Liu Xiaojun, Guo Ying, Li Yexiong, Jiang Yingjun, Chubb Sherri, Azuma Atsushi, Huang Peng, Matsuda Akira, Hittelman Walter, Plunkett William
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Cancer Res. 2005 Aug 1;65(15):6874-81. doi: 10.1158/0008-5472.CAN-05-0288.
2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by beta-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.
2'-C-氰基-2'-脱氧-1-β-D-阿拉伯呋喃糖基胞嘧啶（CNDAC）是一种具有新型作用机制的核苷类似物，目前正在临床试验中进行评估。三磷酸CNDAC掺入DNA并在复制过程中延伸会导致由β消除直接引起的单链断裂。这些断裂或进一步加工产生的损伤会导致细胞停滞在G2期。本研究的目的是确定G2检查点激活的分子基础，并描述其废除的后果。来自不同人类组织的细胞系在CNDAC处理后经历G2期停滞，这表明对所造成损伤的反应存在共同机制。CNDAC诱导的G2期停滞是通过激活Chk1-Cdc25C-Cdk1/细胞周期蛋白B检查点途径实现的。检查点激活不需要Chk2、p38或p53。用7-羟基星孢菌素（UCN-01）抑制Chk1激酶可废除检查点途径，这表现为检查点蛋白的去磷酸化以及细胞通过有丝分裂进入G1期。细胞死亡在进入G1期后首先在血液学细胞系中明显出现。如组蛋白H2AX磷酸化所示，当ML-1细胞停滞在G2期时，由CNDAC掺入引发的DNA损伤转化为双链断裂。当用UCN-01废除CNDAC停滞细胞的G2检查点时，一些断裂表现为染色体畸变，但在仅用CNDAC处理期间逃入有丝分裂的少数细胞群体中也有出现。这些发现为在血液系统恶性肿瘤治疗中结合CNDAC与细胞周期检查点调节抑制剂设计新策略提供了机制依据。
