National Heart, Lung, and Blood Institute, Bethesda, Maryland, 20892-1603
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ABSTRACT |
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We previously found that p53 upregulation by hypertonicity protected renal inner medullary collecting duct (mIMCD3) cells from apoptosis. The purpose of the present study was to investigate the mechanism by which p53 protects the cells. We now find that hypertonicity (NaCl added to a total of 500 mosmol) inhibits DNA replication and delays G1-S transition as concluded from analysis of cell cycle distributions and bromodeoxyuridine (BrDU) incorporation rates. Lowering of p53 with p53 antisense oligonucleotide attenuated such effects of hypertonicity, resulting in an increased number of apoptotic cells in S phase and cells with >4 N DNA. Results with synchronized cells are similar, showing that cells in the early S phase are more sensitive to hypertonicity. Immunocytochemistry revealed that p53 becomes phosphorylated on Ser15 and translocates to the nucleus in S both in isotonic and hypertonic conditions. Caffeine (2 mM) greatly reduces the p53 level and Ser15 phosphorylation, followed by a remarkable increase of DNA replication rate, by failure of hypertonicity to inhibit it, and by reduction of cell number during hypertonicity. Finally, inhibition of DNA replication by the DNA polymerase inhibitor aphidicolin significantly improves cell survival, confirming that keeping cells in G1 and decreasing the rate of DNA replication is protective and that these actions of p53 most likely are what normally help protect cells against hypertonicity.
cell cycle arrest; apoptosis; sodium chloride
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INTRODUCTION |
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CELLS OF THE renal inner medulla are normally exposed to variable and often extreme hypertonicity as the result of the renal mechanism for concentrating the urine. This raises questions about the mechanisms that they employ to survive and function under such adverse conditions. p53 Is a tumor suppressor whose loss of function, observed in many types of cancer, contributes to genomic instability and malignancy (2, 15, 28). Numerous stresses increase p53 activity, producing either growth arrest until damage is repaired or apoptosis, eliminating cells that are potentially dangerous to the organism. p53 is induced by DNA damage caused by cytotoxic drugs, free radical formation, or ionizing radiation (25). p53 Can also be induced in the absence of observed DNA damage by growth factor withdrawal, hypoxia, metabolic change, virus infection, cytokines, or deregulated expression of cell cycle genes (25).
In previous studies, we found that hypertonicity increased the amount of total and phosphorylated Ser15 p53- and p53-dependent transcription in renal inner medullary collecting duct cells (mIMCD3; see Ref. 11). Under these conditions, reducing p53 with p53 antisense oligonucleotide (p53-AS) increased apoptosis, suggesting that activation of p53 is protective (11). Hypertonicity also arrests growth of mIMCD3 cells (7, 21, 26, 32) and increases levels of GADD45 (21), a growth arrest and DNA damage-inducible protein whose transcription is regulated by p53 (40).
The purpose of present study was to analyze the mechanism by which p53 protects against hypertonicity. We found that phosphorylation of p53 on Ser15, previously noted to be protective during hypertonicity, occurs in the S phase of the cell cycle. Furthermore, reducing p53 expression with p53-AS or caffeine reversed both the G1-S arrest and reduction of DNA replication that are caused by hypertonicity. Under those conditions, apoptosis increased mainly in the cells in which DNA content had increased. We conclude that p53 protects cells against hypertonic stress by restricting DNA replication.
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EXPERIMENTAL PROCEDURES |
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Cell culture. Subconfluent cultures of mIMCD3 cells (generously provided by S. Gullans; see Ref. 31) were used in passages 13-17. The medium contained 45% DME low glucose, 45% Coon's Improved Medium mF-12 (Irvine Scientific), and 10% FBS (Life Technologies). Osmolality of control ("isotonic") medium was 320 mosmol/kg. Hypertonic media, prepared by adding NaCl, were substituted for the control medium, as indicated. Cells were incubated at 37°C and gassed with 5% CO2-95% air during growth and during all experiments.
Antisense oligonucleotide experiments. For all experiments with p53-AS (Biognostik), cells were grown on eight-chamber plastic slides (Nalge Nunc International) and preincubated for 16 h with 2 µM of p53-AS (sequence: CGT CAT GTG CTG TGA C) or control (CG-matched randomized-sequence phosphorothioate oligonucleotide: GAC TAC GAC CTA CGT G). Next, the media were changed to iso- or hypertonic ones containing the same oligonucleotides.
