In the present study we evaluated the mechanism of enhanced radiosensitivity in cells with short telomeres. As expected, primary and transformed cells with short telomeres had markedly enhanced sensitivity to IR compared to cells with longer telomeres. These results mirror the results observed in Terc
knock-out mice, which showed that progressive telomere shortening was associated with enhanced cellular and organismal sensitivity to ionizing radiation (7
). Similar results have been observed in human cells (24
). Although some studies have suggested that telomerase expression per se
influences radiosensitivity (8
), our results and the results of other groups suggest that telomere length is the primary determinant of radiosensitivity (7
). In these studies, ectopic telomerase expression or telomerase inhibition had no effect on sensitivity to IR until a measurable change in telomere length was observed.
The biological mechanism underlying the relationship between telomere integrity and radiation sensitivity remains to be determined. A simple model to explain this correlation is that cells with short telomeres possess a baseline level of DNA damage from uncapped telomeres even before they are subjected to IR. In this model, the pre-existing damage would be additive with the extra damage induced by IR, thereby enhancing radiation sensitivity. Arguing against this model is our observation that the baseline number of γH2AX foci in late-passage fibroblasts is equivalent to an ionizing radiation dose of only 0.1 Gy (data not shown). Adding 0.1 Gy to the levels of ionizing radiation that were used in the clonogenic survival assays would be insufficient to explain the enhanced sensitivity to IR that we observed, though the protracted 0.1 Gy-equivalent exposure in late-passage cells may be biologically different from the short-lived exposure associated with a one-time dose of IR. Although the baseline number of γH2AX foci in late-passage cells was elevated compared to early-passage HFF, the late-passage cells had diminished H2AX phosphorylation in response to IR at early time points (). If the enhanced radiation sensitivity in late-passage cells were merely the result of an additive effect of radiation damage to telomere damage, we would have expected the peak levels of H2AX phosphorylation to be higher in the late-passage cells than the early passage cells. A second model that has been proposed is that telomeres serve as repositories of proteins that participate in the DNA damage response, which can readily translocate to DNA breaks (11
). According to this premise, once telomeres become short, repair proteins can no longer bind telomeres and are no longer immediately available to sites of DNA damage. However, the DSB repair proteins that associate with telomeres, such as the MRE11/RAD50/NBS1 (MRN) complex, are uniformly distributed in the nuclei of unirradiated cells (31
). Because the telomere-bound component comprises just a small fraction of the abundant pool of repair proteins, it seems unlikely that a decrease in telomere length would have a major effect on the availability of repair proteins to be recruited to DNA breaks.
The present data support a third model for increased radiation sensitivity in cells with short telomeres. One of the earliest events in the DNA damage response is ATM phopsphorylation/activation and swift phosphorylation of H2AX (γH2AX) on Ser139, primarily by activated ATM and DNA-PK (16
). Upon DSBs, phosphorylated H2AX (γH2AX) rapidly forms DNA repair foci that include the MRN complex, 53BP1 and BRCA1 (32
). Our results showed that late-passage cells had attenuated peak levels of H2AX phosphorylation and that the kinetics of H2AX phosphorylation and dephosphorylation were delayed. Because γH2AX plays a crucial role in the recruitment of proteins to the repair focus and in its stabilization to recruit late factors like cohesins to tether sister chromatids for homologous recombination repair pathway (33
), the altered H2AX phosphorylation kinetics may account for the enhanced radiosensitivity in late-passage cells. Recently, it was shown that telomerase-null and telomerase-inhibited mouse embryo fibroblasts (MEFs) formed fewer γH2AX foci and had delayed appearance and disappearance of such foci following heat shock and IR exposure compared to telomerase positive MEFs (6
). Although these results focused on telomerase expression and not telomere length, they are concordant with our results. Furthermore, delayed kinetics of DNA DSB processing, including the rates of recruitment of DSB repair proteins to γH2AX foci, has been observed in normal and pathological aging (9
). Our data suggest a mechanism for the altered kinetics of γH2AX focus formation and disappearance in late-passage cells. Late-passage chromatin was resistant to micrococcal nuclease digestion and had reduced levels of H3K9 acetylation and higher levels H3K9 dimethylation, indicative of heterochromatinization. These results are consistent with the observation that senescent cells accumulate foci of heterochromatin (37
), though the late-passage fibroblasts employed in our experiments were pre-senescent (Figure S2
). The compacted chromatin could explain why phosphorylation and activation of chromatin-bound DNA damage proteins (H2AX, SMC1, and NBS1) were delayed in late-passage cells, while activation of non-chromatin-bound proteins (p53 and ATM) was unchanged. Moreover, we observed that dephosphorylation of γH2AX was delayed in late-passage cells. Dephosphorylation of γH2AX is achieved by protein phosphatase 2A (38
), by the HTP-C phosphatase complex in budding-yeast (39
) and by Wip1 phosphatase (40
). Like delayed phosphorylation of chromatin-bound proteins, delayed dephosphorylation may be caused by reduced access of phosphatases to late-passage chromatin. Furthermore, we and others have shown that H3K9Ac is a DNA-damage-responsive modification (Zhang and Drissi, unpublished data; (43
), suggesting this histone modification is a mark of heterochromatin and DNA damage.
Telomerase protein has also been described to regulate chromatin state and the DNA damage response (28
). Suppression of hTERT in human fibroblasts led to enhanced radiosensititivity and delayed kinetics of γH2AX focus formation, without measurable changes in telomere length. Abrogation of hTERT was associated with a pattern of euchromatin (enhanced sensitivity to micrococcal nuclease-induced DNA digestion, increased H3K9 acetylation, and decreased H3K9 dimethylation). It is interesting that although the end-result of telomerase suppression (enhanced radiosensitivity and delayed DNA repair kinetics) was similar to the effect we observed with telomere shortening, telomerase suppression increased euchromatin content whereas telomere shortening increased heterochromatin content. These results indicate that although telomerase repression and telomere shortening are closely intertwined, the two processes do not have identical consequences for the cell.
It remains to be determined how telomere length influences chromatin structure and, in turn, how chromatin structure influences the DNA repair process. It has recently been shown that ATM-mediated phosphorylation of heterochromatin-promoting proteins such as KAP-1 increases the repair of DNA breaks within heterochromatin (44
). Our data showed that ATM activation kinetics in late-passage cells were not affected by short telomeres, but that phosphorylation of H2AX, SMC1, and NBS1 was delayed. It is possible that telomere shortening-induced heterochromatin introduces an impediment to ATM-dependent chromatin modifications that facilitate DNA double-strand break repair.
Finally, our results support several predictions regarding the use of telomerase inhibitors as therapeutic agents for human cancer. First, telomerase inhibition in itself can induce apoptosis and growth arrest in cancer cells, though this response may be delayed, as observed in our HeLa cells. Second, the concurrent administration of telomerase inhibitors and ionizing radiation may have a synergistic effect once sufficient telomere shortening has occurred. The concurrent application of DNA-damaging agents and telomerase inhibitors may provide a therapeutic benefit.