Cellular lifespan and senescence: a complex balance

Review

between multiple cellular pathways

David Dolivo, Sarah Hernandez and Tanja Dominko

The study of cellular senescence and proliferative lifespan is becoming increasingly important because of the promises of
autologous cell therapy, the need for model systems for tissue disease and the implication of senescent cell phenotypes in
organismal disease states such as sarcopenia, diabetes and various cancers, among others. Here, we explain the concepts
of proliferative cellular lifespan and cellular senescence, and we present factors that have been shown to mediate cellular
lifespan positively or negatively. We review much recent literature and present potential molecular mechanisms by which
lifespan mediation occurs, drawing from the fields of telomere biology, metabolism, NAD+ and sirtuin biology, growth factor
signaling and oxygen and antioxidants. We conclude that cellular lifespan and senescence are complex concepts that are
governed by multiple independent and interdependent pathways, and that greater understanding of these pathways, their
interactions and their convergence upon specific cellular phenotypes may lead to viable therapies for tissue regeneration
and treatment of age-related pathologies, which are caused by or exacerbated by senescent cells in vivo.

Keywords: continuous proliferation [3]. It is now presumed that the chick

■ antioxidants; lifespan; metabolism; senescence. embryo extract added to the growth medium was contaminated
with additional chick embryonic cells, so that newly added
cells replaced the dead cells at each passage [4].
Somatic cells display a finite replicative Although Carrel’s paradigm was accepted until the 1960s,
lifespan his findings were called into question when Leonard Hayflick
characterized the subcultivation and subsequent growth arrest
The study of proliferative cellular lifespan is more than a cen- of 25 human diploid fibroblast strains. In this study, Hayflick
tury old. In 1912, Alexis Carrel published his findings on the defined a common cause of the degradation of cell strains: “A
exploration of the in vitro cellular lifespan of fetal chick connec- consideration of the cause of the eventual degeneration of
tive tissue. Carrel reported growth in culture for months at a these strains leads to the hypothesis that non-cumulative
time, and he even reported acceleration in the growth rate of external factors are excluded and that the phenomenon is attrib-
these cultures as they aged [1]. The mindset of Carrel and his utable to intrinsic factors, which are expressed as senescence at
colleagues was that, when cells stopped growing and eventu- the cellular level” [5]. Cells that have reached this state of
ally perished, it was because of extrinsic factors such as the growth arrest, known as cellular senescence, have been identi-
buildup of metabolic toxins. In line with this logic, Carrel fre- fied by particular genotypic and phenotypic characteristics,
quently washed his cells and changed his growth media in including a flat, vacuolated morphology, increased activity of
order to ensure that the cells were growing without interference. SA-ßgal at pH 6 (ß-galactosidase), SAHF (senescence-associated
Using Carrel’s method, Albert Ebeling was able to maintain a heterochromatic foci) enriched in H3K9 methylation, arrest in the
culture of chick embryonic heart tissue for 129 passages over G1 phase of the cell cycle and high levels of p53 and p21 protein
the course of a year [2]. The culmination of this view of cellular [6]. It is worth noting, however, that exceptions to some of these
immortality in the absence of extrinsic, lifespan-limiting factors phenotypes exist, including a recently reported transcriptional
occurred when, in 1946, it was reported that the last of Carrel’s down-regulation of p53 upon replicative and oncogene-induced
original chick embryo cultures had died, following 43 years of senescence in human keratinocytes, which appears to be
because of changes in chromatin structure via deacetylation
of histone H3 [7]. Additionally, senescent cells display a
DOI: 10.1002/icl3.1036 characteristic secretory phenotype, characterized by the
secretion of pro-inflammatory cytokines including IL-6 and
Biology and Biotechnology Department, Worcester Polytechnic Institute, IL-8, as well as a host of other differentially regulated factors.
Worcester, MA, USA These secreted factors likely play a role in the development
of age-related disease, as factors secreted from senescent cells
Corresponding author: have been shown to induce senescent phenotypes in autocrine
Tanja Dominko
E-mail: tdominko@wpi.edu
[8] and paracrine [9] manners, and they have also been shown
to promote the epithelial-to-mesenchymal transition in neigh-
Received 17 April 2015; revised 14 September 2015; accepted 17 September boring cells, potentially facilitating the formation of metastatic
2015 tumors in vivo [10]. Recently, senescent cells have also been

36 www.icelljournal.com Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
 D. DOLIVO et al.

shown to communicate through cell-cell contact-dependent the increase in proliferative lifespan afforded by elongation-
protein transfer that is mediated by cytoplasmic bridges, which deficient hTERT mutants is not completely understood, some

