Quantifying pharmacologic suppression of cellular senescence: prevention of cellular hypertrophy versus preservation of proliferative potential
Abstract
Development of agents that suppress aging (aging suppressants) requires quantification of cellular senescence. Cellular senescence in vitro is characterized by a large cell morphology and permanent loss of proliferative potential. When HT-1080 cells were arrested by p21, they continued to grow exponentially in size and became hypertrophic with a 15-fold increase in the protein content per cell. These changes were mirrored by accumulation of GFP (driven by CMV promoter) per cell, which also served as a marker of cellular hypertrophy. Preservation of proliferative potential (competence) was measured by an increase in live cell number, when p21 was switched off. While modestly decreasing hypertrophy in p21-arresrted cells, rapamycin considerably preserved competence, converting senescence into quiescence. Preservation of proliferative potential (competence) correlated with inhibition of S6 phosphorylation by rapamycin. When p21 was switched off, competent cells, by resuming proliferation, became progressively less hypertrophic. Preservation of proliferative potential is a sensitive and quantitative measure of suppression of mTOR-driven senescence.
Introduction
In cell culture, cellular senescence is
usually defined as a state of irreversible cell cycle arrest [1,2]. Hence,
cellular senescence is sometimes confused with growth inhibition. Here we will
use the term ‘growth' as an increase in cellular mass, regardless of whether
cells proliferate or not. Intriguingly, Ras, MEKeIF-4E
and serum, which stimulate growth-promoting pathways, contribute to and
facilitate cellular senescence [3-6]. In theory, cellular senescence is caused
by inappropriate activation
of growth-promoting pathways, when actual
growth is impossible [7,8]. In proliferating cells, growth-promoting mTOR
(Target of Rapamycin) and MAPK (Mitogen-activated Protein Kinase) pathways
drive both cellular mass growth and cell cycle progression. When
the cell cycle is blocked by either p21 or p16, growth-stimulation via mTOR
leads to cellular senescence [9]. Serum withdrawal, PI-3K, mTOR and MEK
inhibitors, all decreased mTOR activity and prevented permanent loss of
proliferative potential [10,11]. The term "permanent loss of proliferative
potential" means that, even when p21 and p16 were shut off, cells cannot resume
proliferation [12]. Inhibitors of mTOR such as rapamycin preserved
proliferative potential [9-11]. To avoid confusions, we stress that rapamycin
does not stimulate proliferation, does not abrogate cell cycle arrest caused by
p21 and does not force cells to by-pass cell cycle arrest. Rapamycin converts
senescence (an irreversible condition) into quiescence (a reversible
condition). It is still unknown whether rapamycin suppresses senescence in a
dose-dependent manner and whether this suppression correlates with the degree
of mTOR inhibition.
Another common marker of cell senescence is a large
cell morphology (hypertrophy). Cellular hypertrophy is usually measured as a
cell diameter. Given that volume (or cell mass) is proportional to the cube of
diameter, then the amount of protein per cell (cell mass) may be a more
sensitive parameter than cell diameter. For example if diameter is increased
2-fold, cell mass is increased 8-fold. In theory, cell mass could be estimated
as an amount of any fluorescent protein such as green fluorescent protein
(GFP), expressed by a constitutive viral promoter such as CMV promoter. If the
cell cycle is blocked but cells continue to grow in size, then GFP should
accumulate. Here we tested this prediction. Independently from our study, a
clone of HT-p21 cells, known as p21-9, had been stably transfected with
CMV-EGFP [13,14,15] and thus expresses enhanced GFP. We predict that
induction of p21 by IPTG should increase GFP per cell, as a marker of cellular
hypertrophy. Given cell-doubling time of 20 hours, there should be a 10-14 fold
increase in GFP/cell in 3 days. Here, we confirmed this prediction. We further
investigated the link between mTOR activity, cellular hypertrophy and loss of
proliferative potential. We found that preservation of proliferative
(competence) was the most sensitive marker of mTOR inhibition, easily
detectable even at concentrations of rapamycin when inhibition of mTOR was
marginal.
