Intracellular Glycation of Nuclear DNA, Mitochondrial DNA, and Cytosolic Proteins During Senescence-like Growth Arrest

Abstract

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(ɛ)-(carboxymethyl)lysine (CML) and N(ɛ)-(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/μg mtDNA) were significantly higher compared with nuclear DNA (4.40 ± 0.64 pg CEdG/μg 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.

Endogenous mitochondrial oxidative stress in MnSOD-deficient mouse embryonic fibroblasts promotes mitochondrial DNA glycation

“…The simultaneous isolation of nDNA, mtDNA, and cytosolic proteins was performed as described previously for NIH 3T3 fibroblasts [21] with minor modifications. For cell disruption, only freeze-thaw cycles were performed.…”

“…The SDH assay was performed as previously described [21]. The suspended pellet, the post-mitochondrial supernatant, or buffer M alone was analyzed in a concentration of 10% v/v in the assay buffer as described.…”

“…Fifteen μg of proteins per lane was loaded onto a 12% acryl amide gel and separated at 150 V for 80 min. After electrophoresis was completed, the gels were stained with Coomassie blue as previously described [21]. …”

“…Very recently, a method was developed to allow simultaneous quantification of AGEs bound to proteins, mtDNA, and nuclear DNA (nDNA). Therewith, significantly higher CEdG levels in mtDNA than in nDNA were detected in NIH 3T3 fibroblasts [21]. …”

Methods for the discovery of new anti-aging products – targeted approaches

“…Increasing in the mouse-senescent embryonic fibroblasts (NIH3T3) [143] DNA cross-linkages DNA--DNA cross-links Increasing in rabbit liver [144] DNA--protein cross-links Enhancing the mouse liver, brain and heart [145] Single-strand DNA breaks Increasing more in old animals especially after g or UV-irradiation [146]; There was no alteration in mice brain, liver and kidney [147] Double-strand DNA breaks (DSB) Chromosomal rearrangements and DNA instability could occur due to decrease of chromosome segments, endangering cell survival and incorrect repaired DSB Frequently occurred in elderly animals [148] and also in cancerous condition [149] network of mechanisms and influence the proteins hemostasis. Some factors can control protein hemostasis, which are briefly described in the Table 2.…”