Abstract
Telomere is a unique loop structure that exists in the end of each chromosome and consists of (TTAGGG)n repeats. Telomeres progressively shorten through each DNA replication by the end replication problem, and finally result in the end-to-end fusion of chromosomes by the destruction of the telomere loop structure. Telomere FISH is a powerful tool to evaluate telomere lengths of each cell or chromosome. In the present study, to evaluate telomere instability, we developed a quantitative telomere FISH using normal human fibroblast (HE7) cells and mouse embryo fibroblast (MEF) cells. For telomere FISH, FITC-labeled 18mer peptide nucleic acid (PNA) probes specific for telomere repeats were applied. The intensity of telomere FISH (T-FISH) signals was quantified in relation to population doubling numbers (PDN). The result indicated that the intensity of T-FISH signals in MEF cells was about two fold stronger than that in HE7 cells, implying the longer telomeres in MEF cells than those in HE7 cells. In contrast to the prediction, however, both MEF cells and HE7 cells showed a similar 10% decrease in telomere lengths after 15~18 PDN, resulting in no difference in a telomere erosion rate between them. Then, we combined centromere FISH (C-FISH) with T-FISH in HE7 cells and used a ratio of the intensity of T-FISH to that of C-FISH as a T/C value to normalize the variation of analyses. The result revealed that HE7 cells showed a 30% decrease in telomere lengths, indicating a clear difference in the telomere erosion rate between them. Therefore, the present study recommends T-FISH normalized with the T/C value for evaluating telomere instability induced by telomere erosion.
