Mechanism of Cancer Cell Death Induced by Depletion of an Essential Replication Regulator

BackgroundDepletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized. Methodology/Principal FindingsWe have used the recently-developed cell cycle indicator, Fucci, to precisely characterize the cell death process induced by Cdc7 depletion. We have also generated and utilized similar fluorescent cell cycle indicators using fusion with other cell cycle regulators to analyze modes of cell death in live cells in both p53-positive and -negative backgrounds. We show that distinct cell-cycle responses are induced in p53-positive and -negative cells by Cdc7 depletion. p53-negative cells predominantly arrest temporally in G2-phase, accumulating CyclinB1 and other mitotic regulators. Prolonged arrest at G2-phase and abrupt entry into aberrant M-phase in the presence of accumulated CyclinB1 are followed by cell death at the post-mitotic state. Abrogation of cytoplasmic CyclinB1 accumulation partially decreases cell death. The ATR-MK2 pathway is responsible for sequestration of CyclinB1 with 14-3-3σ protein. In contrast, p53-positive cancer cells do not accumulate CyclinB1, but appear to die mostly through entry into aberrant S-phase after Cdc7 depletion. The combination of Cdc7 inhibition with known anti-cancer agents significantly stimulates cell death effects in cancer cells in a genotype-dependent manner, providing a strategic basis for future combination therapies. ConclusionsOur results show that the use of Fucci, and similar fluorescent cell cycle indicators, offers a convenient assay system with which to identify cell cycle events associated with cancer cell death. They also indicate genotype-specific cell death modes induced by deficient initiation of DNA replication in cancer cells and its potential exploitation for development of efficient cancer therapies.

背景 复制因子的耗竭常会引发癌细胞死亡。Cdc7作为DNA复制起始所必需的激酶，其耗竭可诱导癌细胞死亡，且不受p53状态的影响，但目前尚未明确其诱导细胞死亡的具体通路。
研究方法与主要结果 本研究借助新近开发的细胞周期指示剂Fucci，精准表征了Cdc7耗竭诱导的细胞死亡过程。同时，我们通过与其他细胞周期调控因子融合构建了同类荧光细胞周期指示剂，用于分析p53阳性与阴性背景下活细胞的细胞死亡模式。研究表明，Cdc7耗竭在p53阳性与阴性癌细胞中诱导了截然不同的细胞周期应答反应。p53阴性癌细胞主要在G2期暂时停滞，伴随CyclinB1及其他有丝分裂调控因子的蓄积。在G2期长期停滞以及在CyclinB1蓄积状态下异常进入M期后，癌细胞会在有丝分裂后状态下发生死亡。阻断细胞质CyclinB1的蓄积可部分降低细胞死亡率。ATR-MK2信号通路负责将CyclinB1与14-3-3σ蛋白结合并隔离。与之相反，p53阳性癌细胞并不会出现CyclinB1的蓄积，其细胞死亡主要通过在Cdc7耗竭后异常进入S期实现。将Cdc7抑制与已知抗癌药物联合使用时，可显著增强癌细胞死亡效应，且该效应具有基因型依赖性，为未来的联合治疗策略提供了理论依据。
结论 本研究结果显示，使用Fucci及同类荧光细胞周期指示剂，可构建便捷的检测系统，用于识别与癌细胞死亡相关的细胞周期事件。同时，本研究还揭示了DNA复制起始缺陷诱导的癌细胞死亡模式具有基因型特异性，这为开发高效的癌症治疗手段提供了潜在方向。