Wang Z, Van Tuyle G, Conrad D, Fisher P B, Dent P, Grant S
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0230, USA.
Cancer Res. 1999 Mar 15;59(6):1259-67.
The effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction of classic apoptotic morphology, DNA fragmentation, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and underphosphorylation of the retinoblastoma protein (pRb) than their empty-vector counterparts. Enhanced susceptibility of antisense-expressing cells to ara-C was accompanied by a corresponding reduction in clonogenic and suspension culture growth. The increased sensitivity of these cells to ara-C-mediated lethality could not be attributed to cytokinetic perturbations, nor did ara-CTP formation or (ara-C)DNA incorporation differ significantly between the cell lines. Moreover, synchronization of p21 antisense-expressing cells in S-phase by aphidicolin block resulted in a further increase in ara-C-mediated apoptosis, suggesting enhanced drug sensitivity of the S-phase cell fraction. After exposure to ara-C, p21 antisense-expressing cells displayed a greater decline in mitochondrial membrane potential (deltapsi(m)) and generation of reactive oxygen species than their empty-vector counterparts, as well as early potentiation (e.g., within 2-4 h) of cytochrome c release into the cytosolic S-100 fraction. Lastly, ara-C-mediated increases in mitogen-activated protein kinase activity over basal levels were attenuated in p21 antisense-expressing cells. Collectively, these findings indicate that dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 increases the susceptibility of U937 human leukemia cells to ara-C-related lethality, and this phenomenon occurs as a relatively early event that is independent of cell cycle or pharmacodynamic factors and is associated with mitochondrial perturbations implicated in activation of the apoptotic protease cascade.
研究了细胞周期蛋白依赖性激酶抑制剂p21WAF1/CIP1失调对U937单核细胞白血病细胞对1-β-D-阿拉伯呋喃糖基胞嘧啶（ara-C）凋亡反应的影响。用1μM ara-C处理6小时后，稳定转染p21WAF1/CIP1反义构建体的细胞比其空载体对照细胞对经典凋亡形态、DNA片段化、caspase-3激活、聚（ADP-核糖）聚合酶降解和成视网膜细胞瘤蛋白（pRb）的低磷酸化诱导更敏感。反义表达细胞对ara-C的易感性增强伴随着克隆形成和悬浮培养生长的相应降低。这些细胞对ara-C介导的致死性增加的敏感性不能归因于细胞动力学扰动，细胞系之间ara-CTP的形成或（ara-C）DNA掺入也没有显著差异。此外，通过阿非科林阻滞使p21反义表达细胞在S期同步化导致ara-C介导的凋亡进一步增加，表明S期细胞部分的药物敏感性增强。暴露于ara-C后，p21反义表达细胞的线粒体膜电位（Δψm）下降和活性氧生成比其空载体对照细胞更大，并且细胞色素c释放到胞质S-100部分的早期增强（例如，在2-4小时内）。最后，在p21反义表达细胞中，ara-C介导的丝裂原活化蛋白激酶活性超过基础水平的增加减弱。总的来说，这些发现表明细胞周期蛋白依赖性激酶抑制剂p21WAF1/CIP1的失调增加了U937人白血病细胞对ara-C相关致死性的易感性，并且这种现象作为一个相对早期的事件发生，独立于细胞周期或药效学因素，并且与参与凋亡蛋白酶级联激活的线粒体扰动有关。