Review
could also contribute to tissue aging and age-related patholo- progress has been made in unraveling this phenomenon.
gies including cancer [11]. Knockdown of the telomere-capping shelterin protein hPOT1
ablated the ability of hTERT to extend proliferative lifespan,
suggesting an effect that, while independent of telomere
elongation, is still connected somehow to the telomeres
Telomeres and telomerase mediate [22]. hTERT and hPOT1 co-localize to telomeric ends [23], sug-
cellular lifespan gesting that localization of hTERT to its proper location along
the telomere may be facilitated by the expression of hPOT,
Initially, cellular senescence was described as being caused by enabling a more robust protection of the telomere in a
attrition to a critical length of telomeric DNA, which is con- manner independent of explicit changes in telomere length.
structed of a noncoding, repeating sequence that shortens In this way, it seems reasonable to hypothesize that hTERT
upon each cell division [12], although many other inducers of and hPOT1 synergistically lead to a protection of the end of
senescence have since been determined [13]. In theory, then, the telomeric strand, suppressing the common p53 response
activity of telomerase, an enzymatic complex that compensates that leads to both the DNA damage repair pathway that
for telomere shortening by addition of copies of a repeating nu- would normally occur at single-stranded DNA (such as those
cleotide sequence to the end of chromosomes, could prevent found at the end of the telomere) via ATR signaling and cellu-
this attrition and prevent or delay the telomere-mediated lar senescence [24]. The localization of hPOT1 to its proper
senescence of cells in culture [14]. Indeed, overexpression of location along the telomere may be facilitated by the expres-
telomerase reverse transcriptase (TERT), the catalytic subunit sion of hTERT, enabling a more robust protection of the
of the telomerase complex, has been successfully employed telomere end independent of telomere lengthening. In further
to significantly extend the lifespan of and/or immortalize support of this hypothesis, another telomere-associated
numerous human cell lines, including endothelial cells, retinal shelterin protein, TRF2, has also been shown to extend the
pigment epithelial cells, human foreskin fibroblasts, T-cell clones replicative lifespan of human diploid fibroblasts when
and skeletal muscle satellite cells [15–19], while allowing these overexpressed by protecting short telomere ends from fusion,
cells to retain a stable karyotype and the capacity to function even though the telomeres of these cells show an extended
normally. Given this broad range of cell types through which rate of shortening compared with control cells expressing
overexpression of TERT can cause cellular immortality, it comes only endogenous TRF2 [25]. TRF2-mediated lifespan extension
as no surprise that 85–90% of human cancers demonstrate may result from protection of the very end of the telomeric
measurable telomerase activity [14]. DNA from the DNA damage response and cell cycle arrest that would
Interestingly, mutant hTERT (human TERT) alleles have otherwise be activated by Ataxia telangiectasia mutated (ATM)
been discovered that fail to elongate telomeres yet retain signaling, as the cell otherwise may improperly recognize the end
their ability to extend proliferative lifespan [20,21]. While of the telomere as a DNA double-stranded break [24] (Fig. 1).

Figure 1. Telomeres, telomerase and the senescent response. While highly replicative cells have long stretches of repetitive telomeric
sequences (TTAGGG, for example, in vertebrates), cells undergoing replicative senescence have, in most cell types, reached a critically short
telomere length, resulting in growth arrest and other senescent phenotypes. The shelterin complex protects the telomere by suppressing p53-
mediated DNA repair, and hTERT (human telomerase reverse transcriptase) appears to be able to enhance this protection, possibly by facilitating
the localization of particular shelterin proteins to their proper place along the telomere and within the shelterin complex.

Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc. 37
 D. DOLIVO et al.

Knockdown of STN1, a recently identified human homologue metabolism has been shown time and time again to be an es-
of a protein involved in budding yeast telomere protection, re- sential determinant of cellular and organismal health and
Review

