Journal: Cell Death & Disease
Article Title: ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors
Figure Lengend Snippet: Inhibition of NF κ B and concomitant activation of JNK during genotoxic stress are critical for inducing ‘resistance to apoptosis' switchover in drug-resistant cancer cells. ( a ) Western blotting and confocal imaging experiments were conducted to determine nuclear translocation of p65NF κ B in cisplatin-treated A549 and MCF-7 cells ( b ) Cisplatin-treated, p65NF κ B-over-expressed MCF-7 cells and I κ B α -SR-constituted A549 cells were subjected to flow cytometric determination of apoptosis (left panel). The effectiveness of transfecting I κ B α -SR-cDNA/p65NF κ B-cDNA, respectively, to A549/MCF-7 cells was determined by western blot analysis (upper right panel). That I κ B α -SR overexpression in A549 cells nullified cisplatin-induced p65NF κ B nuclear translocation was confirmed by western blotting (lower right panel). ( c ) The role of IKK α / β / γ in chemoresistance was evaluated by transfecting A549 cells with dominant negative (DN) clones of IKK α / β / γ followed by cisplatin treatment and subsequent flow cytometric determination of apoptosis. The efficacy of IKK α / β / γ DN transfections (upper panel; inset) as well as expression of I κ B α and nuclear p65NF κ B in un-transfected/transfected/cisplatin-treated A549 cells (lower left panel) was determined by western blotting. Simultaneously co-immunoprecipitation experiment was employed to study the IKK γ ubiquitination (Ub-IKK γ ) in cisplatin-treated cells, and phospho-IKK β (p-IKK β ) expression was determined by western blot analysis in cisplatin-treated cells (lower right panel). ( d ) Un-transfected/p38MAPK/JNK-siRNA-transfected or SB203580/SP600125-pre-treated A549 cells were subjected to western blot analysis for determination of the levels of p38MAPK, p-p38MAPK, JNK and p-JNK in the presence or absence of cisplatin (upper left panel). In parallel, percent cell death was determined by Annexin-V/7-AAD-positivity (upper right panel). The effectiveness of pharmacological and genetic interventions of p38MAPK and JNK was demonstrated both by western blot analysis and RT-PCR (lower panel). α -Actin, histone H1 and GAPDH were used as internal controls. Values are mean±S.E.M. of five independent experiments in each case. * P
Article Snippet: To evaluate the involvement of p38 mitogen-activated protein kinase (p38MAPK), JNK and ATM, cells were pre-treated with a specific p38MAPK inhibitor (SB203580; 10 μ M, Calbiochem, Gibbstown, NJ, USA), JNK inhibitor (SP600125; 10 μ M, Calbiochem) 90 min before treatment and with an ATM inhibitor (Wortmanin; 20 μ M and KU55933; 10 μ M, Calbiochem) 30 min before drug treatment.
Techniques: Inhibition, Activation Assay, Western Blot, Imaging, Translocation Assay, Flow Cytometry, Over Expression, Dominant Negative Mutation, Transfection, Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction