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Tumor Biology |
Institute of General Pathology, Catholic University, 00168 Rome, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2), hydrogen peroxide
(H2O2), hydroxyl radical
(OH·), and nitric oxide (NO) derived from incomplete reduction
of molecular oxygen are highly reactive toward key constituents of
living cells, including DNA, proteins, and lipids. Genotoxicity,
protein denaturation, compromised enzymatic activities, and
lipid peroxidation all represent consequences of cell exposure to
excess amounts of
ROS,3
a condition usually indicated as oxidative stress. Both exogenous
oxygen radicals and oxygen species endogenously derived as a byproduct
of mitochondrial respiration, drug metabolism, or any other
intracellular redox reaction can exert deleterious effects on cell
function and viability, depending on cellular antioxidant defenses and
capability to repair oxidative damage (2)
. Accordingly, a
role for ROS has been postulated in the genesis of a large number of
human and experimental pathological processes, including chronic
inflammation, neurodegeneration, tissue aging, and cancer (3
, 4)
.
Massive cell oxidation leads easily to cell death through a necrotic
process in which a main role is played by bioenergetic failure, ATP
depletion, and loss of cell membrane integrity. However, milder
oxidative insults can also induce cell death, which is mediated mainly
by apoptosis. This observation, together with many findings on
the increase of intracellular ROS during apoptosis and on the
protective effects of antioxidant species in most cellular models of
programmed cell death, recently led to the hypothesis that ROS may play
a central role in determining the fate of cells committed to die
(5
, 6)
. This view has been further strengthened by the
discovery of the role of mitochondria and mitochondrial factors [such
as cytochrome c (7)
and apoptosis-inducing
factor (8)
] in the triggering of the apoptotic
process. As an early event after cell exposure to apoptogenic stimuli
(including oxidants), cytochrome c is released by the
mitochondria to the cell cytosol, where it contributes to the
activation of the caspase cascade. A direct consequence of cytochrome
c translocation is the interruption of the respiratory
chain, leading to excess production of superoxide anion [O2
(9)
], which in turn might contribute to mitochondrial
damage and accelerate the release of proapoptotic substances. ROS are
therefore produced by mitochondria in the early phases of the apoptotic
process and are not necessary for apoptosis triggering but likely play
a central role in amplifying the catastrophic cascade of events
ultimately leading to cell collapse. The general cytoprotective effects
of antioxidant species in most models of apoptosis are in agreement
with this view.
MnSOD, a Mr 85,000 tetrameric
enzyme located in the mitochondrial matrix, is the principal scavenger
for superoxide in mitochondria (10)
. Consistent with the
idea of a role for mitochondrial O2 in amplifying and
accelerating cell response to apoptotic stimuli, this enzyme has been
shown to be induced by and protect from a number of cytotoxic and
proapoptotic agents, including inflammatory cytokines tumor necrosis
factor (11)
and interleukin 1, UV and ionizing radiation,
anticancer drugs (12)
, and pesticides (Paraquat; Ref.
13
). We have recently shown that MnSOD overexpression in
HeLa cells also confers resistance to cell death induced by serum
deprivation (14)
. This evidence, together with recent
reports of reduced survival and mitochondrial dysfunction in mice
genetically deprived of MnSOD (15)
, supports the
notion that SOD2 acts mainly as a survival protein that is required to
maintain mitochondrial integrity in cells exposed to adverse
conditions. Consistent with this interpretation, we and others have
described an increased expression of this enzyme in certain classes of
tumors, such as brain tumors (16)
, thyroid tumors
(17)
, and colon neoplasm (18)
in comparison
with corresponding normal tissues.
ROS have recently been indicated as downstream mediators of the tumor suppressor protein p53. This nuclear phosphoprotein, whose function is very often lost in human cancers (19) , normally acts as a transcription factor, promoting the expression of a number of genes by binding to a specific consensus DNA sequence (20 , 21) and repressing the expression of some others, especially those harboring TATA (22) or CAAT (23) boxes or SP-1 (24) binding sequences in their promoter regions, mainly through protein-protein interactions with the corresponding DNA binding factors.
