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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

Cell context dependent induction of TKI-resistance by KOSR.(A) Imatinib dose-response in BCR-ABL positive CML cell lines treated in regular media (RPMI with 10% FBS) or in KOSR media (RPMI with 20% KOSR). AR230-R cells are imatinib-resistant clones generated from AR230. Relative cell number was measured by MTT assay at 48 hours. Data shown are mean ± SEM (n = 8). (B) Gefitinib dose-response in NSCLC cells treated in the indicated media for 48 hours. Data shown are mean ± SEM values from MTT assays (n = 8).
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pone.0140585.g002: Cell context dependent induction of TKI-resistance by KOSR.(A) Imatinib dose-response in BCR-ABL positive CML cell lines treated in regular media (RPMI with 10% FBS) or in KOSR media (RPMI with 20% KOSR). AR230-R cells are imatinib-resistant clones generated from AR230. Relative cell number was measured by MTT assay at 48 hours. Data shown are mean ± SEM (n = 8). (B) Gefitinib dose-response in NSCLC cells treated in the indicated media for 48 hours. Data shown are mean ± SEM values from MTT assays (n = 8).

Mentions: We then tested the protective effect of KOSR on six CML cell lines across a range of IM concentrations using the MTT assay (Fig 2A). The shift from FBS to KOSR media caused a decrease in MTT values, which measured the cellular reducing activity, in each of the six CML cell lines (Fig 2A). Among the CML lines tested, the AR230-R cells, which were selected from the AR230 cells for resistance to IM [27], became even more resistant in KOSR media. Four other lines, namely K562, EM3, KYO1 and AR230 were also protected by KOSR with a significant right-shift in the IM does-response curves (Fig 2A). By contrast, KOSR did not have a significant effect on the IM does-response in the LAMA-84 cells (Fig 2A). These results suggested that the protective effect of KOSR was not universal but required a permissive cell context that variably manifested among the CML cell lines.


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)

Cell context dependent induction of TKI-resistance by KOSR.(A) Imatinib dose-response in BCR-ABL positive CML cell lines treated in regular media (RPMI with 10% FBS) or in KOSR media (RPMI with 20% KOSR). AR230-R cells are imatinib-resistant clones generated from AR230. Relative cell number was measured by MTT assay at 48 hours. Data shown are mean ± SEM (n = 8). (B) Gefitinib dose-response in NSCLC cells treated in the indicated media for 48 hours. Data shown are mean ± SEM values from MTT assays (n = 8).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140585.g002: Cell context dependent induction of TKI-resistance by KOSR.(A) Imatinib dose-response in BCR-ABL positive CML cell lines treated in regular media (RPMI with 10% FBS) or in KOSR media (RPMI with 20% KOSR). AR230-R cells are imatinib-resistant clones generated from AR230. Relative cell number was measured by MTT assay at 48 hours. Data shown are mean ± SEM (n = 8). (B) Gefitinib dose-response in NSCLC cells treated in the indicated media for 48 hours. Data shown are mean ± SEM values from MTT assays (n = 8).
Mentions: We then tested the protective effect of KOSR on six CML cell lines across a range of IM concentrations using the MTT assay (Fig 2A). The shift from FBS to KOSR media caused a decrease in MTT values, which measured the cellular reducing activity, in each of the six CML cell lines (Fig 2A). Among the CML lines tested, the AR230-R cells, which were selected from the AR230 cells for resistance to IM [27], became even more resistant in KOSR media. Four other lines, namely K562, EM3, KYO1 and AR230 were also protected by KOSR with a significant right-shift in the IM does-response curves (Fig 2A). By contrast, KOSR did not have a significant effect on the IM does-response in the LAMA-84 cells (Fig 2A). These results suggested that the protective effect of KOSR was not universal but required a permissive cell context that variably manifested among the CML cell lines.

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