Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Abstract

Large, long-lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short-lived species. Large, long-lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger-bodied species but not necessarily longer-lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long-lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16(Ink4a) and p21(Cip1/Waf1) cycline-dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter-lived species display continuous rapid proliferation, whereas cells from small long-lived species display continuous slow proliferation. We hypothesize that cells of small long-lived rodents, lacking replicative senescence, have evolved alternative tumor-suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large-bodied species and small but long-lived species have evolved distinct tumor suppressor mechanisms.

Figure 1. Phylogeny and phenotypic data for 15 rodent species

The tree topology is based on molecular phylogenies inferred from (Adkins et al., 2003; Martin et al., 2000; Michaux et al., 2001; Montgelard et al., 2002; Murphy et al., 2001; Steppan et al., 2004). Body mass and maximum lifespan data is obtained from the following (Buffenstein & Jarvis, 2002; de Magalhaes et al., 2005; Nowak, 1999; Turturro et al., 1999; Weigl, 2005). The data on replicative senescence and growth rates is derived from the growth curves shown in Figure 2. The growth curve for each species represents an average of at least 5 independent cultures.

Figure 2. Growth curves of primary rodent fibroblasts

Cells were isolated from skin and lung tissue and grown at 3% oxygen. The number of donors for each species is shown in Figure 1. (A) Species that use replicative senescence. Each curve is an average of at least 5 independent cultures, and error bars show s.d. Beaver cultures entered senescence at PD39±10; porcupine and paca cultures entered senescence at PD22±3; and capybara cultures senesced at PD25±8. Human primary fibroblasts culture HCA2 is shown as a reference. (B) Rodents that do not use replicative senescence fall into two groups: fast growing and slow growing. Each curve is an average of at least 5 independent cultures, and error bars show s.d. (C) Growth of grey squirrel embryonic fibroblasts (SEFs). Cells grow rapidly for the first 30 PDs, and then slow down and attain the growth rate of adult cells. SEFs were isolated from 4 embryos derived from 2 pregnant females.

Figure 3. Telomerase activity in early and late passage rodent fibroblasts

Telomerase activity was examined using modified TRAP assay. Lanes labeled with “+” contains a sample of telomerase positive human cancer cell line. Arrow indicates a primer dimer, which is not dependent on telomerase activity. NMR, naked mole-rat. The mouse sample shown is from a of 129/Sv×BL6 F1 hybrid. The rat sample shown is from a Fischer F344 rat.

Figure 4. Telomere length in early and late passage rodent cultures

Telomere length was analyzed using a terminal restriction fragment assay as described in Experimental procedures. Five µg of genomic DNA is loaded in each lane. The mouse sample shown is from a 129/Sv×BL6 F1 hybrid. The rat sample shown is from a BN rat.

Figure 5. Activation of p16 (A) and p21 (B) in senescent rodent fibroblasts

Western blot was performed using antibodies to conserved regions of the proteins. p16 and p21 are poorly conserved and molecular weights differ between species. Y, young cells; S, senescent cells.

Figure 6. Species that display replicative senescence have larger body mass

Relationship of replicative senescence with body mass (A), and maximum lifespan (B) was analyzed. Replicative senescence correlates with body mass (t=−5.87, df=13, P<0.0001) but not with maximum lifespan (t=−2.06, df=13, P=0.06). “No” and “Yes” indicate presence or absence of replicative senescence in the species. Plots use species data uncorrected for phylogeny.

Figure 7. Relationships of cell growth rate with maximum lifespan (A) and body mass (B)

Cell growth rate correlates with maximum lifespan (F_1,9=10.68, r²=0.54, P=0.0097) but not body mass (F_1,9=0.11, r²=0.012, P=0.746). The analysis is done for the small-bodied species only, and the species having replicative senescence are not included. Plots use species data uncorrected for phylogeny.

Figure 8. Coevolution of body mass, lifespan and tumor-suppressor mechanisms

Increase in body mass leads to increased cancer risk and evolution of replicative senescence. Small, short-lived rodent species have lower cancer risk and fewer tumor-suppressors. Fibroblasts from these species grow rapidly in culture. An increase in lifespan in small rodents also increases cancer risk and drives evolution of alternative tumor-suppressor mechanisms, which restrict cell growth in vitro.