A number of stimuli can trigger p53 activation. DNA damage activates
p53 by phosphorylation and increased resistance to degradation
(25
, 26) . Furthermore, a number of "nongenotoxic"
stresses, such as heat shock and hypoxia (27
, 28)
, can
up-regulate p53 function; hypoxia induces p53
stabilization by the hypoxia-induced factor 
(29)
. The
main physiological outcomes of p53 activation, cell growth arrest and
apoptosis, justify its tumor suppression activity and reflect the
effect of p53 on the transcriptional activation/repression of a
multiplicity of target genes.
At present, whereas many p53 target genes (such as p21/Waf1, GADD45, cyclin G, and MDM2) involved in the inhibitory effects of the tumor suppressor protein on cell cycle progression have been identified, the mechanisms mediating p53-induced apoptosis are still incompletely understood. Recently, convincing evidence has shown that p53 activation is accompanied by a net increase in intracellular ROS concentration and that the removal of oxygen radicals by antioxidant drugs impedes apoptosis induced by p53 (30 , 31) . These data suggest that oxygen radicals may play an important role in the apoptotic process triggered by p53, a role that is also supported by the observation that many of the proteins transcriptionally regulated by p53 are somehow involved in redox metabolism (30) . To date, however, very little is known about the molecular mechanisms linking p53 activation to cell damage by oxygen radicals and, ultimately, to programmed cell death.
Here we show that in p53-deficient cells, resistance to oxidative stress correlates with increased expression of the mitochondrial scavenger MnSOD and that this enzyme is negatively regulated at a transcriptional level by p53. Because MnSOD is also able to prevent the induction of apoptosis by p53, these data outline a novel mechanism whereby down-regulation of the survival protein MnSOD links p53 function to oxidative mitochondrial damage and cell death.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines, Plasmids, and Animals.
Cervical adenocarcinoma HeLa cells (purchased from the American Type
Culture Collection) and p53+/+ and p53-/- E1A/Ras-transformed
fibroblasts (a generous gift of Dr. Scott Lowe, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY) were maintained in RPMI 1640
and DMEM, respectively, supplemented with 10% FCS (Eurobio, Les Ulis
Cedex B, France), antibiotics, and L-glutamine (Life
Technologies, Inc.) at 37°C in 5% CO2.
Human MnSOD cDNA (excised by EcoRI) and human p53 (excised by BamHI) were obtained from the American Type Culture Collection and cloned into PcDNA3/zeo (zeocine resistance) and PcDNA3/neo (neomycin resistance; Invitrogen), respectively. Mutant p53 (C53–175) in pCMV-neo and the synthetic p53-responsive promoter PG13 were generous gifts of Dr. B. Vogelstein (Howard Hughes Medical Institute, Baltimore, MD). The CMV-GFP and promoterless-GFP plasmids were from Clontech.
p53+/+ and p53-/- C57Bl6-J mice (Jackson Laboratories) used as a source of liver tissue were maintained in the local animal facility.
Cell Transfection.
The p53/Neo construct (0.2 µg) or the corresponding empty vector (0.2
µg) was transfected into 50–60 x 104 HeLa cells in a 24-well plate using
Effectene (Qiagen, Valencia, CA) according to the manufacturer’s
recommendations. After 48 h, cells were harvested, and total RNA
was extracted with Trizol (Life Technologies, Inc.). Average
transfection efficiency was around 40%, as assessed by
cytofluorometric analysis of cells transfected with an equivalent
amount of a CMV-GFP construct (Clontech).
Stable cotransfection of HeLa cells with p53 (0.2 µg) and MnSOD (0.8 µg) was performed in duplicate in a 24-well plate using the MnSOD/zeo and p53/neo constructs. Empty vectors were used as appropriate controls. Each well received a total amount of 1 µg of DNA.
The HeLa subclone A12, which overexpresses human MnSOD under the control of the CMV promoter, has been described previously (14) .
p53 Reporter Assay.
To assess the transcriptional activity of p53 in HeLa cells, a reporter
plasmid was created by cloning the synthetic p53-dependent promoter
PG13 in the HindIII site of a promoterless GFP vector
(pEGFP-1; Clontech). The reporter construct was cotransfected with wt
p53, mutant p53, or empty pCDNA3 in HeLa as described above. After
24 h, cell fluorescence was assessed by flow cytometry. A CMV
promoter-driven GFP vector was used as a positive control.
mRNA Evaluation.
