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Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.

Ishii Y, Nhiayi MK, Tse E, Cheng J, Massimino M, Durden DL, Vigneri P, Wang JY - PLoS ONE (2015)

Bottom Line: We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib.Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents.These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology, Department of Medicine, School of Medicine, University of California San Diego, San Diego, California, United States of America; Moores Cancer Center, University of California San Diego, San Diego, California, United States of America.

ABSTRACT
Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

No MeSH data available.


Related in: MedlinePlus

AKT inhibitor prevented the formation of BIM-resistant mitochondria in KOSR-cultured cells.(A) Caspase 3 cleavage in KOSR-cultured K562 cells treated with MK2206 (3 μM) and imatinib (1 μM) for 24 hours. Whole cell lysates were immunolotted with antibody specific to cleaved caspase 3. (B) DEVDase activity (arbitrary units, a.u.) in K562 cells treated with imatinib (1 μM) and the indicated concentrations of MK2206 for 2 days. The values shown are mean± SEM (n = 6). *, p<0.05; **, p<0.01. (C) Cytochrome c release in K562 cells treated with imatinib (1 μM) and MK2206 (3 μM) for 2 days. Cells were lysed, fractionated and then probed for cytochrome c, COX4 and GAPDH in the cytosolic and the mitochondrial fractions. (D) Inhibition of p-AKT by MK2206 in the mitochondrial fraction. K562 cells were cultured in the regular or the KOSR media in the presence or the absence of MK2206 (3 μM) for 18 hours. Mitochondria were isolated and immunoblotted with anti-p-AKT, anti-AKT or anti-COX 4 antibodies. (E) Blocking BIM-resistant mitochondria formation with MK2206. K562 cells were cultured in the regular or the KOSR media with or without MK2206 (3 μM) for 2 days. Mitochondria were isolated and incubated with or without in vitro-translated mouse BIM-EL or mouse BIM-EL-ΔBH3 mutant for 1 hour. The levels of cytochrome c in the supernatant and the pellet fractions were examined by immunoblotting.
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pone.0140585.g008: AKT inhibitor prevented the formation of BIM-resistant mitochondria in KOSR-cultured cells.(A) Caspase 3 cleavage in KOSR-cultured K562 cells treated with MK2206 (3 μM) and imatinib (1 μM) for 24 hours. Whole cell lysates were immunolotted with antibody specific to cleaved caspase 3. (B) DEVDase activity (arbitrary units, a.u.) in K562 cells treated with imatinib (1 μM) and the indicated concentrations of MK2206 for 2 days. The values shown are mean± SEM (n = 6). *, p<0.05; **, p<0.01. (C) Cytochrome c release in K562 cells treated with imatinib (1 μM) and MK2206 (3 μM) for 2 days. Cells were lysed, fractionated and then probed for cytochrome c, COX4 and GAPDH in the cytosolic and the mitochondrial fractions. (D) Inhibition of p-AKT by MK2206 in the mitochondrial fraction. K562 cells were cultured in the regular or the KOSR media in the presence or the absence of MK2206 (3 μM) for 18 hours. Mitochondria were isolated and immunoblotted with anti-p-AKT, anti-AKT or anti-COX 4 antibodies. (E) Blocking BIM-resistant mitochondria formation with MK2206. K562 cells were cultured in the regular or the KOSR media with or without MK2206 (3 μM) for 2 days. Mitochondria were isolated and incubated with or without in vitro-translated mouse BIM-EL or mouse BIM-EL-ΔBH3 mutant for 1 hour. The levels of cytochrome c in the supernatant and the pellet fractions were examined by immunoblotting.

Mentions: We then measured the effect of MK (5 μM) on cytochrome c release and caspase activation in KOSR-cultured K562 cells. Although MK or IM alone did not cause cytochrome c release and caspase activation in KOSR-cultured K562 cells, the combined treatment with MK and IM activated this mitochondrial-dependent apoptotic mechanism (Fig 8A, 8B and 8C). We could detect p-AKT in the mitochondria preparations from KOSR-cultured K562 cells and found that MK treatment blocked this association of p-AKT with the mitochondrial fraction (Fig 8D). Furthermore, we found that MK treatment of KOSR-cultured K562 cells prevented the formation of BIM-resistant mitochondria (Fig 8E). We found that mitochondria isolated from MK-KOSR-cultured K562 cells released cytochrome c when treated with recombinant Bim, but not the mutant Bim-ΔBH3 protein (Fig 8E). Together, these results suggested that KOSR induced p-AKT increase was required for the formation of BIM-resistant mitochondria and the protection from IM-induced apoptosis in CML cells.


Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.

