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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

Rapid induction of resistance to BCR-ABL kinase inhibitors in serum replacement media without cytokines.(A) Quantification of response to imatinib by clonogenic survival assay under different media conditions. K562 cells were plated in the indicated media (S1 Table) +/- 1 μM imatinib. After 72 hours, cells were re-plated in 0.8% methylcellulose in regular media (RPMI+10%FBS) and the numbers of colonies were counted 10 days later. Values are means ± s.d. of three experiments performed in triplicates. Statistical analysis: *, p<0.05; **, p<0.01. (B) Effect of cytokines on the imatinib response. K562 cells were plated in RPMI or StemSpan media plus 10% FBS with or without 1 μM of imatinib in the presence or the absence of cytokines (4 units/mL of EPO, 10ng/mL of IL-3, 100ng/mL of IL-6, 100ng/mL of SCF, and 100ng/mL of Flt-3L). Survival was measured by clonogenic assay as in (A). Values are means ± s.d. of three experiments performed in triplicates. *, p<0.05; **, p<0.01. (C) Inhibition of BCR-ABL tyrosine phosphorylation by imatinib (IM) in different media. Whole lysates from K562 cells cultured in the indicated media treated with IM (1 μM, 24 hours) or not were immunoblotted with anti-pY245, which measures the autophosphorylation of BCR-ABL on the ABL-tyrosine-245 residue. The levels of GAPDH serve as loading controls. BCR-ABL is autophosphorylated on many sites and can thus migrate as multiple bands depending on the stoichiometry of overall phosphorylation. (D) Quantification of response to dasatinib and nilotinib under different media conditions. K562 cells were plated in the regular, StemSpan (no cytokines) or KOSR media with or without 5 nM of dasatinib or 10 nM of nilotinib for clonogenic assay. Representative results are shown as the mean from one experiment performed in triplicate. *, p<0.05; **, p<0.01.
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pone.0140585.g001: Rapid induction of resistance to BCR-ABL kinase inhibitors in serum replacement media without cytokines.(A) Quantification of response to imatinib by clonogenic survival assay under different media conditions. K562 cells were plated in the indicated media (S1 Table) +/- 1 μM imatinib. After 72 hours, cells were re-plated in 0.8% methylcellulose in regular media (RPMI+10%FBS) and the numbers of colonies were counted 10 days later. Values are means ± s.d. of three experiments performed in triplicates. Statistical analysis: *, p<0.05; **, p<0.01. (B) Effect of cytokines on the imatinib response. K562 cells were plated in RPMI or StemSpan media plus 10% FBS with or without 1 μM of imatinib in the presence or the absence of cytokines (4 units/mL of EPO, 10ng/mL of IL-3, 100ng/mL of IL-6, 100ng/mL of SCF, and 100ng/mL of Flt-3L). Survival was measured by clonogenic assay as in (A). Values are means ± s.d. of three experiments performed in triplicates. *, p<0.05; **, p<0.01. (C) Inhibition of BCR-ABL tyrosine phosphorylation by imatinib (IM) in different media. Whole lysates from K562 cells cultured in the indicated media treated with IM (1 μM, 24 hours) or not were immunoblotted with anti-pY245, which measures the autophosphorylation of BCR-ABL on the ABL-tyrosine-245 residue. The levels of GAPDH serve as loading controls. BCR-ABL is autophosphorylated on many sites and can thus migrate as multiple bands depending on the stoichiometry of overall phosphorylation. (D) Quantification of response to dasatinib and nilotinib under different media conditions. K562 cells were plated in the regular, StemSpan (no cytokines) or KOSR media with or without 5 nM of dasatinib or 10 nM of nilotinib for clonogenic assay. Representative results are shown as the mean from one experiment performed in triplicate. *, p<0.05; **, p<0.01.

