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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

Imatinib-induced cytochrome c release was blocked despite inhibition of survival pathways in K562 cells on defined media.(A) Levels of BCR-ABL and proteins in downstream pathways that promote growth and survival. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 24 hours. WCLs were immunoblotted with the indicated antibodies. The levels of GAPDH are shown as loading control. (B) Levels of the anti-apoptotic and pro-apoptotic BCL-2 family proteins. The same sets of samples as used in (A) were probed for the indicated proteins. The levels of X-linked inhibitor of apoptosis protein (XIAP) were also examined. (C) Levels of caspases and PARP1. The same set of samples as used in (A) were immunoblotted with the indicated antibodies to assess caspase activation. The bar graph represents the cell numbers in each sample (n = 5) after 2 days. **, p<0.01. (D) DEVDase activity measurements. K562 cells were cultured in the indicated media ± 1 μM of imatinib. At the indicated time, cells were harvested and the cleavage of Ac-DEVD-AMC determine by fluorescence. Representative results are presented as the mean from one independent experiment performed in triplicate. *, p<0.05; **, p<0.01. (E) Cytochrome c release. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 2 days. Mitochondria and cytosolic fractions were prepared as described in Experimental Procedures and immunoblotted for cytochrome c and COX 4.
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pone.0140585.g004: Imatinib-induced cytochrome c release was blocked despite inhibition of survival pathways in K562 cells on defined media.(A) Levels of BCR-ABL and proteins in downstream pathways that promote growth and survival. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 24 hours. WCLs were immunoblotted with the indicated antibodies. The levels of GAPDH are shown as loading control. (B) Levels of the anti-apoptotic and pro-apoptotic BCL-2 family proteins. The same sets of samples as used in (A) were probed for the indicated proteins. The levels of X-linked inhibitor of apoptosis protein (XIAP) were also examined. (C) Levels of caspases and PARP1. The same set of samples as used in (A) were immunoblotted with the indicated antibodies to assess caspase activation. The bar graph represents the cell numbers in each sample (n = 5) after 2 days. **, p<0.01. (D) DEVDase activity measurements. K562 cells were cultured in the indicated media ± 1 μM of imatinib. At the indicated time, cells were harvested and the cleavage of Ac-DEVD-AMC determine by fluorescence. Representative results are presented as the mean from one independent experiment performed in triplicate. *, p<0.05; **, p<0.01. (E) Cytochrome c release. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 2 days. Mitochondria and cytosolic fractions were prepared as described in Experimental Procedures and immunoblotted for cytochrome c and COX 4.

Mentions: Inhibition of BCR-ABL kinase causes inactivation of the PI3K-AKT, the JAK-STAT, and the RAS-ERK pathways to trigger apoptosis in CML cells [42]. We found that IM treatment reduced the phosphorylation of STAT3, STAT5, ERK and AKT in K562 cells cultured in the regular (RPMI+FBS), the StemSpanTM, the iPSC and the KOSR (RPMI+KOSR) media (Fig 4A, S3A and S3B Fig). IM treatment also led to downregulation of the anti-apoptotic BCL2-family proteins including BCL2, BCLxL, and MCL1 irrespective of the culture media (Fig 4B, S3A and S3B Fig). Furthermore, IM treatment stimulated the expression of the BIM mRNA and the BIM protein in K562 cells in the regular, the StemSpanTM and the KOSR media (Fig 4B, S3A–S3C Fig). While reductions in the anti-apoptotic BCL2-proteins and increase in the BH3-only BIM protein caused the release of cytochrome c from the mitochondria and the activation of caspases in K562 cells cultured in regular media (Fig 4C, 4D and 4E), these pro-apoptotic alterations failed to cause cytochrome c release or caspase activation in K562 cells that were switched to the StemSpanTM or the KOSR media at the time of drug addition (Fig 4C, 4D and 4E). These results showed that the pro-survival effect of KOSR was associated with the inhibition of mitochondria dependent apoptosis.


