target DNA of PI-b (Fig. 2B). These results indicate that both PI-a and PI-b can specifically recognize and bind to their target DNA sequences in vitro.

Since the PI polyamides could be expected to exert gene silencing function through the direct interaction with their target DNA sequence in cells, it should be required to confirm their nuclear distribution. To adequately address this issue, we generated FITC-conjugated PI-a and PI-b. Human fibroblasts-derived from a BWS patient BWS6 cells and Wilms’ tumor-derived G401 cells were cultured in fresh medium containing PI-a or PI-b. Twenty-four hours after incubation, cells were fixed, stained with DAPI and observed under a confocal microscope. As seen in Fig. 3, a significant nuclear access of FITC-labeled PI-a and PI-b was detected under our experimental conditions. These observations strongly suggest that PI-a and PI-b display nuclear localization without any drug delivery systems such as adenovirus.

Next, we examined whether PI-a and/or PI-b could downregulate the transcription of their target gene KCNQ1OT1. To this end, BWS6, BWS9 and G401 cells were incubated in the presence of the indicated concentrations of PI-a, PI-b, PI-a plus PI-b or mismatch PI polyamide. Seventy-two hours after treatment, total RNA was prepared and the expression level of KCNQ1OT1 was analyzed by quantitative real-time PCR. Unexpectedly, a significant downregulation of KCNQ1OT1 was not detected in all cells examined after PI-a or PI-b single treatment (Fig. 4). Notably, the combinatory treatment of cells with PI-a and PI-b resulted in a marked decrease in the expression level of KCNQ1OT1. As expected, mismatch PI polyamide alone had no detectable effect on the expression level of KCNQ1OT1.

Since it has been described that a higher KCNQ1OT1 level is tightly associated with tumor cell growth (25), these observations prompted us to examine whether the simultaneous treatment of PI-a and PI-b could affect cell proliferation of G401 cells. For this purpose, G401 cells were exposed to the indicated concentrations of PI-a plus PI-b or mismatch PI polyamide. At the indicated time points after treatment, cells were subjected to the standard WST8 assay to assess their cell viability. As shown in Fig. 5, the combinatory treatment of PI-a and PI-b caused a remarkable suppression of G401 cell proliferation, whereas mismatch PI polyamide had a negligible effect on their viability. As expected, PI-a or PI-b single treatment did not affect G401 cell proliferation (data not shown). These results imply that the PI polyamide-mediated silencing of KCNQ1OT1 expression might contribute to the suppression of G401 cell proliferation.

Based on our above-mentioned results, we asked whether a combined treatment with PI-a plus PI-b in G401 cells could induce cell death. To this end, we used PI and Annexin V double staining experiments followed by FACS analysis. It has been established that Annexin V-positive cells undergo early apoptotic cell death (26). G401 cells were treated with the indicated concentrations of PI-a plus PI-b or with mismatch PI polyamide. As a positive control, G401 cells were exposed to H₂O₂ for 9 h. It is well-known that Annexin V-positive/ PI-negative cells and Annexin V-positive/PI-positive cells undergo early apoptosis and late apoptosis/necrosis, respectively (27). As seen in Fig. 6A, a large number of cells exposed to H₂O₂ displayed Annexin V-positive/PI-positive, indicating that cells undergo late apoptosis in response to H₂O₂. Apoptotic cells were barely detectable in untreated or mismatch-treated cells. In contrast, substantial amounts of cells were enriched in early and late apoptotic fractions. Average number of cells showing early apoptosis, late apoptosis or necrosis is depicted as a graph in Fig. 6B. According to our present results, the combinatory treatment of cells with PI-a and PI-b markedly induced apoptosis in a dose-dependent manner. In addition, the relatively small number of Annexin V-negative/PI-positive cells was observed in each experiment.