Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture
Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture
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DOI：
10.1046/j.1474-9728.2003.00066.x
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发表时间：
2003-12-01
期刊：
影响因子：
7.8
通讯作者：
Vijg, J
中科院分区：
文献类型：
--
作者：
Busuttil, RA;Rubio, M;Vijg, J
氧化损伤是衰老和癌症的一个致病因素，但目前尚不清楚DNA损伤、细胞对这种损伤的反应以及通过错误修复或错误复制转化为突变如何促成这些过程。使用携带lacZ突变报告基因的转基因小鼠，我们先前已经表明，在体内大多数器官和组织中，突变随着年龄的增长而增加。先前也已经表明，小鼠细胞通过经历细胞衰老来响应标准培养条件下典型的氧化应激。为了更好地了解氧化应激的后果，我们培养小鼠胚胎成纤维细胞（MEFs）从lacZ小鼠在生理氧张力（3%）或高氧张力（20%）与标准文化，并确定突变的频率和频谱。在原代培养时，发现突变频率相对于胚胎增加约三倍。大多数突变是基因组重排。随后在20%的氧气中培养导致衰老，然后自发永生化。永生化伴随着突变的额外三倍增加，其中大多数是G：C到T：A的颠换，这是氧化DNA损伤的标志性突变。相比之下，在3%的氧气中，MEFs没有衰老，突变频率和谱仍然与原代培养物相似。这些发现首次证明了氧化应激对小鼠细胞衰老和永生化过程中基因组完整性的影响。
Oxidative damage is a causal factor in aging and cancer, but it is still not clear how DNA damage, the cellular responses to such damage and its conversion to mutations by misrepair or misreplication contribute to these processes. Using transgenic mice carrying a lacZ mutation reporter, we have previously shown that mutations increase with age in most organs and tissues in vivo. It has also been previously shown that mouse cells respond to oxidative stress, typical of standard culture conditions, by undergoing cellular senescence. To understand better the consequences of oxidative stress, we cultured mouse embryo fibroblasts (MEFs) from lacZ mice under physiological oxygen tension (3%) or the high oxygen tension (20%) associated with standard culture, and determined the frequency and spectrum of mutations. Upon primary culture, the mutation frequency was found to increase approximately three-fold relative to the embryo. The majority of mutations were genome rearrangements. Subsequent culture in 20% oxygen resulted in senescence, followed by spontaneous immortalization. Immortalization was accompanied by an additional three-fold increase in mutations, most of which were G:C to T:A transversions, a signature mutation of oxidative DNA damage. In 3% oxygen, by contrast, MEFs did not senesce and the mutation frequency and spectrum remained similar to primary cultures. These findings demonstrate for the first time the impact of oxidative stress on the genomic integrity of murine cells during senescence and immortalization.