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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

Combined inhibition of BCR-ABL and AKT overcame the protective effect of KOSR.(A) Induction of p-AKT at 1 hour after treatment of cells with the indicated concentrations of H2O2. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (B) Rapid Induction of p-AKT upon media switch. K562 cells were cultured in the regular or KOSR ±1 μM of imatinib for 1 hour. (C) AKT-PH-domain inhibitor MK2206 blocked p-AKT increase. K562 cells were pre-treated with 0, 0.1 or 3 μM of MK2206 in the regular media for 24 hours, then re-plated either in the regular or the KOSR media with the same doses of MK2206 used as in pre-treatments. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (D) Overcoming imatinib-resistance with AKT-PH-domain inhibitor MK2206. K562 cells were pre-treated with MK2206 (0, 0.1, 1, 5 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of MK2206 ± imatinib (1 μM). Survival was measured by clonogenic assay. The values are means ± SEM (n = 6). *, p<0.05. (E) Overcoming imatinib-resistance with PI3 kinase inhibitor, SF1126. K562 cells were pre-treated with SF1126 (0, 20, 40, 60 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of SF1126 ± 1 μM of imatinib. MTT assay was performed after 3 days to determine the relative cell number. The values are means ± SEM (n = 8). *, p<0.05.
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pone.0140585.g007: Combined inhibition of BCR-ABL and AKT overcame the protective effect of KOSR.(A) Induction of p-AKT at 1 hour after treatment of cells with the indicated concentrations of H2O2. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (B) Rapid Induction of p-AKT upon media switch. K562 cells were cultured in the regular or KOSR ±1 μM of imatinib for 1 hour. (C) AKT-PH-domain inhibitor MK2206 blocked p-AKT increase. K562 cells were pre-treated with 0, 0.1 or 3 μM of MK2206 in the regular media for 24 hours, then re-plated either in the regular or the KOSR media with the same doses of MK2206 used as in pre-treatments. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (D) Overcoming imatinib-resistance with AKT-PH-domain inhibitor MK2206. K562 cells were pre-treated with MK2206 (0, 0.1, 1, 5 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of MK2206 ± imatinib (1 μM). Survival was measured by clonogenic assay. The values are means ± SEM (n = 6). *, p<0.05. (E) Overcoming imatinib-resistance with PI3 kinase inhibitor, SF1126. K562 cells were pre-treated with SF1126 (0, 20, 40, 60 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of SF1126 ± 1 μM of imatinib. MTT assay was performed after 3 days to determine the relative cell number. The values are means ± SEM (n = 8). *, p<0.05.

Mentions: It has been shown that ROS, such as hydrogen peroxide (H2O2), can activate AKT to promote cell survival [50]. We therefore tested the effect of H2O2 on p-AKT levels in K562 and LAMA-84 cells. We found that H2O2 treatment raised the levels of p-AKT in K562 but not LAMA-84 cells (Fig 7A). Furthermore, we found that the levels of p-AKT also increased during the first 1 to 3 hours after switching K562 cells to the KOSR-media (Fig 7B, S5A and S5B Fig). This KOSR-induced p-AKT increase was not found in LAMA-84 cells (S5B Fig), consistent with the result that KOSR did not cause ROS increase in these cells (Fig 6C).


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)

Combined inhibition of BCR-ABL and AKT overcame the protective effect of KOSR.(A) Induction of p-AKT at 1 hour after treatment of cells with the indicated concentrations of H2O2. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (B) Rapid Induction of p-AKT upon media switch. K562 cells were cultured in the regular or KOSR ±1 μM of imatinib for 1 hour. (C) AKT-PH-domain inhibitor MK2206 blocked p-AKT increase. K562 cells were pre-treated with 0, 0.1 or 3 μM of MK2206 in the regular media for 24 hours, then re-plated either in the regular or the KOSR media with the same doses of MK2206 used as in pre-treatments. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (D) Overcoming imatinib-resistance with AKT-PH-domain inhibitor MK2206. K562 cells were pre-treated with MK2206 (0, 0.1, 1, 5 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of MK2206 ± imatinib (1 μM). Survival was measured by clonogenic assay. The values are means ± SEM (n = 6). *, p<0.05. (E) Overcoming imatinib-resistance with PI3 kinase inhibitor, SF1126. K562 cells were pre-treated with SF1126 (0, 20, 40, 60 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of SF1126 ± 1 μM of imatinib. MTT assay was performed after 3 days to determine the relative cell number. The values are means ± SEM (n = 8). *, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140585.g007: Combined inhibition of BCR-ABL and AKT overcame the protective effect of KOSR.(A) Induction of p-AKT at 1 hour after treatment of cells with the indicated concentrations of H2O2. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (B) Rapid Induction of p-AKT upon media switch. K562 cells were cultured in the regular or KOSR ±1 μM of imatinib for 1 hour. (C) AKT-PH-domain inhibitor MK2206 blocked p-AKT increase. K562 cells were pre-treated with 0, 0.1 or 3 μM of MK2206 in the regular media for 24 hours, then re-plated either in the regular or the KOSR media with the same doses of MK2206 used as in pre-treatments. The levels of p-AKT (Ser473) and total AKT were determined by western blotting. (D) Overcoming imatinib-resistance with AKT-PH-domain inhibitor MK2206. K562 cells were pre-treated with MK2206 (0, 0.1, 1, 5 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of MK2206 ± imatinib (1 μM). Survival was measured by clonogenic assay. The values are means ± SEM (n = 6). *, p<0.05. (E) Overcoming imatinib-resistance with PI3 kinase inhibitor, SF1126. K562 cells were pre-treated with SF1126 (0, 20, 40, 60 μM) in the regular media. After 24 hours, cells were re-plated in the regular or the KOSR media with indicated doses of SF1126 ± 1 μM of imatinib. MTT assay was performed after 3 days to determine the relative cell number. The values are means ± SEM (n = 8). *, p<0.05.
Mentions: It has been shown that ROS, such as hydrogen peroxide (H2O2), can activate AKT to promote cell survival [50]. We therefore tested the effect of H2O2 on p-AKT levels in K562 and LAMA-84 cells. We found that H2O2 treatment raised the levels of p-AKT in K562 but not LAMA-84 cells (Fig 7A). Furthermore, we found that the levels of p-AKT also increased during the first 1 to 3 hours after switching K562 cells to the KOSR-media (Fig 7B, S5A and S5B Fig). This KOSR-induced p-AKT increase was not found in LAMA-84 cells (S5B Fig), consistent with the result that KOSR did not cause ROS increase in these cells (Fig 6C).

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