Results
Exponential mass-growth precedes senescence
A number of proliferating cells increased
exponentially (with a doubling time 20-24 h). As previously described,
induction of p21 by IPTG caused G1 and G2 arrest [1,4,5], completely blocking
cell proliferation (Figure 1). p21-arrested cells continued to grow in size,
becoming hypertrophic. Since the cells contained CMV-driven EGFP, we measured
both protein and GFP. Per well, amounts of GFP and protein were increased
almost exponentially with or without IPTG (Figure 2). Per cell, amounts of GFP
and protein were increased only for IPTG-treated (non-dividing) cells (Figure 3). For proliferating cells (no IPTG), GFP per cell and protein per cell
remained constant (Figure 3), because mass growth was balanced by cell
division. In contrast, in IPTG-treated cells, protein/cell and GFP/cell
increased almost exponentially for 3 days (Figure 3). During induction of
senescence by IPTG, cellular mass continued to increase but was not balanced by
cell division. In all cases, protein and GFP correlated (Figure 3), making GFP
per cell a convenient marker of cellular hypertrophy.
Figure 1. Inhibition of cell proliferation by IPTG. Closed bars:
HT-p21 cells were treated with IPTG (+IPTG). Cells do not proliferate. Open
bars: Untreated HT-p21 cells. Exponentially proliferating cells. Cells were
counted daily.
Figure 2. Total cellular mass growth during senescence induction.
HT-p21 cells were grown in 60 mm wells and soluble protein and GFP were
measured daily. Closed bars: HT-p21 cells were treated with IPTG (+IPTG).
Open bars: Untreated HT-p21 cells (-IPTG). In both proliferating (-IPTG)
and non-proliferating (+IPTG) conditions, protein per well
and GFP per well
were increasing. In panel B, protein was measured in duplicate and shown
without standard deviations, therefore statistical difference between
–IPTG and + IPTG should not be considered. The panel simply illustrates
exponential growth in both conditions.
Figure 3. Cellular hypertrophy during senescence induction.
HT-p21 cells were grown in 60 mm wells and cell numbers, soluble protein
and GFP were measured daily. Closed bars: HT-p21 cells were treated with
IPTG (+IPTG). Open bars: Untreated HT-p21 cells (-IPTG). Protein per cell
and GFP per cell
were constant in proliferating (-IPTG) cells. Protein per cell
and GFP per cell increased exponentially in non-proliferating (+IPTG) cells.
Although that was not the goal of our study, our data
can explain how induction of p21 can induce GFP without trans-activating CMV
promoter: by inhibiting cell cycle without inhibiting cell growth. Furthermore,
the notion that GFP per cell is a marker of hypertrophy yields 2 predictions.
First, mutant p21 that cannot bind CDKs and thus cannot arrest cell cycle will
not induce GFP. Second, antihypertrophic agents such as rapamycin will reduce
GFP per cell without abrogating cell cycle arrest.
Dose dependent suppression of cellular hypertrophy
We next investigated the effects of
rapamycin on hypertrophy of senescent cells. Cells were induced to senesce by
IPTG in the presence (+R) or the absence of rapamycin. On days 3 and 5 effects
of rapamycin on cellular hypertrophy were evaluated. By microscopy, the
anti-hypertrophic effect of rapamycin was the most evident at low cell
densities (such as 1000 cells per 60-mm dish) because there was a sufficient
space for IPTG-treated cells to grow in size in the absence of rapamycin (Figure 4). However, we could not reliably measure protein levels at such low cell
densities. At regular cell densities, rapamycin (500 nM) reduced cellular
hypertrophy by 30% -40% (Figure 5A and data not shown). Two markers of
hypertrophy (protein/cell and GFP/cell) correlated (Figure 5A). The
anti-hypertrophic effect of rapamycin was not statistically significant at
concentrations of rapamycin below 20 nM. At first, this was puzzling given that
rapamycin inhibits the mTOR pathway at low concentrations in many cell types.
