Analysis of UV-induced damage and repair in young and senescent human dermal fibroblasts using the comet assay.
A major cause of ageing is thought to be the accumulation of damage to macromolecules. Accumulation to DNA damage in cells therefore presupposes that aged cells are unable to repair this damage. We have used the in vitro model of cellular ageing to test the idea that senescent cells are deficient in some aspect of DNA repair. Using the alkaline single cell gel electrophoresis assay (comet assay), we have determined the responses of young and senescent human dermal fibroblasts to DNA damage caused by exposure to UVC light. At low doses of UVC, senescent cells generate smaller comets than young cells whilst at medium doses the situation is reversed. At high doses, young and senescent cells respond similarly to one another. Time course experiments revealing repair of DNA damage show that senescent cells generate larger comets than young cells at early stages of repair suggesting that either senescent cells bear more damage per genome than do young cells or that senescent cells are more efficient at excising bulky adducts from DNA. Cells maintained in low levels of serum irrespective of age are less able to repair DNA damage compared with cells maintained in high levels of serum, and furthermore young and senescent cells maintained in high levels of serum are equally able to repair DNA damage. Our data, therefore, reveal both age-dependent and age-independent responses to UV-induced DNA damage. Use of the comet assay highlights the heterogeneity of cellular responses to genotoxic stress.
Al-Baker
Eman A
EA
Department of Biological Sciences, Faculty of Science, Kuwait University, Safat, Kuwait.
Oshin
Michael
M
Hutchison
Christopher J
CJ
Kill
Ian R
IR
D002478:Cells, Cultured; D016922:Cellular Senescence / Q000528:radiation effects*; D020552:Comet Assay; D004249:DNA Damage / Q000502:physiology / Q000528:radiation effects*; D004260:DNA Repair / Q000502:physiology / Q000528:radiation effects*; D020405:Dermis / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D014466:Ultraviolet Rays