Figure 3
Figure 3. Coadministration of PD184352 and ATO activates the caspase cascade in MM. (A) HMCLs were exposed to 1 μM PD and/ or ATO 2 μM for 24 hours; cell lysates were analyzed by immunoblotting analysis using rabbit polyclonal anticleaved caspase-9, -8, -7, -3, Bid, and PARP, all provided by Cell Signaling Technology (CST, Danvers, MA). Blots were subsequently reprobed with goat polyclonal antiactin (sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA) to ensure equivalent loading and transfer of protein. (B) CD138-purified plasma cells from 3 representative patients with MM were seeded at 2.5 × 105 cells/mL in the presence of DMSO (vehicle) or PD (1 μM) for 3 hours and then incubated with ATO (2 μM) for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor the expression of cleaved caspase-9, -8, -3, PARP, and cleaved PARP. (C) HMCLs were cultured with PD and/or ATO for 24 hours, after which the percentage of apoptotic cells displaying loss of mitochondrial membrane potential (ΔΨm) was monitored by flow cytometry. Values represent mean plus or minus SD for 3 separate experiments performed in triplicate. (D) CD138-purified plasma cells from 2 representative patients with MM were cultured with PD and/or ATO for 24 hours, after which the percentage of apoptotic cells displaying loss of ΔΨm was monitored by flow cytometry. (E) HMCLs cells were treated with PD and/or ATO in the presence or absence of 30 μM of caspase inhibitors for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor the expression of caspase-9 and -8 using rabbit polyclonal anticaspase-9 and -8 (Cell Signaling Technology); blots were subsequently reprobed for actin expression to ensure equivalent loading and transfer of protein. (F) HMCLs were cultured with PD and/or ATO in the presence or absence of 30 μM caspase inhibitors for 48 hours, after which the percentage of apoptotic cells was determined by the annexin V method (**P < .001 vs PD/ATO Z-FA-FMK; Dunnett test). CTR indicates control; PD, PD184352 (1 μM); ATO, arsenic trioxide (2 μM); t-Bid, truncated Bid; Z-FA-FMK, peptide control; Z-LEHD-FMK, selective caspase-9 inhibitor; Z-IETD-FMK, selective caspase-8 inhibitor; Z-VAD-FMK, pancaspase inhibitor.

Coadministration of PD184352 and ATO activates the caspase cascade in MM. (A) HMCLs were exposed to 1 μM PD and/ or ATO 2 μM for 24 hours; cell lysates were analyzed by immunoblotting analysis using rabbit polyclonal anticleaved caspase-9, -8, -7, -3, Bid, and PARP, all provided by Cell Signaling Technology (CST, Danvers, MA). Blots were subsequently reprobed with goat polyclonal antiactin (sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA) to ensure equivalent loading and transfer of protein. (B) CD138-purified plasma cells from 3 representative patients with MM were seeded at 2.5 × 105 cells/mL in the presence of DMSO (vehicle) or PD (1 μM) for 3 hours and then incubated with ATO (2 μM) for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor the expression of cleaved caspase-9, -8, -3, PARP, and cleaved PARP. (C) HMCLs were cultured with PD and/or ATO for 24 hours, after which the percentage of apoptotic cells displaying loss of mitochondrial membrane potential (ΔΨm) was monitored by flow cytometry. Values represent mean plus or minus SD for 3 separate experiments performed in triplicate. (D) CD138-purified plasma cells from 2 representative patients with MM were cultured with PD and/or ATO for 24 hours, after which the percentage of apoptotic cells displaying loss of ΔΨm was monitored by flow cytometry. (E) HMCLs cells were treated with PD and/or ATO in the presence or absence of 30 μM of caspase inhibitors for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor the expression of caspase-9 and -8 using rabbit polyclonal anticaspase-9 and -8 (Cell Signaling Technology); blots were subsequently reprobed for actin expression to ensure equivalent loading and transfer of protein. (F) HMCLs were cultured with PD and/or ATO in the presence or absence of 30 μM caspase inhibitors for 48 hours, after which the percentage of apoptotic cells was determined by the annexin V method (**P < .001 vs PD/ATO Z-FA-FMK; Dunnett test). CTR indicates control; PD, PD184352 (1 μM); ATO, arsenic trioxide (2 μM); t-Bid, truncated Bid; Z-FA-FMK, peptide control; Z-LEHD-FMK, selective caspase-9 inhibitor; Z-IETD-FMK, selective caspase-8 inhibitor; Z-VAD-FMK, pancaspase inhibitor.

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