From the Human diploid fibroblasts eventually lose the
capacity to replicate in culture and enter a viable but
nonproliferative state of senescence. Recently, it has been
demonstrated that retroviral-mediated gene transfer into primary
fibroblasts of an activated ras gene (V12ras)
rapidly accelerates development of the senescent phenotype. Using this
in vitro system, we have sought to define the mediators of
Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular,
mitochondrial reactive oxygen species. The ability of V12Ras to induce
growth arrest and senescence is shown to be partially inhibited by
coexpression of an activated rac1 gene. A more dramatic
rescue of V12Ras-expressing cells is demonstrated when the cells are
placed in a low oxygen environment, a condition in which reactive
oxygen species production is inhibited. In addition, in a 1% oxygen
environment, Ras is unable to trigger an increase in the level of the
cyclin-dependent kinase inhibitor p21 or to activate the
senescent program. Under normoxic (20% O2) conditions, the
V12Ras senescent phenotype is demonstrated to be unaffected by
scavengers of superoxide but rescued by scavengers of hydrogen
peroxide. These results suggest that in normal diploid cells, Ras
proteins regulate oxidant production and that a rise in intracellular
H2O2 represents a critical signal mediating
replicative senescence.
A growing body of evidence suggests that reactive oxygen species
(ROS)1 regulate a number of
diverse intracellular pathways (1). In particular, certain growth
factors and cytokines can trigger a rapid, transient increase in
intracellular ROS levels; ROS, in turn, participates in downstream
signaling (2-8). Similarly, the overexpression of wild type p53 in
several different cell types is accompanied by a sustained rise in ROS
levels, which appears to be required for the subsequently observed
apoptotic cell death (9-10).
Numerous studies suggest that ROS may also participate in aging
(11-14). On a cellular level, replicative senescence is perhaps the
most widely studied model of organismal aging. Consistent with a role
for ROS in senescence, examination of cells in culture suggest that
older cells have higher levels of ROS than younger ones (15). In
addition, cells treated with antioxidants or grown in conditions of low
oxygen appear to have a prolongation of life span (16, 17). Finally,
treatment of primary fibroblast with a sublethal concentration of
H2O2 induces a state resembling senescence (18-20).
Recently, it has been demonstrated that a replicative senescence state
could be rapidly triggered by expression of an activated ras
gene (V12ras) in primary human fibroblasts (21). Oncogenic Ras expression in these experiments also resulted in an increase in the
expression of the cyclin inhibitor proteins p21 and p16, which are
linked to the senescent phenotype (22, 23). Although Ras proteins
activate a number of distinct downstream pathways, some evidence
suggests that these proteins also play a role in the regulation of the
redox state of the cell (24, 25). Based on the previous evidence
linking oxidants to aging, we have explored in this study the
possibility that V12Ras expression induces rapid replicative senescence
by altering the intracellular levels of ROS.
Retroviral Infection--
A V12Ras-expressing retrovirus was
constructed in a bicistronic vector that also encoded the IL-2 receptor
Adenoviral Infection--
Adenoviruses encoding either an
activated Rac1 (Ad.V12Rac1 (28)), a dominant negative Rac1 (Ad. N17Rac1
(28)), Cu,Zn superoxide dismutase (Ad.SOD (29)), or the histological
marker gene lacZ (Ad. ROS Measurements--
Levels of cytostolic ROS were assessed by
loading cells with 5 µg/ml of 2'-7' dichlorodihydrofluorescin
diacetate (DCF) (Molecular Probes) for 5 min and then imaging with a
Leica laser scanning confocal microscope (7). Alternatively, cells were
loaded with DCF and analyzed and sorted by FACS (Epics Elite-ESP
cytometer, Coulter). Direct visualization of mitochondrial ROS was
achieved using dihydrorhodamine 123 (DHR123) as described previously
(32), and the resulting fluorescence was imaged using confocal
microscopy. To confirm the mitochondrial localization of DHR123, cells
were also incubated with Mitotracker red (Molecular Probes).
