Figure 3

Cisplatin inhibition of cell growth and increase of apoptosis and DNA strand breaks is augmented by amino acid deprivation. (A–C) Dih10 cells were cultured in the presence of the indicated concentrations of cisplatin (CDDP), either in complete (full) DMEM (black circles) or in the same medium containing 75 µM lysine (A), 125 µM arginine (B), or 150 µM leucine (C) (grey squares). The lysine, arginine, and leucine concentrations in DMEM are 798, 398, and 801 µM, respectively. Cell number was counted after 72 h, and the data are plotted as percent inhibition compared to untreated control cells in full medium. The cisplatin concentration that yielded 50% inhibition is shown as the IC₅₀. Each symbol is the mean of three independent experiments performed in duplicate, with error bars representing the standard deviation of the three experiments. Amino acid deprivation caused an upward shift of the inhibition curve, indicating a greater degree of inhibition. (D,E) Dih10 cells (D) and IGROV-1 cells (E) were plated at a low density, and cultured in either complete medium (control), medium containing 20 µM lysine (low Lys), complete medium containing 3 µM cisplatin (CDDP), or medium containing 20 µM lysine and 3 µM cisplatin (low Lys/CDDP). Two weeks later, they were fixed, stained, and photographed. The numbers below the wells are the optical absorption after dissolving the stain in acid, and are the mean ± SD of three independent experiments derived from duplicate wells. (F–I) Dih10 cells were cultured for 6 h in complete DMEM (control) in the presence of 100 µM cisplatin (G,I) or the indicated cisplatin concentration (F,H). As noted, some cells were cultured in lysine-deficient medium (Lys-). (F,G) Cells were extracted, and proteins were analyzed by SDS-PAGE/immunoblotting using antibodies against actin and cleaved caspase-3. (F) Protein bands were scanned on a Li-Cor Odyssey instrument, and the sum of the density of the 17 and 19 kDa cleaved caspase-3 bands was normalized to the respective actin bands. Each symbol is from an independent experiment, with the bar height representing the mean value of all experiments and the error bars indicating the standard deviation. (G) A full blot is shown. (H) The medium and cells were harvested separately, and lactate dehydrogenase (LDH) activity in the medium and cell extracts was measured in a coupled spectrophotometric assay. Data are presented as the percent of LDH activity in the medium. (I) Cells were stained using an anti-ɣ-H2A.X antibody and a Texas Red-conjugated secondary antibody, and nuclei were counterstained with DAPI. The scale bar is 50 µm and magnification was 20 × . The number of ɣ-H2A.X-positive foci per cell were counted in 50 cells per condition in four independent experiments, yielding a total of 200 cells counted per condition. The numbers shown are the mean ± SD of the number of foci per cell. *, **, ***, and **** indicate p < 0.05, < 0.01, < 0.001, and < 0.0001, respectively, for the indicated comparisons or, in Panels D and E, for comparison to the control. # and ## in Panels D and E indicate p < 0.05 and < 0.01, respectively, for comparison to the individual conditions of cisplatin treatment or lysine deprivation. CDDP, cisplatin.