The Telomere Hypothesis of Cell Aging and Immortalization

When I started my research on replicative senescence in the early 1990s, the exact nature of the genetic event which accounted for the finite life span of HDFs was still unknown. However, exciting initial data in support of the end replication problem had recently been generated in the lab of my soon-to-be thesis advisor, Calvin Harley (Harley et al. 1990). This early study on the measurement of telomere length in human cells (in this case, HDFs) showed that the size of telomeres gradually shortened during replicative aging in vitro. In fact, prior to this study, there had been some indirect evidence that telomeres shorten during aging in humans. Some of the very earliest evidence in support of the loss of telomeric DNA during aging of primary cells in humans was from a study by Cooke and Smith, who showed the telomeric fragments of chromosomes were longer in the germ line than in the peripheral blood (Cooke and Smith, 1986). Shortly after the sequence of mammalian telomeric DNA was shown to be (TTAGGG)n in the late 1980s, work by de Lange et al. confirmed this earlier finding by directly showing that the terminal (TTAGGG)n tract at the ends of chromosomes is shorter in peripheral blood than the germ line (de Lange et al. 1990). This work by de Lange et al. was soon followed by two other studies which directly demonstrated the reduction in telomere size during replicative aging, one by Harley et al., which demonstrated that the size of the terminal restriction fragment (TRF) gradually shortens during replicative aging of HDF (Harley et al. 1990), and another study which showed that TRF length gradually decreases in size in human colon mucosa and peripheral blood during in vivo aging (Hastie et al. 1990). Another key observation in support of the notion that telomere shortening causes replicative senescence was the initial demonstration of active telomerase, an RNA-protein enzymatic complex that functions to complete the replication of, and even elongate, telomeric DNA, and which was originally discovered in ciliates (Greider and Blackburn, 1985) present in a human immortalized cell line (Morin, 1989).

Together, these initial observations of telomere shortening during replicative aging of primary human cells in vitro and in vivo, along with the end replication problem, led us to propose the telomere hypothesis of cellular aging and immortalization (Fig. 2.2) (Harley, 1991; Harley et al. 1992). This hypothesis made a number of (what were then) predictions: first, due to the end replication problem and possibly other mechanisms, telomeric DNA will shorten during replicative aging of mortal primary cell cultures established from different types of human somatic

Fertilization M1 M2 Replicative age

(senescence) (orisis)

Fig. 2.2 The telomere hypothesis of cell aging and immortalization. The changes in telomere length during replicative aging of cells in vivo, beginning at the time of fertilization, is depicted. The change in telomere length during replicative aging for somatic cells and germ cells are indicated by the solid and dashed gray arrows respectively. The green bar shows the telomere size range for which there is insufficient telomeric DNA to allow a stable telomere complex to form. The upper size limit of this region is the critical telomere length (marked as TC), which is the minimum length of telomeric DNA required to allow formation of a stable telomere complex at the end of a chromosome. The status of telomerase activity is also indicated. See text for further details (See Color Plate)

Fertilization M1 M2 Replicative age

(senescence) (orisis)

Fig. 2.2 The telomere hypothesis of cell aging and immortalization. The changes in telomere length during replicative aging of cells in vivo, beginning at the time of fertilization, is depicted. The change in telomere length during replicative aging for somatic cells and germ cells are indicated by the solid and dashed gray arrows respectively. The green bar shows the telomere size range for which there is insufficient telomeric DNA to allow a stable telomere complex to form. The upper size limit of this region is the critical telomere length (marked as TC), which is the minimum length of telomeric DNA required to allow formation of a stable telomere complex at the end of a chromosome. The status of telomerase activity is also indicated. See text for further details (See Color Plate)

cells; second, telomere length will be maintained in germ line cells from generation to generation by telomerase; third, once a critically short size is reached on one or more telomeres, a signal will be sent to initiate replicative senescence, or the Ml checkpoint (the "Hayflick limit"); fourth, telomeres will continue to shorten in primary cells transformed by DNA tumor viruses, or transforming genes from these viruses (e.g., SV40 large T antigen) until nearly all cells have acquired multiple chromosomal ends with critically short telomeres. At this point, the M2 checkpoint, or crisis, will be signaled; fifth, in the rare cells which survive crisis and acquire replicative immortality, telomere length will be maintained by telomerase.

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