sulted in fragile telomeres and aberrant telomeric structures, lifespan; the magnitude of the effects of even single metabolic
increased amounts of DNA damage signaling in culture and de- enzymes can be staggering. In MEFs (mouse embryonic fibro-
creased replicative lifespan in human diploid fibroblasts. Inter- blasts), overexpression of either two glycolytic enzymes, PGM
estingly, overexpression of hTERT rescued or improved some of or GPI (phosphoglycerate mutase or glucose-6-phosphate
these phenotypes, including the reduction of proliferative isomerase, respectively), was sufficient to enable bypass of
lifespan, to the levels of control fibroblasts, suggesting the senescence induced by growth at atmospheric oxygen [34],
ability of hTERT to confer some degree of protection to conditions under which murine fibroblasts generally senes-
telomeric ends on its own, possibly independent of its interac- cence quickly because of enhanced sensitivity to oxygen and
tions with shelterin proteins [26]. This hypothesis of a protec- oxidative damage [35], or senescence induced by oncogenic
tive role of hTERT falls in line with previous reports indicating stimulus. This senescence bypass and subsequent immortali-
that, in human fibroblasts, hTERT has the ability to partially zation required catalytic activity of the glycolytic enzymes,
rescue growth arrest caused by DNA replication stress induced suggesting a direct link between glycolytic activity and cellular
by oncogene expression or genotoxic drug treatment [27]. senescence [34].
There is more to telomeres and telomerase than their direct Although the mechanism or mechanisms by which overex-
roles in cellular lifespan and senescence, however. Recently, it pression of PGM and GPI allow for bypass of senescence are
was discovered that mTERT (murine telomerase reverse tran- not entirely understood, the requirement of catalytic activity,
scriptase) knockout mice with critically short telomeres demon- as well as the knowledge of enhanced oxygen sensitivity in
strate, in a p53-dependent manner, markedly reduced levels of MEFs, suggests the ability of glycolytic intermediates or prod-
PGC-1α and PGC-1β, transcriptional coactivators that have been ucts to counteract agents of oxidative damage. Indeed, there
implicated in the regulation of genes that modulate cellular is evidence to suggest that pyruvate, the end product of glycol-
metabolism and their downstream targets. Resulting age- ysis, can act as a scavenger of reactive oxygen species in rat
associated deleterious organismal and cellular phenotypes ob- thymocytes [36] and protect against oxidative damage-induced
served in these mice include cardiomyopathy, increased levels cytotoxicity in rat osteoblasts [37], thereby attenuating a stim-
of ROS (reactive oxygen species) and decreased mitochondrial ulus that is known to cause senescence-inducing genotoxic
function and biogenesis [28]. Additionally, hTERT has been damage [38] and has been shown to be important for the main-
shown to modulate expression of a myriad of other genes tenance of senescence [39]. In a similar vein, overexpression of
and genetic pathways. hTERT-immortalized human foreskin fi- hexokinase 2 which, unlike the housekeeping gene hexokinase
broblasts demonstrated high expression levels of the growth 1, is subject to substantial cell type-specific regulation, favored
factor epiregulin which, when inhibited, triggered the cells to escape from oncogene-induced senescence in human epithe-
enter senescence [29]. This finding suggests that the expres- lial cells, as evidenced by continued proliferation, the presence
sion of epiregulin is necessary for cells to demonstrate of proliferative markers including Cyclin A and reduced activity
extended proliferation upon immortalization by hTERT. High of SA-ßgal. This escape from oncogene-induced senescence
throughput analyses have also identified hundreds of genes could be replicated by exogenous addition of glucose-6-
transcriptionally modulated by hTERT, which are associated phosphate, the product of the enzymatic reaction of hexoki-
with cellular processes as diverse as differentiation, metabo- nase 2 with its glucose substrate, suggesting that the
lism and the cell cycle [30], and hTERT has been implicated senescence-evading effects of hexokinase 2 are due to its ki-
specifically in regulation of the Wnt/β-Catenin [31] and NF-κB nase activity. Interestingly, it was shown that supplementation
[32] pathways. Given that the expression of hTERT has the abil- with N-acetylglucosamine also favored escape from oncogene-
ity to confer proliferative immortality in numerous cell types induced senescence, suggesting that the anti-senescent
[15], as well as the implication of hTERT signaling in all six tra- effects of hexokinase 2 overexpression (and glucose-6-
ditional hallmarks of cancer [33], it is unlikely that an idea as phosphate supplementation) may be due to an increase in
broad as activation or overexpression of hTERT will be a useful activity of the hexosamine pathway, which lies downstream of
tool for treatment of age-associated pathologies or as a glucose-6-phosphate and regulates protein glycosylation [40]
method of elongation of proliferative lifespan in order to, for (Fig. 2).
example, obtain sufficient cells to make cellular therapies a Related to the effects of metabolic enzymes on cellular
worthwhile option. Instead, we must understand more specific lifespan is the effect of cellular senescence on metabolism. Re-
details involving the molecular mechanisms of cellular senes- cently, it has been proposed that, contrary to previous assump-
cence and the methods by which we can ameliorate this irre- tions, the specific metabolic profile of senescent cells depends
versible growth arrest, some of which will be explained on the senescence trigger. Ras-induced senescent human
forthwith. fibroblasts displayed substantial increases in fatty acid oxida-
tion and mitochondrial oxygen consumption, as well as a
metabolic profile that was easily distinguishable from that of
replicatively senescent human fibroblasts. Interestingly, the
Metabolism is an integral mediator of ability of Ras-induced fibroblasts to maintain a typical senes-
cellular lifespan cent secretory profile depended largely on the ability of the
cells to transport fatty acids into the mitochondria and, pre-
As cellular processes and energy availability are so intrinsically sumably, catabolize them [41]. The finding that Ras-induced
connected to metabolism, it comes as no surprise that senescent fibroblasts exhibit increased fatty acid oxidation is

38 Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc.
 D. DOLIVO et al.