Fixation and propidium iodide staining for cell cycle and
apoptosis analysis.
Cells on the plastic slides were fixed in 100% methanol at 20°C
for 15 min. After fixation, the cells were permeabilized with 0.1%
Triton X-100, incubated with 1 mg/ml RNase (Sigma) for 15 min, stained
with 20 µg/ml propidium iodide (PI) for 5 min, and then mounted with
150 µl of antifade (no. S-7461; Molecular Probes).
Analysis of mitosis by immunostaining with anti-phospho-histone
H3 and anti--tubulin antibody.
After fixation in 100% methanol at
20°C for 45 min, the cells were
washed three times for 5 min each with 0.1% Triton X-100 in PBS,
followed by blocking buffer (3% BSA-0.1% Triton X-100). Next, they
were incubated with anti-phospho-histone H3 (mitosis marker, no.
06-570; Upstate Biotechnology) or anti-
-tubulin (no. F2168;
Sigma) antibody, washed with 0.1% Triton X-100 in PBS, incubated for
1 h with secondary antibody (Alexa 488 goat anti-mouse or
anti-rabbit IgG, nos. A-11029 and A-11034; Molecular Probes), stained
with 0.7 µg/ml PI, and mounted with 150 µl of antifade (no. S-7461;
Molecular Probes). Cells were observed under a microscope, and the
percentage of phospho-histone H3-positive (mitotic) cells or cells with
anaphase morphology were calculated. At least 2,000 cells were counted
for each point.
Analysis of intracellular localization of p53 by immunostaining
with anti-p53 and anti-phospho-p53(Ser15) antibodies.
The immunostaning procedure was the same as for anti--tubulin,
except that the primary antibodies were anti-p53 (no. 1 413 147;
Boehringer Mannheim) or anti-phospho-p53(Ser15) (no. 9284;
New England Biolabs).
Protein sample preparation, Western blotting, and
immunodetection.
Cells were rinsed with PBS adjusted with NaCl to the same osmolality as
the medium and then were lysed with 300 µl of lysis buffer (100 mmol/l NaF, 50 mmol/l Tris, 250 µmol/l thimerosal, 1% vol/vol
igepal, 16 mmol/l CHAPS, 5 mmol/l activated NaVO4, 50 mg/l
Pefabloc, 100 mg/l leupeptin, and 10 mg/l aprotinin). After
centrifugation for 20 min at 15,000 g and 4°C, the
supernatant was separated into aliquots and stored at 80°C. Protein
content was measured using the bicinchoninic acid protein assay
(Pierce). Proteins were separated by SDS-PAGE. Equal amounts of protein (6 µg) were loaded in each lane of 12% acrylamide-Tris-glycine gels.
Immunodetection procedures were carried out using specific antibodies
against p53 (no. 1 810944; Boehringer Mannheim) or phospho-p53(Ser15) (no. 9284; New England Biolabs).
Densitometry analysis was done with a Molecular Imager FX (Bio-Rad).
Bromodeoxyuridine labeling, immunostaining, and analysis by LSC. Cell cultures were labeled with 10 µM of bromodeoxyuridine (BrDU) for 30 min and processed with a 5-bromo-2-deoxyuridine Labeling and Detection Kit I (catalog no. 1 296 736; Roche) according to the manufacturer's instructions. DNA was stained with 0.7 µg/ml PI.
The slides were analyzed by LSC. Green fluorescence was recorded as a measure of anti-BrDU antibody binding (BrDU incorporation). Red fluorescence was recorded as a measure of PI binding (DNA content). Bivariate distributions of cells showing incorporation of BrDU vs. DNA content were obtained.Statistical analysis. For statistical analysis, experiments were repeated at least three times and analyzed by ANOVA Parametric Repeated Test followed by the Student-Newman-Keuls Test.
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RESULTS |
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During hypertonic stress caused by high NaCl, p53-AS increases the
percentage of cells in early S phase and of cells with >4 N DNA
content, resulting in apoptosis.
To assess p53-dependent cell cycle changes, mIMCD3 cells stressed by
high NaCl were exposed to p53-AS. We have shown previously that the
p53-AS decreases abundance of p53 in mIMCD3 cells (11). A
representative example of cell cycle analysis is shown in Fig. 1, A-C. Cells for all
experimental conditions were grown on the same eight-chamber slide to
ensure that fixation and DNA staining were uniform for all of the
samples. Cells were gated in G1, S, and G2/M
phases of the cell cycle based on DNA content. To establish the gates,
an isotonic sample with nonspecific oligonucleotide was used (Fig.
