H2AX磷酸化型微核的形成机制及生物学意义

Formation of micronuclei (MN) is indicative of genomic instability and exposure to environmental mutagens. We recently characterized a subclass of MN, MN-gamma H2AX, bearing the histone modification mark ?H2AX throughout the MN. We found that MN-gamma H2AX are preferentially induced by replication stress. We hypothesize that the frequency of MN-gamma H2AX will also reflect the capacity of homologous recombination (HR), because collapsed replication forks are primarily repaired by HR. We will therefore determine whether the formation of MN-gamma H2AX also reflects the capacity of homologous recombination (HR). We will test how the formation of MN-gamma H2AX will be affected when representative members in HR pathways are downregulated by RNA interference. Oncogene activation is known to induce DNA hyperreplication, we will determine whether RAS V12, CDC6 and cyclin E will lead to increased formation of MN-gamma H2AX. We will also examine the fates of cells that harbor MN-gamma H2AX. Findings made from this study may provide important insights into the genomic instability usually associated with HR defect and oncogene activation. Formation of MN-gamma H2AX may also serve as a simple indicator of HR capacity in cancer cells, thus facilitating the screening of cancer cells that are sensitive to PARP inhibitors.

微核是常用于评估遗传诱变物和基因组不稳定性的细胞学指标。我们最近发现有一亚类微核呈现均一的H2AX磷酸化，因而我们将其称之为H2AX 磷酸化型微核(MN-gamma H2AX)。这类微核可占总微核数的1/5~1/3, 并可被复制压力特异诱导。因为复制叉塌垮引起的复制压力主要是由同源重组所修复的，我们推测该类微核的频率可能反映了细胞同源重组的能力，我们将通过干预同源重组通路的不同成员检测MN-gamma H2AX的发生情况；并探讨RASV12等癌基因的激活引起的复制压力对MN-gamma H2AX发生的影响；还将观察携带MN-gamma H2AX的细胞趋向凋亡或衰老的生物学后果。本研究会帮助我们认识同源重组缺陷和癌基因激活如何导致基因组不稳定性而诱导MN-gamma H2AX的形成，该类微核将有望作为筛选同源重组缺陷的肿瘤细胞的简易指标，用于判别PARP 抑制剂类抗癌药物的适应症。

微核是基因组不稳定性的常用指标。我们实验室先期报道了一类被我们称之为MN-gamma-H2AX(+)的H2AX磷酸化型微核。这一亚类微核占总微核数的1/5 到1/3。在本课题资助下我们进一步研究了MN-gamma-H2AX(+)的发生机制和生物学意义。我们发现除复制压力外，氧化压力也能特异诱导该亚类微核的发生。降低氧化压力能够削弱复制压力对微核的诱导作用。氧化压力与复制压力相互促进各自对微核的诱导。干扰若干同源重组通路关键成员如RAD51的表达或药物抑制其功能均能特异诱导MN-gamma-H2AX(+)的发生。我们还发现CHK1所介导的S期或G2期停滞是MN-gamma-H2AX(+)的发生的必要条件，但不是充分条件。当S期或G2期停滞伴随DNA双链断裂时， MN-gamma-H2AX(+)才能被有效诱导。这些结果进一步明确了复制压力和氧化压力诱导基因组不稳定性的机制，对同源重组修复和细胞周期等因素在基因组不稳定性中的作用及机制也提供了新的认识。