Therefore, we investigated a dose response of mTOR inhibition by measuring S6
phosphorylation, a marker of mTOR activity. In agreement with anti-hypertrophic
effects, rapamycin inhibited S6 phosphorylation at concentrations 20 nM or
higher, achieving maximal effects at 100 nM-500 nM (Figure 5 B). Thus,
inhibition of S6 phosphorylation and inhibition of hypertrophy correlated,
explaining the requirements of high concentration (100-500 nM) of rapamycin for
anti-hypertrophic effects in this particular cell line.
Figure 4. Visualization of cellular hypertrophy. HT-p21 cells
express enhanced green fluorescent protein (GFP) under the constitutive
viral CMV promoter. Expression of GFP per cell is a marker of cellular
hypertrophy. Low cell density - 2 thousand cells were plated in 100 mm dish
and treated with either IPTG or IPTG + Rapamycin.
Dose-dependent preservation of cellular competence
Rapamycin preserves proliferative potential in
arrested cell, meaning that cells can successfully divide when the arrest is
lifted. But rapamycin does not induce proliferation and in contrast can cause
quiescence (in some cell types). To clearly distinguish the potential
to
proliferate (competence) and actual
proliferation, we introduce terms
competence and incompetence (permanent loss of proliferative potential
associated with cellular senescence). In HT-1080 cells, rapamycin preserves
competence during cell cycle arrest caused by [10]. Unlike senescent cells,
quiescent cells are competent.
Figure 5. Correlation between S6 phosphorylation, hypertrophy and loss of proliferative potential in senescent cells. HT-p21 cells were plated in 6 well
plates and treated with IPTG plus the increasing concentrations of rapamycin
(from 0.16 to 500 nM). At concentration 0, cells were treated with IPTG alone.
(A) Cellular hypertrophy: protein and GFP. After 3 days, soluble protein and
GFP were measured per well. [Note: in non-proliferating cells, protein/well is a
measure of protein/cells]. Results are shown as percent of IPTG alone (0) without
rapamycin. (B) After 3 days, cells were lysed and immunobloted for p-S6, S6
and p21. (C) PC: preservation of proliferative competence. After 3 days, cells
were washed to remove IPTG and RAPA. Cells were incubated for additional 5 days in the
fresh medium and then were counted. Results are shown as percent of IPTG alone (0)
without rapamycin.
We have demonstrated previously that
rapamycin preserved cellular competence (the ability to proliferate after p21
is switched off) in IPTG-arrested HT-p21 cells [10]. We performed these
experiments using rapamycin at concentration 500 nM [10], which completely
inhibited S6 phosphorylation. Here we determined whether preservation of
competence (PC) correlated with inhibition of S6 phosphorylation and the
anti-hypertrophic effect of rapamycin. Cells were treated with IPTG and
increasing concentrations of rapamycin ranging from 0 to 500 nM (Figure 5 C).
After 3 days, IPTG was washed out, thus allowing the cells to proliferate, and
after another 5 days cells were counted. As expected, the IPTG-treated cells
became incompetent, whereas rapamycin suppressed incompetence (Figure 5 C).
Remarkably, preservation of competence was detectable at lower concentrations
of rapamycin than those that inhibited either S6 phosphorylation or cellular
hypertrophy. In part, such a higher sensitivity of a PC-test compared with
inhibition of hypertrophy may be due to the relative magnitudes of the effects
(30% inhibition of hypertrophy versus 800% PC). Perhaps even a transient
inhibition of mTOR (missed by immunoblot) detectably increased competence.
Consistent with this explanation, even when rapamycin was added with delay,
preservation of competence was detectable [10].
Exponential proliferation of competent cells
In the presence of IPTG (with or without rapamycin),
the cells did not proliferate and did not form colonies. When IPTG was washed
out, 3-5% cells remained competent even without rapamycin [10] and Figure 6.
