From the
Lerner Research Institute, the Cleveland
Clinic Foundation, Cleveland, Ohio 44195,
Institute of Carcinogenesis, Russian Cancer
Research Center, Moscow, Russia 115478, and
¶Engelhardt Institute of Molecular Biology,
Moscow, Russia 119991
Received for publication, January 17, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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In addition to the control of the cell cycle and apoptosis, the p53 family
members are involved in other cellular processes. In particular, p53
participates in the DNA repair
(4,
14), whereas p73 is involved
in the neurogenesis (15,
16). Noteworthy, both p53 and
p73 up-regulate a number of genes whose products are implicated in regulation
of cell/matrix interactions and cell motility as was recently revealed by cDNA
microarray hybridizations (9,
1719).
The involvement of p53 in these processes is supported by other reports
relating p53 to transcriptional regulation of the genes encoding smooth muscle
-actin (20), hepatocyte
growth factor/scatter factor
(21) and its receptor
(22), fractalkine
(23),
HB-EGF1
(24), EGF receptor
(25), metalloproteinase-2
(26), etc. Similarly, it was
shown that p73 overexpression is associated with increased expression of
vascular EGF, fibroblast growth factor 2, and platelet-derived growth factor
(27). Although the
physiological significance of the p53/p73-mediated up-regulation of the above
target genes remains unclear, these data suggest involvement of p53 and p73 in
the control of cell migration.
Cell migration is a key aspect of many normal and abnormal biological processes, including embryonic development, protection from infections, wound healing, and metastasis of tumor cells. It is generally accepted that the driving force for the cell movement is provided by the dynamic reorganization of the actin cytoskeleton, directing protrusion at the front of the cell and retraction at the rear. Cooperative action of small GTPases, particularly Rho, Cdc42, and Rac, is required to promote coordinated assembly and disassembly of actin filaments. Rho maintains cell adhesion during the movement and regulates the assembly of contractile actin-myosin filaments to form stress fibers. Cdc42 is required to maintain cell polarity, which includes localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of the movement. Rac is necessary for the protrusion of lamellipodia and for the forward movement (reviewed in Refs. 28 and 29).
Previously, we demonstrated that overexpression of exogenous p53 in mouse
fibroblasts causes diminution of focal contacts and an increase in lamellar
activity of the cell edge that was paradoxically accompanied by inhibition of
cell migration into the wound in vitro rather than by its stimulation
(1). We explained such an
effect by "skidding" of fibroblasts expressing high levels of
exogenous p53 due to their inability to form effective focal contacts because
of p53-induced repression of fibronectin production
(1). The goal of this work was
to study the effect of physiological levels of p53 on the Rho GTPase activity
and cell migration in various cell contexts. Here we present the data
demonstrating the ability of p53 as well as its homologue p73 to affect
migration ability of different cell types through activation of the
PI3-kinase/Rac1 pathway.
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EXPERIMENTAL PROCEDURES |
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All the cells except keratinocytes were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Fetaclone). The keratinocytes were cultured in keratinocyte/serum-free medium (Invitrogen) containing bovine pituitary extract (30 µg/ml, Invitrogen), recombinant EGF (0.1 ng/ml, Invitrogen), and 0.02 mM CaCl2. The H1299tet/wt-p53 and 10(1)tet/wt-p53 cells were grown in the presence of tetracycline (2 µg/ml).
Boyden Chamber AssayBoyden chamber cell migration assay was performed using transwell chambers with 8-µm pore size membranes (BD Biosciences). The chambers were inserted into 24-well culture plates containing Dulbecco's modified Eagle's medium with either 0.5% bovine serum albumin, 10% FBS, 100 ng/ml EGF (Invitrogen), or conditioned media. To prepare the conditioned media, equal quantities of the cells were plated for 48 h before harvesting the culture media. After collection of the conditioned media, the cells were harvested and counted again. To equalize the conditioned media they were diluted in proportion to the cell quantities.
