Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination

Abstract

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny-based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co-evolved with body size. Multiple independent times smaller, shorter-lived species changed to having longer telomeres and expressing telomerase. Trade-offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human-like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.

Fig. 1

Evolutionary distribution of telomere length, telomerase activity and stasis (stress or aberrant signaling induced senescence) in the mammalian evolutionary tree. Telomere length, telomerase activity, and stasis induction run in evolutionary clades. Telomere length was measured by terminal restriction fragment (TRF) analysis: Values < 20 kb are shaded black, while values > 20 kb are shaded pink. Nonplacental mammalian orders such as Marsupials and Monotremes show the presence of restriction enzyme recognition sites intercalated between the telomeric (TTAGGG)_n sequences and thus are labeled as being of indeterminate size (I). Telomerase activity was detected with TRAP: Values are expressed as a%of the activity in the reference lung adenocarcinoma tumor line H1299, and shaded black if absent and pink if any activity was detected. Stasis: (N) with black shading = cells grew beyond 15 doublings, (Y) with pink shading = cells growth arrested in culture before population doubling 15. Based on these characteristics, some taxonomic orders could be described as exhibiting uniformly human-like telomere phenotypes or having discontinuous telomeres. Arsenite resistance: (Y) with black shading = LD90 > 20 mm sodium arsenite after 4 h exposure, (N) with pink shading = LD90 < 5 mm (see Fig. 5). Published data on Primates (Steinert et al., 2002), Lagomorphs (Forsyth et al., 2005), and Muntjacs (Zou et al., 2002) are from our laboratory, and thus are directly comparable. Data from other laboratories are indicated by (*) [cow (Lanza et al., 2000), sheep (Cui et al., 2002), pig (Fradiani et al., 2004; Oh et al., 2007), rat (Mathon et al., 2001)]. Scientific names and specific data for each species (scientific name, growth curves, TRF gels, telomerase assays, mass, lifespan) are provided in Table S1 and Fig. S1 (Supporting Information). The telomere lengths for giraffe, rhinoceros, and anteater are adjusted for a large digestion-resistant subtelomeric region using the rate of disappearance of the telomeric signal with increased cell doublings (see Fig. S1, Supporting Information). Cladogram adapted from Bininda-Emonds et al. (2007, 2008).

Fig. 2

Relationship of telomeres and telomerase to mass and lifespan. Dark arrows indicate significance while dotted arrows show lack of significance. Phylogenetic generalized least squares framework (PGLS) analysis demonstrates that telomerase expression inversely co-evolves with increased mass, while telomere length inversely correlates with increased lifespan.

Fig. 3

Nonplacental mammals have discontinuous telomeres. Telomere restriction fragment analysis (TRF) involves digestion of genomic DNA with a mixture of six 4-base restriction enzymes (HaeIII, AluI, HinfI, MspI, RsaI, and CfoI) and yields telomeres of a variety of sizes, examples of which are shown. Nonplacental mammalian repeats are interrupted by DNA containing restriction sites, so their size depends on which enzyme is used. Dashes corresponding to the 19- and 6.2-kb size markers were digitally fixed to the image for each species, and then the size of each image adjusted so that all the marks aligned to allow a direct visual comparison between the different species. The DNA from species with very long telomeres was analyzed on FIGE gels.

Fig. 4

Telomere length, telomerase, lifespan, and body mass distributions. Telomere length (A and B) or telomerase (C and D) vs. log body mass (A and C) or lifespan (B and D). The probability of a significant relationship as an independent variable is indicated by the phylogenetic generalized least squares (PGLS) analysis, which takes into account phylogenetic relationships (which are not indicated in the figure). Telomerase is significantly inversely related to mass while telomere length is inversely correlated with lifespan. Most of the variability occurs within smaller shorter-lived species.

Fig. 5

Resistance to tert-butyl hydroperoxide and sodium arsenite. The LD90 of a 4-h treatment with different oxidative damage-inducing agents was examined for 15 different species. Data are shown for telomere length (A and B), maximum lifespan (C and D), tert-butyl hydroperoxide (A and C), and sodium arsenite (B and D). Phylogenetic generalized least squares (PGLS) analysis demonstrates that resistance is significantly associated with telomere length independent of the effects on lifespan or mass. Virtually identical patterns are observed if plotted against body mass (data not shown) instead of lifespan. Table S3 (Supporting Information) gives the actual values for the specific species analyzed. ±SEM of 2–6 titration curves.