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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

KOSR does not cause growth arrest and is continuously required to induce imatinib resistance.(A) K562 cells proliferate in KOSR-media without or with imatinib (IM). Cells were seeded (2x105cells/ml) in RPMI+10%FBS, RPMI+20%KOSR or RPMI+20%KOSR+10%FBS ± 1 μM of imatinib on day 0 and live cell numbers counted every 24 hours. After 3-days, the cultures were split (2x105cells/ml) into fresh media and counting continued for 3 more days. The K562 cells cultured in the RPMI+FBS+IM media and the LAMA-84 cells cultured in the three media containing IM were not split due to low live cell numbers and simply carried forward (indicated by the continuity of the curves). The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment. (B) KOSR does not cause cell cycle arrest. K562 cells were cultured in RPMI+FBS or RPMI+KOSR media ± 1 μM of imatinib for 3 days. Cells were fixed, stained with propidium iodide and the DNA contents (cell cycle distributions) were determined by flow cytometry. The data shown are the mean from four experimental samples. (C) KOSR-induced resistance to imatinib is reversible. K562 cells were pre-cultured with 1 μM of imatinib (IM) in RPMI+KOSR or RPMI+KOSR+FBS media for 6 days with splitting on day-3. Cells were then collected and re-plated in RPMI+KOSR, RPMI+KOSR+FBS or RPMI+FBS media in the presence of 1 μM of IM (day 0). Live cell numbers were counted daily for 3 days. The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment.
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pone.0140585.g003: KOSR does not cause growth arrest and is continuously required to induce imatinib resistance.(A) K562 cells proliferate in KOSR-media without or with imatinib (IM). Cells were seeded (2x105cells/ml) in RPMI+10%FBS, RPMI+20%KOSR or RPMI+20%KOSR+10%FBS ± 1 μM of imatinib on day 0 and live cell numbers counted every 24 hours. After 3-days, the cultures were split (2x105cells/ml) into fresh media and counting continued for 3 more days. The K562 cells cultured in the RPMI+FBS+IM media and the LAMA-84 cells cultured in the three media containing IM were not split due to low live cell numbers and simply carried forward (indicated by the continuity of the curves). The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment. (B) KOSR does not cause cell cycle arrest. K562 cells were cultured in RPMI+FBS or RPMI+KOSR media ± 1 μM of imatinib for 3 days. Cells were fixed, stained with propidium iodide and the DNA contents (cell cycle distributions) were determined by flow cytometry. The data shown are the mean from four experimental samples. (C) KOSR-induced resistance to imatinib is reversible. K562 cells were pre-cultured with 1 μM of imatinib (IM) in RPMI+KOSR or RPMI+KOSR+FBS media for 6 days with splitting on day-3. Cells were then collected and re-plated in RPMI+KOSR, RPMI+KOSR+FBS or RPMI+FBS media in the presence of 1 μM of IM (day 0). Live cell numbers were counted daily for 3 days. The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment.

Mentions: It has been shown that quiescent CML stem cells are resistant to BCR-ABL kinase inhibitors [19]. Because KOSR caused a decrease in MTT readings (Fig 2 and S1D Fig) and clonogenic survival (Fig 1), we determined its effect on the growth of K562 (KOSR-responsive) and LAMA-84 (KOSR-non-responsive) cells. We cultured these two lines of CML cells in RPMI+FBS, RPMI+KOSR, and RPMI+FBS/KOSR media in the presence or the absence of IM (1 μM) and counted the number of live cells daily over six days during which cultures with sufficient live cells were split on day-3 (Fig 3A). We found that K562 cells proliferated in each of the three media, although the growth rate was reduced in KOSR-media without FBS (Fig 3A, upper left panel). Addition of IM inhibited K562 growth in the RPMI+FBS media (Fig 3A, upper right panel). However, addition of IM did not inhibit K562 growth in RPMI+KOSR, or RPMI+FBS/KOSR media (Fig 3A, upper right panel). We also determined the cell cycle distribution of K562 cells by FACS analysis of DNA content (Fig 3B). IM treatment increased the fraction of sub-G1 cells and decreased the fractions of G1 and G2/M cells in RPMI+FBS media. IM treatment did not increase the sub-G1 fraction in the RPMI+KOSR media. The FACS analysis showed an increase in the S/G2/M fractions when K562 cells were cultured in RPMI+KOSR media. When treated with IM in this media, the G1-fraction increased (Fig 3B), but cell proliferation was not inhibited (Fig 3A). These results show that KOSR protected K562 cells from IM without causing growth arrest.


