Our study shows that fibroblast growth characteristics have coevolved with species body mass and lifespan. Previously we showed that repression of telomerase activity in rodent tissues coevolves with large body mass in rodents (Seluanov et al., 2007
). Here we extend these observations to include a correlation between larger body mass and the presence of replicative senescence: the largest rodents — beaver, capybara, porcupine, and paca — have evolved replicative senescence (). The sizes of these species suggest that, to the level of resolution of our study, body mass greater than 8,000 g favors the evolution of replicative senescence (). Evolutionary increases in body mass increase cancer risk, as larger animals contain more cells in their body, and any cell may potentially turn cancerous. Increased mortality rate due to cancer then drives the adaptive evolution of repression of telomerase activity in species with large body mass. It has been predicted that larger animals should have multiple, redundant tumor-suppressor systems so that their cells require more steps for tumor formation (Graham, 1983
; Leroi et al., 2003
; Nunney, 1999
). Our data on rodents provide experimental support for this theory.
Coevolution of body mass, lifespan and tumor-suppressor mechanisms
Our study is focused exclusively on rodent species and it remains unclear if the same rules apply to other groups of mammals. Telomere biology in mammals studied so far supports the model of coevolution of replicative senescence and body mass (reviewed in (Gorbunova & Seluanov, 2008
)). For instance, large mammals such as cow, sheep, and horse do not express telomerase in somatic tissues, and their fibroblasts have finite lifespan in culture (Argyle et al., 2003
; Davis et al., 2005
; Hornsby et al., 1986
). Replicative senescence has also been documented in large primates such as chimpanzee, orangutan, gorilla, baboon, and several macaque species (Gardner et al., 2007
; Herbig et al., 2006
; Steinert et al., 2002
). An intermediate situation where fibroblasts do not express telomerase activity but their cultures fail to undergo growth arrest has been detected in smaller species, such as rabbits and the ring-tailed lemur (Forsyth et al., 2005
; Steinert et al., 2002
). A general trend seems to be that mammals with a body mass greater than 8,000 g evolve stringent replicative senescence, mammals smaller than 2,000 g do not use replicative senescence, and species with body mass between 2,000 and 8,000 g display a spectrum of intermediate phenotypes.
Even less is known about telomere biology in birds. Long-lived species, such as the storm petrel and common tern, express telomerase throughout their lives (Haussmann et al., 2007
). Both of these species are small with an adult body mass below 200 g. Telomerase activity has also been detected in the somatic tissues of a larger bird, the domestic chicken (Venkatesan & Price, 1998
). However, chicken fibroblasts do not express telomerase and undergo a clear-cut replicative senescence (Dinowitz, 1977
; Ponten, 1970
). Little is known about telomere biology in the largest bird species such as ostrich or emperor penguin. The number of population doublings before senescence has been correlated to maximum lifespan (Rohme, 1981
), or when phylogenetic correction was applied, to species body mass (Lorenzini et al., 2005
). Our data set does not allow for such analysis as only four species undergo replicative senescence with three of them showing similar replicative lifespans.
Long lifespan, like body mass, is expected to increase cancer risk. It may therefore seem puzzling that the long-lived rodents in our study have not evolved replicative senescence. While being a potent tumor suppressor, replicative senescence has many tradeoffs, such as slower wound healing and less robust immune response. Furthermore, replicative senescence is only one of many possible tumor-suppressor mechanisms, and it is plausible that these species rely on other mechanisms to mitigate the cancer risk conferred by their long lifespan. Indeed we found that fibroblasts of small, long-lived species such as grey squirrel, naked mole-rat, chinchilla, musk-rat, and chipmunk exhibit a novel in vitro phenotype: their cells do not enter replicative senescence but instead proliferate slowly in culture (). We show that, for small rodent species that have not evolved replicative senescence, in vitro fibroblast proliferation rate negatively correlates with longevity. We hypothesize that the slow in vitro growth rate is a manifestation of those alternative tumor suppressor mechanisms that evolve in small, long-lived species lacking replicative senescence ().
Interestingly, embryonic squirrel fibroblasts, proliferated rapidly up to PD30, after which the culture slowed down and attained the adult growth phenotype. Thus, the growth control mechanisms that restrict proliferation of squirrel cells are characteristic of an adult but not embryonic cells. This scenario is reminiscent of the repression of hTERT expression during human embryonic development. Telomerase is expressed in early embryogenesis, but its expression is progressively shut off in later development (Bekaert et al., 2004
). Thus, the mechanisms that restrict proliferation of somatic cells are inactive during early development when they would otherwise interfere with rapid cell division.
In vitro culture forces cells to proliferate under non-physiological conditions. In vivo the majority of cells in adult tissues are non-dividing. When placed in culture, cells are stimulated to divide by mitogens provided by fetal serum. The ability of cells to proliferate in culture dishes can be considered a measure of their tumorigenic potential. We hypothesize that long-lived rodents evolve mechanisms that make their cells acutely sensitive to any environmental or physiological imbalances, and arrest cell proliferation in inappropriate conditions. The same mechanisms will prevent inappropriate cell division in vivo, protecting the organism from tumor growth and metastasis.
What are the potential cues that slow proliferation of adult fibroblasts of small long-lived rodents? Perhaps, it could be unrestrained mitogenic stimulation, disrupted cell-cell contacts, sensitivity to DNA damage, or some other as yet undetermined cue. It seems likely that these proliferation control mechanisms differ among species because, as can be inferred from rodent phylogeny (), they have evolved with slow aging independently at least three times. In-depth studies of the individual small long-lived species are required to understand the molecular mechanisms responsible for the different anti-cancer adaptations that have evolved among them.
In summary, our analysis of fifteen rodent species has uncovered an intricate picture of how increased cancer risk conferred by large body mass or long lifespan drives evolution of tumor suppressor mechanisms (). Body mass has previously been linked to the evolution of several characteristics such as repression of telomerase activity (Seluanov et al., 2007
), more efficient DNA repair (Promislow, 1994
), and the number of population doublings before senescence (Lorenzini et al., 2005
). Here we show that both body mass and lifespan contribute to the evolution of tumor suppressor mechanisms, but in two different ways. Large body mass coevolved with replicative senescence, while long lifespan is associated with evolution of alternative mechanisms that increase the sensitivity of the cells to growth conditions and slow cell proliferation in culture.