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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

Mitochondria from KOSR-cultured cells did not release cytochrome c when stimulated with recombinant BIM-EL in vitro.(A) In vitro-translated BIM-EL (extra long variant of BIM). Human BIM-EL, mouse Bim-EL and mouse Bim-EL-ΔBH3 translated in vitro using the TNT Quick coupled Transcription/Translation System reacted with the anti-BIM antibody. (B) Induction of cytochrome c release from mitochondria FBS-cultured K562 cells in the test tubes. Mitochondria isolated from FBS-cultured K562 cells were incubated with the indicated amounts of in vitro translated human, mouse and mutant proteins for 1 hour. The reactions were centrifuged and the levels of cytochrome c and COX4 in the supernatant and the pellet fractions were determined by immunoblotting. (C) Mitochondria from KOSR-cultured K562 cells were resistant to BIM-induced cytochrome c release. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated without or with in vitro translated human BIM-EL and the release of cytochrome c determined as in (B). (D) cBID stimulated cytochrome c release from isolated mitochondria. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated with the indicated concentrations of cBid and the release of cytochrome c determined as in (B). (E) Levels of endogenous and exogenous BCL2-family proteins in isolated mitochondria. Mitochondria isolated from K562 cells grown in the indicated culture media were incubated with in vitro translated human BIM-EL protein for 1 hour. The reaction mixtures were centrifuged and the supernatant and pellet fractions were immunoblotted with the indicated antibodies to detect the levels of the endogenous BCL2, BCLxL, MCL1 proteins and the exogenously added BIM-EL protein.
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pone.0140585.g005: Mitochondria from KOSR-cultured cells did not release cytochrome c when stimulated with recombinant BIM-EL in vitro.(A) In vitro-translated BIM-EL (extra long variant of BIM). Human BIM-EL, mouse Bim-EL and mouse Bim-EL-ΔBH3 translated in vitro using the TNT Quick coupled Transcription/Translation System reacted with the anti-BIM antibody. (B) Induction of cytochrome c release from mitochondria FBS-cultured K562 cells in the test tubes. Mitochondria isolated from FBS-cultured K562 cells were incubated with the indicated amounts of in vitro translated human, mouse and mutant proteins for 1 hour. The reactions were centrifuged and the levels of cytochrome c and COX4 in the supernatant and the pellet fractions were determined by immunoblotting. (C) Mitochondria from KOSR-cultured K562 cells were resistant to BIM-induced cytochrome c release. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated without or with in vitro translated human BIM-EL and the release of cytochrome c determined as in (B). (D) cBID stimulated cytochrome c release from isolated mitochondria. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated with the indicated concentrations of cBid and the release of cytochrome c determined as in (B). (E) Levels of endogenous and exogenous BCL2-family proteins in isolated mitochondria. Mitochondria isolated from K562 cells grown in the indicated culture media were incubated with in vitro translated human BIM-EL protein for 1 hour. The reaction mixtures were centrifuged and the supernatant and pellet fractions were immunoblotted with the indicated antibodies to detect the levels of the endogenous BCL2, BCLxL, MCL1 proteins and the exogenously added BIM-EL protein.

Mentions: Previous studies have established that IM-induced upregulation of the pro-apoptotic BH3-only BIM protein is required for the induction of apoptosis in K562 cells and in CML patient cells [43–46]. Because KOSR-cultured K562 cells escaped apoptosis despite the down-regulation of BCL2-proteins and the up-regulation of BIM, it is possible that the mitochondria in KOSR-cultured cells became resistant to BIM-induced MOMP. To test this possibility, we isolated and then incubated mitochondria in the test tubes with recombinant BIM-EL protein generated by in vitro transcription translation (IVT&T) [30, 31] (Fig 5A). In control experiments, we showed that in vitro translated BIM-EL (human BIM and mouse Bim) but not the mouse ΔBH3-Bim-EL caused cytochrome c release from mitochondria isolated from K562 cells cultured in the regular media (RPMI+ FBS) (Fig 5B and 5C). The amount of released-cytochrome c increased with increasing amount of IVT&T reactions added to the isolated mitochondria (Fig 5B). However, with mitochondria isolated from KOSR-cultured K562 cells, treatment with hBIM failed to cause cytochrome c release (Fig 5C). We also tested the effect of recombinant cleaved-BID (cBID) purified from bacteria on the release of cytochrome c from isolated mitochondria. In contrast to BIM-EL, cBID caused cytochrome c release from mitochondria derived from either the FBS-cultured or the KOSR-cultured K562 cells (Fig 5D), suggesting that the mitochondria from KOSR-cultured K562 cells were selectively resistant to BIM-induced cytochrome c release. As shown in Fig 5E, we found similar levels of BCL2, BCLxL and MCL1 proteins in the mitochondrial preparations from FBS-cultured and KOSR-cultured K562 cells. We also detected similar levels of the exogenously added BIM-EL proteins in these two mitochondrial pellet fractions after incubation (Fig 5E). These results showed that mitochondria from FBS-cultured and KOSR-cultured K562 cells contain similar levels of anti-apoptotic BCL2-family members that bind to BIM with similar capacity. However, the mitochondria from KOSR-cultured cells do not release cytochrome c at a level of BIM that caused mitochondria from FBS-cultured cells to release cytochrome c.


