Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE, USA.
Breast Cancer Res. 2012 Apr 11;14(2):R60. doi: 10.1186/bcr3164.
In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G2/M cell-cycle arrest. Previous studies from our laboratory showed that the G2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR.
With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor.
IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis.
Studies in this report suggest that Rac1 GTPase plays an essential role in the activation of IR-induced ERK1/2 signaling and subsequent G2/M checkpoint response. Furthermore, results also support a role for Rac1 in promoting cell survival after irradiation treatment.
为了应对γ射线（IR）诱导的双链 DNA 断裂，细胞会经历细胞周期停滞，以便在重新进入细胞周期之前有时间进行 DNA 修复。G2/M 检查点的激活涉及到ataxia telangiectasia mutated (ATM)/ATM-和 rad3-related (ATR) 激酶的激活和 Cdc25 磷酸酶的抑制，从而抑制 Cdc2 激酶，随后导致 G2/M 细胞周期停滞。我们实验室之前的研究表明，MCF-7 乳腺癌细胞在受到 IR 照射后 G2/M 检查点的激活依赖于细胞外信号调节蛋白激酶 1 和 2 (ERK1/2) 信号的激活。在本研究中，我们研究了 Ras-related C3 botulinum toxin substrate 1 (Rac1) 鸟嘌呤三磷酸酶 (GTPase) 在 IR 诱导的 G2/M 检查点反应和 ERK1/2 激活中的作用，以及在 IR 照射后细胞存活中的作用。
使用 Rac1 特异性抑制剂、显性负突变体 Rac1 (N17Rac1) 和特异性小干扰 RNA，研究 Rac1 对人乳腺癌细胞中 IR 诱导的 G2/M 检查点反应和 ERK1/2 激活的影响。此外，还使用 Rac1 特异性抑制剂评估 Rac1 对照射后细胞存活的影响。
IR 暴露于 MCF-7 乳腺癌细胞会导致 Rac1 GTPase 的显著激活。此外，使用特异性抑制剂、显性负 Rac1 突变体或特异性 siRNA 抑制 Rac1，会导致 IR 诱导的 G2/M 阻滞减弱，同时伴随 ATM、ATR、Chk1 和 Chk2 激酶的激活以及 Cdc2-Tyr15 的磷酸化减弱。此外，Rac1 抑制或 Rac1 表达减少也会消除 IR 诱导的丝裂原激活蛋白激酶激酶 1 和 2 (MEK1/2) 和 ERK1/2 的磷酸化。最终，Rac1 的抑制显著增加了细胞对 IR 暴露的敏感性，这涉及到细胞凋亡的诱导。
本研究表明 Rac1 GTPase 在激活 IR 诱导的 ERK1/2 信号和随后的 G2/M 检查点反应中起着至关重要的作用。此外，结果还支持 Rac1 在促进照射后细胞存活中的作用。