Mentions: Previous studies have shown that cytokines can activate survival pathways to protect CML cells from TKI-induced death [18, 33–35]. With K562 cells, which can differentiate along the erythroid and the megakaryocytic pathways [36–39], we found that erythropoietin (EPO) partially inhibited imatinib (IM) induced death (S1A–S1C Fig). We then tried to enhance EPO protection by culturing K562 cells in media that contained other cytokines and growth factors formulated to support stem cells (Fig 1A). We tested the serum-free StemSpanTM media (supplemented with IL3, IL6, SCF and Flt3L) and the serum-free iPSC (induced Pluripotent Stem Cell) media (supplemented with KnockOut™ Serum Replacement (KOSR) and b-FGF) (S1 Table). In regular culture media (RPMI plus 10% fetal bovine serum, FBS), treatment with IM (1 μM, 72 hours) was sufficient to kill off >90% of K562 cells (Fig 1A). Interestingly, we found that switching to the StemSpanTM or the iPSC media at the time of IM addition abrogated this cytotoxic effect (Fig 1A and 1B). The protective effect of the StemSpanTM media could not be attributed to the cytokines because addition of IL3, IL6, SCF and Flt3L to the regular media (RPMI+10%FBS) did not abrogate the cytotoxic effect of IM (Fig 1B), most likely because K562 cells do not express receptors for these cytokines. Furthermore, removal of those cytokines from the StemSpanTM did not abrogate the protective effect of this media (Fig 1B). Because the formulation of StemSpanTM was not available, we could not identify the media components that were required for the induction of IM resistance. In the iPSC media, KOSR and b-FGF are used to replace serum. We therefore tested two components and found that the addition of KOSR to RPMI (without FBS), or to the regular media (RPMI+FBS) was sufficient to induce IM resistance (Fig 1A and S1D Fig, left panel). These results showed that KOSR can cause IM resistance and this protective effect is observed without b-FGF and occurs in the absence or the presence of serum. Addition of TGF-beta, a major anti-stemness factor in serum [40], also failed to abrogate the pro-survival effect of KOSR (S1E Fig). The media shift did not interfere with the inhibition of BCR-ABL tyrosine kinase activity by IM (Fig 1C). The media switch also protected K562 cells from two other BCR-ABL kinase inhibitors, dasatinib and nilotinib (Fig 1D).


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)

Rapid induction of resistance to BCR-ABL kinase inhibitors in serum replacement media without cytokines.(A) Quantification of response to imatinib by clonogenic survival assay under different media conditions. K562 cells were plated in the indicated media (S1 Table) +/- 1 μM imatinib. After 72 hours, cells were re-plated in 0.8% methylcellulose in regular media (RPMI+10%FBS) and the numbers of colonies were counted 10 days later. Values are means ± s.d. of three experiments performed in triplicates. Statistical analysis: *, p<0.05; **, p<0.01. (B) Effect of cytokines on the imatinib response. K562 cells were plated in RPMI or StemSpan media plus 10% FBS with or without 1 μM of imatinib in the presence or the absence of cytokines (4 units/mL of EPO, 10ng/mL of IL-3, 100ng/mL of IL-6, 100ng/mL of SCF, and 100ng/mL of Flt-3L). Survival was measured by clonogenic assay as in (A). Values are means ± s.d. of three experiments performed in triplicates. *, p<0.05; **, p<0.01. (C) Inhibition of BCR-ABL tyrosine phosphorylation by imatinib (IM) in different media. Whole lysates from K562 cells cultured in the indicated media treated with IM (1 μM, 24 hours) or not were immunoblotted with anti-pY245, which measures the autophosphorylation of BCR-ABL on the ABL-tyrosine-245 residue. The levels of GAPDH serve as loading controls. BCR-ABL is autophosphorylated on many sites and can thus migrate as multiple bands depending on the stoichiometry of overall phosphorylation. (D) Quantification of response to dasatinib and nilotinib under different media conditions. K562 cells were plated in the regular, StemSpan (no cytokines) or KOSR media with or without 5 nM of dasatinib or 10 nM of nilotinib for clonogenic assay. Representative results are shown as the mean from one experiment performed in triplicate. *, p<0.05; **, p<0.01.
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Related In: Results  -  Collection

