PMC:101407 / 3210-8096 JSONTXT

{"project":"2_test","denotations":[{"id":"11914144-233137-8811532","span":{"begin":439,"end":441},"obj":"233137"}],"text":"Materials and methods\n\nChemicals\nHemin, ALA, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and L-glutamine were obtained from Sigma Chemical Co. (St. Louis, MO). D-Glucose, PBS, Dulbeco's Modified Eagles Medium (DMEM) and antibiotics were obtained from GIBCO BRL.\n\nCell lines and cell culture\nHEP G2 and HEP 3B cell lines (American Type Culture Collection, Rockville, MD) were derived from a human hepatocellular carcinoma [11-13]. HEP G2 contained wild-type p53 [14] and HEP 3B cells contained deleted p53. Cell lines were grown in total darkness, at 37°C in 5% CO2 atmosphere in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 10,000 U penicillin, 10,000 μg streptomycin and 2 mM L-glutamine.\n\nMTT assay\nAfter exposure to drugs, the viability was determined by the MTT assay following published procedures [15]. Briefly, 40,000–50,000 cells in 500 μl of medium were plated onto each well of 24-well tissue culture plate. After 24 h of culture, ALA (0.5–5 mM), Hemin (8–24 μg/ml) or D-glucose (2–3 mg/ml) dissolved in PBS immediately prior to use, were added to the medium. Cells were cultured for 24 h. After exposure, the medium was aspirated and replaced with 300 μl of fresh drug-free medium containing 0.5 mg/ml (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide) MTT. The cells were incubated for an additional 4 h. This medium containing MTT was removed by inverting the plate and 200 μl of dimethylsulphoxide (DMSO) were then added to each well. After 10 minutes of shaking, absorbance in each well was read in a microplate reader at 570 nm. All experiments included untreated control cells, they were repeated three times and each concentration was tested in sextuplicate. Viability was expressed in terms of population growth, as percent of an untreated control population with standard error of the mean (SEM).\n\nAnalysis of DNA fragmentation\nTreated cells were grown in 75 cm3 flasks. Both attached and unattached cells were harvested, washed with PBS, resuspended in TNE solution (10 mM Tris-HCI, pH 7.6; 140 mM sodium chloride; 1 mM EDTA), and lysed at 37°C in 2 ml of extraction buffer (10 mM Tris-HCI, pH8.0; 100 mM EDTA, pH 8.0; 20 μg/ml pancreatic RNase; 0.5% SDS). After 2 h, proteinase K was added at a final concentration of 100 μg/ml and the mixture was incubated for another 3 h at 50°C. The DNA was extracted twice with equal volumes of phenol and once with chloroform. The DNA was then precipitated with 0.1 vol of sodium acetate (pH 4.8) and 2.5 vol of ethanol at -20°C overnight and pelleted at 13,000 g for 30 min. Samples were electrophoresed in 2% (w/v) agarose gel and DNA was visualized by ethidium bromide staining.\n\nNorthern blot analysis\nTotal RNA (40 μg), from exponentially growing cells treated with different concentrations of ALA (0.5–5 mM) during 24 h, was prepared by Chomsinsky and Sacchi [16] method, size-fractionated on 1% agarose/5% formaldehyde gels, transferred onto nylon membrane Hybond-N+ (Amersham Pharmacia Biotech), and ultraviolet cross-linked. The blots were then prehybridized and hybridized with pTRI-p53-Human or pTRI-GAPDH-Mouse (Ambion) to 65°C with Ultrahyb solution (Ambion) and washed as manufacturer described. Blots were exposed to AGFA films at -70°C and developed. cDNA probes were labeled with [α-32P]ATP (3,000 Ci/mmol) using Kleanow (Biolabs) and random primers, for 2 hours at 37°C. Intensity of bands were analysed with ImageMaster (Amersham Pharmacia Biotech).\n\nWestern blot analysis\nExponentially growing cells (5 × 106/25 ml) were treated with different concentrations of ALA (0.5–5 mM). After 24 h of incubation, cells were washed with PBS and lysed in 500 μl of RIPA buffer (50 mM Tris pH 7.4; 150 mM NaCI; 20 mM EDTA pH 8; 0.1% SDS). After sonication (3 × 15 s) and 30 min incubation with PMSF (10 mg/ml) at 4°C, cell lysates were centrifuged at 10,000 g for 10 min at 4°C. Protein content of supernatants was determined by Bradford protein assay. Cell lysates containing equal amounts of protein (50 μg) were resolved on 12 % SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto nitrocellulose membranes Hybond ECL (Amersham Pharmacia Biotech) at 300 mA for 1 hour at 4°C using Towbin buffer (25 mM Tris; 192 mM glycine; 3.5 mM SDS; 10% Methanol; pH 8.3). Membranes were blocked by incubation in TBS (20 mM Tris; 137 mM NaCl; 0.05 % Tween 20; pH 7.6) containing 5% dry low fat milk for 1 hour at room temperature. Blots were probed with either anti-CDK2 (1:300), anti-CDK4 (1:300) or anti-actin (1:100) goat polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA). Immune complexes were detected using donkey anti goat secondary antibody (1:1,500) (Santa Cruz Biotech, Santa Cruz, CA) and were visualized using ECL reagents (Amersham Pharmacia). Intensity of bands was analyzed with Image Master (Amersham Pharmacia Biotech)."}