Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells
Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells
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DOI：
10.1002/stem.1312
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发表时间：
2013-04-01
期刊：
影响因子：
5.2
通讯作者：
Rotrekl, Vladimir
中科院分区：
文献类型：
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作者：
Kruta, Miriama;Balek, Lukas;Rotrekl, Vladimir
人胚胎干细胞（hESCs）在体外扩增过程中不可避免的染色体异常积累是细胞替代疗法的一个相当大的障碍。为了确定染色体异常的来源，我们检测了培养超过55个月的hESCs在端粒维持和DNA修复方面的缺陷。虽然长时间培养既不影响端粒酶活性，也不影响非同源端连接，但碱基切除修复（BER）的效率显著降低，并与BER所需的主要核酸酶APE1（无嘌呤/无嘧啶内切酶1）的表达减少相关。有趣的是，其他BER酶的表达没有变化。将人重组APE1蛋白加入到晚期hESCs的核提取物中，使BER效率提高到早期hESCs的典型水平。在早期传代hESCs中，通过siRNA下调APE1后，DSB释放减少，证明了BER与双链断裂（DSB）之间的联系。相应的，较低的APE1水平导致电离辐射（IR）下DSB释放较慢，强度较小，但持续时间较长。早期传代hESCs中APE1的下调也导致IR后-H2AX信号减少约30%，与晚期hESCs相似。我们认为，在hESCs的长期培养过程中，APE1的下调显著促进了BER的失败，并且BER的失败是通过改变BER依赖的DSB释放和细胞周期/检查点信号传导来影响hESCs基因组不稳定性的因素之一。干细胞2013；31:693702
The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in -H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling. STEM CELLS 2013;31:693702