Quantification of DCF of DHR123 fluorescence was achieved by measuring
fluorescent intensity of approximately 60 random cells from six
different fields (7).
For manipulation of ambient O2 levels, cells were placed in
sealed chambers (Billups-Rothenberg) 1 day after immunoaffinity sorting. The chambers were flushed for 20 min prior to sealing with the
desired O2 concentration, and the gas mixture was
replenished every 4 days thereafter. The antioxidants Mn(111) tetrakis
1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP) (Calbiochem) and
N-acetylcysteine (NAC) (Sigma) were added at the indicated
concentrations from day 6 onward and exchanged with fresh media and
antioxidants every 3 days.
We first sought to ascertain whether in our primary cultures of
human diploid fibroblasts, levels of ROS differed between young and old
cells. Cells from mid- or late passage were loaded with the
peroxide-sensitive fluorophore DCF and assayed by FACS analysis. As
demonstrated in Fig. 1A, older
cells had markedly increased levels of DCF fluorescence, consistent
with higher levels of intracellular hydrogen peroxide. The relationship
between intracellular levels of H2O2 and
senescence was further extended by analyzing mid-passage cells by FACS
analysis and sorting cells based on the level of DCF fluorescence. The
senescent phenotype was assessed by subsequently analyzing the
percentage of cells exhibiting We next infected low passage human diploid fibroblasts with a
bicistronic retrovirus encoding V12Ras and the subunit of the IL-2
receptor. Infected cells were purified by magnetic immunoaffinity sorting using an antibody to the IL-2 Cardiology Branch and the ¶ Pathology
Section,
Laboratory of Molecular Growth
Regulation,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
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MATERIALS AND METHODS
-chain as a cell surface tag. Primary human diploid fibroblasts were
infected with a retrovirus encoding both V12Ras and the surface tag or,
as a control, with a retrovirus encoding the surface tag alone. Five days after infection, infected cells were magnetically immunoaffinity sorted using an IL-2 receptor antibody coupled to magnetic beads (26).
Using this system, growth arrest was evident from day 6 onward. By day
15 following infection, cells had developed an enlarged and flattened
morphology and demonstrated endogenous
-galactosidase activity (pH
6.0), which was assessed as described previously (27). Low passage
cells (<25 population doublings) were routinely used for retroviral
infection. When noted, mid-passage (25-35 population doublings) or
late passage (>35 population doublings) primary human fibroblasts were
employed. Western blot analysis for protein expression was performed
using 10 µg of protein lysate and detected by enhanced
chemiluminescence (7). Antibodies to Ras proteins and the
cyclin-dependent kinase inhibitor p21 were obtained from
Santa Cruz Biotechnology.
GAL) (30) have been previously
described. An additional adenovirus, Ad.d1312, which is E1-negative but
lacks a transgene, was used as a control (31). All viruses were
amplified in 293 cells and purified by double cesium gradients (30).
Adenoviral infections were on day 6 following retroviral infection and
were performed at an multiplicity of infection of 200. Detection by Western blot analysis of the Myc epitope-tagged form of V12Rac or
N17Rac was as described previously (28).
RESULTS
-galactosidase activity at a
neutral pH. Cells exhibiting the highest DCF fluorescence (top 10%
fluorescence) had a significantly higher percentage of senescent cells
when compared with cells of the identical passage number but containing
the lowest decile of DCF fluorescence (Fig. 1B).
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Fig. 1.
Levels of intracellular
H2O2 correlate with senescence.
A, mid- or late passage fibroblasts were loaded with DCF and
analyzed by FACS. B, mid-passage cells were sorted based on
DCF fluorescence intensity, replated, fixed, and stained for -gal.
Percentage of
-gal-positive cells in the bright (top 10% DCF
fluorescence) and dim (bottom 10% DCF fluoroscence) groups are shown.