Colonies grew in size, while the number of colonies was almost unchanged (Figure 6). Rapamycin increased a number of colonies (a number of competent cells)
almost 10- fold. We further compared the proliferative quality of competent
cells remained after treatment with IPTG either without or with rapamycin (I/w
and I+R/w, respectively). In I/w and I+R/w conditions, the number of cells
started to increase exponentially after 1 day and 3 days, respectively (Figure 7).
After 6 days, both curves (I/w and I+R/w) became parallel. The curve "I+R/w"
was just shifted to the right on approximately 3 days (Figure 7). This corresponded
to a 10-fold difference in an initial number of competent cells, if their
doubling time was around one day. Noteworthy, this also corresponds to the
initial difference in the number of competent cells as determined by colony
formation (Figure 6). Also, both in I/w and I+R/w conditions, doubling time of
the competent cells was around 20-24 hours, similar to the proliferative rate
of the untreated cells.
Reversal of hypertrophy during proliferation of competent cells
Rapamycin decreased cellular hypertrophy approximately
30% in IPTG treated cells (Figure 5A). When IPTG and rapamycin were washed out,
there was a lag period about 24-30 hrs for competent cells to undergo first
division (supplementary movie will be available at). During the lag period, cells
grew in size, because rapamycin was washed out. Consequently, as measured by
GFP per cell (Figure 8A), rapamycin-treated cells reached the size of the cells treated with IPTG alone (Figure 8A: I/w and I+R/w at day one).
Similarly, as measured by protein per cell, the cells treated with IPTG plus
rapamycin become fully hypertrophic at day one after wash (data not shown).
Despite regaining hypertrophy, IPTG+rapamycin-treated cells remained competent
(Figures 6, 7). This indicates that hypertrophy was not a cause of
proliferative incompetence in IPTG-treated cells. When competent cells divided,
GFP per cell decreased (Figure 8 B). In agreement, there was a marked
difference in cell morphology of typical cells in both conditions (Figure 9).
Under I/w conditions, most of the cells were still large and flat, expressing
beta-Gal staining. Under I+R/w conditions, predominant cells were with a
small-cell morphology and beta-Gal-negative. These cells formed colonies,
indicating that they acquired non-senescent morphology due to proliferation (Figure
10 C, example 1). In contrast, senescent cells that did not resume proliferation
remained large (Figure 10 C, example 2). Competent cells, while proliferating
and forming colonies, became smaller in size (Figure 10 C, example 1).
Eventually, the average cell size dropped to normal levels under I+R/w
conditions, coincident with a decrease in both the amount of protein/cell and
GFP/cell coincided (Supplemental Figure 2), indicating that both are markers
of cellular hypertrophy. Despite reversal of hypertrophy and a drop in
GFP/cell, the amount of total GFP and protein per well increased due to cell
proliferation (Figure 8 B and data not shown).
Figure 6. Clonal proliferation of competent cells. HT-p16 cells
were plated in 100-mm plates. The next day, 50 μM IPTG with or without
rapamycin, if indicated (RAPA), was added. After 3 days, the plates were
washed to remove IPTG and RAPA. (A) Photographs. Upper panel: On
days 5 and 8 (after IPTG removal), plates were fixed, stained and
photographed. Lower panel: On days 5 and 8 (after IPTG removal), plates
were fixed, stained and photographed. (B) Number of colonies. On
days 6, 7, 8 and 9 (after IPTG removal), plates were fixed, stained and
photographed. The number of colonies was counted and results are shown as
percent of plated cells in log-scale.
Figure 7. The dynamics of cell numbers. 500 HT-p21 cells were plated in
12 well plates. On the next day, either IPTG alone (I) or IPTG plus
rapamycin (I+R) were added. After 3 days, plates were washed (I/w and
I+R/w) or left unwashed. Cells were counted at days 1, 3, 6 and 9. Upper
panel: linear-scale. Lower panel: log-scale. Open and closed squares: IPTG
and IPTG plus Rapa, respectively. Open and closed circles: IPTG washed
(I/w) and IPTG plus Rapa washed (I+R/w), respectively. In the presence of
IPTG (open squares) and IPTG plus rapamycin (closed squares), the cells did
not proliferate.
