Increased common fragile site expression, cell proliferation defects, and apoptosis following conditional inactivation of mouse Hus1 in primary cultured cells.
Increased common fragile site expression, cell proliferation defects, and apoptosis following conditional inactivation of mouse Hus1 in primary cultured cells.
复制标题
在原代培养细胞中条件性灭活小鼠 Hus1 后，常见脆弱位点表达、细胞增殖缺陷和细胞凋亡增加。
DOI：
10.1091/mbc.e06-10-0957
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发表时间：
2007
影响因子：
3.3
通讯作者：
Weiss,RobertS
中科院分区：
文献类型：
--
作者：
Zhu,Min;Weiss,RobertS
有针对性地破坏小鼠Hus1细胞周期检查点基因导致胚胎死亡和培养细胞的增殖停滞。为了研究Hus1的基本功能，我们开发了一个系统，用于在原代成纤维细胞中调节小鼠Hus1的失活。通过使用表达cre的腺病毒灭活loxP位点侧翼的条件Hus1等位基因导致细胞倍增减少、细胞周期改变和细胞凋亡增加。这些表型与显著增加的总染色体异常频率和磷酸化组蛋白H2AX（双链DNA断裂的指标）的S期特异性积累相关。为了确定这些染色体异常是随机发生的还是发生在特定的基因组区域，我们评估了常见脆性位点的稳定性，这些染色体位点在经历复制应激的细胞中易于断裂。发现Hus1对脆性位点的稳定性至关重要，因为自发性染色体异常在条件Hus1失活后优先发生在常见脆性位点。尽管p53水平在Hus 1缺失后增加，但p53缺失未能挽救细胞倍增缺陷或增加条件Hus 1敲除细胞的凋亡。综上所述，我们认为Hus 1缺失导致DNA复制过程中染色体不稳定，通过p53非依赖性机制引发细胞凋亡增加和增殖受损。
Targeted disruption of the mouseHus1cell cycle checkpoint gene results in embryonic lethality and proliferative arrest in cultured cells. To investigate the essential functions ofHus1, we developed a system for the regulated inactivation of mouseHus1in primary fibroblasts. Inactivation of a loxP site-flanked conditionalHus1allele by using a cre-expressing adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase–specific accumulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile sites, chromosomal loci that are prone to breakage in cells undergoing replication stress.Hus1was found to be essential for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites upon conditionalHus1inactivation. Although p53 levels increased afterHus1loss, deletion ofp53failed to rescue the cell-doubling defect or increased apoptosis in conditionalHus1knockout cells. In summary, we propose thatHus1loss leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation through p53-independent mechanisms.