Nonsorted cells at this passage exhibited approximately 7%
-gal-positive cells.
receptor. Compared with cells
infected with a control retrovirus expressing only the IL-2
receptor, fibroblasts expressing V12Ras had elevated levels of DCF
fluorescence (Fig. 2, A and
B). This rise in ROS was evident by 7 days after retroviral
infection and therefore coincided with the time when cells underwent
growth arrest but preceded by at least 1 week the development of
senescence markers, such as endogenous
-galactosidase activity and
an enlarged and flattened morphological phenotype (27, 33). In an
attempt to identify the source of the Ras-induced increase in ROS,
cells were incubated with DHR123. This nonfluorescent compound
selectively accumulates in mitochondria, where it can be oxidized by
mitochondria-derived ROS to a fluorescent rhodamine derivative. As
demonstrated in Fig. 2, C and D, V12Ras expression resulted in a significant increase in DHR123 fluorescence, consistent with the notion that the mitochondria are a source of the
Ras-induced rise in ROS levels. To confirm the mitochondrial localization of DHR123, cells were simultaneously labeled with another
fluorescent dye (Mitotracker red), which also localizes to the
mitochondria. As demonstrated in Fig. 2E, an overlapping pattern of fluorescence was seen using either fluorophore.
Quantification of both DCF and DHR123 fluorescence demonstrated that
although there was considerable variation in fluorescence, Ras
expression resulted in an approximate 2-4-fold increase in mean ROS
levels (Fig. 2F).
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Fig. 2.
V12Ras expression increases levels of
ROS. Cells were infected with either a control retrovirus
(A and C) or a retrovirus encoding V12Ras
(B, D, and E). Shown are levels of DCF
fluorescence in control cells (A) and in V12Ras-expressing
cells (B) 7 days after retroviral infection and levels of
mitochondrial oxidant production as assessed by DHR123 fluorescence in
control cells (C) and in V12Ras-expressing cells
(D). E, the cells shown in D were
simultaneously incubated with Mitotracker red to confirm the
mitochondrial localization of DHR123. F, quantification
(mean ± S.D.) of fluorescence intensity from approximately 60 random cells. Levels of fluorescence were quantitated on a gray scale
(0-256), and values were normalized for each fluorophore to the levels
observed in control cells. *, p < 0.05 compared with
control cells.
It has been previously demonstrated that the Rac family of GTPases may also regulate ROS production in nonphagocytic cells (24, 25, 34, 35). To test whether Rac proteins could modulate the phenotype observed in Ras-expressing normal human fibroblast, we superinfected retroviral-infected cells with recombinant adenovirus encoding either an activated (Ad.V12Rac1) or dominant negative (Ad. N17Rac1) rac1 gene product. Similar infection with an adenovirus encoding a marker gene (lacZ) revealed evidence of successful gene transfer in over 90% of V12Ras-expressing cells (data not shown). Western blot analysis from lysates of cells infected with adenoviruses encoding the epitope-tagged form of Rac1 readily demonstrated recombinant protein expression (Fig. 3A).
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We next asked what the effect of N17Rac1 or V12Rac1 expression was in
the setting of concomitant V12Ras expression. Consistent with its known
ability in primary human fibroblasts to induce growth arrest and
senescence, V12Ras-expressing cells demonstrated little change in cell
number over a one week period from day 7 to day 14 after retroviral
infection (Fig. 3B). In contrast, over that same period of
time, control transduced cells exhibited a greater than 10-fold
increase in cell number. Co-expression of N17Rac1 along with V12Ras
resulted in a decrease in cell number (Fig. 3B).
Morphologically, the V12Ras- and N17Rac-expressing cells appeared to
undergo apoptosis (data not shown). In contrast, the simultaneous
expression of V12Rac1 resulted in an increase in cell growth compared
with V12Ras expression alone. Similarly, compared with control cells,
V12Ras expression appeared to produce a 5-7-fold increase in the
number of cells exhibiting the senescence-associated endogenous
-galactoside activity (Fig. 3C). This increase in
-galactosidase activity was reduced in the presence of V12Rac coexpression.
