Cartee L, Wang Z, Decker R H, Chellappan S P, Fusaro G, Hirsch K G, Sankala H M, Dent P, Grant S
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298, USA.
Cancer Res. 2001 Mar 15;61(6):2583-91.
Interactions between the cyclin-dependent kinase inhibitor (CDKI) flavopiridol (FP) and phorbol 12-myristate 13-acetate (PMA) were examined in U937 human leukemia cells in relation to differentiation and apoptosis. Simultaneous, but not sequential, exposure of U937 cells to 100 nM FP and 10 nM PMA significantly increased apoptosis manifested by characteristic morphological features, mitochondrial dysfunction, caspase activation, and poly(ADP-ribose) polymerase cleavage while markedly inhibiting cellular differentiation, as reflected by diminished plastic adherence and CD11b expression. Enhanced apoptosis in U937 cells was associated with an early caspase-independent increase in cytochrome c release and accompanied by a substantial decline in leukemic cell clonogenicity. Moreover, PMA/FP cotreatment significantly increased apoptosis in HL-60 promyelocytic leukemia cells and in U937 cells ectopically expressing the Bcl-2 protein. In U937 cells, coadministration of FP blocked PMA-induced expression and reporter activity of the CDKI p21WAF/CIP1 and triggered caspase-mediated cleavage of the CDKI p27KIP1. Coexposure to FP also resulted in a more pronounced and sustained activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase cascade after PMA treatment, although disruption of this pathway by the mitogen-activated protein kinase kinase 1 inhibitor U0126 did not prevent potentiation of apoptosis. FP accelerated PMA-mediated dephosphorylation of the retinoblastoma protein (pRb), an event followed by pRb cleavage culminating in the complete loss of underphosphorylated pRb (approximately Mr 110,000) by 24 h. Finally, gel shift analysis revealed that coadministration of FP with PMA for 8 h led to diminished E2F/pRb binding compared to the effects of PMA alone. Collectively, these findings indicate that FP modulates the expression/activity of multiple signaling and cell cycle regulatory proteins in PMA-treated leukemia cells and that such alterations are associated with mitochondrial damage and apoptosis rather than maturation. These observations also raise the possibility that combining CDKIs and differentiation-inducing agents may represent a novel antileukemic strategy.
在U937人白血病细胞中，研究了细胞周期蛋白依赖性激酶抑制剂（CDKI）黄酮哌啶醇（FP）与佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）之间关于分化和凋亡的相互作用。U937细胞同时（而非序贯）暴露于100 nM FP和10 nM PMA时，显著增加了凋亡，表现为特征性形态学特征、线粒体功能障碍、半胱天冬酶激活和聚（ADP - 核糖）聚合酶裂解，同时明显抑制细胞分化，这可通过塑料贴壁和CD11b表达减少反映出来。U937细胞中凋亡增强与细胞色素c释放早期非半胱天冬酶依赖性增加相关，并伴有白血病细胞克隆形成能力的显著下降。此外，PMA/FP联合处理显著增加HL - 60早幼粒细胞白血病细胞和异位表达Bcl - 2蛋白的U937细胞中的凋亡。在U937细胞中，FP的共同给药阻断了PMA诱导的CDKI p21WAF/CIP1的表达和报告活性，并触发了半胱天冬酶介导的CDKI p27KIP1的裂解。共同暴露于FP还导致在PMA处理后丝裂原活化蛋白激酶激酶/细胞外信号调节蛋白激酶级联反应更明显和持续的激活，尽管丝裂原活化蛋白激酶激酶1抑制剂U0126对该途径的破坏并未阻止凋亡的增强。FP加速了PMA介导的视网膜母细胞瘤蛋白（pRb）的去磷酸化，这一事件随后是pRb裂解，最终在24小时时完全丧失低磷酸化的pRb（约110,000分子量）。最后，凝胶迁移分析表明，与单独使用PMA的效果相比，FP与PMA共同给药8小时导致E2F/pRb结合减少。总体而言，这些发现表明FP调节PMA处理的白血病细胞中多种信号和细胞周期调节蛋白的表达/活性，并且这种改变与线粒体损伤和凋亡而非成熟相关。这些观察结果还提出了联合使用CDKIs和分化诱导剂可能代表一种新型抗白血病策略的可能性。