Levels of MnSOD and actin mRNAs were evaluated by RT-PCR using a kit
from Promega according to the manufacturer’s recommendations. Primer
sequences were as follows: (a) MnSOD forward primer
sequence, 5'-GGTAGCACCAGCACTAGCAG-3'; (b) MnSOD reverse
primer sequence, 5'-CTGCAGTACTCTATACCACTACA-3'; (c)
ß-actin forward primer sequence, 5'-TGAGGCTCTTTTCCAGCCTT-3'; and
(d) ß-actin reverse primer sequence,
5'-CTAGAAGCACTTGCGGTGCA-3'.
Protein Expression.
MnSOD activity was evaluated by an "in gel" SOD assay on 50 or 100
µg of total protein lysate as described by Beauchamp and Fridovich
(32)
. Immunoreactive MnSOD, CuZnSOD, and p53 were assessed
by standard Western blotting analysis of mitochondrial (MnSOD) or total
(CuZn, p53) protein lysates, using the appropriate Abs (see above)
followed by enhanced chemiluminescence detection. Mitochondria were
obtained as described previously (33)
.
Analysis of ROS Generation.
Intracellular ROS concentration under different growth conditions was
assessed by flow cytometry on cells loaded with the oxygen
radical-sensitive probe dichlorodihydrofluorescein diacetate
(Molecular Probes). The dye was added to cell cultures 30 min
before analysis, at a concentration of 10 µg/ml. Cells were then
trypsinized, and green fluorescence (FL-1 channel, 520 band-pass
filter) was analyzed immediately with a Coulter Epics flow cytometer
equipped with a 480 nM emission argon laser.
Averages ± SD of mean cell fluorescence in duplicate
experiments were calculated.
MTT Reduction Test for Cell Toxicity.
To assess cell survival after different cytotoxic treatments,
cells were seeded at 4 x 104
cells/well (E1A-Ras fibroblasts) or 2 x 104
cells/well (HeLa cells) in 96-well plates in
the presence of the indicated amounts of FCS, Adriamycin, and Paraquat.
Forty-eight h later, surviving cells were stained with MTT as described
by Mossman (34)
.
Selection of G418-resistant Cells.
Effects of transfected MnSOD and p53 on HeLa cell growth potential were
evaluated by counting neomycin-resistant cells after transfection of
the selection marker alone or with p53, MnSOD, or
both genes. After 2 weeks of selection in medium containing 800 µg/ml
G418, live cells were trypsinized and counted.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) as assessed by the MTT reduction test on cells derived from either wt
p53 or p53-null mice and exposed to scalar strengths of the
above-mentioned treatments. However, protection was not complete, and
the effect of p53 was less evident at the highest levels of cell
insult.
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).
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Because oxygen radicals are almost universal mediators of cell damage (1) , and evidence exists that p53 is involved in the apoptotic response to oxidative stress (37) , these findings are consistent with a causal role for ROS in the cytotoxic effects observed on oncogene-transformed fibroblasts, and by extension, with the idea that the relative resistance in p53-deficient cells may depend, at least in part, on increased cell resistance to oxidative stress.
We have reported recently that overexpression of the mitochondrial superoxide scavenger MnSOD increases the resistance of HeLa cells to apoptosis on serum withdrawal. This observation contributed to the idea that MnSOD may act as a survival protein in maintaining cellular and mitochondrial integrity under adverse growth conditions, a role already recognized for other mitochondrial proteins such as bcl-2 and bcl-XL (38) . MnSOD has also been reported to protect mouse fibroblasts from Paraquat cytotoxicity (13) and cardiomyocites from the adverse effect of Adriamycin (39) , probably by removing harmful oxygen radicals generated by the intracellular metabolism of these drugs.
The above-mentioned considerations, together with the finding of increased resistance of p53-defective cells to typical superoxide-producing cytotoxic treatments, prompted us to investigate SOD2 expression in wt and p53-deficient cells.
