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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

Effects of KOSR on the mitochondria.(A) Mitochondrial membrane potential (ΔΨm) in cells at the indicated time after imatinib (1 μM) addition in the indicated culture media. Data shown are mean ± SEM (n = 6). Note that the KOSR media did not affect ΔΨm, but prevented imatinib from causing ΔΨm dissipation in K562 but not LAMA84 cells. *, p<0.05; **, p<0.01. (B) Electron micrographs of K562 cells cultured for 24 hours in the indicated media. Scale bar: 500nM. (C) ROS levels (arbitrary units) in cells after switching to the regular (R) or the KOSR (K) media for 2 hours. Data shown are mean ± SEM (n = 5). *, p<0.05. (D) Menadione dose-response in K562 or LAMA-84 cells in the indicated media. Relative cell number determined by MTT assay is shown as mean ± SEM (n = 8).
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pone.0140585.g006: Effects of KOSR on the mitochondria.(A) Mitochondrial membrane potential (ΔΨm) in cells at the indicated time after imatinib (1 μM) addition in the indicated culture media. Data shown are mean ± SEM (n = 6). Note that the KOSR media did not affect ΔΨm, but prevented imatinib from causing ΔΨm dissipation in K562 but not LAMA84 cells. *, p<0.05; **, p<0.01. (B) Electron micrographs of K562 cells cultured for 24 hours in the indicated media. Scale bar: 500nM. (C) ROS levels (arbitrary units) in cells after switching to the regular (R) or the KOSR (K) media for 2 hours. Data shown are mean ± SEM (n = 5). *, p<0.05. (D) Menadione dose-response in K562 or LAMA-84 cells in the indicated media. Relative cell number determined by MTT assay is shown as mean ± SEM (n = 8).

Mentions: Because KOSR caused the formation of BIM-resistant mitochondria, we examined its effect on the mitochondria in the KOSR-responsive K562 and the KOSR-non-responsive LAMA-84 cells. We found that IM treatment reduced the mitochondrial membrane potential (ΔΨm) in K562 cells cultured in RPMI+FBS media (Fig 6A). However, IM did not reduce the ΔΨm in K562 cells cultured in RPMI+KOSR media (Fig 6A). By contrast, IM reduced the ΔΨm in LAMA-84 cells cultured in FBS or KOSR supplemented media (Fig 6A). Examination of the mitochondrial morphology by transmission electron microscopy (TEM) did not reveal any discernable differences in K562 cells cultured in the FBS or the KOSR media (Fig 6B). Staining with MitoTracker-Green, the intensity of which reflects the overall dimension of the mitochondrial compartment [47], showed a transient reduction in MitoTracker signal within one hour after switching to the KOSR media (S4A Fig), indicating that KOSR might have caused some mitochondrial compaction or fusion that was not detectable by TEM. We also found a rapid reduction in the ability of cells to reduce the MTT-dye within one hour of switching to the KOSR media, indicating an alteration in the redox homeostasis (S4B Fig). Measurement with the reactive oxygen species (ROS)-sensitive DCF (5-(and-6)-chloromethyl-2’,7’-dichlorodihydro fluorescein diacetate acetyl ester) dye showed that switching to the KOSR media caused a small but significant increase in ROS in K562 cells (Fig 6C). By contrast, switching to KOSR media did not increase ROS in LAMA-84 cells (Fig 6C) that did not acquire IM-resistance in KOSR.


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)

Effects of KOSR on the mitochondria.(A) Mitochondrial membrane potential (ΔΨm) in cells at the indicated time after imatinib (1 μM) addition in the indicated culture media. Data shown are mean ± SEM (n = 6). Note that the KOSR media did not affect ΔΨm, but prevented imatinib from causing ΔΨm dissipation in K562 but not LAMA84 cells. *, p<0.05; **, p<0.01. (B) Electron micrographs of K562 cells cultured for 24 hours in the indicated media. Scale bar: 500nM. (C) ROS levels (arbitrary units) in cells after switching to the regular (R) or the KOSR (K) media for 2 hours. Data shown are mean ± SEM (n = 5). *, p<0.05. (D) Menadione dose-response in K562 or LAMA-84 cells in the indicated media. Relative cell number determined by MTT assay is shown as mean ± SEM (n = 8).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608728&req=5

pone.0140585.g006: Effects of KOSR on the mitochondria.(A) Mitochondrial membrane potential (ΔΨm) in cells at the indicated time after imatinib (1 μM) addition in the indicated culture media. Data shown are mean ± SEM (n = 6). Note that the KOSR media did not affect ΔΨm, but prevented imatinib from causing ΔΨm dissipation in K562 but not LAMA84 cells. *, p<0.05; **, p<0.01. (B) Electron micrographs of K562 cells cultured for 24 hours in the indicated media. Scale bar: 500nM. (C) ROS levels (arbitrary units) in cells after switching to the regular (R) or the KOSR (K) media for 2 hours. Data shown are mean ± SEM (n = 5). *, p<0.05. (D) Menadione dose-response in K562 or LAMA-84 cells in the indicated media. Relative cell number determined by MTT assay is shown as mean ± SEM (n = 8).
Mentions: Because KOSR caused the formation of BIM-resistant mitochondria, we examined its effect on the mitochondria in the KOSR-responsive K562 and the KOSR-non-responsive LAMA-84 cells. We found that IM treatment reduced the mitochondrial membrane potential (ΔΨm) in K562 cells cultured in RPMI+FBS media (Fig 6A). However, IM did not reduce the ΔΨm in K562 cells cultured in RPMI+KOSR media (Fig 6A). By contrast, IM reduced the ΔΨm in LAMA-84 cells cultured in FBS or KOSR supplemented media (Fig 6A). Examination of the mitochondrial morphology by transmission electron microscopy (TEM) did not reveal any discernable differences in K562 cells cultured in the FBS or the KOSR media (Fig 6B). Staining with MitoTracker-Green, the intensity of which reflects the overall dimension of the mitochondrial compartment [47], showed a transient reduction in MitoTracker signal within one hour after switching to the KOSR media (S4A Fig), indicating that KOSR might have caused some mitochondrial compaction or fusion that was not detectable by TEM. We also found a rapid reduction in the ability of cells to reduce the MTT-dye within one hour of switching to the KOSR media, indicating an alteration in the redox homeostasis (S4B Fig). Measurement with the reactive oxygen species (ROS)-sensitive DCF (5-(and-6)-chloromethyl-2’,7’-dichlorodihydro fluorescein diacetate acetyl ester) dye showed that switching to the KOSR media caused a small but significant increase in ROS in K562 cells (Fig 6C). By contrast, switching to KOSR media did not increase ROS in LAMA-84 cells (Fig 6C) that did not acquire IM-resistance in KOSR.

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