Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release

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.

敲除血清替代物（Knockout serum replacement，KOSR）是一种常用于替代血清进行干细胞培养的营养补充剂。本研究证实，KOSR对BCR-ABL癌基因转化的慢性髓系白血病（chronic myelogenous leukemia，CML）细胞具有促存活活性。BCR-ABL酪氨酸激酶抑制剂可通过激活促凋亡蛋白BIM，同时抑制抗凋亡蛋白BCL2、BCLxL与MCL1来杀伤CML细胞。本研究发现，KOSR可使CML细胞免受BCR-ABL抑制剂——伊马替尼（imatinib）、达沙替尼（dasatinib）及尼罗替尼（nilotinib）——的杀伤作用。KOSR的这种保护作用具有可逆性，且并非源于耐药克隆的选择性增殖。在经KOSR保护的CML细胞中，伊马替尼仍可抑制BCR-ABL酪氨酸激酶活性，降低STAT、ERK及AKT的磷酸化水平，下调BCL2、BCLxL与MCL1的表达并上调BIM的表达。然而，上述促凋亡相关变化并未引发线粒体细胞色素c的释放。对从KOSR培养的CML细胞中分离的线粒体进行实验，结果显示加入重组BIM蛋白同样无法引发细胞色素c的释放。除酪氨酸激酶抑制剂外，KOSR还可保护细胞免受氧化应激诱导剂甲萘醌（menadione）的损伤，但无法保护细胞免受DNA损伤剂的侵害。将培养基从血清更换为KOSR后，在受KOSR保护的CML细胞中会出现活性氧水平与AKT磷酸化的一过性升高，而在未受该营养补充剂保护的细胞中则无此现象。使用PH结构域抑制剂MK2206处理KOSR培养的细胞，可阻断AKT磷酸化、消除BIM耐药线粒体的形成并诱导细胞死亡。上述结果表明，KOSR具有细胞背景依赖性的促存活活性，该活性与AKT激活及BIM诱导的线粒体细胞色素c释放抑制密切相关。