An in gel SOD assay as described by Beauchamp and Fridovich
(32)
was used to assess SOD2 activity in protein extracts
from wt and p53-/- ras-transformed mouse fibroblasts. As shown in
Fig. 3A
, MnSOD activity, seen as an unstained area in the context
of a dark nondenaturing gel, was significantly higher in p53-deficient
cells than in wt controls. This difference was mirrored by the content
of immunoreactive protein, which was evaluated by Western blot analysis
of protein lysates obtained from purified mitochondria (Fig. 3B
). No significant differences were found in the expression
of CuZnSOD (Fig. 3B
) or of the antiapoptotic protein bcl-2
between p53+/+ and p53-/- cells (data not shown). These data
suggest that MnSOD enzyme is selectively up-regulated in p53-deficient
cells, consistent with the finding of increased resistance of these
cells to the cytotoxic effects of ROS-producing stimuli.
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),
suggesting that this difference is not limited to transformed cells and
is not likely to reflect different cell distribution through the cell
cycle but is directly linked to the functional status of p53.
To further confirm that the negative control of p53 on MnSOD expression
represents a general regulatory mechanism, SOD2 activity was evaluated
in a number of human cancer cell lines with different p53 functional
status. This analysis revealed that MnSOD content is in an inverse
correlation with p53 function, with the enzyme being expressed at
considerable levels in cells in which p53 is either mutated (HT29 colon
carcinoma) or virtually absent (HeLa cervical carcinoma) but barely
detectable in cell lines expressing high amounts of wt p53 such as the
breast carcinoma MCF7 cell line (Ref. 27
; data not shown).
In HeLa cells, in particular, very low p53 expression is due to protein
degradation driven by human papillomavirus protein E6 (43)
. However, these cells retain normal responsiveness to
p53-dependent regulatory mechanisms and rapidly undergo apoptosis when
p53 is artificially overexpressed by transfection (44
, 45)
. Consistent with a previous report (46)
, HeLa
cells transiently transfected with wt p53 cDNA display p53 protein
accumulation and increased activity of a p53-dependent reporter
construct, despite accelerated p53 turnover in this cell line (Fig. 4A
, top and bottom panels).
Concomitantly, SOD2 mRNA was significantly reduced in comparison with
mock control (Fig. 4B
). Densitometry of the RT-PCR SOD2
bands consistently revealed a 30–40% decrease of the steady-state
MnSOD messenger level; the percentage of inhibition becomes at least
70% when normalized for the efficiency of cell transfection, which was
reproducibly around 40%, according to GFP expression (Fig. 4D
.) This finding therefore supports the hypothesis that
MnSOD expression is negatively regulated at a transcriptional level by
tumor suppressor protein p53. Accordingly, MnSOD activity was also
significantly decreased in HeLa cells by p53 overexpression as assessed
by in gel SOD assay, whereas no obvious effect was observed on the
activity of cytosolic CuZnSOD (Fig. 4C
).
|
). When challenged with the above-mentioned stimuli, HeLa
cells overexpressing MnSOD showed significantly improved survival in
comparison with mock-transfected cells (Fig. 5 and C
). Intriguingly, this difference closely resembles the
effect of p53 deletion on the survival of E1A-Ras-transformed
fibroblasts (Fig. 1
).
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), suggesting that this
mitochondrial superoxide scavenger can protect cells from p53-induced
growth suppression/apoptosis, albeit with some variability among
different experiments. MnSOD cDNA alone did not affect cell selection
in G418-containing medium (Fig. 6
), or significantly influence
the efficiency of p53 transfection, as assessed by Western blot
analysis 48 h after transfection (data not shown). However, it
should be noted that the protective effect of MnSOD was not complete
(Fig. 6
) and was not sufficient to allow the generation of stable
clones overexpressing p53. Despite these limitations, the
beneficial effect of MnSOD on survival of p53-transfected cells
confirms that ROS are not only potent inducers but are also downstream
effectors of p53 activity (30
, 31)
and identify the
superoxide anion as an important radical species involved in p53
cytotoxicity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The major findings of the present work are the identification of the mitochondrial superoxide scavenger MnSOD as an enzyme whose cellular content is negatively regulated by p53 and the ability of this enzyme to counteract the growth-inhibitory effects of p53 on cervical carcinoma HeLa cells.