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)

KOSR does not cause growth arrest and is continuously required to induce imatinib resistance.(A) K562 cells proliferate in KOSR-media without or with imatinib (IM). Cells were seeded (2x105cells/ml) in RPMI+10%FBS, RPMI+20%KOSR or RPMI+20%KOSR+10%FBS ± 1 μM of imatinib on day 0 and live cell numbers counted every 24 hours. After 3-days, the cultures were split (2x105cells/ml) into fresh media and counting continued for 3 more days. The K562 cells cultured in the RPMI+FBS+IM media and the LAMA-84 cells cultured in the three media containing IM were not split due to low live cell numbers and simply carried forward (indicated by the continuity of the curves). The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment. (B) KOSR does not cause cell cycle arrest. K562 cells were cultured in RPMI+FBS or RPMI+KOSR media ± 1 μM of imatinib for 3 days. Cells were fixed, stained with propidium iodide and the DNA contents (cell cycle distributions) were determined by flow cytometry. The data shown are the mean from four experimental samples. (C) KOSR-induced resistance to imatinib is reversible. K562 cells were pre-cultured with 1 μM of imatinib (IM) in RPMI+KOSR or RPMI+KOSR+FBS media for 6 days with splitting on day-3. Cells were then collected and re-plated in RPMI+KOSR, RPMI+KOSR+FBS or RPMI+FBS media in the presence of 1 μM of IM (day 0). Live cell numbers were counted daily for 3 days. The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment.
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Related In: Results  -  Collection

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pone.0140585.g003: KOSR does not cause growth arrest and is continuously required to induce imatinib resistance.(A) K562 cells proliferate in KOSR-media without or with imatinib (IM). Cells were seeded (2x105cells/ml) in RPMI+10%FBS, RPMI+20%KOSR or RPMI+20%KOSR+10%FBS ± 1 μM of imatinib on day 0 and live cell numbers counted every 24 hours. After 3-days, the cultures were split (2x105cells/ml) into fresh media and counting continued for 3 more days. The K562 cells cultured in the RPMI+FBS+IM media and the LAMA-84 cells cultured in the three media containing IM were not split due to low live cell numbers and simply carried forward (indicated by the continuity of the curves). The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment. (B) KOSR does not cause cell cycle arrest. K562 cells were cultured in RPMI+FBS or RPMI+KOSR media ± 1 μM of imatinib for 3 days. Cells were fixed, stained with propidium iodide and the DNA contents (cell cycle distributions) were determined by flow cytometry. The data shown are the mean from four experimental samples. (C) KOSR-induced resistance to imatinib is reversible. K562 cells were pre-cultured with 1 μM of imatinib (IM) in RPMI+KOSR or RPMI+KOSR+FBS media for 6 days with splitting on day-3. Cells were then collected and re-plated in RPMI+KOSR, RPMI+KOSR+FBS or RPMI+FBS media in the presence of 1 μM of IM (day 0). Live cell numbers were counted daily for 3 days. The data shown are the mean ± s.d. from three independent experiments with duplicates in each experiment.
Mentions: It has been shown that quiescent CML stem cells are resistant to BCR-ABL kinase inhibitors [19]. Because KOSR caused a decrease in MTT readings (Fig 2 and S1D Fig) and clonogenic survival (Fig 1), we determined its effect on the growth of K562 (KOSR-responsive) and LAMA-84 (KOSR-non-responsive) cells. We cultured these two lines of CML cells in RPMI+FBS, RPMI+KOSR, and RPMI+FBS/KOSR media in the presence or the absence of IM (1 μM) and counted the number of live cells daily over six days during which cultures with sufficient live cells were split on day-3 (Fig 3A). We found that K562 cells proliferated in each of the three media, although the growth rate was reduced in KOSR-media without FBS (Fig 3A, upper left panel). Addition of IM inhibited K562 growth in the RPMI+FBS media (Fig 3A, upper right panel). However, addition of IM did not inhibit K562 growth in RPMI+KOSR, or RPMI+FBS/KOSR media (Fig 3A, upper right panel). We also determined the cell cycle distribution of K562 cells by FACS analysis of DNA content (Fig 3B). IM treatment increased the fraction of sub-G1 cells and decreased the fractions of G1 and G2/M cells in RPMI+FBS media. IM treatment did not increase the sub-G1 fraction in the RPMI+KOSR media. The FACS analysis showed an increase in the S/G2/M fractions when K562 cells were cultured in RPMI+KOSR media. When treated with IM in this media, the G1-fraction increased (Fig 3B), but cell proliferation was not inhibited (Fig 3A). These results show that KOSR protected K562 cells from IM without causing growth arrest.

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