1A). For the isotonic sample with no treatment, the position
of the gates was the same (data not shown). The same gates were used
for analysis of all samples. In isotonic conditions, p53-AS do not
affect the cell cycle. Hypertonicity, as previously observed (7,
21, 26, 32), increases the proportion of cells in
G2. In the presence of p53-AS after 6 h of hypertonic stress (500 mosmol/kg with added NaCl), the proportion of cells in
early S phase and of cells with >4 N DNA content increases greatly
(Fig. 1A). Because it is conceivable that cell doublets could be mistaken for cells with >4 N DNA content, representative cells were examined microscopically, which confirmed that they were in
fact individual cells (Fig. 1B). These changes of the cell
cycle caused by p53-AS resulted in an increase in the proportion of
apoptotic cells, identified by the high red max pixel values (Fig.
1, A and B) that are associated with condensed
chromatin (5, 11). Cell cycle analysis of these
apoptotic cells shows that they are predominantly the cells that
have progressed into early S phase or that have >4 N DNA content (Fig.
1C). Analysis of the percentage of apoptotic cells
confirmed this observation (Fig. 1D). In the presence of
p53-AS, the increase in the percentage of apoptotic cells is much
bigger in the S and G2 phases compared with the
G1 phase. Among those cells that have >4 N DNA content, 27 ± 7% were apoptotic. Of note even without p53-AS under
hypertonicity, there was a higher percentage of apoptotic cells in
S and G2 phases compared with the G1 phase
(Fig. 1D).
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p53-AS does not abrogate G2 and mitotic arrest caused
by hypertonicity.
Appearance of cells with >4 N DNA content and an increased level of
apoptosis among these cells suggest some disregulation of
G2-M progression in the presence of p53-AS. Hypertonicity
normally induces G2 arrest (7, 21, 26, 32).
Staining of the cells with the mitotic marker phospho-histone H3 (Fig.
2A) revealed that p53-AS do
not abrogate the G2 arrest that takes place after hypertonic stress; there is a great decrease in the percentage of
mitotic cells both with nonspecific oligonucleotide and with p53-AS. To
evaluate morphology of the mitotic cells, we stained the cells with
antibody to -tubulin. As
-tubulin polymerizes to form a mitotic
spindle, it becomes concentrated, resulting in bright green staining
with anti-
-tubulin antibodies. At 320 mosmol/kg, ~2.5% of the
cells are in normal anaphase with mitotic spindles partitioning
chromosomal DNA (Fig. 2B). After 1-4 h of hypertonicity
(500 mosmol/kg with added NaCl), none of the cells are in normal
anaphase with partitioned DNA, indicating complete mitotic arrest (Fig.
2, B and C). p53-AS does not change the result; there are normal mitoses at 320 mosmol/kg, but there still are no
mitoses at 500 mosmol/kg. Therefore, p53-AS abrogates neither G2 nor mitotic arrests caused by hypertonicity, suggesting
a different mechanism of its action.
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Cells in early S phase of the cell cycle are most sensitive to
hypertonic stress caused by NaCl.
Analysis of p53-AS effects on the cell cycle (Fig. 1) showed that a
proportion of cells, which become apoptotic under hypertonicity, is
greatest in the S and G2 phases of the cell cycle, both in control conditions and with p53-AS. p53-AS increases apoptosis in all phases of the cell cycle, but this increase is greatest in S and
G2. This observation suggests that cells in the S and G2 phases have increased sensitivity to hypertonic stress.
To further test this conclusion, we analyzed the survival under
hypertonic conditions of cells synchronized in different phases of the
cell cycle. We used an inhibitor of DNA polymerase, aphidicolin, to achieve synchronization. After 9 h of incubation with aphidicolin (1 µM), the majority of cells are in late G1-early S
(Fig. 3A). After 14 h of
incubation with aphidicolin, the cells are mostly in late
S-G2 (Fig. 3A). After the synchronization,
aphidicolin was washed out, and the medium was made hypertonic (500 mosmol/kg with added NaCl) for 6 h. Next, the number of
nonapoptotic ("healthy") and the percentage of apoptotic
cells were counted by LSC after PI staining. Hypertonicity decreased
the number of healthy cells (Fig. 3B). The decrease in cell
number and increase in the percentage of apoptotic cells were
greater when the cells had been synchronized in early S by 9 h of
previous exposure to aphidicolin than when the cells were
unsynchronized or synchronized in late S-G2 (Fig. 3,
B and C). This result confirms the observation
from Fig. 1 that cells are more sensitive to hypertonicity in early S. We conclude that increased p53 activity might directly protect them in
early S, indirectly protect them by arresting them in G1,
or both.