as evidenced by decreased levels of NADH. The extracellular

metabolomes of these senescent fibroblasts also resemble those

Review
of aging fibroblasts with significant DNA damage [45]. Knockdown
of p53, an essential effector protein in the senescent response,
led to increases in the expression of malic enzymes ME1 and
ME2, which are responsible for converting malate into pyruvate.
These findings suggest a positive feedback loop by which p53
sustains its own expression via down-regulation of malic
enzymes, thereby alleviating malic enzyme-mediated self-
repression, resulting in higher levels of p53 overall. This regula-
tory pathway is thought to involve AMPK (AMP-activated protein
kinase) and E3 ubiquitin ligase (MDM2) in the stabilization and
degradation of p53 via phosphorylation [46] and ubiquitination,
respectively [47]. p53 has been shown to be involved directly or in-
directly in other metabolic processes implicated in tumor biology
ranging from glycolysis [48,49] to oxidative phosphorylation
[50,51] and others, reviewed in [52], suggesting the possibility of
other roles for p53 in the establishment of a senescence-specific
metabolic profile. A more complete understanding of cross-talk
and reciprocal regulation between proteins that are associated
with the initiation and maintenance of senescence and proteins
that are involved in metabolism, and a better understanding of
how these categories are not necessarily mutually exclusive, will
Figure 2. Metabolism as a regulator of cellular lifespan. Metabolic allow us to make more definitive statements about causality in
processes comprise some of the most important functions of cellu- senescence and use this logic to plan further studies and develop
lar machinery. Overexpression of GPI (glucose-6-phosphate isom- therapies for senescence-associated organismal phenotypes and
erase) and PGM (phosphoglycerate mutase) enables bypass of disease states.
senescence induced by oncogene activation and oxidative damage
in a manner dependent on catalytic activity. This suggests that this
bypass is dependent on some aspect of glycolysis, possibly the
ability of the glycolytic end product pyruvate to scavenge ROS
(reactive oxygen species). Similarly, overexpression of HK2 leads to
Dietary restriction, sirtuins and nicotin-
bypass of oncogene-induced senescence, which is also observed amide adenine dinucleotide, together and
upon addition of glucose-6-phosphate and N-acetylglucosamine, separately, mediate lifespan
suggesting that this senescence bypass may be mediated by the
hexosamine pathway, which is known to regulate protein
Dietary restriction is considered one of the most promising
glycosylation.
strategies for increasing lifespan and healthspan in humans
[53]. The mechanisms by which dietary restriction extends
made more interesting by previous findings, which suggest lifespan in diverse organisms have been reviewed extensively
that free fatty acid levels may serve as a determinant of elsewhere [54] but, in mammals, the mechanistic target of
lifespan in lower eukaryotes [42]. It has also recently been rapamyacin (mTOR) appears to be particularly important, which
shown that Ras-induced senescent fibroblasts show a decrease is unsurprising because of its role as a metabolic regulator and
in dNTP levels that is mediated by oncogene-induced repres- essential component of energy sensing pathways [55].
sion of the dNTP synthesis protein RRM2. This decrease in Although mTOR and its roles in aging have also been subject
dNTP levels is both necessary and sufficient for induction and to review recently as well [56], it is worth noting here that mTOR
maintenance of the oncogene-induced senescent response, has been shown to antagonize senescence and/or increase
as both exogenous addition of nucleotides and ectopic expres- cellular lifespan as well, as seen in MEFs [57], human retinal
sion of RRM2 can overcome the senescent growth arrest, and pigment epithelial cells [58] and murine dendritic cells [59].
knockdown of RRM2 is sufficient to decrease dNTP levels and The full picture of mTOR’s relationship to cellular senescence is
induce cellular senescence in human fibroblasts [43]. The also not completely understood and is likely very complex [60].
metabolic changes associated with oncogene-induced senes- A recent explanation that attributes a major role in the
cence, including the reduction in dNTP levels, were shown to senescence program to mTOR signaling is the hypothesis of
be due at least in part to the activity of the DNA damage gerocentric conversion, or geroconversion, as a means to
response agent ATM, which is responsible for inhibition of better explain the differences between reversible “quiescent”
glucose and glutamine consumption and pentose phosphate growth arrest and irreversible “senescent” growth arrest. The
pathway activity under conditions of replicative stress [44]. geroconversion hypothesis implicates mTOR and its myriad
Human fibroblasts induced to senesce under conditions of pro- activators in cellular senescence by proposing a senescent
liferative exhaustion (replicative senescence) and DNA damage state in which the cell cycle is inhibited (via, for example, p16
(DNA damage-induced senescence) display increased levels of or p21) but mTOR and MAPK signaling are still active. The result
glycolytic metabolism and lower levels of oxidative metabolism, is a cell that continues to grow without the ability to proliferate,

Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc. 39
 D. DOLIVO et al.

resulting in the establishment of cellular senescence. The exogenous nicotinamide was shown to increase proliferative
hypothesis of geroconversion aims to explain many of the lifespan slightly in human diploid fibroblasts [80]. At first, com-
Review

