Agyeman Akwasi, Mazumdar Tapati, Houghton Janet A
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
Oncotarget. 2012 Aug;3(8):854-68. doi: 10.18632/oncotarget.586. Epub 2012 Aug 20.
Transcriptional regulation of the Hedgehog (HH) signaling response is mediated by GLI genes (GLI1, GLI2) downstream of SMO, that are also activated by oncogenic signaling pathways. We have demonstrated the importance of targeting GLI downstream of SMO in the induction of cell death in human colon carcinoma cells. In HT29 cells inhibition of GLI1/GLI2 by the small molecule inhibitor GANT61 induced DNA double strand breaks (DSBs) and activation of ATM, MDC1 and NBS1; γH2AX and MDC1, NBS1 and MDC1 co-localized in nuclear foci. Early activation of ATM was decreased by 24 hr, when p-NBS1(Ser343), activated by ATM, was significantly reduced in cell extracts. Bound γH2AX was detected in isolated chromatin fractions or nuclei during DNA damage but not during DNA repair. MDC1 was tightly bound to chromatin at 32 hr as cells accumulated in early S-phase prior to becoming subG1, and during DNA repair. Limited binding of NBS1 was detected at all times during DNA damage but was strongly bound during DNA repair. Transient overexpression of NBS1 protected HT29 cells from GANT61-induced cell death, while knockdown of H2AX by H2AXshRNA delayed DNA damage signaling. Data demonstrate following GLI1/GLI2 inhibition: 1) induction of DNA damage in cells that are also resistant to SMO inhibitors, 2) dynamic interactions between γH2AX, MDC1 and NBS1 in single cell nuclei and in isolated chromatin fractions, 3) expression and chromatin binding properties of key mediator proteins that mark DNA damage or DNA repair, and 4) the importance of NBS1 in the DNA damage response mechanism.
刺猬索尼克（HH）信号应答的转录调控由SMO下游的GLI基因（GLI1、GLI2）介导，这些基因也被致癌信号通路激活。我们已经证明，在人结肠癌细胞中诱导细胞死亡时，靶向SMO下游的GLI具有重要意义。在HT29细胞中，小分子抑制剂GANT61对GLI1/GLI2的抑制作用诱导了DNA双链断裂（DSB）以及ATM、MDC1和NBS1的激活；γH2AX与MDC1、NBS1共定位于核灶。当ATM早期激活在24小时时下降，由ATM激活的p-NBS1（Ser343）在细胞提取物中显著减少。在DNA损伤期间而非DNA修复期间，在分离的染色质组分或细胞核中检测到结合的γH2AX。随着细胞在进入亚G1期之前在早S期积累以及在DNA修复期间，MDC1在32小时时紧密结合于染色质。在DNA损伤期间始终检测到NBS1的有限结合，但在DNA修复期间NBS1强烈结合。NBS1的瞬时过表达保护HT29细胞免受GANT61诱导的细胞死亡，而H2AXshRNA对H2AX的敲低延迟了DNA损伤信号传导。数据表明在GLI1/GLI2抑制后：1）在对SMO抑制剂也具有抗性的细胞中诱导DNA损伤；2）γH2AX、MDC1和NBS1在单细胞核和分离的染色质组分中的动态相互作用；3）标记DNA损伤或DNA修复的关键介导蛋白的表达和染色质结合特性；4）NBS1在DNA损伤应答机制中的重要性。
