Intracellular Glycation of Nuclear DNA, Mitochondrial DNA, and Cytosolic Proteins During Senescence-like Growth Arrest
Intracellular Glycation of Nuclear DNA, Mitochondrial DNA, and Cytosolic Proteins During Senescence-like Growth Arrest
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DOI：
10.1089/dna.2011.1236
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发表时间：
2011-09-01
影响因子：
3.1
通讯作者：
Pischetsrieder, Monika
中科院分区：
文献类型：
--
作者：
Breyer, Viola;Becker, Cord-Michael;Pischetsrieder, Monika
为了研究细胞内晚期糖基化终产物（AGEs）的积累，建立了一种同时分析细胞质蛋白、核DNA和线粒体DNA （mtDNA）糖基化产物的方法。通过差速离心和机械和化学细胞破坏相结合的方法，同时从一个细胞裂解液中分离出核DNA、mtDNA和细胞质蛋白。ELISA法测定核DNA和mtDNA中主要DNA- age N(2)-羧乙基-2′-脱氧鸟苷（CEdG）， western blot法测定蛋白质- ages N(epsilon)-（羧甲基）赖氨酸（CML）和N(epsilon)-（羧乙基）赖氨酸（CEL）。采用该方法对NIH3T3成纤维细胞进行分析。在未处理的细胞中，mtDNA的CEdG水平（14.84 +/- 3.07 pg CEdG/ μ g mtDNA）显著高于核DNA （4.40 +/- 0.64 pg CEdG/ μ g DNA, p < 0.001）。然后，在衰老样生长停止7天后对成纤维细胞进行分析。衰老成纤维细胞细胞核DNA中CEdG含量显著增加25%。但mtDNA的CEdG水平显著下降至52%；同时，观察到线粒体质量和mtDNA的增加。衰老不会导致蛋白质- ages的普遍积累，但在32和34 kDa的两个蛋白带中，衰老成纤维细胞的CML/CEL修饰率与对照细胞相比显著增加（208%,p < 0.001; 196%, p = 0.0016）。
To investigate the accumulation of intracellular advanced glycation end products (AGEs), a method was established for the simultaneous analysis of glycation products of cytosolic proteins, nuclear DNA, and mitochondrial DNA (mtDNA). Nuclear DNA, mtDNA, and cytosolic proteins were simultaneously isolated from one cell lysate by differential centrifugation and combined mechanical and chemical cell disruption methods. The major DNA-AGE N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) was quantified in nuclear DNA and mtDNA by ELISA, whereas the protein-AGEs N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) were determined by western blot. The method was used to analyze NIH3T3 fibroblasts. In untreated cells, CEdG levels of mtDNA (14.84 +/- 3.07 pg CEdG/mu g mtDNA) were significantly higher compared with nuclear DNA (4.40 +/- 0.64 pg CEdG/mu g DNA; p < 0.001). Then, fibroblasts were analyzed after 7 days of senescence-like growth arrest. In senescent fibroblasts, the CEdG content of nuclear DNA significantly increased by 25%. However, the CEdG level of mtDNA significantly decreased to 52%; in parallel, an increase in mitochondrial mass and mtDNA was observed. Senescence did not lead to general accumulation of protein-AGEs, but two protein bands at 32 and 34 kDa showed a significant increase in the CML/CEL modification rate (208%, p < 0.001; 196%, p = 0.0016) in senescent fibroblasts compared with control cells.