title: Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.
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.
authors.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.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ishii
authors.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.
authors.forename: May Keu
authors.initials: MK
authors.lastname: Nhiayi
authors.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.
authors.forename: Edison
authors.initials: E
authors.lastname: Tse
authors.affiliation: Division of Biological Sciences, University of California San Diego, San Diego, California, United States of America.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Clinical and Molecular Bio-Medicine, University of Catania, Catania, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Massimino
authors.affiliation: Moores Cancer Center, University of California San Diego, San Diego, California, United States of America; Department of Pediatrics, School of Medicine, University of California San Diego, San Diego, California, United States of America.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Durden
authors.affiliation: Department of Clinical and Molecular Bio-Medicine, University of Catania, Catania, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Vigneri
authors.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; Division of Biological Sciences, University of California San Diego, San Diego, California, United States of America.
authors.forename: Jean Y J
authors.initials: JY
authors.lastname: Wang
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism*; D000069439:Dasatinib / Q000494:pharmacology; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000068877:Imatinib Mesylate / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D003470:Culture Media; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D045304:Cytochromes c; D016044:Fusion Proteins, bcr-abl; C498826:nilotinib; D000069439:Dasatinib