Figure 2
Figure 2. SAHA-induced cell death is augmented by chloroquine or 3-methyladenine. (A) Effects of drug treatments on Bcr-Abl autophosphorylation. p210- and T315I-expressing Ba/F3 cells were treated for 24 hours with 25 μM CQ, 2 mM 3-MA, 1 μM SAHA, or the indicated combinations. K562 and LAMA 84 cells were treated for 24 hours with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations. Protein lysates were subjected to SDS-PAGE, blotted, and probed with phospho-Bcr– or c-Abl–specific antibodies. Actin was used as a loading control. C+S indicates CQ plus SAHA; M+S, 3-MA plus SAHA. (B) SAHA-induced mitochondrial depolarization is enhanced by CQ or 3-MA. Ba/F3 p210 and T315I cells were treated with 25 μM CQ, 2 mM 3-MA, and 1 μM SAHA, and K562 and LAMA 84 cells were treated with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations for 6, 12, and 24 hours. Mitotracker Red CMXRos was used to assess the mitochondrial transmembrane potential status. Bars represent the mean of 3 independent experiments; error bars indicate the SEM. (C) CQ and 3-MA enhance SAHA-induced caspase-9 and -3 activation. Ba/F3 p210- and T315I-expressing cells were treated for 24 hours with 25 μM CQ, 2 mM 3-MA, and 1 μM SAHA, and K562 and LAMA 84 cells were treated for 24 hours with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations. Protein lysates were subjected to SDS-PAGE, blotted, and probed with caspase-9– and cleaved caspase-3–specific antibodies. Actin was used as a loading control. (D) Chloroquine and 3-MA enhance SAHA-induced caspase-3 activation. Ba/F3 cells expressing wild-type (p210) or imatinib-resistant (T315I) BCR-ABL were treated with 25 μM CQ, 2 mM 3-MA, and 1 μM SAHA, and K562 and LAMA 84 cells were treated with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations for 24 hours or 48 hours. The percentage of cells containing the active (cleaved) form of caspase-3 was quantified using flow cytometry. Bars represent the mean of 3 independent experiments. Error bars indicate the SEM. (E) Effects of CQ, 3-MA, SAHA, and combinations on overall cell growth and viability. Ba/F3 p210 and Ba/F3 T315I cells were treated with 25 μM CQ, 2 mM 3-MA, 1 μM SAHA, or the indicated combinations for 48 hours. K562 and LAMA 84 cells were treated with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations for 48 hours. Cell viability was determined by MTT assay. n = 3; error bars indicate the SEM. *P < .05.

SAHA-induced cell death is augmented by chloroquine or 3-methyladenine. (A) Effects of drug treatments on Bcr-Abl autophosphorylation. p210- and T315I-expressing Ba/F3 cells were treated for 24 hours with 25 μM CQ, 2 mM 3-MA, 1 μM SAHA, or the indicated combinations. K562 and LAMA 84 cells were treated for 24 hours with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations. Protein lysates were subjected to SDS-PAGE, blotted, and probed with phospho-Bcr– or c-Abl–specific antibodies. Actin was used as a loading control. C+S indicates CQ plus SAHA; M+S, 3-MA plus SAHA. (B) SAHA-induced mitochondrial depolarization is enhanced by CQ or 3-MA. Ba/F3 p210 and T315I cells were treated with 25 μM CQ, 2 mM 3-MA, and 1 μM SAHA, and K562 and LAMA 84 cells were treated with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations for 6, 12, and 24 hours. Mitotracker Red CMXRos was used to assess the mitochondrial transmembrane potential status. Bars represent the mean of 3 independent experiments; error bars indicate the SEM. (C) CQ and 3-MA enhance SAHA-induced caspase-9 and -3 activation. Ba/F3 p210- and T315I-expressing cells were treated for 24 hours with 25 μM CQ, 2 mM 3-MA, and 1 μM SAHA, and K562 and LAMA 84 cells were treated for 24 hours with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations. Protein lysates were subjected to SDS-PAGE, blotted, and probed with caspase-9– and cleaved caspase-3–specific antibodies. Actin was used as a loading control. (D) Chloroquine and 3-MA enhance SAHA-induced caspase-3 activation. Ba/F3 cells expressing wild-type (p210) or imatinib-resistant (T315I) BCR-ABL were treated with 25 μM CQ, 2 mM 3-MA, and 1 μM SAHA, and K562 and LAMA 84 cells were treated with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations for 24 hours or 48 hours. The percentage of cells containing the active (cleaved) form of caspase-3 was quantified using flow cytometry. Bars represent the mean of 3 independent experiments. Error bars indicate the SEM. (E) Effects of CQ, 3-MA, SAHA, and combinations on overall cell growth and viability. Ba/F3 p210 and Ba/F3 T315I cells were treated with 25 μM CQ, 2 mM 3-MA, 1 μM SAHA, or the indicated combinations for 48 hours. K562 and LAMA 84 cells were treated with 25 μM CQ, 5 mM 3-MA, 2 μM SAHA, or the indicated combinations for 48 hours. Cell viability was determined by MTT assay. n = 3; error bars indicate the SEM. *P < .05.

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