The cells (5 x 104) were loaded into the upper volume of the Boyden chambers. Non-migrated cells were removed with a cotton swab, and the cells were fixed with methanol for 15 min and stained with crystal violet. The migration activity was quantified by blind counting of the migrated cells on the lower surface of the membrane of at least 10 fields per chamber using a x20 objective.
Western Blot Analysis of Protein ExpressionWhole cell
extracts were lysed in ice-cold RIPA buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1%
SDS, 100 mM phenylmethylsulfonyl fluoride, 1 mM
pepstatin A, and 1 mM E64). Protein concentration in the extracts
was determined with a protein assay system (Bio-Rad). 100 µg of protein was
separated on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene
difluoride membrane (Amersham Biosciences). The membranes were probed with
antibodies specific to p53 (monoclonal antibody 421, a gift of Dr. A. J.
Levine, or DO-1, Santa Cruz Biotechnology), p73 (Ab-1, Oncogene
Research Products), PI3-kinase subunits p85
(polyclonal, Sigma) or p110
(polyclonal, Santa Cruz Biotechnology). The membranes were treated with
secondary sheep anti-mouse horseradish peroxidase antibodies or anti-rabbit
horseradish peroxidase (Jackson Immuno-Research). The filters were developed
by ECL chemiluminescence reagents (PerkinElmer Life Sciences) according to the
manufacturer's protocol.
Detection of Transcriptional Activity of p53 and
p73For luciferase assay, cells were transiently
transfected using LipofectaAMINE reagent (Invitrogen) with 1 µg of the
pWWp-GL2 expressing luciferase under the control of the wild type
p21waf1/cip1 gene promoter
(33). To control the
efficiency of the transfection, the cells were co-transfected with 0.5 µg
of pCH100 (Amersham Biosciences) expressing
-galactosidase. The
luciferase activity was measured using Dual-Light luminescent reporter gene
assay system (Tropix) 48 h after the transfection.
-Galactosidase staining of HCT116-Waf1ConA cells expressing
-galactosidase reporter gene under the p53-responsive promoter was
performed as described previously
(34).
Measuring Activity of the Rho GTPasesThe cells were incubated in serum-free media for 24 h. Thirty minutes or 2 h after stimulation with 10% FBS or EGF (100 ng/ml), the cells were lysed in the MLB buffer (Upstate Biotechnology, Inc.). The amounts of GTP-bound Rac1 or GTP-bound Rho were determined using the Rac1 activation assay kit and the Rho activation assay kit (Upstate Biotechnology, Inc.), respectively, according to the manufacturer's protocol.
Measuring Activity of PI3-KinaseThe serum-starved cells
were stimulated with 10% FBS and lysed at 4 °C in the lysis buffer (150
mM NaCl, 1% Triton X-100, 4 mM
Na3VO4, 200 mM NaF, 10 mM EDTA, 2
mM phenylmethylsulfonyl fluoride, and 10% glycerol, pH 7.4). The
lysates were incubated overnight at 4 °C with the anti-p85
antibodies (Sigma) and protein A-agarose. The beads were pelleted at 14,000
x g, washed three times with Buffer A (Tris-buffered saline, pH
7.5, 0.1% Nonidet P-40, and 100 µM
Na3VO4), three times with the Buffer B (100
mM Tris, pH 7.5, 500 mM LiCl2, and 100
µM Na3VO4), and twice with the Buffer C
(10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA,
and 100 µM Na3VO4). The pellets were
resuspended in the Buffer D (50 mM Hepes, pH 7.5, 1 mM
EDTA, and 100 µM NaH2PO3), and the
activity of PI3-kinase was assessed by phosphorylation of a mixture of
phosphatidylinositols in the presence of 40 µM ATP (1 µCi of
[
-32P]ATP). The reaction was carried out for 15 min and
stopped with 150 µl of ice-cold 1 N HCl. The lipids were
extracted by vortexing with 450 µl of chloroform/methanol (1:1) mixture.
