The process by which cells regulate the onset of cellular senescence represents an important question for understanding replicative aging and the mechanisms by which tumor cells escape these limits and become immortal. We have previously demonstrated that the timing of senescence is dependent on the shortest telomeres (Ouellette et al., 2000
; Steinert et al., 2000
), but these studies did not distinguish between one or a few short sentinel telomeres or a more general monitoring of short telomeres and did not identify the specific telomeres involved. The present results show that the shortest telomeres are found in γH2AX/53BP1 foci at senescence and that in a cell type with strong oxidative protection mechanisms the frequency of chromosomal abnormalities at senescence are very low. If checkpoint arrest is blocked by the papilloma virus proteins E6+E7, then ~10% of the telomeres are found to be responsible for >90% of the telomeric end-association events at the population-doubling level when senescence would have occurred. Thus, a substantial subset rather than one or two telomeres is being used to time the onset of replicative arrest in the population of cells. These results also indicate that although blocking p53/pRB checkpoints with E6+E7 bypasses senescence, it does not prevent end-associations involving the shortest telomeres. der-Sarkissian et al.
) have shown that the shortest telomeres drive karyotypic changes in transformed human embryonic kidney cells. Our results are consistent with their observations.
Approximately 85% of metaphase spreads from near-senescent cultures contained more than one signal-free end. This establishes that normal cells are still able to divide in the presence of telomeres sufficiently short to fail to give a hybridization signal (which may or may not be short enough to initiate a DNA damage response). Furthermore, many interphase nuclei from near-senescent cultures had γH2AX/53BP foci colocalizing with more than one of the shortest telomeres (). It is as yet unknown whether the initial failure to growth arrest is due to a graded response from individual telomeres (the strength of the damage signal is initially low when the telomere is sufficiently short to initiate any signal and increases as that telomere gets progressively shorter) or if more than one short telomere is required to initiate growth arrest (for example, a short telomere has to find a partner and form an end-association before growth arrest occurs). Telomeric end-associations were not seen in metaphases from near-senescent BJ cultures. The fact that end-associations first appear in E6+E7 expressing BJ cells at the same population doubling level when senescence occurred in control cultures suggests that end-associations were correlated with growth arrest in the normal cells (and thus failure to form metaphases). This supports the hypothesis that cells can divide in the presence of signal-free telomeres until an end-association occurs.
Although there is a general correlation between chromosome size and telomere length (Suda et al., 2002
), the fact that there is variation between maternal and paternal telomere length on any given chromosome (Londono-Vallejo et al., 2001
) suggests that the control of telomere length in the germline is imprecise. A 3-kb variation in size between a 15-kb maternal and an 18-kb paternal telomere would make little difference in the germline, but would make an enormous difference in which specific telomere became limiting when shortening reduced the respective lengths to 0.5 and 3.5 kb. Furthermore, if telomere shortening/processing of C-strands is much more extensive than G-strands, a several kilobase distribution of sizes develops over time even for a single telomere of defined length (Levy et al., 1992
). Given the importance of counting cell divisions in order for replicative aging to function as a brake against the progression of cancer, it thus makes biological sense for cells to monitor a group of short telomeres to control the timing of replicative aging as found here, rather than attempting to have one or two specified telomeres function as “sentinels” that count divisions and determine when senescence should occur.
Although there is a statistically significant difference in the length of telomeres at particular chromosome ends when multiple individuals are analyzed (Martens et al., 2000
; Graakjaer et al., 2003
), there is considerable variability between individuals. This is consistent with the variability in telomere sizes between maternal and paternal chromosomes (; Londono-Vallejo et al., 2001
). We would thus predict that the 10 shortest telomeres that regulate replicative aging in BJ foreskin fibroblasts should be enriched for ends that have been found to be short in other studies, but that the overlap should not be complete and a different subset of ends would produce senescence in other cell strains. This analysis is complicated by the fact that the published studies on chromosome-specific telomere length have averaged maternal and paternal chromosomes, whereas our results and those of others (; Londono-Vallejo et al., 2001
; der-Sarkissian et al., 2004
) suggest that only one of each pair is among the 10 shortest. Nonetheless, 8 of our 10 shortest telomeres were found in the shortest 50% of chromosome ends from an analysis of the average of 20 older individuals (Graakjaer et al., 2003
). This study also showed that although there is an overall pattern of telomere length on different chromosome ends, there is an individual pattern superimposed on the average pattern such that the particular telomeres that are the shortest will vary between individuals.
