Smith Hannah L, Prendergast Lisa, Curtin Nicola J
Newcastle Centre for Cancer, Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
Cancer Research UK Drug Discovery Unit, Newcastle Centre for Cancer, Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
Cancers (Basel). 2020 Apr 4;12(4):878. doi: 10.3390/cancers12040878.
PARP inhibition results in the accumulation of DNA SSBs, causing replication stress (RS) and lesions that can only be resolved by homologous recombination repair (HRR). Defects in HRR, e.g., due to mutation, confer profound sensitivity to PARP inhibitor (PARPi) cytotoxicity. In response to RS, CHK1 is activated to signal to S and G2/M cell cycle checkpoints and also to HRR. To determine the relative contribution of these two functions of CHK1 to survival following PARPi exposure, we investigated the effects of rucaparib (a PARPi) and PF-477736 (a CHK1 inhibitor) alone and in combination in cells with mutated and corrected . The mutated V-C8 cells were 1000× more sensitive to rucaparib cytotoxicity than their matched corrected V-C8.B2 cells, but no more sensitive to PF-477736 despite having seven-fold higher levels of RS. PF-477736 caused a five-fold enhancement of rucaparib cytotoxicity in the V-C8.B2 cells, but no enhancement in the V-C8 cells. This differential sensitivity was not due to a difference in PARP1 or CHK1 expression or activity. PF-477736 increased rucaparib-induced RS (γH2AX foci) and completely inhibited RAD51 focus formation, indicating a profound suppression of HRR. Our data suggested that inhibition of HRR was the main mechanism of sensitisation to rucaparib, compounded with an inhibition of cell cycle checkpoints by PF-477736.
聚（ADP - 核糖）聚合酶（PARP）抑制导致DNA单链断裂（SSB）积累，引发复制应激（RS）以及只能通过同源重组修复（HRR）解决的损伤。HRR缺陷，例如由于突变导致的，会使细胞对PARP抑制剂（PARPi）的细胞毒性产生高度敏感性。响应RS时，CHK1被激活，向S期和G2/M期细胞周期检查点以及HRR发出信号。为了确定CHK1的这两种功能对PARPi暴露后细胞存活的相对贡献，我们研究了鲁卡帕尼（一种PARPi）和PF - 477736（一种CHK1抑制剂）单独及联合使用对具有突变和校正的细胞的影响。突变的V - C8细胞对鲁卡帕尼细胞毒性的敏感性比其匹配的校正后的V - C8.B2细胞高1000倍，但尽管RS水平高7倍，对PF - 477736并不更敏感。PF - 477736使V - C8.B2细胞中鲁卡帕尼的细胞毒性增强了5倍，但在V - C8细胞中没有增强。这种差异敏感性并非由于PARP1或CHK1表达或活性的差异。PF - 477736增加了鲁卡帕尼诱导的RS（γH2AX焦点）并完全抑制了RAD51焦点形成，表明对HRR有显著抑制作用。我们的数据表明，抑制HRR是对鲁卡帕尼敏感化的主要机制，同时PF - 477736对细胞周期检查点也有抑制作用。