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At the G1-S interface of the cell cycle, p53 becomes
phosphorylated on Ser15 and translocates to the nucleus,
unaffected by tonicity.
Because the effects of p53-AS and hypertonicity by itself appeared to
be dependent on cell cycle position, we attempted to relate the
expression and localization of p53 and phospho-p53(Ser15)
with the position of the cell in the cell cycle. For this analysis, we
stained cells for p53 or phospho-p53(Ser15) with specific
antibodies and with PI for DNA content and used LSC for the analysis in
a way that has been described in several publications
(8-10). Images of cells stained against p53 are shown in Fig. 4A. It can be seen
that the intensity of green fluorescence representing p53 concentration
and its localization varies between cells. Figure 4B shows
representative cytograms plotting green fluorescence intensity from the
nuclear area (Fn) or from cytoplasmic area (Fc)
vs. DNA content. Hypertonicity only slightly increased Fc
and Fn for p53 and phospho-p53 if all cells are taken in
analysis together (Fig. 4C). This increase was significant
only for nuclear fluorescence for phospho-p53(Ser15).
Because on the basis of differences in DNA content it was possible to
distinguish G1, S, and G2/M cells,
Fc, Fn, and the ratio
Fn/Fc were estimated for cells in each of these
phases by gating analysis (as it was done on Fig. 1; see Fig.
4D). As is evident, hypertonicity significantly increased
Fn for phospho-p53(Ser15) in the S phase.
Fn/Fc also have a trend to increase with
hypertonicity in S and G2, meaning that phosphorylated p53
is localized in the nucleus. Moreover, even in isotonic conditions,
Fn and Fn/Fc for phospho-p53(Ser15) are higher in S and G2
cells. The higher level of Fn/Fc is found also
in isotonic conditions for total p53. These results suggest that p53
translocates to the nucleus and becomes phosphorylated as cells move
from G1 to the S phase. This approach did not give us a
large increase in p53 and phospho-p53(Ser15) levels as seen
by Western analysis (6). The possible explanation for such
inconsistency is that, under hypertonicity, p53 translocates to very
localized spots on DNA (Fig. 4A) that could lead to
underestimation of integrated fluorescence. Such translocation of p53
is detected by the increase of the maximal pixel of p53
immunofluorescence (Fig. 4E and Ref. 8). We
previously found by Western analysis that survival of mIMCD3 cells
exposed to hypertonicity correlates with the amount of
phospho-p53(Ser15) (11). The fact that the
p53(Ser15) phosphorylation occurs at the G1-S
interface and during the S phase of the cell cycle suggests that p53
may protect against hypertonicity by affecting DNA replication.
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During hypertonicity, p53 slows the G1-S transition and
reduces the rate of DNA synthesis.
Hypertonicity slows all phases of the cell cycle, including the
G1-S transition and mitosis (26). To
investigate further the role of p53 in these cell cycle changes, we
labeled cells with BrDU, which is incorporated in place of thymidine
during DNA replication (Fig. 5). Cells
were labeled with BrDU during the last 30 min of 1-, 2-, and 4-h
periods of hypertonicity. The BrDU content of S phase cells was taken
as a measure of DNA replication rate. Hypertonicity decreased the DNA
replication rate. p53-AS increased the DNA replication rate both in
isotonic and hypertonic conditions (Fig. 5, A and
C). To test the effect of p53-AS on the G1-S
transition, we analyzed the percentage of cells outside the
G1 phase (outside the area of G1 cells shown in
Fig. 5A). This parameter was found to be very convenient for
the G1-S transition analysis in hypertonic stress
conditions because there is absolute G2-M arrest (Fig. 2),
preventing any cell entering in G1 from G2. If
progression from G1 to S were to continue under those
conditions, the percentage of cells in G1 should decrease,
and the percentage outside of G1 should increase. In case
of G1 arrest, the percentage of cells outside
G1 should not increase. As seen from Fig. 5B, there is no increase in the percentage of cells outside G1
during the first 2 h with hypertonicity, meaning that
G1 arrest takes place. The decrease in this percentage is
probably caused by predominant death from the S phase (Figs. 1 and 2).