Our results differ from those reported by Martens et al.
), which failed to find a correlation between the telomere length determined by Q-FISH and replicative aging. There are a variety of possible explanations. Their study averaged the telomere length between homologous chromosomes, thus the influence of particular parental telomeres would have been diminished. Furthermore, they used a strain of fetal rather than neonatal skin fibroblasts. We have found considerable differences in the culture requirements of fetal vs. adult lung fibroblasts. Fetal lung fibroblasts exhibited stasis (growth arrest due to inadequate culture conditions; Ramirez et al., 2001
; Drayton and Peters, 2002
; Wright and Shay, 2002
) rather than pure telomere-based replicative senescence using standard tissue culture media in room oxygen (Forsyth et al., 2003
). If fetal skin fibroblasts exhibit similar differences, the short telomeres in the above study may have failed to predict the onset of growth arrest because the cells never reached telomere-based replicative senescence.
The shortest telomeres preferentially formed end-associations with other short telomeres rather than random chromosome ends (). Short telomeres might be metastable, spending progressively larger fractions of time in a partially unprotected configuration. The presence of one unprotected telomere could then trap a second metastable telomere in an end-association before it could be repackaged into a protected conformation, explaining the preponderance of short-short rather than short-random associations.
One report has suggested that M1 is caused by the disappearance of the G-rich 3′ telomeric overhang and that it is the global loss of this overhang on all telomeres with progressive cell divisions rather than telomere length that causes replicative aging (Stewart et al., 2003
). Furthermore, this and a subsequent report (Masutomi et al., 2003
) suggested that telomerase immortalizes cells by restoring the overhang rather than by elongating telomeres. Our results are largely incompatible with these reports because we establish that specific telomeres with a high fraction of signal-free ends become localized to γH2AX/53BP1 foci at the time of senescence, whereas telomeres with a very low fraction of signal-free ends do not (). Furthermore, if cell cycle arrest is blocked, only a small subset of telomeres accounts for almost all of the end-association events that occur (). Not only are these specifically the shortest telomeres, in any given cell it is only one or two of the shortest telomeres that are producing end-associations. One should thus not see an 85% reduction in overhangs in BJ cells (Stewart et al., 2003
) using an assay that measures all 92 ends at once if only one or two telomeres per cell are limiting for growth. These authors also demonstrated that the induction of DNA damage in young fibroblasts could induce a shortening of the overhangs (Stewart et al., 2003
). Given the known recruitment to telomeres of a large number of DNA repair factors, it would not be surprising if the activation of a DNA damage response influenced telomere end-processing. A reasonable hypothesis is that 1 of the 10 shortest telomeres induces a DNA damage signal, and any subsequent global shortening of overhangs then occurs as a simple consequence of signal-induced altered processing. Short overhangs would thus represent an epiphenomenon, a secondary consequence of short telomeres, rather than a proximate cause of senescence. We cannot exclude the possibility that the consequences of short overhangs contribute to a greater deprotection of the shortest telomeres or that there might be cis
-acting effects so that overhang loss is greatest on the shortest telomeres.
Studies of telomere lengths on specific human chromosomes have shown a progression of sizes rather than one or a few individual telomeres that are dramatically shorter than the rest. Our increasing knowledge of stochastic processes that influence telomere lengths suggest that it would be difficult to have a few sentinel telomeres that could time replicative aging. The demonstration that relatively large groups of short telomeres are functionally monitored to determine the timing of cellular senescence now provides a sound biological rationale for understanding this process.