Cuneo Kyle C, Morgan Meredith A, Davis Mary A, Parcels Leslie A, Parcels Joshua, Karnak David, Ryan Caila, Liu Na, Maybaum Jonathan, Lawrence Theodore S
Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
Int J Radiat Oncol Biol Phys. 2016 Jun 1;95(2):782-90. doi: 10.1016/j.ijrobp.2016.01.028. Epub 2016 Jan 22.
Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC).
Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization.
Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation.
Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.
Wee1激酶抑制剂在缺乏G1期检查点的细胞中是有效的放射增敏剂。在本研究中，我们检测了抑制Wee1激酶对诱导肝细胞癌（HCC）复制应激的潜在影响。
合并来自Oncomine数据库的五个独立数据集，比较HCC与正常组织中的基因表达，并分析与Wee1敏感性相关的特定标志物。然后，我们进行了一系列体外实验，以研究抑制Wee1对不同p53突变状态的受辐照HCC细胞系的影响。使用抗γH2AX和磷酸化组蛋白H3抗体与碘化丙啶进行克隆形成存活分析和流式细胞术，以研究AZD1775对存活、细胞周期和DNA修复的影响。此外，使用富含核苷的培养基来检测改变核苷酸库对Wee1靶向放射增敏的影响。
我们对Oncomine数据库的分析发现，HCC中存在高水平的CDK1和其他细胞周期调节因子，表明其对Wee1敏感。在我们的体外实验中，用AZD1775处理使Hep3B、Huh7和HepG2细胞系产生放射增敏和化学增敏作用，并与γH2AX灶延迟消退和全核γH2AX染色诱导相关。抑制Wee1减弱了辐射诱导的Hep3B（TP53缺失）和Huh7（TP53突变）细胞系中的G2期阻滞，但在TP53野生型细胞系HepG2中未减弱。补充核苷可逆转AZD1775的放射增敏作用，并减少辐射后全核γH2AX染色的细胞数量。
无论细胞的p53突变状态如何，抑制Wee1均会产生放射增敏作用。在本研究中，我们首次表明，核苷酸过度消耗引起的复制应激在AZD1775诱导的放射增敏中起重要作用。