canonical hallmarks of senescent cells as, for example, continued pared with the previous results, this finding seems paradoxi-
growth without cell division would be expected to yield large, cal, as nicotinamide is known to reduce sirtuin deacetylase
flattened cells characteristically seen in senescent cultures, activity via inhibition of NAD+ hydrolysis [81]. However, this ap-
and as increased lysosomal activity would be necessary in parent discrepancy may be explained by the NAD+ salvage
order to counterbalance cellular hypertrophy, resulting in pathway. If sufficient NAMPT was available in these cells, and
increased βgal activity commonly used as a marker of senescent the levels of nicotinamide were kept well below the IC50 of nic-
cells in vitro [61]. otinamide on SIRT1, the ability of nicotinamide to act as a sub-
In addition to mTOR, the lifespan-extending effects of die- strate for NAD+ synthesis would likely outweigh its suppression
tary restriction also appear to involve the sirtuin family of of sirtuin activity, resulting in a net lifespan extension of the
proteins [62]. The sirtuins are class III, NAD+ (nicotinamide treated cells (Fig. 3). Recently, it has also been shown that ex-
adenine dinucleotide)-dependent protein deacetylases that pression of SIRT1, which is normally lost over time, mediates
are known to interact with a wide variety of targets to mediate cellular senescence in mesenchymal stem cells by protecting
the lifespan and healthspan of many distantly related organ- the telomeres from DNA damage and downstream signaling.
isms [63]. Although lifespan-extending effects of other sirtuins It appears that this effect operates at least in part via up-
have been uncovered, such as the ability of SIRT6 to regulate regulation of several shelterin proteins including TPP1, as well
the lifespan of male mice [64], the most rigorously studied as via down-regulation of DNA damage-associated tumor sup-
sirtuin in mammals is SIRT1. Alongside its homologues in yeast pression proteins [82].
[65], Drosophila, and Caenorhabditis elegans, although the Reduction in NAD+ levels has also been associated with ag-
effects of these homologues on C. elegans and Drosophila ing. It has been shown that the levels of NAD+ are lower in sam-
lifespan have been a subject of debate [66–69], SIRT1 has been ples taken from aged tissues in both rats and humans,
linked to mammalian lifespan as well, likely at least in part suggesting that there may be a causal relationship between
because of its role in dietary restriction [70], although the NAD+ and age-related cellular and organismal pathologies.
results of studies of dietary restriction on the lifespan of The reduction of NAD+ levels over time is thought to be, at least
primates remain controversial as well [71,72]. in part, a result of depletion via NAD+-dependent enzymes such
The effects of SIRT1 are not limited to organismal longevity, as PARPs (poly ADP-ribose polymerases) [83,84], as these en-
however. It has been shown that, in human diploid fibroblasts, zymes are involved in DNA damage repair, and DNA damage
overexpression of and/or activation of SIRT1 by resveratrol sup- has been shown to accumulate with age in murine [85] and
plementation led to an increase in lifespan and decrease or de- human cells, and in vivo in aging mice [86]. This suggests that
lay in senescence-associated phenotypes including G1 arrest, decreasing NAD+ levels over time may inhibit a cell’s ability to
SA-ßgal activity, p16 expression, pRb activity and ID1 repres- repair its genome, potentially leading to genomic instability,
sion [73]. Additionally, SIRT1 has been shown to suppress se- cellular stress responses such as senescence or apoptosis
nescent phenotypes in part via modulation of the acetylation and deleterious phenotypes in aging organisms.
state of the liver kinase LKB1 in porcine aortic endothelial cells Alongside the involvement of NAD+ in the processes of DNA
[74]. The effects of SIRT1, and indeed other mammalian damage repair and sirtuin-mediated deacetylase activity, NAD+
sirtuins, on cellular lifespan are not completely understood, also has roles in oxidative phosphorylation [87] and ß-oxida-
but recent studies have begun to unravel at least some of the tion [88], both of which are irrevocably intertwined with bioen-
mechanisms underlying these effects. The repression of ergetic homeostasis and, by extension, longevity (Fig. 4). It has
senescence-induced phenotypes is likely due at least in part also recently been shown that age-associated declines in levels
to the fact that p53, one of the most important players in senes- of cellular NAD+ may lead to defects in nuclear-mitochondrial
cent growth arrest, is a direct deacetylation target of SIRT1 [75]. communication through reduction of SIRT1 activity and subse-
The deacetylation of p53 allows for MDM2-mediated quent stabilization of the hypoxia-inducible transcription factor
ubiquitination at the very same lysine residues [76], implicat- HIF-1α via a reduction in the levels of the E3 ubiquitin ligase
ing SIRT1 in p53 protein stability and turnover and, by associa- Von Hippel-Lindau tumor suppressor (VHL). This leads to a
tion, the senescent growth arrest. Additionally, the deacetylase deficiency in mitochondrially encoded proteins and a loss of
activity of mammalian sirtuins is dependent on their cofactor mitochondrial homeostasis, thereby suggesting another man-
NAD+ [77], which is a coenzyme that, while implicated predom- ner by which age-associated decline in cellular NAD+ may lead
inantly in cellular metabolism, has also been shown to partici- to deleterious phenotypes in aging tissues and organisms.
pate in cellular activities including regulation of gene Intriguingly, this NAD+-dependent decline in mitochondrial
expression and cell death [78]. It is unsurprising, then, that ma- function was rescued by addition of exogenous NAD+, restoring
nipulation of the levels of NAD+ leads to variations in the mitochondrial homeostasis and muscle health in aged mice,
lifespan elongation effect of SIRT1. Ectopic expression of SIRT1 suggesting therapeutic potential for treating this particular bio-
alone was shown to increase the proliferative lifespan of hu- chemical pathway [89]. Maintenance of NAD+ homeostasis has
man smooth muscle cells modestly (~18% extension) but the also been shown to increase the lifespan of Saccharomyces
effect became much more pronounced (~129% extension) cerevisiae via the SPS (Ssy1-Ptr3-Ssy5) amino acid-sensing
when SIRT1 was co-expressed with NAMPT (nicotinamide pathway [90]. A more holistic understanding of the pathways
phosphoribosyl transferase), an enzyme responsible for a in which NAD+ is a cofactor and the regulation of its synthesis
rate-limiting step in the salvage pathway that converts nicotin- and degradation may provide promising avenues by which to
amide to, ultimately, NAD+ [79]. In another study, addition of pursue additional research into pathologies of aging.

40 Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc.
 D. DOLIVO et al.

Review
Figure 3. Antisenescent effects of SIRT1 and its partners. Overexpression of the sirtuin SIRT1 leads to an increase in cellular and, in some
cases organismal, lifespan. Similarly, resveratrol leads to increased activation of SIRT1 and downstream phenotypes, such as increased
lifespan and suppression of senescence. Without overexpression of SIRT1 and/or resveratrol supplementation, only wild-type levels of SIRT1
activity are available to activate downstream phenotypes which, to a lesser extent, increase lifespan and suppress senescence. Addition of
+
nicotinamide and/or overexpression of NAMPT (nicotinamide phosphoribosyl transferase) ultimately leads to increased production of NAD
(nicotinamide adenine dinucleotide), a cofactor essential for the deacetylase activity of SIRT1, resulting in a further increase in cellular lifespan
+
and resistance to senescent phenotypes. Without the addition of the SIRT1 cofactor NAD , SIRT1 activity is dependent on endogenous levels
+
of NAD , resulting in a lesser effect on lifespan and senescence.

Growth factors influence the rate of lifespan witnessed between FGF2-treated and untreated pri-
mary human fibroblasts grown at atmospheric oxygen levels
cellular senescence and the substantial extension of temporal lifespan under con-
ditions of sub-atmospheric oxygen, which also appears to be
Because of their essential roles in mitotic regulation and me- independent of hTERT [91]. According to this hypothesis, an ex-
tabolism, it should come as no surprise that numerous growth ternal factor (e.g. accumulation oxidative damage to genomic
factors have been implicated as determinants of cellular DNA in the nucleus or accumulation of ROS and subsequent
lifespan. Previously, our lab and others have demonstrated a DNA damage in the mitochondria) drives cells towards senes-
significant extension in proliferative cellular lifespan (>50% in- cence at a time-dependent rate, and the lifespan extension re-
crease in cumulative population doublings) in primary human sults from an increased rate of doubling during the limited
dermal fibroblasts treated with exogenous FGF2 (fibroblast chronological lifespan afforded by the external stressor. Addi-
growth factor 2) [91,92], a mitogen that is critically important tional evidence for this hypothesis includes the finding that,
to the medium for in vitro culture of human embryonic stem in human diploid fibroblasts, overexpression of ERK2 and/or
cells [93]. Although exogenous supplementation of FGF2 has targeting of ERK2 to the nucleus results in a significant exten-
been shown to up-regulate hTERT transcription and telomerase sion of proliferative lifespan [94]. Because FGF2 leads to the
activity in HUVECs (human umbilical vein endothelial cells) phosphorylation and subsequent activation of ERK2 [95,96], it
[94], we have not witnessed a comparable increase in hTERT seems plausible to suggest that some downstream result of
expression in primary human fibroblasts. Although the mecha- this mechanism may be able to explain the effect of FGF2 on
nism of an hTERT-independent lifespan extension has not been proliferative lifespan, one of which may be mitogenesis. Inter-
determined, there are several plausible explanations. One pos- estingly, in mouse fibroblasts, FGF2 stimulation has been
sibility is that the senescence stressor in human dermal fibro- shown to signal down the RAS/RAF/MEK/MAPK pathway to
blasts, under typical culture conditions, may be more tightly increase protein levels of MDM2, an E3 ubiquitin ligase that
correlated to temporal lifespan than replicative lifespan. This is responsible for the nuclear export and degradation of the
explanation is supported by the similarity in chronological senescence-initiator p53 [97,98]. These potential mechanisms

Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc. 41
 D. DOLIVO et al.
Review

+
Figure 4. Diverse functions of NAD (nicotinamide adenine dinucle-
+
otide). Although well-known for its metabolic importance, NAD
partakes in many other essential cellular functions. Aside from its
role as the oxidation product of NADH, an electron donor in oxidative Figure 5. Fibroblast growth factor-2 signaling and the cell cycle. The
+
phosphorylation, NAD is also involved in DNA damage signaling via growth factor FGF2 (fibroblast growth factor-2) can signal via trans-
the PARPs (poly ADP-ribose polymersases), in deacetylase activity membrane RTKs (receptor tyrosine kinases) down the RAS/RAF/
as an essential cofactor of the sirtuins, and in control of ß-oxidation, MEK/ERK pathway. Upon stimulation with FGF2, this signaling
among other processes. cascade leads to phosphorylation of ERK, enabling its nuclear
translocation. Nuclear translocation of ERK leads to cell growth via
increased expression of cyclin D and subsequent progression from
the G1 to the S phase of the cell cycle.

are not mutually exclusive, and it is altogether possible that

the pronounced effect of FGF2 on proliferative lifespan
observed in fibroblasts is a result of several independent or Additionally, genome-wide association studies have been
interdependent mechanisms, some of which may not yet be used to investigate the role of IGF-1 signaling in the longevity-
understood (Fig. 5). In fact, the substantial number of genes associated effects of dietary restriction on mice [103], as the
differentially expressed in fibroblasts treated with FGF2 com- downstream effectors of IGF signaling are numerous. Myriad
pared with untreated fibroblasts suggests that we may have genes were also implicated in lifespan modulation through a
only begun to scratch the surface of the effects of FGF2 on transcriptome analysis of long-lived C. elegans mutants lacking
cellular lifespan in fibroblasts, especially because many of functional DAF-2, the C. elegans homologue of the mammalian
these genes regulate metabolism and the cell cycle [99]. IGF-1 receptor [104]. In addition to genes encoding stress re-
Another growth factor that has been implicated in lifespan sponse proteins, like catalase and heat shock proteins, as well
regulation is IGF-1 (insulin-like growth factor-1), in conjunction as genes that code for several insulin-like peptides, several
with its associated signaling pathway (also known as the antibacterial-associated genes were also uncovered which,
IGF/growth hormone axis). At the organismal level, decreased despite their heightened importance in C. elegans given that
levels of serum IGF-1 have been associated with increased lon- bacterial proliferation in the pharynx and the intestines is a
gevity in women, or in both men and women who have a history frequent cause of death in laboratory-grown C. elegans, may
of cancer [100]. FIRKO mice, which have an adipose tissue- still be important factors in modulating the lifespan of higher
specific knockout of IGF-1, show a reduction in fat accumula- animals and of their cellular constituents [105]. Additional
tion and in metabolic abnormalities, while exhibiting an ap- proteins with less well-defined roles were also implicated in
proximately 14–18% increase in mean and maximum lifespan lifespan mediation, including a particularly large increase in
[101]. Female mice heterozygous for the IGF-1R (IGF-1 receptor) lifespan (>30% increase) upon knockdown of the C. elegans
display about half the number of functional receptors of wild- gene dod-18, the protein product of which shares significant
type mice, and they exhibit a statistically significant increase homology to the human protein ASMTL (N-acetylserotonin
in lifespan (the male mouse lifespan increase is about half O-methyltransferase-like protein). To our knowledge, ASMTL
the magnitude of that of females and trends towards signifi- has yet to be investigated in mammalian systems in the context
cance). These heterozygous mice also demonstrate increased of organismal aging or cellular lifespan. In C. elegans, the
resistance to oxidative stress relative to wild-type mice [102]. lifespan effects of dysfunctional DAF-2 have been linked to

42 Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc.
 D. DOLIVO et al.

activity of the forkhead transcription factor DAF-16, as this gene organismal level), independent of telomerase activity and/or
is required for lifespan extension in the daf-2 mutant. Interest- telomere length. Perhaps more relevant, and at the cellular

Review
ingly, forkhead transcription factors have been implicated in level, the culture of primary MEFs grown in low oxygen drasti-
the regulation of human lifespan as well, as variants of FOXO1 cally increases their proliferative lifespan, even though MEFs
[106,107] and FOXO3A [106,108–111] have been shown to be have unusually long telomeres and constitutively express both
strongly correlated with increased lifespan in various human of the primary components of murine telomerase [35]. The
cohorts. senescent phenotype of MEFs in atmospheric oxygen closely
Insulin-like growth factor-1 signaling has been implicated in resembles, morphologically and genetically, that of human
lifespan at the cellular level as well. While short-term treatment fibroblasts that have been made to senesce prematurely by
with exogenous IGF-1 promotes mitosis in primary human fibro- treatment with hydrogen peroxide, further supporting the
blasts, sustained treatment with IGF-1 causes premature senes- notion that murine cells grown under ambient oxygen likely
cence at least in part via inhibition of SIRT1-mediated p53 senesce as a result of chronic oxidative damage, rather than
deacetylation [112]. An increase in activity of the AKT pathway, telomeric attrition because of replication stress [35,122].
which can be activated via signaling through IGF-1 and PI3K It makes sense to hypothesize, then, that the extension of
[113], has also been shown to negatively regulate proliferative the lifespan of human fibroblasts grown in physiological oxy-
lifespan in human endothelial cells in a p53/p21-dependent gen in vitro may be a direct consequence of the evasion of se-
manner [114]. As we learn more about growth factor signaling, nescence from the low-grade, chronic oxidative stress that
we may uncover promising targets for pharmacological agents results from culture in oxygen tension (atmospheric oxygen),
in order to alleviate pathologies of aging. One of the many which is higher than that experienced by cells in their native
questions that remains about growth factor signaling and its physiological environment. Rather than simply activating an
relationship to cellular senescence is how signaling via various endogenous mechanism enabling them to live longer, these
growth factor-associated pathways, such as the FGF2/MAPK cells may be, at least in part, bypassing a premature, oxidative
and IGF-1/PI3K/AKT pathways, induce seemingly opposite damage-induced growth arrest (Fig. 6). This hypothesis is sup-
effects on cellular lifespan, and whether this explanation ported by identification of various compounds and enzymes
involves concentration-dependent effects, interplay with other with demonstrated antioxidant activity that have been shown
molecular pathways or downstream factors, aspects of the to extend cellular lifespan. Long-term supplementation of cul-
aforementioned geroconversion model or something different ture media with ascorbic acid has been shown to extend the
entirely. proliferative lifespan via reduction in ROS and mitochondrial
damage and via a decrease in the rate of attrition of telomeres
in human embryonic fibroblasts in vitro [123,124]. Addition to
the culture medium of juglanin, levorotary loliolide, sun gin-
Oxygen and antioxidants mediate lifespan seng, epifriedelanol, quercetin-3-O-ß-D-glucuronide (a deriva-
and senescence tive of quercetin), various plant extracts and coenzyme Q,
many of which have demonstrated antioxidant activity, have
Oxygen tension has been implicated heavily in the behavior of all been shown to have anti-senescent effects as determined
cells as they age. For decades, culturing cells at sub- by SA-ßgal staining, expression of senescence-associated pro-
atmospheric, physiological oxygen has been shown to increase teins and/or extension of proliferative lifespan in vitro [125–131].
their proliferative lifespan significantly beyond control cells The ability of numerous antioxidants to suppress senescent
grown in atmospheric oxygen [91,115,116], but only fairly re- phenotypes and extend the proliferative lifespan of various types
cently has progress been made in elucidating the effect of oxy- of human cells, along with reports of increased lifespan in
gen on the timing of cellular senescence. Part of this effect may response to overexpression of antioxidant enzymes such as
be because of an increase in hTERT transcription, as the hyp- catalase [132] seems to suggest that ROS are important stimuli
oxia inducible transcription factor HIF-1 has been shown to up- in the initiation and maintenance of cellular senescence, which
regulate hTERT transcription in human choriocarcinoma and has also been documented elsewhere [39].
cervical cancer cells via hypoxia response elements at the Interestingly, it has been suggested that the antioxidant
hTERT promoter [117,118], although work in our laboratory has effects of some compounds added to culture media actually
demonstrated a large increase in proliferative lifespan under cause an increase in ROS, likely because of the ability of anti-
growth at physiological oxygen in the absence of substantial oxidants to become oxidized in the growth medium, as
transcriptional up-regulation of hTERT [91]. Although choriocar- reviewed in [133]. This effect of antioxidant supplementation
cinoma and cervical cancer cell lines may not be expected to on reactive oxygen species in vitro may seem counterintuitive
function normally relative to non-neoplastic cells, these reports when taken together with the ability of antioxidant supplemen-
show that HIF-1 at least has the potential to regulate the tran- tation to suppress senescent phenotypes in cultured cells, but
scription of hTERT. However, there are several other lines of ev- there is precedent for this kind of observation. For example, it
idence that suggest that the ability of culture in physiological has been shown that growth under hypoxia, which has been
oxygen to extend lifespan is far more robust than this. For shown to enhance the proliferative lifespan of many cell types
one, HIF-1 has also been shown to modulate the lifespan of in vitro, is associated with an increase in the production of
C. elegans [119,120], which has been shown to be independent mitochondrial ROS in the short term [134–136]. Although they
of telomere length in wild-type worms [121], which is unsurpris- have roles as important signaling molecules in a variety of
ing for a post-mitotic organism. This suggests a mechanism of cellular processes [137], ROS can, in excess quantities, cause
lifespan elongation that is, at least in part (although at the damage to cellular components. Indeed, oxidative damage to

Inside the Cell, 2016, 1, 36–46, © 2015 The Authors. Inside the Cell published by WILEY Periodicals, Inc. 43
 D. DOLIVO et al.
Review

Figure 6. Oxygen is a mediator of cellular senescence. It is well-demonstrated that cell culture under sub-atmospheric oxygen extends
lifespan substantially and in many cell types. In vitro, cells are typically cultured under atmospheric oxygen, the level of which is artificially
high when compared with physiological oxygen. This increase in oxygen likely causes chronic oxidative stress, leading to DNA damage. When
cells are cultured under conditions that more closely mimic the physiological environment, it is likely that these cells bypass the premature,
oxidative damage-induced proliferative arrest that would otherwise occur via the MRN complex and the ATM and ATR signal transducers.

mitochondrial components and DNA has been implicated in Conclusion

premature cellular senescence and the deregulation of bioenergetic
homeostasis [138–143]. The value of studying cellular aging and senescence is not
The theory of mitochondrial hormesis, or “mitohormesis,” limited to its importance in in vitro studies and the potential
reviewed in [144] aims to resolve some of these paradoxes for autologous cellular therapies, as senescence has recently
and to explain why low-grade oxidative stress is associated been implicated in diseases as varied as sarcopenia, dyskeratosis
in some cases with an increase in cellular and organismal lon- congenita, diabetes, atherosclerosis, neurodegenerative disease
gevity, rather than cellular damage and a loss of homeostasis. and certain cancers, as senescence-associated secretory
Briefly, triggers of cellular stress are able to induce factors have been shown to induce the epithelial-to-mesenchymal
mitochondrial-related responses via enzymes associated with transition [150–155]. Our understanding of the paradigms
antioxidant activity, such as catalase or superoxide dismutase, and molecular mechanisms underlying cellular and organismal
or the mitochondrial UPRmt (unfolded protein response), aging has been developed greatly in recent years, and further
resulting in increased tolerance to misfolded proteins, reactive research into the causes and effects of cellular senescence at
oxygen species and toxins, among others. This may help both the single cell and the whole organism level will prove
explain findings in which transient up-regulations in ROS levels fruitful in fields ranging from gerontology to oncology.
have been associated with elongated lifespan and healthspan
in C. elegans insulin signaling-deficient, calorie restriction
mimetic and low-grade arsenite stress models [145–148]. In
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