Here we present results showing that SIRT1, the NAD-utilizing deacetylase enzyme is a negative regulator of growth under normal and restrictive conditions in certain cell lineages. Consistent with this notion, efficient inhibition of SIRT1 deacetylase was associated with an increase in telomerase activity that is required for survival and long-term cell growth. Our data indicate that the effect of SIRT1 on telomerase activity is mediated through the catalytic subunit of telomerase, hTERT. On SIRT1 inhibition there is a small increase in hTERT mRNA level and a significant increase in levels of hTERT protein. This increase in mRNA correlated with lack of SIRT1 at proximal regions of hTERT promoter and an increase in total H4 acetylation at the hTERT promoter. Cell lines expressing either endogenous hTERT under its native promoter or primary human diploid fibroblasts expressing ectopic hTERT showed increased levels of hTERT protein and activity upon SIRT1 suppression. We find that the suppression of SIRT1 and its effects on telomerase are independent of how hTERT is expressed (i.e., under native or ectopic viral promoters). Interestingly, we also observed an hTERT doublet in BJT cells in which SIRT1 was expressed suggesting a posttranslational role for SIRT1 in regulation of hTERT protein stability. However, further experimentation is required to investigate if this is caused by increased hTERT acetylation.
The increased telomerase activity and cell growth phenotype observed could be rescued by a silent mutant SIRT1-R protein that is resistant to repressive effect of shRNA directed to SIRT1, showing that the effect observed was specific. Our results point toward a functional interaction between SIRT1 and hTERT; however, the basis for this genetic interaction is unknown, and it is possible that the effect of SIRT1 on hTERT is not direct and is mediated via other proteins. Furthermore, given the diverse range of SIRT1 targets the effects observed on hTERT maybe one factor that contributes to the observed cellular phenotype.
Overexpression of SIR2 extends replicative lifespan of single-cell eukaryotes such as S. cerevisae and chronological lifespan of multicellular protostomes such as C. elegans. We reasoned that if the lifespan-inducing functions of the mammalian SIR2 homolog SIRT1 is conserved, this should reflect itself in either survival or replicative lifespan of vertebrate cells with long life spans, such as somatic cells of Homo sapiens.
When we overexpressed wild-type SIRT1 in mortal normal human diploid BJ fibroblasts, we observed no significant effect on replicative lifespan, consistent with published data (Michishita et al., 2005
). We also performed the reverse experiments by suppressing SIRT1 to near detection limits in primary BJ fibroblasts, and we still did not observe any effects on replicative life span. Because it has been shown before by us and others that ectopic expression of hTERT and reconstitution of its activity causes life span extension in human cells, we reasoned that inhibition of SIRT1 may have an effect on telomerase-induced extension of lifespan. If primary BJ cells were first infected with an hTERT-expressing virus and sequentially were subjected to SIRT1 inhibition, there was an increased efficiency in cell growth reflected by a decrease in the population doubling time. This effect could be mediated through telomeres or other indirect effects on cell survival. It is however clear from our data that SIRT1 suppression promotes cell growth in the presence of ectopic telomerase activity. Our findings in human cells are consistent with that of others who have shown that murine fibroblasts deficient for Sirt1(Sir2α) have a higher frequency of immortalization (Chua et al., 2005
). In contrast, others have shown that in different cell types such as endothelial cells SIRT1 suppression has the opposite effect: its loss induces cell cycle arrest (Ota et al., 2007
). Given the range of substrates currently identified for SIRT1 and their increasing number, it is possible that the contradicting growth-promoting and growth-suppressing properties observed are cell type or species specific.
Extension of our in vitro results to hematopoiesis under adverse conditions caused by lack of growth factors is consistent with the notion that SIRT1 is a growth suppressor. Although we observed no appreciable difference in HSCs or progenitor frequencies in young Sirt1−/−
mice, the in vitro proliferative capacity of Sirt1-deficient HSCs were significantly elevated in both complete media and under cytokine-deprived conditions containing a single growth factor. These results were consistent with that of immortalized human BJT cells lacking SIRT1 expression that showed higher proliferation under normal or glucose-deprived conditions. We found that consistent with the role of activated AMPK in response to low glucose (Salt et al., 1998
), cells lacking SIRT1 showed an earlier peak in both total levels and activated phospho-AMPK-α protein upon glucose deprivation. Activation of AMPK hence may allow survival in response to an energy shortage. Although this finding suggests that SIRT1 may regulate AMPK, others have found that induction of AMPK by the SIRT1 activator resveratrol is SIRT1 independent (Dasgupta and Milbrandt, 2007
). Although a useful marker of energy status and survival, AMPK induction observed here maybe due to a complex and indirect effect of SIRT1 on cell survival under ATP-limiting conditions.
It is possible that under nutrient-restrictive conditions, SIRT1 acts as a growth suppressor to limit division in high-capacity progenitor cells. This limitation may be a physiological response to save on usage of macromolecules required for survival of pre-existing stem cells. Hence, SIRT1 can modulate the division and survival capacity of stem cells in response to nutrient availability. Our results have significant implications for survival of adult stem cells under stress and would be of interest to examine whether SIRT1 has similar effects in other types of stem cells. They also indicate that specific chemical inhibitors of SIRT1 may enhance survival or pluripotency in adult or embryonic human or murine stem cells.
Evidence suggests that calorie restriction is associated with decreased age-associated tumor incidence (Weindruch, 1992
). Furthermore, the beneficial biological effects of calorie restriction in increasing lifespan have been well documented. Therefore, it is possible that in human cells, calorie restriction can increase SIRT1 activity, which in turn can suppress immortalizing genes such as telomerase. Therefore increased SIRT1 activity would then suppress tumor incidence and therefore only indirectly leads to extension of lifespan. Hence the effects of induction of molecules such as SIRT1 on longevity of complex multicellular vertebrates may be mediated indirectly via stimulating its tumor suppressor functions and hence reduce death due to cancer. We predict that overexpression of SIRT1 in mice would primarily result in suppression of certain types of tumors. Based on our results and models, SIRT1 overexpression may have no functional effect on the network of human genes promoting somatic cell chronological/replicative survival, leading directly to increased longevity. Current lack of a unifying evolutionary conservation in longevity functions of SIR2 however should not detract from its fundamental roles in cellular survival and growth from yeast to mammals.
Our findings underscore the importance of nutrient-dependent pathways and propose that SIRT1 is a nutrient-sensitive growth suppressor that may act as an important barrier to retard the growth of certain nutrient-sensitive immortal tumor cells.