Contribution Of DNA Strand Breaks To Telomere Shortening In Human Fibroblasts

Chromosome ends in eukaryotes are capped with tandem copies of a simple DNA sequences termed telomeric repeat. Telomeric repeats are predominately double stranded, with a 3' single-stranded overhang. In mammalian cells, the length of the double-stranded repeats range from a few to tens of kbp, whereas the length of single-stranded overhang - (TTAGGG)n- can be a few hundred nucleotides long. Telomeric DNA and its associated proteins prevent illegitimate recombination and repair, establishing a stable linear chromosome end. Telomeres in human fibroblasts shorten by about 30-200bp with each cell division. This shortening triggers replicative senescence in human cells when a critical point is reached. The inability of DNA polymerases to replicate linear DNA molecular completely is one cause of telomere shortening. Other causes, such as preferential vulnerability of telomeres to oxidative stress, also contribute to telomere shortening.In this work, we attempt to evaluate the amount of telomere strand breaks in cultured human fibroblast MRC-5 by alkaline denaturing and electrophoresis of cells embedded in agarose. After blotting the telomere fragments on to membrane, the quantity of telomere fragments was detected by probe specific to telomere sequence.In MRC-5 cells exposed to H2O2 at 37℃, a significant increase in the amount of telomere fragments occurred at concentration between 9.1 to 291 μM. To further examine the potential of this technique, different amount of cells were treated with 291 μM H2O2 and 0.1 mM Fe2+, and an approximately linear increase in the amount of telomere fragments was observed for cell number between 5xl04 and 2xl06. This technique appears to be sensitive and useful for detecting telomere fragments.Using this same technique, the telomere fragments of MRC-5 cells kept in confluence for one day and three weeks were measured. Results show that thefrequency of telomere strand breaks in these cells is 0.58+0.41 and 0.63+0.55 breaks /cell respectively. This represents about 22 bp loss of telomere per population doubling in normal cultured conditions and much less than 50-100 bp of telomere loss per population doubling observed in fibroblast MRC-5. It shows that DNA strand breaks only has a small contribution to telomere shortening in human cells even the stranded breaks can be accelerated by a modification of the amount of oxidative stress.