Adaphostin-induced p53 up-regulation, DNA strand breaks, and ROS production in ML-1 cells is inhibited by NAC. (A) ML-1 cells were treated with 3 μM adaphostin for up to 6 hours. Whole-cell lysates were subjected to immunoblotting with antibodies that detect p53. (B) After ML-1 cells were treated with the indicated concentrations of adaphostin for 3 hours, whole-cell lysates were probed using an antibody recognizing phosphorylation on Ser15 (left) or Ser20 (right) of p53. In the indicated lanes, the proteasome inhibitor ALLnL was included at 50 μM to stabilize p53 and ensure that the increased signal for phospho-p53 was not simply due to increased amounts of p53. (C) After ML-1 cells were treated with varying concentrations of adaphostin for 3 hours in the presence and absence of 24 mM NAC, DNA single-strand breaks were analyzed by alkaline elution.²²  Elution of DNA through the filters was compared to elution after irradiation of ML-1 cells with 300 to 900 cGy ionizing radiation. (D) After a 15-minute pretreatment with 24 mM NAC (dotted line) or diluent, ML-1 cells were treated with diluent (solid line) or 3 μM adaphostin (heavy solid line or dotted line) for 90 minutes and stained with CM-H₂DCFDA to assess intracellular peroxides. (E) After treatment with the indicated adaphostin concentration in the presence of 0 (-), 500 (+), or 1000 (++) U/mL catalase, ML-1 cells were fixed, stained with PI, and examined for subdiploid cells by flow microfluorimetry. Error bars, ± 1 SD from 3 independent experiments. (F) ML-1 cells were pretreated with 24 mM NAC for 15 minutes prior to addition of 3 μM adaphostin for up to 8 hours. Whole-cell lysates were subjected to immunoblotting with antibodies that detect p53 or, as a loading control, actin. (G) After ML-1 cells were treated with varying adaphostin concentrations for 8 hours in the absence or presence of 24 mM NAC, cells were stained with PI and assayed for the presence of subdiploid cells by flow microfluorimetry. (H) Adaphostin was incubated for 1 hour at 37°C in RPMI medium in the absence (-) or presence (+) of 24 mM adaphostin and subjected to HPLC. The areas of the adaphostin peaks are indicated.