Cell line and culture conditions
The HCT 116 cell line was acquired from ATCC (American Type Cell Culture, Manassas, VA). The HCT 116 was created by the integration of a pEGFP-N3 vector (Clontech, Palo Alto, CA) containing a mutated eGFP gene, as described by Hu et al. [11]. A nonsense mutation at position +67 results in nonfunctional eGFP protein; the oligonucleotide directs the conversion of the stop codon to a tyrosine (wild-type eGFP), allowing the expression of functional eGFP. HCT 116 cells were grown in McCoy's 5A Modified medium (Sigma-Aldrich, St. Louis, MO) and 10% fetal bovine serum. The mutant eGFP gene target was integrated into these cells and a clonal line containing a single copy was isolated by neo-selection (Geneticin, GIBCO, Invitrogen, Carlsbad, CA). DLD-1 integrated cell line was created and transfected as described by Hu et al. [11].
Selenomethionine treatment
Cells were seeded at a density of 1.5 × 106 in a 100 mm dish in complete medium supplemented with 10% FBS and desired concentration of selenomethionine (Sigma-Aldrich, St. Louis, MO) 24 h prior to electroporation of the oligonucleotide. All cells were washed twice in 1X PBS (GIBCO, Invitrogen, Carlsbad, CA) immediately prior to harvesting for electroporation.
MTT viability assay
Sensitivity to selenomethionine was analyzed by a 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO) viability assay. Cells were plated in a 24-well plate at a density of 1 × 105 cells per well using complete medium supplemented with 10% FBS and selenomethionine at concentrations of 50, 200, 400, and 1000 μM, respectively. After 24 hours of treatment, cells were washed in PBS and 500 μl (5 mg/ml) MTT in RPMI was added to each well and incubated for 2 hours at 37°C, in the dark. This assay is based on a reaction in which MTT is reduced and converted into purple formazan only by living cells. After the incubation period, the media containing MTT was removed, cells were washed in PBS, and 300 ml DMSO (Fisher Scientific, Pittsburgh, PA) was added to each well followed by incubation for 1 hour at room temperature, on an orbiting shaker protected from light. Samples were then transferred to 96-well plate and absorbance at 570 nm was determined using a Wallac 1420 Victor3V micro-plate reader (PerkinElmer, Shelton, CT). Each data point represents three (± S.D.) independent results; all data points were normalized to un-treated cells.
eGFP gene targeting
Cells grown in complete medium supplemented with 10% FBS and experimental treatment where necessary were trypsinized and harvested by centrifugation. 2 × 106 cells were resuspended in 100 μl serum-free medium and transferred to a 4 mm gap cuvette (Fisher Scientific, Pittsburgh, PA). The oligonucleotide was added to a final concentration of 8 μM and the cells were electroporated (250 V, 13 ms, 2 pulses, 1s interval) using a BTX Electro Square Porator™ ECM 830 (BTX Instrument Division, Holliston, MA). The electroporated cells were then transferred to a 60 mm dish, recovered in complete medium supplemented with 10% FBS, and incubated at 37°C for 24 h prior to FACS analysis.
Flow cytometry analysis
eGFP fluorescence was measured by a Becton Dickinson FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Forty-Eight hours after electroporation, the cells were harvested and resuspended in FACS buffer (0.5% BSA, 2 mM EDTA, 2 μg/ml propidium iodide in PBS) and immediately processed. To analyze cell cycle, cells were seeded at a density of 1.5 × 106 in a 100 mm dish in complete medium supplemented with 10% FBS and the desired concentration of selenomethionine. After 24 h cells were trypsinized, resuspended in 300 μl cold PBS and fixed by addition of 700 μl cold 95% ethanol. Cells were incubated a 4°C for 16 hours, subsequently washed and resuspended in 500 μl of PBS containing 50 μg/ml propidium iodide and analyzed by FACScalibur for DNA content.
Pulsed-field gel electrophoresis (PFGE)
Cells were seeded at a density of 1.5 × 106 in a 100 mm dish in complete medium supplemented with 10% FBS and selenomethionine at concentrations of 0, 100, 200, and 400 μM respectively, or MMS at 150 μM to serve as a positive control. After 24 h, the cells were trypsinized and 1 × 106 cells were melted in 1% low melt agarose (GIBCO, Invitrogen, Carlsbad, CA) in 50 mM EDTA. These agarose inserts were incubated in 50 mM EDTA, 1% N-laurosylsarcosine, 1 mg/ml proteinase K at 50°C, shaking, for 48 hours and subsequently washed four times in 1X TE buffer before loading onto a 1% pulsed field certified agarose gel (Bio-Rad, Hercules, CA). Samples were separated by electrophoresis for 24 h using a 120° field angle, 60 to 240s switch time, 4 V/cm (Bio-Rad, Hercules, CA), and visualized by ethidium bromide staining on an AlphaImager 2200 (Alpha Innotech Corp., San Leandro, CA).
Western blot analyses
Cell extracts were prepared 24 h after the plasmids were introduced to the cells by electroporation. The cells were first washed with PBS and then suspended in lysis buffer (25 mM Tris, pH 7.5, 5 mM EDTA, 600 mM NaCl, 1 mM DTT, 0.1% NP40, 5 μg/ml leupeptin, 2 μM pepstatin, 1 mM PMSF, 10% glycerol) and incubated for 30 min followed by centrifugation at 15,000 × g for 30 min. The supernatant was analyzed for protein concentration using the BioRad Protein Assay. Proteins were separated on a 10% SDS-PAGE gel and electroblotted to a nitrocellulose membrane (BioRad, Richmond, CA). The membrane was incubated with α-p53 (DO-1), a mouse monoclonal antibody against total (wild-type and mutant) human p53 (Santa Cruz Inc., Santa Cruz, CA) at a 1:200 dilution. The secondary antibody, a bovine anti-mouse horse radish peroxidase conjugated-antibody (Santa Cruz Inc., Santa Cruz, CA) was used to visualize the primary reaction at a 1:5000 dilution, followed by the addition of Chemiluminescence ECL™ Reagents (Amersham, Piscataway, NJ) and hyperfilm (Kodak). Activated p53 was detected by α-p53 Ser15 (PC386, Calbiochem, San Diego, CA) at a 1:1000 dilution, visualized by a goat α-rabbit secondary at a 1:2000 dilution. Ref-1 protein was detected using the Ref-1 (C-20): sc-334 affinity purified rabbit polyclonal antibody (Santa Cruz Inc., Santa Cruz, CA) at a 1:400 dilution; visualized by a goat α-rabbit secondary at a 1:2000 dilution. Actin expression was used as an internal control to normalize for protein levels; a goat β-actin antibody (Santa Cruz Inc., Santa Cruz, CA) in a 1:500 dilution, visualized by anti-goat IgG-HRP (Sigma, St. Louis, MI) with the Chemiluminescence ECL™ Reagents.
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