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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

Gefinitib-resistant AKT phosphorylation in NSCLC cells.(A) The effect of gefitinib on p-AKT, p-tyrosine and BIM levels in NSCLC cells. H1650 and HCC827 cells were cultured in the regular media with the indicated concentrations of gefitinib for 24 hours. WCLs were immunoblotted with anti-p-AKT, anti-BIM and p-tyrosine (4G10) antibodies. (B) MK2206 sensitized H1650 cells to gefitinib. Relative cell number was measured by MTT assay at 4 days after incubations with the indicated drugs. Values are means ± SEM (n = 9). **, p<0.01.
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pone.0140585.g009: Gefinitib-resistant AKT phosphorylation in NSCLC cells.(A) The effect of gefitinib on p-AKT, p-tyrosine and BIM levels in NSCLC cells. H1650 and HCC827 cells were cultured in the regular media with the indicated concentrations of gefitinib for 24 hours. WCLs were immunoblotted with anti-p-AKT, anti-BIM and p-tyrosine (4G10) antibodies. (B) MK2206 sensitized H1650 cells to gefitinib. Relative cell number was measured by MTT assay at 4 days after incubations with the indicated drugs. Values are means ± SEM (n = 9). **, p<0.01.

Mentions: We also tested the effect of MK on the NSCLC cells. We found that gefitinitib treatment reduced tyrosine-phosphorylated proteins and upregulated BIM in both the more resistant H1650 and the more sensitive HCC827 cells (Fig 9A). However, a higher level of p-AKT remained in H1650 than HCC827 cells following treatment with gefinitib (Fig 9A). Furthermore, the combined treatment of H1650 cells with gefitinib and MK enhanced the cytotoxic response (Fig 9B). This result suggested that the cytotoxic response to gefinitib could also be enhanced by the inhibition of p-AKT.


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)

Gefinitib-resistant AKT phosphorylation in NSCLC cells.(A) The effect of gefitinib on p-AKT, p-tyrosine and BIM levels in NSCLC cells. H1650 and HCC827 cells were cultured in the regular media with the indicated concentrations of gefitinib for 24 hours. WCLs were immunoblotted with anti-p-AKT, anti-BIM and p-tyrosine (4G10) antibodies. (B) MK2206 sensitized H1650 cells to gefitinib. Relative cell number was measured by MTT assay at 4 days after incubations with the indicated drugs. Values are means ± SEM (n = 9). **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140585.g009: Gefinitib-resistant AKT phosphorylation in NSCLC cells.(A) The effect of gefitinib on p-AKT, p-tyrosine and BIM levels in NSCLC cells. H1650 and HCC827 cells were cultured in the regular media with the indicated concentrations of gefitinib for 24 hours. WCLs were immunoblotted with anti-p-AKT, anti-BIM and p-tyrosine (4G10) antibodies. (B) MK2206 sensitized H1650 cells to gefitinib. Relative cell number was measured by MTT assay at 4 days after incubations with the indicated drugs. Values are means ± SEM (n = 9). **, p<0.01.
Mentions: We also tested the effect of MK on the NSCLC cells. We found that gefitinitib treatment reduced tyrosine-phosphorylated proteins and upregulated BIM in both the more resistant H1650 and the more sensitive HCC827 cells (Fig 9A). However, a higher level of p-AKT remained in H1650 than HCC827 cells following treatment with gefinitib (Fig 9A). Furthermore, the combined treatment of H1650 cells with gefitinib and MK enhanced the cytotoxic response (Fig 9B). This result suggested that the cytotoxic response to gefinitib could also be enhanced by the inhibition of p-AKT.

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