We next sought to better understand the importance of the rise in ROS levels in the development of the Ras-induced senescent phenotype. We therefore cultured Ras-expressing cells in varying oxygen environments, an established method to regulate the production of intracellular ROS (36). As demonstrated previously (21), as well as in Fig. 4, V12Ras-expressing cells maintained in the standard tissue culture environment of 20% oxygen undergo near complete growth arrest. In contrast, lowering ambient oxygen to 3 or 1% enabled cells expressing V12Ras to continue to proliferate.
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Previous studies (21) have demonstrated that expression of V12Ras in
primary fibroblast results in an increase in the levels of the cyclin
inhibitor p21, which in turn appears to be essential for the
development of the senescent phenotype (22). As demonstrated in Fig.
5, although reducing the ambient oxygen
concentration did not alter the expression of Ras proteins, it did,
however, inhibit the ability of Ras to signal an increase in the level of p21. Consistent with these results, when the senescence-associated -galactosidase (
-gal) activity was assessed 15 days after
infection, as previously shown, V12Ras expression resulted in an
approximate 5-7-fold increase in
-gal-positive cells in 20%
oxygen, whereas only a 1-1.5-fold increase was observed when cells
were maintained in a 1% oxygen environment (Fig.
6).
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The mitochondrial redox state is maintained primarily by manganese
superoxide dismutase (Mn-SOD) and glutathione, which allows for the
coordinated dismutation of O2 and the subsequent elimination of hydrogen peroxide. To attempt to understand the specific
contribution of each of these reactive oxygen species to Ras-induced
senescence, we treated cells with NAC, which replenishes intracellular
glutathione, and MnTMPyP, a member of a class of SOD mimetics that have
been demonstrated to be capable of rescuing mice made homozygous
deficient in Mn-SOD (37).
Treatment of cells expressing V12Ras with the SOD mimetic MnTMPyP did not restore growth, and in fact, these cells exhibited toxicity in a concentration-dependent fashion (Fig. 7A). At low concentrations of MnTMPyP (0.1 µM), approximately 50% of V12Ras-expressing cells died, and the remaining cells were viable but remained growth-arrested.
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In contrast to the effects of an SOD mimetic, addition of the peroxide
scavenger NAC to the medium rescued V12Ras-expressing cells from growth
arrest (Fig. 7A). Surprisingly, although MnTMPyP was
incapable by itself of restoring proliferation, the combination of an
SOD mimetic and a peroxide scavenge (NAC) proved slightly more
effective than NAC alone (Fig. 7A). Similarly, assessment of
the effects of antioxidants on the subsequent development of the
senescent phenotype demonstrated that V12Ras-expressing cells that
survived treatment with MnTMPyP had a higher percentage of -gal-positive cells than untreated cells. In contrast, treatment with NAC or the combination of NAC and MnTMPyP substantially reduced the rate of senescence (Fig. 7B).
To further confirm the inability of superoxide scavengers to rescue
cells, we coexpressed V12Ras with Cu, Zn-SOD using a recombinant adenovirus encoding superoxide dismutase. We have previously shown that
this construct allows for a 3-5-fold increase in SOD activity in cells
(29). As seen in Fig. 7C, consistent with the effects of
MnTMPyP, Ad.SOD infection failed to rescue V12Ras-induced senescence.
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DISCUSSION |
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Our results suggest that V12Ras expression leads to an increase in intracellular H2O2, the source of which appears to be the mitochondria. The ability of Ras proteins to increase ROS levels is essential for the Ras phenotype because V12Ras-expressing cells grown in the presence of either a peroxide-scavenging antioxidant or low oxygen are rescued from senescence. Take together, these results provide strong support for the concept that a rise in ROS is an important intracellular trigger for replicative senescence.
