Figure 1.
Effects of K2CrO4 on a bronchoalveolar carcinoma isogenic H358 cell line system. (A) Clonogenic assay. Cells were plated at 1,000 cells/well in 60-mm dishes in complete medium for 24 h, then rinsed twice with salts-glucose medium (SGM: 50 mM Hepes [pH 7.2], 10 mM NaCl, 5 mM KCl, 2 mM Cacl2, 5 mM glucose), and treated for 2 h with different concentrations of K2CrO4 diluted in SGM medium. After drug removal (three washes in SGM), cells were cultured in complete medium. Colonies were scored after 10 days. Data (median ± SE) are representative of three replicate experiments yielding similar results. (B) DNA-SSB induction. H358 p53−/− and H358p3+/+ cells (2.5 × 106 cells/150 cm2) were labeled for 24 h with [3H]thymidine (0.2 μCi/ml) (New England Nuclear, Boston, MA) and, after 4 h incubation in a label-free medium, treated with 200 μM of K2CrO4 in SGM. Samples were withdrawn immediately after damage induction and analyzed by the alkaline filter elution assay under deproteinizing conditions (proteinase K 0.5 mg/ml). Data (median ± SE) are representative of three replicate experiments yielding similar results. (C) Effects of p53 or p21waf1 protein levels. Cells were treated with 200 μM of K2CrO4 in SGM for 2 h, washed, incubated in drug-free medium for a range of times, and then collected. Equal amounts of total protein were resolved on 12% SDS-PAGE gels, transferred onto nitrocellulose membranes (ECL; Amersham Biosciences, Piscataway, NJ), and immunoblotted. Blots were developed with ECL chemiluminescence (Amersham) in this and in all Western blotting experiments. Lanes: 1: control; 2: 1 h; 3: 2 h; 4: 4 h of recovery. Data are representative of three replicate experiments yielding similar results.