Many studies suggest that MnSOD (or SOD2) could play a general role as
a cell survival protein; in fact, in cell culture models, SOD2
overexpression has proven to be protective against the cytotoxicity
(either by apoptosis or necrosis) of a number of agents including
lipopolysaccharide and tumor necrosis factor , ionizing and UV
radiations, anticancer drugs, pro-oxidant chemicals, and serum
deprivation (12
, 14)
. Interestingly, many of these
treatments are well-known inducers of p53. As for in vivo
studies, mice genetically deprived of this enzyme exhibit severe
cardiomyopathy and neurodegenerative lesions, likely related to
impaired mitochondrial function by unscavenged oxygen radicals. Recent
discoveries on the central role of mitochondria in the initiation of
apoptosis and on the generation of ROS in mitochondria after cytochrome
c release have added new support to the notion that MnSOD
can play an important role in the maintenance of cell viability under
stressful conditions and, through the surveillance on mitochondrial
integrity, can directly influence cell fate toward different possible
outcomes represented by recovery from stress, apoptosis, or necrosis
(47)
. In view of these properties of SOD2, the finding
that this enzyme is negatively controlled by p53 could significantly
contribute to the understanding of how p53 activation is coupled to the
triggering of the apoptotic process and, eventually, to cell death.
Important evidence exists that modification of the cellular redox balance represents a crucial event downstream of p53 activation. p53 retroviral transfer to cultured cells is accompanied by a significant production of oxygen radicals (30 , 31) ; furthermore, the expression of several redox active proteins is transcriptionally regulated by p53 (31) . Among these, the mitochondrial scavenger glutathione peroxidase is transcriptionally induced by the tumor suppressor protein (48) . To date, however, inhibition of antioxidant defenses by p53 activation has not been reported, despite the fact that this is a very common mechanism of oxidative stress in mammalian cells.
HIV protein Tat, for example, determines an oxidative stress in
infected cells by down-regulating the expression of MnSOD
(49)
. We suggest that, in a similar fashion, MnSOD
down-regulation contributes, at least in part, to the increase of
oxygen radicals reported in p53-transduced cells as necessary for
p53-mediated apoptosis. Whereas previous studies have not identified
the source and nature of these radical species, the present data
showing decreased MnSOD expression in p53-transfected cells together
with a protective effect of SOD2 toward p53-mediated apoptosis indicate
mitochondrial superoxide (O2) as a potentially critical
effector in this process.
Increased expression of the mitochondrial scavenger MnSOD in
p53-deficient fibroblasts could appear somehow inconsistent with the
fact that we did not detect significant differences in the
intracellular content of oxygen radicals between p53-/- and wt cells.
This apparent contradiction can be easily reconciled when one considers
that in presence of stimuli such as serum deprivation or redox cycling
drugs, massive amounts of ROS are generated in the cells, but only one
part of those is likely to involve mitochondria and can be targeted by
SOD2. Nevertheless, as shown in Fig. 5
, improved mitochondrial
antioxidant defense translates into a dramatic increase in cell
resistance to the above-mentioned treatments in HeLa cells. In view
of this evidence, we conclude that overall cellular capability to
generate ROS is unaffected by loss of p53, whereas cellular resistance
to oxidative stress is markedly increased in p53-deficient cells,
likely due to up-regulation of MnSOD.
The molecular mechanism through which p53 represses MnSOD gene expression has not been addressed in the present study. p53 has been reported to negatively regulate a number of promoters, especially those harboring either TATA (22) or CAAT (23) boxes; moreover, p53 interaction with SP-1 results in reduced transcriptional activity by this factor, thereby inhibiting the expression of many SP-1 target genes (24) . Human MnSOD promoter does not contain TATA or CAAT regions but harbors seven potential SP-1 binding sites (50) that are probably involved in the basal level expression of the enzyme. Although not proven in this study, it is therefore conceivable that the inhibitory effects of p53 on SOD2 expression are mediated by transcriptional complex SP-1, as shown, for instance, for insulin receptor expression in mouse liver (51) .
MnSOD is a highly inducible gene, and its expression is strongly up-regulated by oxidative stress (52) . Whereas increased content of MnSOD in p53-deficient tissues could reflect an increase in cell exposure to oxygen radicals, this mechanism appears to be unlikely because intracellular ROS are not significantly different in wt and p53-/- cells (see above) and in view of recent findings suggesting the pro-oxidant effects of p53 (30) .
