McNeely Samuel C, Taylor B Frazier, States J Christopher
Department of Pharmacology and Toxicology, Center for Environmental Genomics and Integrative Biology, Center for Genetics and Molecular Medicine and Brown Cancer Center, University of Louisville, 570 S. Preston St. Suite 221, Louisville, KY 40202, USA.
Toxicol Appl Pharmacol. 2008 Aug 15;231(1):61-7. doi: 10.1016/j.taap.2008.03.020. Epub 2008 Apr 9.
A375 human malignant melanoma cells undergo mitotic arrest-associated apoptosis when treated with pharmacological concentrations of sodium arsenite, a chemotherapeutic for acute promyelocytic leukemia. Our previous studies indicated that decreased arsenite sensitivity correlated with reduced mitotic spindle checkpoint function and reduced expression of the checkpoint protein BUBR1. In the current study, arsenite induced securin and cyclin B stabilization, BUBR1 phosphorylation, and spindle checkpoint activation. Arsenite also increased activating cyclin dependent kinase 1 (CDK1) Thr(161) phosphorylation but decreased inhibitory Tyr15 phosphorylation. Mitotic arrest resulted in apoptosis as indicated by colocalization of mitotic phospho-Histone H3 with active caspase 3. Apoptosis was associated with BCL-2 Ser70 phosphorylation. Inhibition of CDK1 with roscovitine in arsenite-treated mitotic cells inhibited spindle checkpoint maintenance as inferred from reduced BUBR1 phosphorylation, reduced cyclin B expression, and diminution of mitotic index. Roscovitine also reduced BCL-2 Ser70 phosphorylation and protected against apoptosis, suggesting mitotic arrest caused by hyperactivation of CDK1 directly or indirectly leads to BCL-2 phosphorylation and apoptosis. In addition, suppression of BUBR1 with siRNA prevented arsenite-induced mitotic arrest and apoptosis. These findings provide insight into the mechanism of arsenic's chemotherapeutic action and indicate a functional spindle checkpoint may be required for arsenic-sensitivity.
用治疗急性早幼粒细胞白血病的化疗药物亚砷酸钠的药理浓度处理时，A375人恶性黑色素瘤细胞会经历有丝分裂停滞相关的凋亡。我们之前的研究表明，亚砷酸盐敏感性降低与有丝分裂纺锤体检查点功能降低以及检查点蛋白BUBR1表达减少相关。在当前研究中，亚砷酸盐诱导了securin和细胞周期蛋白B的稳定、BUBR1磷酸化以及纺锤体检查点激活。亚砷酸盐还增加了活性细胞周期蛋白依赖性激酶1（CDK1）苏氨酸（161）磷酸化，但降低了抑制性酪氨酸15磷酸化。有丝分裂停滞导致凋亡，这可通过有丝分裂磷酸化组蛋白H3与活性半胱天冬酶3的共定位来表明。凋亡与BCL-2丝氨酸70磷酸化相关。在亚砷酸盐处理的有丝分裂细胞中用roscovitine抑制CDK1，从BUBR1磷酸化减少、细胞周期蛋白B表达降低和有丝分裂指数减小可推断出其抑制了纺锤体检查点维持。Roscovitine还降低了BCL-2丝氨酸70磷酸化并防止凋亡，表明CDK1过度激活直接或间接导致的有丝分裂停滞会导致BCL-2磷酸化和凋亡。此外，用小干扰RNA抑制BUBR1可防止亚砷酸盐诱导的有丝分裂停滞和凋亡。这些发现为砷的化疗作用机制提供了见解，并表明功能性纺锤体检查点可能是砷敏感性所必需的。
