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Techniques to Induce and Quantify Cellular Senescence
Published on: May 1, 2017
Augmented nuclease activity during cellular senescence in vitro
M A Dayton1, P Nahreini, A Srivastava
1Department of Medicine, Indiana University School of Medicine, Indianapolis 46223.
Abstract:
The molecular correlates of the limited proliferative potential of normal human diploid fibroblasts and extensive single-strand breaks in the genomic DNA of these cells were examined by transfection analyses in which DNA replication could be uncoupled from DNA damage and repair. Both supercoiled (fmI), and restriction endonuclease-cleaved, linear (fmIII) molecules of a well-defined bacterial plasmid DNA, pBR322, were transfected into, and subsequently recovered from, early and late passage fibroblasts. Southern blot analysis revealed that fmI DNA was converted by random nicks into fmII DNA slightly more rapidly in late passage cells compared with cells at early passage. Similarly, fmII and fmIII DNAs also sustained multiple random nicks and no appreciable net religation of free ends of fmIII DNA could be detected at either passage. In addition, the efficiency of in vitro ligation of fmIII DNA recovered from late passage cells was also reduced, compared with that from early passage cells, as determined by Southern blotting. These data suggest that in the absence of DNA replication, a putative nuclease activity may contribute to DNA damage observed in senescent cells, which, in turn, may be causally related to their limited replicative potential.
Insights
Senescent fibroblasts exhibit increased DNA damage and reduced DNA repair capacity, potentially explaining their limited proliferation. This suggests a role for nucleases in cellular aging.
Area of Science:
- Molecular Biology
- Cellular Senescence
- Genetics
Background:
- Normal human diploid fibroblasts have limited proliferative potential, a hallmark of cellular senescence.
- Genomic DNA in senescent cells shows extensive single-strand breaks, but the underlying molecular mechanisms are not fully understood.
Purpose of the Study:
- To investigate the molecular basis of limited fibroblast proliferation and DNA damage in senescent cells.
- To determine if DNA damage and repair processes are altered in late-passage fibroblasts, independent of DNA replication.
Main Methods:
- Transfection of bacterial plasmid DNA (pBR322) in supercoiled (form I) and linear (form III) forms into early and late passage human diploid fibroblasts.
- Analysis of plasmid DNA recovery and integrity using Southern blotting to assess nicking, religation, and ligation efficiencies.
Main Results:
- Late passage fibroblasts showed slightly faster conversion of supercoiled to nicked plasmid DNA (form II).
- Both early and late passage cells exhibited nicking of plasmid DNA, with no significant religation of linear DNA ends.
- Reduced in vitro ligation efficiency of plasmid DNA recovered from late passage cells was observed.
Conclusions:
- A putative nuclease activity, active even without DNA replication, may contribute to DNA damage in senescent cells.
- This increased DNA damage, potentially mediated by nucleases, could be causally linked to the limited replicative potential of senescent fibroblasts.
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