The G1 arrest is transient, and by 4 h with
hypertonicity (500 mosmol/kg) cells resume G1-S transition
that leads to their accumulation in G2 by 6 h with hypertonicity (Fig. 1A). However, when the amount of p53 was
reduced by a specific antisense oligonucleotide, hypertonicity
increased the percentage of cells outside of G1 (Fig.
5B), confirming that p53 is necessary for the
hypertonicity to block movement of cells from G1 into S.
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When caffeine reduces p53, hypertonicity-induced cell cycle delay
is inhibited, cell number decreases, and apoptosis increases.
Caffeine inhibits gamma and ultraviolet radiation-induced
phosphorylation of p53 on Ser15 by inhibition of ataxia
telangiectasia, mutated and ataxia telangiectasia and Rad 3-related
kinases (33). Therefore, we used caffeine as an
additional way to test the role of p53-induced cell cycle delay in the
response to hypertonicity. Caffeine (2 mM) reduces the level of p53 and
phospho-p53(Ser15), both under control and hypertonic
conditions (Fig. 6A).
Densitometry analysis showed that the level of p53 phosphorylation was
reduced by caffeine both under control and hypertonic conditions (Fig. 6A). Associated with the decrease in p53, BrDU incorporation
increases both at 320 and 500 mosmol/kg (Fig. 6, D and
E). Also, the number of cells decreases by 6 h with
hypertonicity, especially at 500 mosmol/kg (Fig. 6B), and
many cells in early S and with >4 N DNA content become apoptotic
at 500 mosmol/kg (Fig. 6C). Thus the effect of caffeine is
similar to that of p53-AS, further supporting the hypothesis that
p53-dependent cell cycle delay protects the cells against
hypertonicity.
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Inhibition of DNA replication protects cells from hypertonicity.
The results thus far support the hypothesis that ongoing DNA
replication sensitizes cells to hypertonicity. As a further test, we
used the DNA polymerase inhibitor aphidicolin to test how inhibition of
DNA replication affects cell survival under hypertonicity. We added
aphidicolin 1 h before the stress to ensure that replication was
inhibited at the time of stress application (time 0), and then aphidicolin was present in the media during the remainder of the
experiment. As seen from Fig.
7A, after 6 h with
hypertonicity without aphidicolin, cells accumulated in G2
because, after the initial period of G1 and S arrest, they
resumed DNA replication. In the same conditions with aphidicolin, the
cell cycle did not change because DNA synthesis was inhibited. Analysis
of changes in cell number showed that aphidicolin prevented the
increase in cell number under isotonic conditions and significantly
improved survival after hypertonic stress (Fig. 7). Note that, on Fig. 7, the cell number is presented as a percentage of the cell number at
time 0. Because absolute G2-M arrest takes place
during the first hours with hypertonicity (Fig. 2), cell number should
not change in the absence of cell death, which is the case at 500 mosmol/kg aphidicolin. There is no improvement in cell survival at 700 mosmol/kg (the osmolality that cells cannot survive). This might
indicate that the mechanism of cell death at 700 mosmol/kg is not
related to DNA replication rate, which is consistent with the absence
of p53 phosphorylation at Ser15 at this osmolality
(11).
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DISCUSSION |
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p53 protects cells from hypertonicity by inhibiting DNA replication and transition from G1 to S. Hypertonicity inhibits cell cycle progression (7, 21, 26, 32). In the present studies, we found that this inhibition was overcome when expression of p53 was reduced by a p53-AS oligonucleotide or by caffeine. With reduced expression of p53, hypertonicity no longer slowed the G1-S transition or decreased the rate of DNA synthesis. Also, with reduced p53 expression, hypertonicity caused some abnormal DNA synthesis in cells in G2/M, resulting in >4 N DNA content. We previously found (11) that reduction of p53 expression in the face of hypertonicity causes apoptosis. Now we find that the apoptosis occurs predominantly in the cells with ongoing DNA replication in early S and with >4 N DNA content. We conclude that cells exposed to hypertonicity are protected by p53 in two ways. First, p53 prevents progression from G1 to S, and, second, it reduces DNA replication rate in S. Similar conclusions were previously reached for other stresses; a number of studies demonstrated that, during S phase and especially at the G1-S border, cells are particularly vulnerable to DNA damage and consequent mutagenesis upon exposure to genotoxic agents (18, 19).