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

pone.0140585.g001: Rapid induction of resistance to BCR-ABL kinase inhibitors in serum replacement media without cytokines.(A) Quantification of response to imatinib by clonogenic survival assay under different media conditions. K562 cells were plated in the indicated media (S1 Table) +/- 1 μM imatinib. After 72 hours, cells were re-plated in 0.8% methylcellulose in regular media (RPMI+10%FBS) and the numbers of colonies were counted 10 days later. Values are means ± s.d. of three experiments performed in triplicates. Statistical analysis: *, p<0.05; **, p<0.01. (B) Effect of cytokines on the imatinib response. K562 cells were plated in RPMI or StemSpan media plus 10% FBS with or without 1 μM of imatinib in the presence or the absence of cytokines (4 units/mL of EPO, 10ng/mL of IL-3, 100ng/mL of IL-6, 100ng/mL of SCF, and 100ng/mL of Flt-3L). Survival was measured by clonogenic assay as in (A). Values are means ± s.d. of three experiments performed in triplicates. *, p<0.05; **, p<0.01. (C) Inhibition of BCR-ABL tyrosine phosphorylation by imatinib (IM) in different media. Whole lysates from K562 cells cultured in the indicated media treated with IM (1 μM, 24 hours) or not were immunoblotted with anti-pY245, which measures the autophosphorylation of BCR-ABL on the ABL-tyrosine-245 residue. The levels of GAPDH serve as loading controls. BCR-ABL is autophosphorylated on many sites and can thus migrate as multiple bands depending on the stoichiometry of overall phosphorylation. (D) Quantification of response to dasatinib and nilotinib under different media conditions. K562 cells were plated in the regular, StemSpan (no cytokines) or KOSR media with or without 5 nM of dasatinib or 10 nM of nilotinib for clonogenic assay. Representative results are shown as the mean from one experiment performed in triplicate. *, p<0.05; **, p<0.01.
Mentions: Previous studies have shown that cytokines can activate survival pathways to protect CML cells from TKI-induced death [18, 33–35]. With K562 cells, which can differentiate along the erythroid and the megakaryocytic pathways [36–39], we found that erythropoietin (EPO) partially inhibited imatinib (IM) induced death (S1A–S1C Fig). We then tried to enhance EPO protection by culturing K562 cells in media that contained other cytokines and growth factors formulated to support stem cells (Fig 1A). We tested the serum-free StemSpanTM media (supplemented with IL3, IL6, SCF and Flt3L) and the serum-free iPSC (induced Pluripotent Stem Cell) media (supplemented with KnockOut™ Serum Replacement (KOSR) and b-FGF) (S1 Table). In regular culture media (RPMI plus 10% fetal bovine serum, FBS), treatment with IM (1 μM, 72 hours) was sufficient to kill off >90% of K562 cells (Fig 1A). Interestingly, we found that switching to the StemSpanTM or the iPSC media at the time of IM addition abrogated this cytotoxic effect (Fig 1A and 1B). The protective effect of the StemSpanTM media could not be attributed to the cytokines because addition of IL3, IL6, SCF and Flt3L to the regular media (RPMI+10%FBS) did not abrogate the cytotoxic effect of IM (Fig 1B), most likely because K562 cells do not express receptors for these cytokines. Furthermore, removal of those cytokines from the StemSpanTM did not abrogate the protective effect of this media (Fig 1B). Because the formulation of StemSpanTM was not available, we could not identify the media components that were required for the induction of IM resistance. In the iPSC media, KOSR and b-FGF are used to replace serum. We therefore tested two components and found that the addition of KOSR to RPMI (without FBS), or to the regular media (RPMI+FBS) was sufficient to induce IM resistance (Fig 1A and S1D Fig, left panel). These results showed that KOSR can cause IM resistance and this protective effect is observed without b-FGF and occurs in the absence or the presence of serum. Addition of TGF-beta, a major anti-stemness factor in serum [40], also failed to abrogate the pro-survival effect of KOSR (S1E Fig). The media shift did not interfere with the inhibition of BCR-ABL tyrosine kinase activity by IM (Fig 1C). The media switch also protected K562 cells from two other BCR-ABL kinase inhibitors, dasatinib and nilotinib (Fig 1D).

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