In some experiments cells were preincubated with either 100 M of Z VAD FMK, 100 M of Ac VDVAD CHO, 100 M cathepsin B inhibitor, 150 M cathepsin L inhibitor, or 100 M cathepsin D inhibi tor for 1 h prior to exposure to the cytotoxic drugs. Cells were visualized on an Olympus IX70 microscope equipped with Hoffmann Modulation Contrast. Images were acquired using a digital Spot Imaging System. Cell Viability Assays Cells seeded in 96 well plates were treated with cytotoxic drugs in the presence and absence of inhibitors, washed with phosphate buffered saline, and fixed in 4% paraformaldehyde for 1 h at 4 C. Cells were then washed three times with distilled water, and Accustain Crystal Violet solution was added to each well followed by incubation for 20 minutes at room temperature.

Plates were washed with distilled water to remove excess dye and then dried at room temperature. Acetic acid was added to each well for 10 minutes and absorbance was measured at 570 nanometers using a Quant microplate reader. Cell viability was also determined using a modified 2,5 diphenyltetrazolium bromide assay. Briefly, cells were seeded in 96 well plates and then treated with cyto toxic drugs in the presence and absence of inhibitors. MTT was then added to each well and plates were incubated in a 5% CO2 incubator at 37 C for 1 h. Plates were centrifuged at 2,000 rpm for 30 minutes to avoid loss of floating cells. Supernatants were discarded and 150 l of dimethyl sulfoxide, were added to each well. Absorbance was measured at 450 nm. Caspase Activity Assays Caspase activity assays were performed as described previ ously.

Briefly, cells were seeded in black, clear bot tomed 96 well plates. At the conclusion of treatment with cytotoxic drugs in the pres ence and absence of inhibitors, cells were incubated with 50 l of 3�� caspase buffer, 30 mM dithiothreitol, 3 mM phenylmethanesulphonylflu oride, and 75 M of the fluorogenic peptide sub strates Ac DEVD AMC or Ac VDVAD AMC for 2 h at 37 C, followed by incubation at room temperature for 12 h. In these experiments, TRAIL actinomycin D treatment was used as a control for cas pase 3 7 activation, whereas STS was used as a control for caspase 2 activation. Absorbance was then Brefeldin_A read in a FLX800 Microplate Fluorescent Reader at excitation of 360 nm and emission of 460 nm. Fold activity was determined by normalizing to one the absorbance values for untreated cells.

Immunoblotting Equal amounts of protein from whole cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted onto polyvinyl dif luoride membranes, and analyzed by immunob lotting as described previously. Analysis of Mitochondrial Membrane Potential Cells were grown in 6 well plates. After treatment with DTX, 7. 5 l of 5,5,6,6 tetrachloro 1,1,3,3 tetraethyl benzimidazoly carbocyanine iodide were added per ml to each well, fol lowed by incubation at 37 C for 30 minutes.