After centrifugation the organic phase was washed twice with 200 µl of 1
N HCl, and 40-µl aliquots were separated on potassium
oxalate-pretreated TLC plates (Whatman) with 35 ml of 2 N acetic
acid and 65 ml of 1-propanol as a mobile phase. The TLC plates were dried and
exposed to Fuji film.
The Wound Healing ExperimentsEight-week-old wild type and
homozygous p53 gene knockout C57BL mice were anesthetized and shaved, and
full-thickness skin wounds were inflicted by excising 0.5 x 0.5-cm
patches of the skin. The wounds were then allowed to dry and to form a scab.
The wound closure was measured daily. The area of wounds was measured by
covering each wound of an anesthetized animal with a transparent plastic film
and recording the wound contour. The wound areas were quantified and expressed
as a percentage of the initial wound size (100%). The mean values (n
= 10 animals) were plotted for each time point.
Expression of p53 was analyzed in 5-day skin wounds by immunohistochemistry of paraffin-embedded wounded skin sections using standard protocol. The FL-293 antibodies (Santa Cruz Biotechnology) were used for the immunostaining of p53.
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RESULTS AND DISCUSSION |
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The decreased cell motility of various p53-deficient cells was further confirmed by Boyden chamber assay. Indeed, the HCT116 p53/ cells showed lower ability to migrate through both uncoated and Matrigel-coated 8-µm pore-size membranes (Fig. 2). The increased number of p53-positive HCT116 cells on the lower side of the membrane as compared with their p53-negative counterparts could not be explained by differences in cell attachment or cell proliferation/survival on the upper side of the filter (see Supplemental Material, Fig. 4). The difference in migration level between HCT116 and HCT116 p53/ cells remained unchanged at different time points of incubation (Supplemental Material, Fig. 5). So the p53 gene knockout in HCT16 cells caused an inhibition rather than a delay of cell migration.
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To exclude that the effect observed in HCT116 cells was due to variations between different clones (the construction of the HCT116 p53-knockout cell line involved selection of individual cell clones), we used retroviral gene transfer to produce mass cultures of HCT116 cells expressing C-terminal dominant-negative fragment of p53 (GSE22 (31)). Introduction of GSE22 into the HCT116 cells decreased p53 transcriptional activity 5 times as measured by the luciferase reporter assay without significant changes in the levels of expressed p53 protein, whereas all HCT116 p53/ derivatives showed background level of luciferase activity and no expression of the p53 protein (Fig. 3, A and B). Boyden chamber assay revealed that GSE22 expression dramatically decreased migration of the HCT116 cells without affecting motility of the HCT116 p53/ cells (Fig. 3C). Hence, there is direct correlation between the level of p53 activity and the migration ability of the HCT116 cells.
The influence of p53 expression on cell motility was also observed in different cell systems. Inhibition of p53 expression in human BJ fibroblasts by siRNA-p53 resulted in decreased cell motility (Fig. 4A). On the other hand the tetracycline-regulated expression of the exogenous p53 in p53-deficient human lung carcinoma H1299 cell line considerably stimulated cell migration (Fig. 4B). Similar correlation between cell migration activity and p53 state was observed in various cells types derived from wild type and homozygous p53 gene knockout mice, in particular for lung and spleen fibroblasts, peritoneal macrophages, and keratinocytes. The most pronounced difference was observed for the peritoneal macrophages (Fig. 4B). Thus, the effect of p53 on cell motility was characteristic for both tumor and normal cells, although the extent of this effect depends on particular cell context.
Overexpression of the p53 Homologue p73 Stimulates Cell
MigrationThe p53 family member p73
displays a high degree
of structural and functional similarity with p53
(10,
11,
13,
16) suggesting that p73
can also affect cell migration. Transcriptionally active p73
was
overexpressed in the p53-positive HCT116 and in the p53-deficient H1299 cell
lines (Fig. 5A).