Cell cycle dependence of nuclear vs. cytoplasmic p53 distribution and p53 phosphorylation on Ser15. We find that the distribution of p53 between the nucleus and cytoplasm and the phosphorylation of p53 on Ser15 are cell cycle dependent (Fig. 4). The p53 becomes phosphorylated on Ser15 at the G1-S interface, coincident with its movement into the nucleus, and remains phosphorylated throughout S, independent of hypertonicity. Reduction of the p53 level by p53-AS or caffeine causes an increase in DNA replication rate both under hypertonicity and in control conditions (Figs. 5 and 6). This is consistent with the possibility that p53 participates in coordination of S phase events, most likely related to DNA replication even in the absence of stress. Cell cycle checkpoint genes are essential for cell and organism survival (6, 24, 37), implying that these pathways are not only surveyors of occasional damage but function in normal cellular physiology. Chromosome structural defects occur during normal cell duplication, even in the absence of exposure of DNA-damaging agents. For example, during DNA replication, errors, such as double-strand breaks, arise from stalled replication forks and require attention by the DNA damage response pathway (42). Dual roles have been proposed for several proteins, such as proliferating cell nuclear antigen (PCNA; see Ref. 23) and p53 (16), in maintaining genomic integrity by checking fidelity of DNA structure both at the basal level of stress that occurs normally and after exposure to external stress.
Experiments with caffeine also demonstrate effects both in unstressed and stressed cells. Among its other effects, caffeine inhibits ATM kinase (33), which is defective in cells from patients with ataxia telangiestasia (AT). In response to DNA damage, AT cells fail to increase phospho-p53(Ser15) (33), fail to reduce both initiation of DNA replication and elongation of DNA (29), and fail to activate G1 and G2 checkpoints (17, 30). We find that caffeine reduces phospho-p53(Ser15), even under isotonic conditions, and prevents its increase by hypertonicity (Fig. 6), similar to the effect of caffeine on the responses to ultraviolet radiation (33). Furthermore, we find that caffeine dramatically increases DNA replication rate, even under isotonic conditions, and prevents the inhibition of DNA replication that hypertonicity would otherwise cause. Thus, p53, in concert with other factors, apparently monitors progression of DNA replication and inhibits replication when DNA is damaged. Hypertonicity causes DNA double-strand breaks (20). The mechanism is unclear, but one possibility is that hypertonicity may induce oxidative stress (41), which can in turn produce single- and double-stranded DNA breaks, DNA base and sugar modification, DNA B protein cross-links, and depurination and depyrimidination (2, 34). p53-Dependent cell cycle arrest presumably promotes cell survival by providing time for accurate repair of damaged DNA. Recent reports suggest that p53 not only regulates cell cycle checkpoints but also activates DNA repair (42), supporting the proposed dual role for p53 in maintaining genomic integrity (16). According to this model, p53 acts to maintain genomic integrity whether or not it is activated by DNA damage. When p53 is activated, its major function is as a transcription factor for other genes. The target genes include waf1, which codes for the kinase inhibitor p21 that directs p53-dependent G1 arrest (13). p21 Also acts in the S phase to slow DNA replication by binding to PCNA and blocking DNA elongation (38). In addition to acting as a transcription factor when it is activated, p53 may also be directly involved in DNA repair, even when it is not activated. Cells in which p53 is lacking or mutated are deficient in repair of genomic DNA (35, 14, 39). p53 Interacts specifically with proteins that are components of DNA repair pathways. Most of these proteins are the members of the transcription factor IIH multiprotein complex, which regulates transcription initiation, nucleotide excision repair, and cell cycle progression (4, 12). p53 may be directly involved in DNA repair by binding to single- or double-stranded DNA breaks (36), to ends of double-strand breaks (3), and to DNA mismatches (22). Furthermore, the core domain of p53 has 3',5'-exonuclease activity (27). The binding and exonuclease activities of p53 exist whether it is activated or not. These findings help put our observations into perspective. Thus we find that p53 translocates to the nucleus at the G1-S interface, putting it into position to participate in monitoring DNA replication and to delay replication long enough to repair any damaged DNA that may have occurred. Hence, when DNA is damaged by hypertonicity, the increased abundance of phospho-p53(Ser15) in the nucleus may reinforce its role in counteracting the stress. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Dmitry Bulavin and Albert Fornace for useful discussions.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Dmitrieva, Bldg. 10, Rm. 6N260, National Institutes of Health, Bethesda, MD 20892-1603 (E-mail: dmitrien{at}nhlbi.nih.gov).
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.
Received 8 February 2001; accepted in final form 14 May 2001.
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