Development of R2d and R2N1d cells
Previously, we have reported the development of immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a human breast epithelial cell type with stem cell characteristics after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of C-erbB2/neu oncogene [25, 28]. These M13SV1R2 cells lost their tumorigenicity concomitant with the expression of two tumor suppressor genes, maspin and alpha-6 integrin, after culturing in a growth factor/hormone-deprived medium for >10 passages (referred to as R2d) . In this study, M13SV1R2N1 cells were cultured in the MSU-1 medium supplemented with growth factors/hormones and 5% fetal bovine serum (FBS) . After one week culture in this medium, M13SV1R2N1 cells were subcultured in the basic MSU-1 medium with 5% FBS without other growth factors/hormones and passaged more than 10 times (referred to as R2N1d cells) (Figure 1A).
Immunocytochemical analysis of gene expression
For immunostaining, cells were fixed by 4% paraformaldehyde in phosphate buffered saline (PBS). After rinsing with PBS, the cells were permeabilized (0.5% triton X-100) for 10 min. These cells were then incubated with primary antibodies (ant-HER2 or anti-ER-α) at 25℃ overnight. The following day, these cells were incubated with a secondary antibody conjugated with fluorescein isothiocyanate (FITC) (50 μg/ml, Sigma, USA) for 1 hr at 25℃. For nuclear staining, the cells were washed with PBS before incubation with 4', 6 diamidino-2-phenylindole (DAPI, Sigma, USA) (1 μg/ml in PBS) for 5 min.
Flow cytometric analysis of gene expression
Following trypsinization, cells were strained through a 40 μM nylon mesh to ensure the obtaining of single cells and suspended in ice-cold solution for a density of 1 × 106 cells/ml. Antibodies (Notch-4, Msi1, Notch-1, CK-18, CK-19, Oct-4, HDAC6, COX2 and HER2; CD24 conjugated with FITC; CD44 conjugated with phycoerythrin, PE) were added to the cell suspension at concentrations suggested by the manufacturer and cells were incubated at 4°C in the dark for 45 min. Then the cells were incubated with a secondary antibody conjugated with FITC or PE for 1 hr at 4°C. These labeled cells were washed twice, suspended in PBS and analyzed using a flow cytometer (FACS Calibur, Becton Dickinson). As negative controls, cells were stained with either isotype-matched control antibodies or with no primary antibody. No difference was observed between these two controls.
The proteins were extracted with 20% SDS lysis solution containing several protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM antipain, 0.1 mM aprotinin, 0.1 mM sodium orthovanadate, 5 mM sodium fluoride). Protein concentrations were measured using Biorad Protein Quantification kit (Biorad, CA, USA). Equal amounts of protein (15 μg/lane) were separated by 12% SDS-PAGE and transferred from the gel to PVDF membranes (Millipore Corp, Bedford, MA). Immunoblotting was carried out using monoclonal antibody (anti-COX-2, anti-COX-1, anti-AKT1, anti-HER2, anti-Oct4, anti-HDAC6, anti-alpha-tubulin and anti-β-Actin). This was then followed by incubation with horseradish peroxidase-conjugated secondary antibody and detected with the ECL chemiluminescent detection reagent (Amersham Co., IL, USA). The membranes were exposed to X-ray film for 15 s to 3 min.
Reverse transcription-polymerase chain reaction (RT-PCR)
5 μg of total RNA extracted from cells were used to synthesize the first-strand cDNA, using the Reverse Transcription System (Promega, A3500) according to the manufacturer's protocol. PCR amplification was carried out by using 1 μL of the first-strand cDNA as a template in a total volume of 15 μL containing 1 μL of each primer (10 pmol/L) and 7.5 μL of EconoTaq ® PLUS GREEN Master Mix Kit (Lucigen, F93481-1). The primers used are as listed for HER2 (forward, 5'-CCCGAAACGTGCTAGTCAAGAG-3'; reverse, 5'-TGCAGATTGGAGGCTGAG GTAG-3') and HDAC6 (forward, 5'-CCAGCTAACCCACCTGCTCATG-3'; reverse, 5'-GGGCTTCCAGAGCACAGGAAAC-3'). Following 1 minute denaturation at 95°C, the reactions were cycled 30 times with 45 seconds denaturation at 95°C and 30 seconds annealing at 55°C and then extension at 72°C for 1 min. The reactions were performed in the DNA Thermal Cycler 480 (Takara). The last polymerization step was at 72°C for 10 min.
Cells were inoculated into 24-well Matrigel™ Invasion inserts (2.5 × 105 cells/well) (8 μm pore size) (BD Biosciences, USA). Inserts were placed into Falcon companion plates and incubated for 24 hr for invasion. Following incubation, media plus cells were removed from the top chamber using cotton swabs and PBS. The number of cells invading to the underside of the membrane was determined. The data are presented as the average number of invading cells per well in triplicate.
Tumorigenicity in SCID mice
Female immune-deficient (SCID) mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl, 4 to 6-week-old) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Different numbers of R2d, R2N1d, non-adherent R2N1d and re-attached R2N1d cells (1 × 105, n = 6; 1 × 106, n = 6; 1 × 107, n = 6), were inoculated subcutaneously into female immune-deficient (SCID) mice, 2 sites for each mouse. Tumors developed were dissected, measured and histologically examined.
Immunohistochemical study of gene expression in tumor tissues
Serially cut tumor sections (4 um thick) were processed and incubated with primary antibodies against Ki-67 (1:75), COX-2 (1:50) (Dako, Denmark), matrix metalloproteinase-9 (MMP-9) (1:75), (Neomarkers, USA), HER2 (Dako, Denmark) (1:75) and vascular endothelial growth factor (VEGF) (1:150) (Santa Cruz Biotechnology, USA) at room temperature for 1 hr. The sections were then incubated in 3, 3-diaminobenzidine solution for 5 min, followed by Mayer's haematoxylin counterstaining and mounting. Negative controls were treated with non-immune serum instead of primary antibody.
The classification and evaluation of the expression of pathological markers in tumor tissues were as described .
Results shown are representative of at least three separate experiments. The significance of difference between treatments was assessed by the Mann-Whitney test of nonparametric statistics and was carried out using SPSS for Windows 13.0 statistics program (SPSS Inc., Chicago, USA). The p value < 0.05 was considered to be significant. All statistical data are presented as mean ± SD.