Discussion
Acting in concert, three conditions can contribute to
cellular hypertrophy: cell cycle arrest, continuous protein synthesis and
insufficient autophagy. When the cell cycle was blocked by p21, HT-p21 cells
grew in size almost exponentially for 3 days, eventually becoming senescent. In
parallel with protein content, the amount of GFP (driven by the CMV promoter)
per cell was increased up to 15-20-fold in senescent cells, an increase that
may be a marker of cellular hypertrophy.
Why cells did not grow in size
indefinitely while turning into senescent cells? First, cellular growth may
become counter-balanced by autophagy. This is likely, given the increase in
beta-Gal staining and vacuolarization in senescent cells and the recent
finding that autophagy is activated several days after senescence induction,
coincident with spontaneous deactivation of the PI-3K/mTOR pathway [16]. We
also observed dephosphorylation of S6, when IPTG-treated cells became
terminally-senescent (MS in preparation). Also, senescent cells may become
compensatory insensitive to growth factors.
Figure 8. Loss of hypertrophy during proliferation of competent cells. 500 HT-p21 cells
were plated in 12 well plates. The next day, either IPTG alone or IPTG plus
rapamycin were added. After 3 days, plates were washed (I/w and I+R/w) or
left unwashed. GFP per well was measured and cells were counted at days 1,
3, 6 and 9. GFP per cell was calculated (upper panel). Results are shown in
arbitrary units (M±m). Open and closed squares: IPTG and IPTG plus Rapa,
respectively. Open and closed circles: IPTG washed (I/w) and IPTG plus Rapa
washed (I+R/w), respectively. When cells resumed exponential proliferation,
GFP per cell dropped to normal levels. Due to robust proliferation, there
was an increase of GFP per well.
Rapamycin modestly (30-40%) suppressed cellular
hypertrophy and dramatically (10-fold) increased the number of competent (for
proliferation) cells. When competent cells were released from p21-induced
block, they first grow in size for one day (before division) and then divided.
This indicates that hypertrophy per se does not preclude normal mitosis. While
dividing and proliferating, such cells became progressively smaller. This
recovery phase is a mirror image of the senescence-induction phase, in which
cells grow without division.
Figure 9. The morphology of cells during recovery. 500 HT-p21 cells
were plated in 12 well plates. The next day, IPTG (A) or IPTG plus
rapamycin (B) was added. After 3 days, plates were washed and
microphotographs were taken after additional 3 days. Cells were stained for
beta-Gal. A: I/w; B: I+R/w.
Figure 10. Visualization of loss of hypertrophy during proliferation of competent cells. 500
HT-p21 cells (A) were treated with IPTG (B) or IPTG plus
rapamycin (C), as indicated, or left untreated. After 3 days, plates
were washed and incubated without drugs to allow proliferation. (A)
Normal size of proliferating cells. (B) Cellular hypertrophy of
senescent cells. (C) Example 1. Clonal proliferation of competent
cells results in loss of hypertrophy. (C) Example 2. Cells that
remained arrested remained hypertrophic.
How can we explain preservation of mitotic competence
by rapamycin? This unlikely results from the anti-hypertrophic effect of
rapamycin, given that after rapamycin removal competent cells ‘catch up' in
size with other cells. We suggest that mitotic incompetence is not caused by
hypertrophy but rather hypertrophy and incompetence are independent hallmarks
of cellular aging. We hypothesize that mitotic incompetence may result from
cellular hyper-activation during cell cycle arrest. Activated mTOR and MAPK
pathways may force cell cycle progression despite p21-induced arrest, causing
abortive S-phase entry. In fact, cyclin D1 is highly elevated in senescent
cells [9] and Rb is depleted [17].
