探討WRN和BLM在BMVC4所誘導之DNA損傷反應和老化機制中所扮演之角色

Abstract

四股螺旋結構 (G-quadruplex) 是一非典型B form DNA結構，在生理上具重要意義，其參與端粒、DNA複製和轉錄上的調控。我們實驗室和中研院原分所張大釗老師合作，合成能穩定G-quadruplex的小分子藥物BMVC4。已知BMVC4對於非小細胞肺癌細胞H1299處理後，會使H1299端粒酶活性下降、端粒縮短、老化、DNA damage response和DNA strand breaks，然而，藉由免疫螢光染色實驗，我們發現DNA斷裂的區域不只出現在端粒，也出現在染色體其他區域，所以我們推測BMVC4所造成的影響不只在端粒和端粒酶上，可能和染色體其他區域出現的G-quadruplex的穩定有關。WRN和BLM屬於RecQ helicase family，其已被證實對於G-quadruplex、replication fork和Holliday junction的結構有特異性、有偏好性的去解開這些結構；此外，其在DNA repair、DNA replication和telomere maintenance也是重要的調控因子。所以，我們想知道，BMVC4穩定G-quadruplex後所造成細胞的DNA damage是否和WRN和BLM有關。首先，我們將WRN和BLM表現抑制後，不管是否處理BMVC4，pATM和total ATM的表現都下降，接著當WRN和BLM表現恢復時，pATM和total ATM的表現也跟著恢復，推測WRN和BLM在BMVC4所造成的DNA damage response中可能是必須的，而且其會互相調控，缺一不可。我們也發現，當WRN和BLM表現量下降，不管是否處理BMVC4的情況下，都會使H1299生長遲緩，接著想知道是否會影響細胞老化，藉由SA-β-galactosidase活性測定，發現BMVC4的處理與否，WRN和BLM的表現一但受到抑制，就會使H1299產生老化的現象。因此，WRN和BLM不僅在BMVC4造成的DNA damage response中扮演重要調控者，其也在細胞生長和細胞老化上有重要影響。

G-quadruplex is a non-canonical B form DNA structure, it plays an important role in the sense of physiology. For example, G-quadruplex participates in the regulation of telomere, DNA replication and transcriptional regulation. BMVC4 is a novel G-quadruplex stabilizer. It was synthesized through the collaboration with Dr. Chang, Academic Sinica. We found that BMVC4 could not only inhibit telomerase activity in H1299, non-small cell lung cancer, but also shorten telomere length, eventually lead to cellular senescence. BMVC4 also caused DNA damage response and DNA strand breaks. After the measurement of immunofluorescence, we found only 20% DNA damage response that arose from telomere. Therefore, the occurrence of DNA damage response maybe come from the other site of the chromosomes. WRN and BLM belong to RecQ helicase family. It is well established that WRN and BLM have capability in preferentially binding to G-quadruplex, replication fork and holliday junction structure. Both of them have high efficiency in resolving these special structures. They play a vital role in DNA repair, DNA replication and telomere maintenance. Because WRN and BLM could resolving G-quadruplex structure, we test if there is a correlation between WRN or BLM and the stabilization of G-quadruplex by BMVC4. We knocked down the expression of WRN and BLM respectively and found that the expression of pATM and total ATM was in concert with the expression of WRN or BLM. It is possible that WRN or BLM may function as the upstream signal of DNA damage response, which caused by the effect of BMVC4. That is, WRN or BLM might be required for eliciting the DNA damage response. We also found that WRN or BLM depletion caused H1299 cells to stop proliferating after a short period, no matter the treatment of BMVC4 or not. In addition, through SA-β-gal analysis, we found that the depletion of WRN or BLM induced cellular senescent phenotype. In conclusion, WRN and BLM are not simply the pivotal regulator of DNA damage response caused by BMVC4, but likewise have deeply influence in cell growth and cellular senescence.