Aging and Replicative Senescence

Cellular or replicative senescence (in vitro) is often utilized as a model for the aging process (in vivo) due to the hypothesis that cellular aging recapitulates organismal aging (Wadhwa et al., 2005). The central dogma of repli-cative senescence holds that cultures of vertebrate fibroblasts have a limited capacity for proliferation. After a finite number of cell divisions, proliferation slows and culture arrest ensues. The barrier represented by culture arrest, termed the Hayflick Limit, is accompanied by a number of morphological changes including increased cell size, increased nuclear and nucleolar sizes, increased vacuolation of the cytoplasm and endo-plasmic reticulum, expression of senescence-associated markers such as beta-galactosidase, and other changes in morphology and gene expression (Cristafalo et al., 2004 and references therein).

A genomic alteration associated with cellular or replicative senescence in a variety of organisms, including the chicken, is the shortening of telomeres (Prowse and Greider, 1995; Taylor and Delany, 2000; Swanberg and Delany, 2003). Shortened telomeres induce a DNA damage response, signaling cell cycle arrest. If the damage cannot be repaired, a checkpoint response results in further arrest or apoptosis. An alternative or complementary model for telomere-induced replicative senescence is loss of the protective effect of accessory proteins, such as TRF2, at the telomeres (Karlseder et al., 2002). Reactivation of telomerase or induction of the ALT (alternate lengthening of telomeres) pathway may provide protection against apoptosis or senescence and facilitate transformation and immortalization by stabilizing telomeres (Swanberg and Delany, 2003 and references therein).

The prevailing explanation for telomere shortening, the end-replication problem, is based on the inability of DNA polymerase to replicate the ends of a linear chromosome, resulting in the incomplete replication of the 5' end of the daughter strand. Telomerase is able to offset telomere shortening by adding telomere repeats to the parent strand which generates a longer telomere in the daughter strand. The telomerase holoenzyme is composed of two elements, telomerase rNA, TR, which contains the template for addition of telomeric repeats (Greider and Blackburn, 1989) and telomerase reverse transcriptase, TERT, the component which catalyzes the addition of repeats to the parent-strand chromosome end (Lingner et al., 1997). Most normal, adult vertebrate somatic cells, with the exception of cells from the lab mouse (Mus musculus), do not exhibit telomerase activity (Levy et al., 1992; Kim et al., 1994; Wright and Shay 2002; Levy et al., 1992). Not only does telomerase maintain telomeres of proliferating cells, it is also implicated in oncogenesis (Greider and Blackburn, 1989).

In addition to the end-replication problem and the compensating function of telomerase, telomere length is impacted by proteins that bind to and contribute to the architecture of the telomere. The thousands of duplex DNA telomere repeats are, for the most part, packaged in closely-spaced nucleosomes (Blackburn, 2001). However, the G-rich 3' overhang assumes a terminal loop (t-loop), which displaces one of the duplex strands forming a related structure (D-loop). The D-loop t-loop is stabilized by telomere-binding proteins and their interaction partners (Greider 1999; Griffith et al., 1999; Wei and Price, 2003). Closed chromatin loops resembling t-loops have been observed in chicken using electron microscopy (Nikitina and Woodcock, 2004).

Telomere-repeat-binding factors 1 and 2 (TRF1 and 2) bind to double-stranded telomeric DNA (Wei and Price, 2003). TRF1, which induces telomeric DNA strands to bend, loop and pair (Bianchi et al., 1997; Smogorzewska et al., 2000), may produce shortening of telomeres by sequestering the 3' overhang from telomerase (van Steensel and de Lange, 1997). TRF2 is described as protective of telomeres in some studies (Karlseder, 2003) and as a negative regulator of telomere length in other studies (Smogorzewska et al., 2000; Stansel et al., 2001). Overexpression of TRF1 or TRF2 produces a progressive shortening of telomeres (Ohki and Ishikawa, 2004 and references therein). Tankyrase 1 and 2 have the ability to bind TRF1, resulting in the ADP-ribosylation of TRF1 and the release of TRF1 from telomeric DNA. Overexpression of tankyrase 1 results in the removal of TRF1 from the telomeres followed by telomere elongation (Smith and de Lange, 2000).

In addition to the tankyrases, TRF1 and TRF2, Rap 1 and Pot 1 are involved in telomere maintenance. Rap1

interacts with TRF2, and Pot 1 may coat and protect both G-strand overhangs and the displaced G strand of a t loop (Bauman and Cech, 2001; Tan et al., 2003). Other proteins known to be relevant to telomere length regulation include c-myc, an oncogenic transcription factor which regulates cell proliferation, differentiation and apoptosis (Piedra et al., 2002). Down-regulation of c-myc is believe to be a prerequisite to differentiation (Skerka et al., 1993; Baker et al., 1994) and c-myc reactivates telomerase in transformed cells by inducing expression of its catalytic subunit TERT (Wu et al., 1999).

Chicken orthologs of TRF1 and 2, tankyrase 1 and 2, TR, TERT, c-myc, Rap 1 and Pot 1 have been characterized. In addition, chicken orthologs of the helicases that are missing or mutated in the progeroid disorders, Werner and Bloom Syndrome, have been identified but not studied. The Werner (WNR) and Bloom (BLM) proteins, both Req-Q helicases, have been implicated in telomere maintenance pathways (Du et al., 2004). Table 29.1 lists chicken genes related to telomere length regulation, their human orthologs and relevant references.

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