TRAIL-induced apoptosis of authentic myeloma cells does not correlate with the procaspase-8/cFLIP ratio by Andrew Spencer, Sung-Lin Yeh, Karly Koutrevelis,

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TRAIL-induced apoptosis of authentic myeloma cells does not correlate with the procaspase-8/cFLIP ratio by Andrew Spencer, Sung-Lin Yeh, Karly Koutrevelis, and Cindy Baulch-Brown Blood Volume 100(8):3049-3050 October 15, 2002 ©2002 by American Society of Hematology

Immunoblot analysis of procaspase-8 activation in TRAIL-treated myeloma cells.The myeloma cells were incubated with 1 μg/mL leucine-zipper TRAIL (Immunex, Seattle, WA) and harvested at 30 and 60 minutes following incubation. Immunoblot analysis of procaspase-8 activation in TRAIL-treated myeloma cells.The myeloma cells were incubated with 1 μg/mL leucine-zipper TRAIL (Immunex, Seattle, WA) and harvested at 30 and 60 minutes following incubation. The proportion of cells undergoing apoptosis, shown by the percentage in parentheses at 60 minutes, was determined by annexin-V staining. Cytosolic protein fractions (25 μg) were separated on 4% to 16% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose, and probed with a caspase-8–specific goat polyclonal antibody C-20 (Santa Cruz, Santa Cruz, CA). Proteins were visualized by enhanced chemiluminescence (ECL). Membranes were stripped and reprobed with monoclonal antibody to α-tubulin (Sigma, St Louis, MO). The sensitive myeloma cell lines RPMI 8226, LP-1, and OPM-2 all show early cleavage of procaspase-8 with generation of the intermediate p43/41 product (*) and the active P18 form. Resistant cell lines NCI H929 and U266 demonstrate delayed procaspase-8 cleavage with minimal P18 generation. Andrew Spencer et al. Blood 2002;100:3049-3050 ©2002 by American Society of Hematology

Determination of cFLIP and procaspase-8 levels in untreated myeloma cells.Untreated myeloma cells were lysed in whole cell lysate buffer and 80 μg protein separated on a 12% SDS-PAGE gel. Determination of cFLIP and procaspase-8 levels in untreated myeloma cells.Untreated myeloma cells were lysed in whole cell lysate buffer and 80 μg protein separated on a 12% SDS-PAGE gel. Procaspase-8 was detected as described above. Membranes were then stripped and reprobed with the cFLIP monoclonal antibody G-11 (Santa Cruz). Tubulin detection was used to confirm equal protein loading. The procaspase-8/cFLIP ratios were calculated for each cell line densitometrically and do not correlate with the amount of TRAIL-induced apoptosis (r = 0.7,p = 0.19, Spearman rank correlation). Andrew Spencer et al. Blood 2002;100:3049-3050 ©2002 by American Society of Hematology