Chaetocin induces oxidative stress in A549 cells without substantial depletion of intracellular reduced glutathione levels, whereas the selective cytotoxicity of chaetocin in freshly collected myeloma cells appears to be attributable to their increased sensitivity to oxidative stressors. Treatment of A549 cells with 200 μM hydrogen peroxide (A; positive control) or 400 nM chaetocin (B) for 24 hours resulted in increased intracellular oxidative species as assessed by FACS analyses. Increased chaetocin-induced oxidative species are attenuated by cotreatment with 10 mM NAC (C). (D) Summary of results from FACS indicating changes of oxidative species induced in response to various treatments. Results shown are representative of 3 independent FACS experiments as described in “Patients, materials, and methods” (*P < .05, **P < .01). (E) Chaetocin alone does not appreciably alter intracellular concentrations of reduced glutathione. A549 cells were exposed to the indicated concentration of chaetocin for 24 hours, with addition of glutathione or diluent 30 minutes prior to chaetocin treatment. Glutathione levels were assessed by spectrophotometric assay as described in “Patients, materials, and methods.” (F) Effects of chaetocin or hydrogen peroxide (in comparison to diluent) on ROS (superoxide) levels in U266 myeloma cells as assessed using FACS analyses as described in “Patients, materials, and methods.” (G) Relative intracellular chaetocin levels in patient CD138⁺ myeloma cells relative to that attained in matched normal patient CD138⁻ bone marrow cells. Cells were treated with 10 μM chaetocin for 20 minutes prior to assay; calculated intracellular chaetocin levels were measured by HPLC, with adjustments for differences in average cell volume (calculated from measured cell radii ascertained via light microscopy). Data points are plotted as •, whereas the shaded area represents a 1–standard deviation confidence interval, with the central bar reflecting the mean relative intracellular chaetocin level. (H) CD138⁺ patient myeloma cells are more sensitive to the cytotoxic effects of hydrogen peroxide than matched patient CD138⁻ bone marrow leukocytes. Cells from 4 myeloma patients were exposed to 200 μM hydrogen peroxide for 24 hours prior to assay, with trypan blue exclusion used to assess surviving cells. (D, F, H) Error bars indicate 1 sample standard deviation and results are replicated in triplicate.