The MRE11-NBS1-RAD50 pathway is perturbed in SV40 large T antigen-immortalized AT-1, AT-2 and HL-1 cardiomyocytes.
The MRE11-NBS1-RAD50 pathway is perturbed in SV40 large T antigen-immortalized AT-1, AT-2 and HL-1 cardiomyocytes.
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MRE11-NBS1-RAD50 通路在 SV40 大 T 抗原永生化 AT-1、AT-2 和 HL-1 心肌细胞中受到干扰。
DOI：
10.1093/nar/28.15.2882
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发表时间：
2000
影响因子：
14.9
通讯作者：
Claycomb,WC
中科院分区：
文献类型：
--
作者：
LansonJr,NA;Egeland,DB;Royals,BA;Claycomb,WC
为了研究心肌细胞增殖的分子控制，我们利用SV40大T抗原（T-ag）诱导心肌细胞无限增殖。在T-ag永生化的AT-1、AT-2和HL-1心肌细胞中，与T-ag和p53相关的正常细胞蛋白被鉴定、分离并进行微测序。肽段测序结果显示，90、100和160 kDa的蛋白分别与MRE11、NBS1和RAD50同源。这三种蛋白在DNA双链断裂的检测和修复、细胞周期检查点的激活和端粒维护中起着至关重要的作用。在这篇报道中，我们描述了mre11， nbs1和rad50的大鼠同源物的cDNA克隆和双链测序。我们还测定了心脏发育不同阶段和不同组织中mre11、nbs1和rad50的mRNA和蛋白水平。MRE11mRNA仅在永生化心肌细胞和睾丸中检测到。尽管90 kDa的MRE11蛋白在大多数检测的样本中都可以看到，但它只在增殖的心肌细胞（正常和永生化）中检测到极低的水平。在所有检测的样本中均可见6.0 kbmre11相关mRNA转录物（MRT）。nbs1mrna和rad50mrna转录本水平在出生后第10天达到峰值。在t -ag永生化的at -1、at -2和HL-1细胞中，NBS1mRNA水平极低，但NBS1蛋白水平极高。我们提出SV40大T抗原与MRE11-NBS1-RAD50通路和p53的相互作用会破坏关键的细胞周期检查点，这是参与该癌蛋白使心肌细胞不朽能力的主要因素之一。
To investigate molecular controls of cardiomyocyte proliferation, we utilized cardiomyocytes induced to proliferate indefinitely by SV40 large T antigen (T-ag). In the T-ag-immortalized AT-1, AT-2 and HL-1 cardiomyocytes, normal cellular proteins associating with T‐ag and p53 were identified, isolated and microsequenced. Peptide sequencing revealed that proteins of 90, 100 and 160 kDa were homologs of MRE11, NBS1 and RAD50, respectively. These three proteins play critical roles in the detection and repair of DNA double-strand breaks, activation of cell cycle checkpoints and telomere maintenance. In this report, we describe the cDNA cloning and double-strand sequencing of the rat homologs ofMRE11,NBS1andRAD50. We also determined the mRNA and protein levels ofMRE11,NBS1andRAD50at different stages of heart development and in different tissues.MRE11mRNA was only detected in the immortalized cardiomyocytes and in the testes. Although the 90 kDa MRE11 protein was seen in most samples examined, it was only detected at extremely low levels in proliferating cardiomyocytes (normal and immortalized). The 6.0 kbMRE11-related mRNA transcript (MRT) was seen in all samples examined. Levels of bothNBS1andRAD50mRNA transcripts peaked in the heart at postnatal day 10.NBS1mRNA levels were at very low levels in the T-ag-immortalized AT-1, AT-2 and HL-1 cells but NBS1 protein was observed at extremely high levels. We propose that SV40 large T antigen’s interaction with the MRE11-NBS1-RAD50 pathway and with p53 ablates critical cell cycle checkpoints and that this is one of the major factors involved in the ability of this oncoprotein to immortalize cardiomyocytes.