Ishii Y, Nhiayi MK, Tse E, Cheng J, Massimino M, Durden DL, Vigneri P, Wang JY - PLoS ONE (2015)

AKT inhibitor prevented the formation of BIM-resistant mitochondria in KOSR-cultured cells.(A) Caspase 3 cleavage in KOSR-cultured K562 cells treated with MK2206 (3 μM) and imatinib (1 μM) for 24 hours. Whole cell lysates were immunolotted with antibody specific to cleaved caspase 3. (B) DEVDase activity (arbitrary units, a.u.) in K562 cells treated with imatinib (1 μM) and the indicated concentrations of MK2206 for 2 days. The values shown are mean± SEM (n = 6). *, p<0.05; **, p<0.01. (C) Cytochrome c release in K562 cells treated with imatinib (1 μM) and MK2206 (3 μM) for 2 days. Cells were lysed, fractionated and then probed for cytochrome c, COX4 and GAPDH in the cytosolic and the mitochondrial fractions. (D) Inhibition of p-AKT by MK2206 in the mitochondrial fraction. K562 cells were cultured in the regular or the KOSR media in the presence or the absence of MK2206 (3 μM) for 18 hours. Mitochondria were isolated and immunoblotted with anti-p-AKT, anti-AKT or anti-COX 4 antibodies. (E) Blocking BIM-resistant mitochondria formation with MK2206. K562 cells were cultured in the regular or the KOSR media with or without MK2206 (3 μM) for 2 days. Mitochondria were isolated and incubated with or without in vitro-translated mouse BIM-EL or mouse BIM-EL-ΔBH3 mutant for 1 hour. The levels of cytochrome c in the supernatant and the pellet fractions were examined by immunoblotting.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608728&req=5

pone.0140585.g008: AKT inhibitor prevented the formation of BIM-resistant mitochondria in KOSR-cultured cells.(A) Caspase 3 cleavage in KOSR-cultured K562 cells treated with MK2206 (3 μM) and imatinib (1 μM) for 24 hours. Whole cell lysates were immunolotted with antibody specific to cleaved caspase 3. (B) DEVDase activity (arbitrary units, a.u.) in K562 cells treated with imatinib (1 μM) and the indicated concentrations of MK2206 for 2 days. The values shown are mean± SEM (n = 6). *, p<0.05; **, p<0.01. (C) Cytochrome c release in K562 cells treated with imatinib (1 μM) and MK2206 (3 μM) for 2 days. Cells were lysed, fractionated and then probed for cytochrome c, COX4 and GAPDH in the cytosolic and the mitochondrial fractions. (D) Inhibition of p-AKT by MK2206 in the mitochondrial fraction. K562 cells were cultured in the regular or the KOSR media in the presence or the absence of MK2206 (3 μM) for 18 hours. Mitochondria were isolated and immunoblotted with anti-p-AKT, anti-AKT or anti-COX 4 antibodies. (E) Blocking BIM-resistant mitochondria formation with MK2206. K562 cells were cultured in the regular or the KOSR media with or without MK2206 (3 μM) for 2 days. Mitochondria were isolated and incubated with or without in vitro-translated mouse BIM-EL or mouse BIM-EL-ΔBH3 mutant for 1 hour. The levels of cytochrome c in the supernatant and the pellet fractions were examined by immunoblotting.
Mentions: We then measured the effect of MK (5 μM) on cytochrome c release and caspase activation in KOSR-cultured K562 cells. Although MK or IM alone did not cause cytochrome c release and caspase activation in KOSR-cultured K562 cells, the combined treatment with MK and IM activated this mitochondrial-dependent apoptotic mechanism (Fig 8A, 8B and 8C). We could detect p-AKT in the mitochondria preparations from KOSR-cultured K562 cells and found that MK treatment blocked this association of p-AKT with the mitochondrial fraction (Fig 8D). Furthermore, we found that MK treatment of KOSR-cultured K562 cells prevented the formation of BIM-resistant mitochondria (Fig 8E). We found that mitochondria isolated from MK-KOSR-cultured K562 cells released cytochrome c when treated with recombinant Bim, but not the mutant Bim-ΔBH3 protein (Fig 8E). Together, these results suggested that KOSR induced p-AKT increase was required for the formation of BIM-resistant mitochondria and the protection from IM-induced apoptosis in CML cells.

Bottom Line: We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib.Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents.These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology, Department of Medicine, School of Medicine, University of California San Diego, San Diego, California, United States of America; Moores Cancer Center, University of California San Diego, San Diego, California, United States of America.

ABSTRACT
Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

No MeSH data available.


Related in: MedlinePlus