In the current study, the fates of cells undergoing senescence were investigated by placing expression of the EST2 polymerase subunit under control of a modified galactose-inducible promoter (GAL1-V10) that allowed precise modulation of telomerase expression. These experiments revealed that most senescent cells, though they had stopped dividing, were greatly enlarged and contained degraded telomeres, could be rescued by reactivation of telomerase. The efficiency of rescue by telomerase reactivation was high immediately after cells reached late senescence and declined only slowly with time. The results demonstrated that most senescing cells had not accumulated lethal DNA lesions. In further support of this conclusion, haploid senescent cells could also be rescued by mating to telomerase-proficient cells to form diploid strains, with mating efficiencies similar to that of normal haploid cells.
Checkpoint response-deficient est2
cells exhibited normal senescence kinetics even though G2
-arrest, which increases opportunities for repair of damaged DNA, was impaired. This result is in accord with previous observations [29
]. A large fraction of the senescent checkpoint-defective cells could be rescued by restoration of telomerase expression (~25% compared to 40% for est2
single mutants), indicating that they had not accumulated lethal DNA lesions that committed them to cell death. Growth and subsequent replicative senescence of cultured human cells was originally divided into phases I, II and III by Hayflick [6
]. Phase III was defined as the stage when cells stop dividing but remain metabolically active and viable for an extended period of time. Shay and Wright referred to this period as M1. Stable ectopic expression of the catalytic subunit of human telomerase (hTERT) overcomes senescence when it is expressed prior to or during M1 phase [22
]. Checkpoint-deficient human cells (e.g., cells with p53 inactivated) continue dividing beyond the normal senescence period (proceeding into M2), display increased recombination events and multiple types of chromosomal DNA aberrations, and undergo cell death [22
]. Athough M1 appears to have similar characteristics in yeast and human cells, the efficient rescue of yeast checkpoint mutants by telomerase reactivation is inconsistent with an M2-equivalent period in these cells.
mutants exhibited rapid senescence kinetics, halting growth after ~ 40 generations versus ~ 60 for est2
cells, and displayed much lower plating efficiencies on glucose (0.003% versus 0.5%). Reactivation of telomerase by transfer of cells to galactose media increased the plating efficiency of est2 rad52
cells by 3 orders of magnitude, but the overall efficiency remained 5-fold lower than that of normal, non-senescent rad52
cells (8% versus 40%). These results indicate that, in contrast to est2
cells, a large fraction of the senescent est2 rad52
cells had become irreversibly growth-arrested during senescence. This finding adds further support to the idea that homologous recombination proteins prolong survival during senescence by promoting repair/stabilization of telomeric DNA ends [28
]. These ends become shortened, partially uncapped, and resected by nucleases such as Exo1 and therefore are likely to generate recombinogenic structures [19
]. Interestingly, no survivors were ever detected among est2 rad52
cells containing the GAL1-V10p::EST2
plasmid grown in either glucose liquid culture- or plate-based senescence assays. This observation is consistent with previous findings [28
] and also indicates that mutations within the ~ 650 bp GAL1-V10
promoter on the plasmid, potentially leading to leaky expression of EST2
in glucose media, occurred at low frequencies undetectable in the assays.
cells rescued by resumption of telomerase expression on galactose plates displayed normal growth rates, but chromosomal DNA purified from the colonies retained short telomeres. Thus, after 30 generations of growth with telomerase reactivated, telomeres in the rescued cells were still shorter than normal by approximately 100 bp. Restoration of the shortened telomeres back to normal lengths required propagation of the rescued cells for ~ 70 generations in the presence of telomerase. This finding is consistent with past reports indicating that telomerase extends only a few base-pairs per generation in yeast cells [58
]. Observation that the cells were phenotypically normal though their telomeres were short is not unprecedented: several yeast mutants have been identified that have shortened telomeres but do not exhibit obvious growth or cytological abnormalities [59
Past studies in both yeast and human cells have indicated that the presence of telomerase in cells, even if it is catalytically impaired, can stabilize critically short telomeres [61
]. Thus, reactivated telomerase may perform two separate functions in the rescued cells: protection of the shortened telomere ends from further degradation, with potential alleviation of the DNA damage checkpoint arrest response, as well as extension of the telomeres by several nucleotides during each cell cycle.
The molecular changes that ultimately lead to the death of cells undergoing replicative senescence are not known, though several mechanisms have been suggested. Proposed models include the possibilities that (i) uncapped, reactive DNA ends may promote lethal chromosome rearrangements such as end-to-end fusions, which have been detected in DNA of senescent cells, (ii) chromosomes may lose essential genetic information due to shortening and 5’-to-3 exonuclease degradation at uncapped ends, (iii) aneuploidy (chromosome loss or gain) may decrease cellular fitness, or (iv) senescence may initiate an apoptosis-like commitment to cell death [15
]. Several studies have also pointed to the importance of strand breaks induced in telomeric DNA by oxidation and these findings must also be incorporated into proposed models of telomere-initiated senescence [21
Greider and colleagues [18
] demonstrated that chromosome loss rates and mutations within telomere-proximal genes, caused primarily by terminal chromosomal DNA deletions, are increased during yeast cell senescence. Although such events were increased, they remained rare, and were largely dependent upon the presence of the nucleases Exo1 and Rad1. Based on these results, the authors posited that exonucleolytic end resection is the major mechanism causing chromosome instability during telomere shortening [18
]. Our experiments demonstrating that most yeast cells can be rescued efficiently by telomerase reactivation, during and long after cells have reached senescence, is in accord with their observation that such potentially lethal mutations and chromosome loss events occur in only a small fraction of the cells.
A model that is consistent with our observations and with those cited above is that replicative senescence involves two classes of events: (i) nonlethal changes that occur at most or all chromosome ends in the cell and are largely reversible upon reactivation of telomerase expression; these phenomena potentially include telomere shortening due to lack of end-replication by telomerase, partial loss of cap proteins, ssDNA resection by exonucleases, and activation of DNA damage checkpoint responses, plus (ii) lethal telomere shortening-induced events that initiate at one or more DNA ends in only a small percentage of cells. The latter events are likely to include end-to-end fusions, recombination-induced genome rearrangements, chromosome loss events and various types of mutations, especially large terminal deletions. The nonlethal changes described above are undoubtedly only a partial list. For example, shortened telomeres exhibit changes in their localization and interactions with nuclear pore complexes [35
] and modifications in telomere-associated structures such as T-loops and G-quadruplexes remain unexplored.
The major conclusion of the current work, that cellular senescence is largely reversible by reactivation of telomerase, has implications for higher eukaryotes since cells with characteristics similar to senescent cells have been detected in aged animals [21
]. It is not clear whether such senescent cells have been permanently altered or if they might be induced to regain youthful characteristics by transient or long-term expression of telomerase. Interestingly, constant high-level expression of telomerase has been found to increase median lifespans in transgenic mice [11
]. In addition, a recent study demonstrated that short-term telomerase reactivation in telomerase-deficient mice can reverse several measures of tissue atrophy without promoting carcinogenesis [68
]. It is possible that the constitutive or temporary production of telomerase in these animals exerts its beneficial effects, in part, by preventing and/or reversing accumulation of senescent cells within critical tissues and organs.
- Reversibility of senescence in yeast cells was tested using two approaches.
- Despite telomere damage, most cells were viable after telomerase reactivation.
- Mating of senescent cells with normal cells also restored growth capability.
- Rescue by telomerase differed in checkpoint mutants and recombination mutants.
- Cells rescued by telomerase exhibited shortened telomeres.