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)

Mitochondria from KOSR-cultured cells did not release cytochrome c when stimulated with recombinant BIM-EL in vitro.(A) In vitro-translated BIM-EL (extra long variant of BIM). Human BIM-EL, mouse Bim-EL and mouse Bim-EL-ΔBH3 translated in vitro using the TNT Quick coupled Transcription/Translation System reacted with the anti-BIM antibody. (B) Induction of cytochrome c release from mitochondria FBS-cultured K562 cells in the test tubes. Mitochondria isolated from FBS-cultured K562 cells were incubated with the indicated amounts of in vitro translated human, mouse and mutant proteins for 1 hour. The reactions were centrifuged and the levels of cytochrome c and COX4 in the supernatant and the pellet fractions were determined by immunoblotting. (C) Mitochondria from KOSR-cultured K562 cells were resistant to BIM-induced cytochrome c release. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated without or with in vitro translated human BIM-EL and the release of cytochrome c determined as in (B). (D) cBID stimulated cytochrome c release from isolated mitochondria. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated with the indicated concentrations of cBid and the release of cytochrome c determined as in (B). (E) Levels of endogenous and exogenous BCL2-family proteins in isolated mitochondria. Mitochondria isolated from K562 cells grown in the indicated culture media were incubated with in vitro translated human BIM-EL protein for 1 hour. The reaction mixtures were centrifuged and the supernatant and pellet fractions were immunoblotted with the indicated antibodies to detect the levels of the endogenous BCL2, BCLxL, MCL1 proteins and the exogenously added BIM-EL protein.
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Related In: Results  -  Collection

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pone.0140585.g005: Mitochondria from KOSR-cultured cells did not release cytochrome c when stimulated with recombinant BIM-EL in vitro.(A) In vitro-translated BIM-EL (extra long variant of BIM). Human BIM-EL, mouse Bim-EL and mouse Bim-EL-ΔBH3 translated in vitro using the TNT Quick coupled Transcription/Translation System reacted with the anti-BIM antibody. (B) Induction of cytochrome c release from mitochondria FBS-cultured K562 cells in the test tubes. Mitochondria isolated from FBS-cultured K562 cells were incubated with the indicated amounts of in vitro translated human, mouse and mutant proteins for 1 hour. The reactions were centrifuged and the levels of cytochrome c and COX4 in the supernatant and the pellet fractions were determined by immunoblotting. (C) Mitochondria from KOSR-cultured K562 cells were resistant to BIM-induced cytochrome c release. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated without or with in vitro translated human BIM-EL and the release of cytochrome c determined as in (B). (D) cBID stimulated cytochrome c release from isolated mitochondria. K562 cells were cultured in the indicated media for 2 days. Mitochondria were isolated and incubated with the indicated concentrations of cBid and the release of cytochrome c determined as in (B). (E) Levels of endogenous and exogenous BCL2-family proteins in isolated mitochondria. Mitochondria isolated from K562 cells grown in the indicated culture media were incubated with in vitro translated human BIM-EL protein for 1 hour. The reaction mixtures were centrifuged and the supernatant and pellet fractions were immunoblotted with the indicated antibodies to detect the levels of the endogenous BCL2, BCLxL, MCL1 proteins and the exogenously added BIM-EL protein.
Mentions: Previous studies have established that IM-induced upregulation of the pro-apoptotic BH3-only BIM protein is required for the induction of apoptosis in K562 cells and in CML patient cells [43–46]. Because KOSR-cultured K562 cells escaped apoptosis despite the down-regulation of BCL2-proteins and the up-regulation of BIM, it is possible that the mitochondria in KOSR-cultured cells became resistant to BIM-induced MOMP. To test this possibility, we isolated and then incubated mitochondria in the test tubes with recombinant BIM-EL protein generated by in vitro transcription translation (IVT&T) [30, 31] (Fig 5A). In control experiments, we showed that in vitro translated BIM-EL (human BIM and mouse Bim) but not the mouse ΔBH3-Bim-EL caused cytochrome c release from mitochondria isolated from K562 cells cultured in the regular media (RPMI+ FBS) (Fig 5B and 5C). The amount of released-cytochrome c increased with increasing amount of IVT&T reactions added to the isolated mitochondria (Fig 5B). However, with mitochondria isolated from KOSR-cultured K562 cells, treatment with hBIM failed to cause cytochrome c release (Fig 5C). We also tested the effect of recombinant cleaved-BID (cBID) purified from bacteria on the release of cytochrome c from isolated mitochondria. In contrast to BIM-EL, cBID caused cytochrome c release from mitochondria derived from either the FBS-cultured or the KOSR-cultured K562 cells (Fig 5D), suggesting that the mitochondria from KOSR-cultured K562 cells were selectively resistant to BIM-induced cytochrome c release. As shown in Fig 5E, we found similar levels of BCL2, BCLxL and MCL1 proteins in the mitochondrial preparations from FBS-cultured and KOSR-cultured K562 cells. We also detected similar levels of the exogenously added BIM-EL proteins in these two mitochondrial pellet fractions after incubation (Fig 5E). These results showed that mitochondria from FBS-cultured and KOSR-cultured K562 cells contain similar levels of anti-apoptotic BCL2-family members that bind to BIM with similar capacity. However, the mitochondria from KOSR-cultured cells do not release cytochrome c at a level of BIM that caused mitochondria from FBS-cultured cells to release cytochrome c.

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