Erlotinib induces caspase-independent mitochondrial outer membrane permeabilization (MOMP) and disruption of the JAK2/STAT-5 pathway. (A) Representative fluorescence microphotographs of KG-1 cells treated with 10 μM erlotinib or DMSO as a control, after staining for the visualization of nuclear chromatin, cytochrome c, endonuclease G, and activated caspase-3 (caspase 3a). Note the mitochondrial distribution of cytochrome c and endonuclease G as cytoplasmic (nonnuclear) dots at the same location as the mitochondrial matrix marker Hsp60 or their diffuse distribution throughout the cell. (B) Quantitative assessment of the data obtained as in panel A, for KG-1 cells cultured for 3 days in the absence or presence of erlotinib and/or the pan-caspase inhibitor Z-VAD-fmk. (C,D) Immunofluorescence assessment of MOMP and caspase-3 activation in erlotinib-treated P39 cells. The same technology as in panels A and B was used on P39 cells. Results are means plus or minus SD of triplicates of 1 experiment representative of 3. (E) The pan-caspase inhibitor Z-VAD-fmk blocks pyknosis and karyorrhexis in erlotinib-treated KG-1 cells. Cells were treated as described in Figure 4A,B. (F-I) Erlotinib disrupts signaling of the JAK2/STAT-5 pathway. (F) Erlotinib decreases phosphorylation of JAK2 and STAT-5. KG-1 cells treated for 30 minutes or 3 hours with erlotinib were subjected for immunoblot detection of JAK2 and STAT-5 phosphorylation. (G,H) Impact of erlotinib and JAK2 on STAT-5 phosphorylation. After depletion of JAK2 with 2 distinct siRNAs (see panel H inset) and 24 hours of erlotinib treatment (10 μM), cells were permeabilized and subjected to the immunofluorometric quantitation of STAT-5 phosphorylation. Representative FACS histograms obtained for the first of 2 control and JAK2-specific siRNAs are shown in panel G (gray curves indicate isotype controls), and quantitative data (expressed as percentage of positive cells, X ± SD, n = 3) are depicted in panel H. Note the decrease in STAT-5 activation (shift toward the isotype) upon erlotinib treatment alone and upon down-regulation of JAK2. (I) Impact of erlotinib and JAK2 on cell death. KG-1 cells were transfected with control siRNAs or JAK2-specific siRNAs (day 0) and then cultured in the absence or presence of 10 μM erlotinib (from day 1–2 of transfection), followed by determination of the frequency (X ± SD, n = 3) of dying and dead cells using the annexin V/PI staining method used in Figure 1B. This experiment has been repeated twice, yielding similar results.