Expression of an activated ras gene has been previously demonstrated to result in growth arrest in several types of nontransformed cells (21, 38, 39). Recently, it has been demonstrated in Swiss 3T3 cells that although when expressed alone V12Ras leads to G1 arrest, the simultaneous coexpression of an activated rhoA GTPases leads to S-phase progression (40). These results are similar to what we observed with coexpression of V12Rac in Ras-induced growth arrest and senescence in normal fibroblasts. Nonetheless, in our case, V12Rac was only able to partially rescue the V12Ras phenotype. The basis for this decreased efficacy is not entirely clear, although it may reflect a difference in activity of RhoA versus Rac1.
Expression of V12Ras has been previously demonstrated to increase ROS levels in cells (24, 25), and this property has been linked to the ability of ras to act as a transforming gene (25). Initially, it would appear contradictory that V12Ras expression leads to transformation of NIH 3T3 cells and senescence of primary fibroblasts. Nonetheless, both states are thought to be a result of the accumulation of somatic mutations. Given the known ability of ROS to induce DNA damage, it is conceivable that in the context of a normal cell, Ras-induced increases in ROS levels may result in oxidative modifications of DNA and, as previously demonstrated (21), the subsequent up-regulation of p53 and p21. Continued high level oxidative stress may eventually result in senescence. In contrast, in immortalized NIH 3T3 cells, oxidatively modified DNA may not be appropriately sensed or repaired, and the cell may respond to DNA damage not through growth arrest but by transformation. In this regard, it is interesting to note that Ras does not induce growth arrest in primary fibroblasts devoid of p53 (21). In addition, the overexpression of p53 can by itself increase ROS levels (9, 10) and, in certain cell types, induce senescence (41).
Our results with the SOD mimetic MnTMPyP and the peroxide scavenger NAC
suggest that H2O2 is the most important
mediator of Ras-induced growth arrest. Treatment with an SOD mimetic
alone raised H2O2
levels2 and resulted in the
death of some V12Ras cells and an increased rate of senescence in the
remaining viable cells. This is consistent with previous data
suggesting that cells treated with high levels of exogenous
H2O2 undergo cell death, whereas moderate,
nonlethal concentrations can induce a senescent phenotype (19).
Nonetheless, because the combination of MnTMPyP and NAC produced the
most efficient rescue of V12Ras-expressing cells, it is conceivable
that hydroxyl radicals, the formation of which requires both
H2O2 and O2, may also participate.
Although our results suggest that Ras proteins may regulate the level
of mitochondrial ROS, based on the subcellular location of Ras, it is
most likely that such regulation is indirect. One potential mediator is
ceramide, a lipid linked to the senescent phenotype (42, 43), that
recently has been demonstrated to affect directly the mitochondrial
electron transport chain, leading to an increase in the release of
reactive oxygen species (44). This suggests that mitochondria may be
direct targets of signaling molecules and that leakage of mitochondrial
ROS may be more intricately regulated than has been previously
appreciated. As described in this study, the use of a simple, rapid,
genetic model should provide the framework to further dissect the
relevant signaling molecules in senescence and potentially provide
important insights and new therapeutic strategies to combat human aging.
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ACKNOWLEDGEMENTS |
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We are grateful to Martha Kirby for help in FACS analysis, S. Gutkind for the V12Ras cDNA, R. Crystal for Ad.SOD, and Ilsa Rovira for aiding in preparation of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Howard Hughes Medical Institute National Institutes of Health Research Scholars program.
** To whom correspondence should be addressed: Cardiology Branch, NHLBI, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892-1650. Tel.: 301-402-4081; Fax: 301-402-0888; E-mail: finkelt{at}gwgate.nhlbi.nih.gov.
2 A. C. Lee and T. Finkel, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
ROS, reactive oxygen
species;
-gal,
-galactosidase;
DCF, dichlorodihydrofluorescin
diacetate;
FACS, fluorescence-activated cell sorter;
MnTMPyP, Mn(111)
tetrakis 1-methyl 4-pyridyl porphyrin pentachloride;
NAC, N-acetylcysteine;
SOD, superoxide dismutase;
DHR123, dihydrorhodamine 123;
IL, interleukin.
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