Cell culture and tumor tissues
The ERBB2-amplified human breast cancer cell line SKBr3, was purchased from American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 100 μg/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA).
Tumor tissue samples were from 45 patients submitted to routine histopathological examination at the Anatomic Pathology Unit of La Maddalena Hospital in Palermo. All experiments using human tissues were performed with the written patients’ informed consent and with the approval of Institutional Review Boards from La Maddalena Hospital.
Reporter and effector plasmid constructs
The construction of the effector plasmid pFlag-MBP-1 has been described previously . For the reporter constructs, the relevant regions of the ERBB2 promoter, including 44 base pairs (bp) of the first exon, were obtained by PCR amplification of genomic DNA from a human-mouse hybrid cell line containing only chromosome 17 . Three DNA fragments, spanning 306-, 558- and 787-bp, were amplified with primers containing restriction sites and cloned into the luciferace vector pGL3-basic (Promega, Madison, WI). In order to confirm the nucleotide sequence and the correct orientation of the cloned fragments, the three reporter plasmids, pG-E300, pG-E500 and pG-E700 were subjected to cycle-sequencing on an ABI 3130 genomic analyzer, according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA),
Cell transfection and luciferase reporter assay
SKBr3 cells were transfected with Lipofectamine LTX reagent in OptiMem medium as instructed by the manufacturer (Invitrogen). For RT-PCR, western blot and ChIP analyses 1.5x106 cells in 10 mm culture dishes were transfected with either the pFlag-MBP1 (3.5 or 7.5 μg) or pFlag-CMV plasmid (7.5 μg) and cell extracts were prepared 48 hrs after transfection. An aliquot of the transfected cells was routinely monitored for transfection efficiency by immunofluorescence assay and Western blot analysis with anti-Flag antibodies. Only samples yielding more than 70% transfected cells and lysates with no detectable Flag-MBP-1 breakdown products were used for further analysis.
For immunofluorescence assays, 1.5x105 SKBr3 cells were grown onto glass coverslips in 12-well culture plates for 24 hrs, then transfected with either 750 ng of pFLAG-MBP1 or pEGFPN1 plasmid (Clontech, Mountain View, CA), as described previously .
For reporter assays cells (6×105) were transfected with 750 ng of the pGL-cmp luciferase reporter construct and 250 ng of the β-galactosidase expressing vector pSVβ-gal (Promega, Madison, WI), the latter used as an internal control plasmid to monitor transfection efficiency. In cotransfection experiments with the pFLAG-MBP1 effector vector (1.25 μg), the total amount of DNA was kept constant by addition of the empty expression plasmid. Luciferase and beta-galactosidase activities were measured independently in duplicate using the Bright-Glo Luciferase Assay and Beta-Glo Assay Systems (Promega, Madison, WI) and a Turner 20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA). Luciferase activity was normalized with respect to beta-galactosidase activity. All transfections were performed in triplicate and results from three independent experiments are expressed as mean ± SD.
Total RNA isolation and quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacture’s instructions. RNA was reverse-transcribed with the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and cDNA amplified as described previously  using either c-MYC or ERBB2 specific primers (Qiagen, Hilden, Germany) and Power SYBER Green PCR ready-mix in a 7300 thermal cycler (Applied Biosystems, Foster City, CA), primer sequences are listed in (Additional file 1: Table S1). PCR conditions were: denaturation at 95C° for 3 minutes, followed by 35 cycles at 95C° for 20 seconds, 60C° for 15 seconds, and 72C° for 15 seconds, and a final extension at 72°C for 7 minutes. Reaction specificity was controlled by post-amplification melting curve analysis and agarose gel electrophoresis of the amplified products. To correct for the experimental variations between samples, Ct value of TBP mRNA was determined in each PCR reaction using specific primers (Qiagen, Hilden, Germany). Data shown were generated from three independent experiments performed in triplicates and are expressed as mean ± SD. Comparison and statistical analysis were performed using Student t test.
