Telomere Shortening and Cellular Aging

Telomeres were brought to the forefront of the cellular aging discussion when Olovnikov's hypothesis turned into experimental fact in the model organism S. cerevisiae. At the time, it had been suggested that due to the inability of conventional polymerases to fully replicate the 3' terminus of the G-rich strand, a sequence-specific enzyme would elongate telomeres. Therefore, a mutation screen was designed to identify enzymatic activities responsible for telomere replication via the isolation of mutants defective for the telomere elongation reaction (Lundblad and Szostak 1989). The authors made several predictions, which all turned out to be correct:

• The nature of the screen would allow the identification of enzymatic functions without having to make assumptions about the exact enzymatic activities.

• The identified mutations should not lead to the cells' immediate demise, but rather to lethality or a senescence phenotype over time.

• The S. cerevisiae telomeric tracts would become shorter with each generation, and eventually become unable to protect the chromosome ends, resulting in the accumulation of chromosomal abnormalities (Lundblad and Szostak 1989).

The genetic screen, based on the linearization and retention of a circular plasmid carrying telomeric repeats, led to the discovery of a new gene, EST1, which stands for Ever Shorter Telomeres 1. Exactly as predicted by the investigators, a strain mutant for EST1 (the strain est1-1) displays a gradual senescence phenotype, associated with a progressive decrease in telomere length. After 25 generations est1-telomeres are 100 bp shorter than wild type telomeres, and continue to lose terminal sequence for many more generations. Strikingly, the gradual telomere loss is accompanied by a decrease in growth rate and an increase in the frequency of cell death. Finally, after approximately 100 generations the est1-1 strains cease to divide.

The authors had also predicted that mutants in the telomere maintenance pathway should accumulate chromosomal aberrations. Analysis of chromosome loss pheno-types in a strain lacking EST1 demonstrates that mutants do not suffer from chromosome loss in early generations, but as the cells approach senescence, chromosomes are lost at a high frequency, validating the authors' prediction. This landmark publication ends with a statement that finally links Hayflick's observations with telomere biology: " „.a loss of telomeric DNA during each somatic cell division may play a role in determining the number of cell divisions possible in a given lineage."

EST1, a protein that binds single-stranded telomeric DNA, turned out to be a subunit of the telomerase complex and to play an essential role in mediating access of the catalytic subunit of telomerase to telomeres (Evans and Lundblad 1999,

Lundblad 2003). It acts as an adapter between the catalytic core of the enzyme and Cdc13, a single-stranded telomeric DNA binding factor in yeast (Nugent et al. 1996, Pennock et al. 2001).

Discovery of the catalytic subunit of telomerase in model organisms allowed the identification of the human telomerase reverse transcriptase subunit (hTERT) via sequence homology (Kilian et al. 1997, Meyerson et al. 1997, Nakamura et al. 1997). Once identified, it became possible to study the effects of hTERT overexpression on telomere length and the life span of human fibroblasts, which usually do not exhibit telomerase activity, and, as a result, suffer from replication-associated telomere shortening. Since the telomere hypothesis of cellular aging holds that cells become senescent when critically short and dysfunctional telomeres appear, the investigators predicted that expression of active telomerase would extend the replicative life span of such cells (Bodnar et al. 1998). Normal human retinal pigment epithelial cells (RPE) and human BJ foreskin fibroblasts, which both lack telomerase activity, were transfected with a vector expressing the catalytic subunit of human telomerase hTERT, leading to detectable telomerase activity. When telomere length was measured to determine whether in vitro telomerase activity translates into in vivo telomere elongation, the researchers found that telomere length had increased significantly, demonstrating that hTERT reconstituted telom-erase activity extends endogenous telomeres in normal human cells. The key experiment was to investigate the effects of hTERT expression on the replicative life span of the hTERT reconstituted cells. hTERT negative RPE or BJ clones showed the expected slowing of growth and consecutive senescence between 50 and 70 population doublings. hTERT-positive RPE and BJ clones, however, exceeded this limit by many population doublings, and kept cycling at the rate of young cells (Bodnar et al. 1998). No senescence markers could be detected in the telomerase-active clones, whereas such markers were highly prevalent in the telom-erase-negative populations, demonstrating that the reconstitution of telomerase activity is sufficient to extend the replicative life span of primary human cells. These results corroborate that telomere loss in the absence of telomerase activity is the mitotic clock that controls the number of cell divisions before terminal differentiation in primary human cells.

These life-extending experiments trigger an important question: Would cells that expressed hTERT retain a diploid genome? Immortality in cancer cells is almost always associated with telomerase activity and aneuploidy. In an effort to characterize telomerase-immortalized cells, researchers had a closer look at BJ foreskin fibroblasts that had been maintained in continuous culture for 280 population doublings (Morales et al. 1999). No loss of contact inhibition or acquisition of serum independent growth was noted in the hTERT-expressing clones, suggesting that they had not adopted such features of transformed cells. Another common attribute of cancer cells is the loss of cell cycle checkpoints, hence the investigators scrutinized the pRB and the p53 cascade for functionality in hTERT immortalized primary cells, and found both pathways intact. The final experiment was to monitor chromosomal aberrations, since accumulation of translocations, amplifications, deletions, inversions, and chromosome multiplications is a common feature of cancer cells (Lengauer et al. 1998). However, cells expressing ectopic telomerase activity maintain a normal karyotype. These datasets indicate that introduction of hTERT into normal somatic human cells is not sufficient for transformation, does not bypass cell cycle checkpoint control, and does not lead to genomic instability (Morales et al. 1999).

The discovery of telomerase, and the demonstration that telomere maintenance is required for continued proliferation, was recognized by the 2006 Albert Lasker Award for Basic Medical Research, whereby Elizabeth Blackburn, Carol Greider, and Jack Szostak were honored (Blackburn et al. 2006, de Lange 2006).

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