However, whereas we favor the hypothesis of SP-1 involvement in MnSOD down-regulation by p53, this regulatory circuit certainly deserves more detailed investigation.
The finding of deregulated MnSOD expression in p53-deficient cells
might have important ramifications for the understanding of some
aspects of tumor biology. Loss of function of p53 in tumors is
associated with increased resistance to adverse growth conditions, as
those created by exposure to anticancer drugs, radiotherapy,
and nutrient restrictions. Many of these conditions are accompanied by
increased production of oxygen radicals. We suggest that this
resistance could be due, at least in part, to increased expression
of the mitochondrial scavenger SOD2 (Fig. 7
). In keeping with this
model, we and others have described exaggerated expression of MnSOD in
some classes of tumors, including brain tumors, cervical carcinoma, and
colon and thyroid carcinoma. Moreover, high expression of MnSOD has
been associated with an accumulation of mutated p53 in cervical
carcinoma samples (53)
, and a similar association has also
been observed in brain
tumors.4
More intriguingly, because SOD2 can at least partially counteract the
growth-suppressive effect of p53 (Fig. 6
) abnormal expression of MnSOD could confer a "p53 deficient-like"
phenotype on tumor cells, even in the presence of a normal tumor
suppressor protein. Data reported in Fig. 5
showing increased
resistance to Paraquat, Adriamycin, and serum deprivation in HeLa cells
overexpressing MnSOD, together with previously reported observations
(11
, 12 , 14)
, clearly support this hypothesis.
|
In view of the presented data, delayed aging and increased
susceptibility to Ras transformation of p53-/- fibroblasts could be a
consequence, at least in part, of MnSOD up-regulation and increased
resistance of mitochondria to oxidative stress (Fig. 7
).
Whereas many of these considerations are still speculative, the finding of SOD2 modulation by p53 strongly supports the role of this enzyme as a survival protein involved in cell resistance to stress and, as such, as a potential tumor-promoting factor involved in the mechanism linking loss of function of p53 to altered cell growth regulation, tumorigenesis, and resistance to chemotherapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Ministero
dell’Università e Ricerca Scientifica e
Tecnologica/Consiglio Nazionale delle Ricerche Biotechnology
Program L. 95/95 Grant 98.00079.PF31. G. P. and B. B. contributed
equally to this work. 
2 To whom requests for reprints should be
addressed, at Institute of General Pathology, Catholic University,
Largo Francesco Vito #1, 00168 Rome, Italy. Fax: 39-06-3386446;
E-mail: tgaleotti{at}rm.unicatt.it
3 The abbreviations used are: ROS, reactive oxygen
species; MnSOD, manganese superoxide dismutase; SOD2, superoxide
dismutase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CMV,
cytomegalovirus; GFP, green fluorescent protein; CuZnSOD,
copper/zinc-dependent, cytosolic superoxide dismutase; wt, wild-type;
RT-PCR, reverse transcription-PCR; Ab, antibody; DCF-DA,
dichlorodihydrofluorescein diacetate.
4 F. Ria, H. Landriscina, F. Remiddi, R. Rosselli,
H. Scerrati, G. Pani, S. Borrello, and T. Galeotti. MnSOD is a
prognostic factor for glioblastoma, manuscript in preparation.
Received 11/29/99. Accepted 6/ 8/00.
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) and p53-/-
(clone A9, •) E1A/Ras-transformed fibroblasts were seeded at 40,000
cells/well in a 96-well plate in the presence of decreasing amounts of
FCS (a) or increasing concentrations of either Paraquat
(b) or Adriamycin (c). Forty-eight h
later, cytotoxicity was evaluated by MTT reduction test as described by
Mossman 
; p53-/-, 
) were
exposed to the indicated stimuli (3% FCS, 0.3 µg/ml Adriamycin, and
125 µM Paraquat) for 16 h, followed by 30-min
loading with 5 µg/ml DCF-DA, trypsinization, and cytofluorometric
analysis. Values are the mean ± error spreading of
duplicate samples. Increases in intracellular ROS after different
treatments were all significant (at least P < 0.5, Student’s t test), whereas differences
between p53+/+ and p53-/- cells where not significant.