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)

Imatinib-induced cytochrome c release was blocked despite inhibition of survival pathways in K562 cells on defined media.(A) Levels of BCR-ABL and proteins in downstream pathways that promote growth and survival. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 24 hours. WCLs were immunoblotted with the indicated antibodies. The levels of GAPDH are shown as loading control. (B) Levels of the anti-apoptotic and pro-apoptotic BCL-2 family proteins. The same sets of samples as used in (A) were probed for the indicated proteins. The levels of X-linked inhibitor of apoptosis protein (XIAP) were also examined. (C) Levels of caspases and PARP1. The same set of samples as used in (A) were immunoblotted with the indicated antibodies to assess caspase activation. The bar graph represents the cell numbers in each sample (n = 5) after 2 days. **, p<0.01. (D) DEVDase activity measurements. K562 cells were cultured in the indicated media ± 1 μM of imatinib. At the indicated time, cells were harvested and the cleavage of Ac-DEVD-AMC determine by fluorescence. Representative results are presented as the mean from one independent experiment performed in triplicate. *, p<0.05; **, p<0.01. (E) Cytochrome c release. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 2 days. Mitochondria and cytosolic fractions were prepared as described in Experimental Procedures and immunoblotted for cytochrome c and COX 4.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140585.g004: Imatinib-induced cytochrome c release was blocked despite inhibition of survival pathways in K562 cells on defined media.(A) Levels of BCR-ABL and proteins in downstream pathways that promote growth and survival. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 24 hours. WCLs were immunoblotted with the indicated antibodies. The levels of GAPDH are shown as loading control. (B) Levels of the anti-apoptotic and pro-apoptotic BCL-2 family proteins. The same sets of samples as used in (A) were probed for the indicated proteins. The levels of X-linked inhibitor of apoptosis protein (XIAP) were also examined. (C) Levels of caspases and PARP1. The same set of samples as used in (A) were immunoblotted with the indicated antibodies to assess caspase activation. The bar graph represents the cell numbers in each sample (n = 5) after 2 days. **, p<0.01. (D) DEVDase activity measurements. K562 cells were cultured in the indicated media ± 1 μM of imatinib. At the indicated time, cells were harvested and the cleavage of Ac-DEVD-AMC determine by fluorescence. Representative results are presented as the mean from one independent experiment performed in triplicate. *, p<0.05; **, p<0.01. (E) Cytochrome c release. K562 cells were cultured in the indicated media ± 1 μM of imatinib for 2 days. Mitochondria and cytosolic fractions were prepared as described in Experimental Procedures and immunoblotted for cytochrome c and COX 4.
Mentions: Inhibition of BCR-ABL kinase causes inactivation of the PI3K-AKT, the JAK-STAT, and the RAS-ERK pathways to trigger apoptosis in CML cells [42]. We found that IM treatment reduced the phosphorylation of STAT3, STAT5, ERK and AKT in K562 cells cultured in the regular (RPMI+FBS), the StemSpanTM, the iPSC and the KOSR (RPMI+KOSR) media (Fig 4A, S3A and S3B Fig). IM treatment also led to downregulation of the anti-apoptotic BCL2-family proteins including BCL2, BCLxL, and MCL1 irrespective of the culture media (Fig 4B, S3A and S3B Fig). Furthermore, IM treatment stimulated the expression of the BIM mRNA and the BIM protein in K562 cells in the regular, the StemSpanTM and the KOSR media (Fig 4B, S3A–S3C Fig). While reductions in the anti-apoptotic BCL2-proteins and increase in the BH3-only BIM protein caused the release of cytochrome c from the mitochondria and the activation of caspases in K562 cells cultured in regular media (Fig 4C, 4D and 4E), these pro-apoptotic alterations failed to cause cytochrome c release or caspase activation in K562 cells that were switched to the StemSpanTM or the KOSR media at the time of drug addition (Fig 4C, 4D and 4E). These results showed that the pro-survival effect of KOSR was associated with the inhibition of mitochondria dependent apoptosis.

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