Durkin S G, Arlt M F, Howlett N G, Glover T W
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.
Oncogene. 2006 Jul 27;25(32):4381-8. doi: 10.1038/sj.onc.1209466. Epub 2006 May 29.
Common fragile sites are specific regions of the genome that form gaps and breaks on metaphase chromosomes when DNA synthesis is partially inhibited. Fragile sites and their associated genes show frequent deletions and other rearrangements in cancer cells, and may be indicators of DNA replication stress early in tumorigenesis. We have previously shown that the DNA damage response proteins ATR, BRCA1 and FANCD2 play critical roles in maintaining the stability of fragile site regions. To further elucidate the pathways regulating fragile site stability, we have investigated the effects of depletion of the cell cycle checkpoint kinases, CHK1 and CHK2 on common fragile site stability in human cells. We demonstrate that both CHK1 and CHK2 are activated following treatment of cells with low doses of aphidicolin that induce fragile site breakage. Furthermore, we show that depletion of CHK1, but not CHK2, using short-interfering RNA (siRNA) leads to highly destabilized chromosomes and specific common fragile site breakage. In many cells, CHK1 depletion resulted in extensive chromosome fragmentation, which was distinct from endonucleolytic cleavage commonly associated with apoptosis. These findings demonstrate a critical role for the CHK1 kinase in regulating chromosome stability, and in particular, common fragile site stability.
常见脆性位点是基因组中的特定区域，当DNA合成受到部分抑制时，这些区域会在中期染色体上形成间隙和断裂。脆性位点及其相关基因在癌细胞中频繁出现缺失和其他重排，可能是肿瘤发生早期DNA复制应激的指标。我们之前已经表明，DNA损伤反应蛋白ATR、BRCA1和FANCD2在维持脆性位点区域的稳定性方面发挥着关键作用。为了进一步阐明调节脆性位点稳定性的途径，我们研究了细胞周期检查点激酶CHK1和CHK2的缺失对人类细胞中常见脆性位点稳定性的影响。我们证明，在用低剂量的阿非迪霉素处理诱导脆性位点断裂的细胞后，CHK1和CHK2都会被激活。此外，我们表明，使用短干扰RNA（siRNA）耗尽CHK1而不是CHK2会导致染色体高度不稳定和特定的常见脆性位点断裂。在许多细胞中，CHK1的缺失导致广泛的染色体片段化，这与通常与细胞凋亡相关的内切核酸酶切割不同。这些发现证明了CHK1激酶在调节染色体稳定性，特别是常见脆性位点稳定性方面的关键作用。