Immunofluorescence and microscopy
SKBr3 breast cancer cells were seeded onto glass coverslips in a 12-well plate culture vessel, 48–72 hrs post-transfection cells were fixed with 3.7% paraformaldehyde in phosphate buffered saline (PBS) and then permeabilized with 0.3% Triton X-100 in PBS. To detect endogenous ErbB2 and ectopically expressed Flag-MBP-1 proteins cells were incubated with 1 ug/ml of mouse anti-ErbB2 (sc-80898, Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-Flag (F7425, Sigma Chemical Company, St Louis, MO) primary antibodies in PBS containing 0.2% Tween 20. AlexaFluor 488-conjugated goat anti-rabbit IgG and AlexaFluor 594-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) at a dilution of 1:600 were used as secondary antibodies. DNA was counterstained with 4′6-diamidino-2-phenylindole (DAPI) and the coverslips were mounted onto glass slides with Slowfade reagent (Invitrogen, Carlsbad, CA). Primary-antibody-omission demonstrated the specificity of the immunostaining. Immunofluorescence microscopy was performed with either a Leica DM-RA2 microscope, or a Leica TCS SP5 confocal laser-scanning microscope and confocal optical sections were created using Leica confocal software.
Immunoblotting and immunohistochemistry
Total cell lysates from transfected cells were prepared in RIPA buffer (50 mM TrispH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodiumdeoxycholate, 1 mM EDTA, 0.5 mM DTT) supplemented with protease and phosphatase inhibitors (Sigma Chemical Company, St Louis, MO). Frozen normal and tumor tissues were homogenized and lysates prepared as described previously . Protein concentrations of tissue and cell lysates were determined by the Bradford protein assay (BioRad, Hercules, CA). Samples (30–40 ug) were separated on 4-12% polyacrylamide gradient gels (Invitrogen, Carlsbad, CA), and transferred to PVDF membrane, according to the manufacturer’s instructions (Amersham Biosciences, Sweden). Membranes were probed with primary antibodies: rabbit anti-Flag (F7425, Sigma Chemical Company, St Louis, MO, dilution 1:200), rabbit anti-ErbB2, (18299-1-AP, Proteintech, dilution 1:100), mouse anti-Myc (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, dilution 1:200) rabbit anti-HDAC1 (ab7028, Abcam, Cambridge, UK, dilution 1:500) and horseradish peroxidase-conjugated secondary antibodies (Amersham Bioscience, Sweden). Membranes were additionally probed with mouse beta-actin antibody (AC-15, Sigma Chemical Company, St Louis, MO) as a loading control. Detection was performed with a chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) and signals were quantified by densitometric analysis employing the AlphaEasyFc software (Alpha Innotech Corporation, Johannesburg, South Africa).
Immunohistochemistry was performed on tissue serial sections of archived formalin-fixed, paraffin-embedded tissue blocks from patients as described previously , using primary antibodies against ErbB2 (4B5, Ventana Medical System, dilution 1:500), MBP-1/alpha-enolase (monoclonal antibodies ENO-19/8 and ENO-276/3, 1.0 ug/ml, ) and HDAC1 (ab7028, Abcam, dilution 1:1000). To confirm the specificity of immunoreactions, the primary antibody was either omitted or replaced by non-immune IgG. Tissue slides were evaluated blindly by two authors (ER and CL). The imunohistochemical grading scale used to evaluate the intensity and percentage of MBP-1-positive cells has been described previously . Tumors were graded as ErbB2-positive with a score of 3+ and negative with a score of 0 or 1+, according to common pathological guidelines. Tumors ErbB2-positive 2+ were further evaluated by in situ hybridization (FISH) with a dual-color probe (PathVysion ErbB2/CEP17; Vysis, Downers Grove, IL, USA), according to manufacturer’s instructions, and scored positive when ErbB2 gene amplification was found. Immunohistochemical score for HDAC1 expression in each tissue section was calculated as the percentage of positively stained cells on total cells.
Chromatin immunoprecipitation (ChIP) assay
In vivo MBP-1 and HDAC1 occupancy at the ERBB2 and c-MYC promoter was investigated using a ChIP assay kit (Upstate Biotech, Billerica, MA). Sheared chromatin samples from either pFlag-MBP1- or pFlag-CMV-transfected SKBr3 cells were separately immunoprecipitated with rabbit anti-Flag, anti-HDAC1 or anti-acetylated Histone H4 polyclonal antibodies (Upstate Biotech, Billerica, MA). The recovered DNA was analyzed by quantitative real-time PCR as described previously , using primers specific to either ERBB2 or c-MYC promoter, and to unrelated sequences as a negative control (Additional file 1: Table S1). A DNA sample representing 10% of the total input chromatin was also included as a positive control. The data shown are means ± standard deviations (SD) from three independent experiments performed in triplicates and are expressed as percentage of total input DNA.
Group comparison and statistical analyses were performed using the software tools in GraphPad Prism version 4.02 for Windows (GraphPad Software, Inc. La Jolla, CA, USA). All tests of statistical significance were two-tailed and p-values less than 0.05 were considered statistically significant.