In principle elevation of cyclins and
depletion of Rb may allow p21-arrested cells to enter S-phase, thus damaging
the cell. Perhaps, premature cell cycle progression and mitotic incompetence
are two sides of the same coin: overactivation of growth promoting and
mitogen-activated pathways during cell cycle arrest. Then unscheduled S phase
re-entry might be preventable by rapamycin. This hypothesis is under
investigation. Noteworthy, rapamycin blocks pseudo-DNA damage response,
associated with cellular overactivation [18]. Another hallmark of cellular
over-activation in senescent cells is hypersecretory and pro-inflammatory
phenotype, characterized by production of cytokins, mitogens and proteases
[19-26]. Needles to say, rapamycin is an anti-inflammatory drug and is labeled
for use (at high doses) as immunosuppressant in the clinic. It was suggested
that rapamycin as an anti-aging drug will extend healthy and maximal lifespan
in humans [27-31].
Materials and Methods
Cell lines and reagents
. In HT-p21 cells, p21 expression can be turned on or
off using isopropyl--thio-galactosidase (IPTG) [14,15].
HT-p21 cells were cultured in DMEM medium supplemented with FC2 serum. Rapamycin was obtained
from LC Laboratories and dissolved in DMSO as 2 mM solution and was used at
final concentration of 500 nM, unless otherwise indicated. IPTG and FC2 were
obtained from Sigma-Aldrich (St. Louis, MO). IPTG was dissolved in water as 50
mg/ml stock solution and used in cell culture at final concentration of 50
μg/ml.
Immunoblot analysis
. Cells were lysed and soluble proteins were harvested as previously
described [9]. Immunoblot analysis was performed using mouse monoclonal
anti-p21, mouse monoclonal anti-phospho-S6 Ser240/244 (Cell Signaling, MA,
USA), rabbit polyclonal anti-S6 (Cell Signaling, MA, USA) and mouse monoclonal
anti-tubulin Ab as previously described [9].
Cell counting.
Cells were counted on a Coulter Z1 cell counter (Hialeah, FL).
Colony formation assay
. Two thousand HT-p21 cells were plated per 100 mm
dishes. On the next day, cells were treated with 50 μg/ml IPTG and/or 500 nM
rapamycin, as indicated. After 3 days, the medium was removed; cells were
washed and cultivated in the fresh medium. When colonies become visible, plates
were fixed and stained with 0.1% crystal violet (Sigma). Plates were
photographed and the number of colonies were determined as previously described
[9].
SA-β-Gal staining
. Cells were fixed for 5 min in β-galactosidase fixative (2 %
formaldehyde; 0.2% glutaraldehyde in PBS), and washed in PBS and stained in
β-galactosidase solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-beta-gal (X-gal) in
5 mM potassium ferricyamide, 5 mM potassium ferrocyamide, 2 mM MgCl2
in PBS) at 37 ºC until beta-Gal staining become visible in either experiment or
control plates. Thereafter, cells were washed in PBS, and the number of
-galactosidase activity-positive cells (blue staining) were counted under
bright field illumination.
Supplementary Materials
Figure S1. Induction of p21 by IPTG. HT-p21 cells were plated in
6 well plates and treated with IPTG with or without rapamycin as indicated.
The next day, cells were lysed and immunoblot for p-S6, S6 and p21 was
performed as described in Methods. IPTG dramatically induced p21, without
affecting S6 phosphorylation, whereas rapamycin inhibited S6
phosphorylation, without affecting p21 induction.
Figure S2. Loss of hypertrophy following release. HT-p21 cells were treated
with IPTG plus 500 nM rapamycin for 3 days. Then the cells were washed and the cells
were incubated in the fresh medium without drugs. At indicated days, soluble protein,
GFP and cell numbers were measured per well. Protein (pr) per cell and GFP per cell
were calculated and plotted in arbitrary units.
Acknowledgments
We thank Lioubov Korotchkina (RPCI) for help with
microphotographs shown in figure 10, members of Department of Cell Stress
Biology (RPCI, Buffalo, NY) for helpful discussion and assistance, Dr. David
Sinclair (Harvard Univ., Boston, MA) for editing of the first version of the
manuscript.
Conflicts of Interest
MVB is a founder of Oncotarget.
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