Overexpression of the p73
in the H1299 cells considerably enhanced both
the p53-dependent luciferase reporter expression and the migration activity
(Fig. 5, B and
C). In the p53-positive HCT116 cells the effect of
p73
was detectable but less significant as compared with the
p53-deficient H1299 cells (Fig. 5,
B and C). Hence, as with p53, the increase in
p73
transcriptional activity was accompanied by stimulation of cell
migration.
Transcriptional Activity Is Required for the p53- or
p73-dependent Stimulation of Cell MigrationTo
study whether p53 or p73
affects cell motility through their ability to
activate transcription of the target genes, we introduced transcriptionally
inactive mutants p53His175
(35,
36) or
p73
His293
(10) into H1299 cells
(Fig. 6A). Expression
of either p53 or p73
mutant had no effect on cell motility
(Fig. 6B), suggesting
that transcriptional up-regulation of certain p53/p73
target genes is
involved in the modulation of cell migration.
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Because transcriptional activity of p53 and its family members can be
modulated at post-translational level by various stresses, we tested whether
p53 can affect motility under unstressed conditions. We investigated the
following: (i) whether conditions of Boyden chamber assay or wounding in
vitro induce transcriptional activation of p53 and (ii) whether the
fraction of the migrated cells is enriched by the cells with transcriptionally
activated p53. We used HCT116 cells carrying -galactosidase reporter
gene under the control of the p53-responsive promoter. We found that below 2%
of the cells were positively stained for
-galactosidase 12 h after
plating into Boyden chambers, whereas over half the cells became
-galactosidase-positive after treatment with the DNA-damaging drug EMS
(see Supplemental Material, Fig.
6). Moreover, we observed the same proportion of the
-galactosidase-positive cells on both sides of the porous membrane (data
not shown). No considerable increase of activity of p53 or its homologues was
observed at the wound edge 24 h after wounding (see Supplemental Material
Fig. 6). This result suggests
that conditions of Boyden chamber assay or wound closure in vitro do
not induce stress-related activation of p53. So even the "latent"
form of p53 is capable of affecting cell migration. This idea is in agreement
with the data that the unstressed latent form of p53 is bound to p53-binding
sites within the p53-responsive genes, including the
p21waf1/cip1 gene
(37). Moreover, p53 expression
under unstressed conditions induces significant changes in the gene expression
profiles revealed by cDNA microarray hybridization
(9,
18,
19,
38). The genes responsible for
the p53-dependent modulation of cell motility might belong to the group of
genes that p53 regulates under normal physiological conditions.
To test whether stress-induced activation of p53 can additionally stimulate cell motility, we analyzed migration of the cells treated with the DNA-damaging agent EMS or with the hypoxia-mimicking agent deferoxamine mesylate. Either of the treatments increased the expression level and transcriptional activity of p53 (data not shown). Boyden chamber assay carried in the presence of the drugs showed proportional increase in migration of both p53-positive and p53-negative cells (Fig. 7) suggesting significant p53-independent component in stress-induced activation of cell motility. Thus, the contribution of activated p53 into stimulation of cell migration under stress conditions can hardly be assessed.
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The Rac1 and PI3-Kinase Activity Correlates with the Expression of p53
and p73The GTPase proteins of the Rho family are
known as key regulators of cell motility (reviewed in Refs.
28 and
29). To study possible
mechanisms of the p53/p73
-mediated cell migration, we tested the effect
of p53 or p73
expression on activity of the Rho family GTPases. We
assessed the Rac1 activity by its ability to bind the effector Pak1 protein.
In serum-starved HCT116 and HCT116 p53/ cells, the active form
of Rac1 was detectable at low level, and unlike Guo et al.
(39) we failed to establish
clear differences between p53-positive and p53-negative cells (see
Supplemental Material, Fig. 7).
After serum stimulation for 30 min or 2 h, the amount of the active GTP-bound
Rac1 was considerably higher in p53-positive HCT116 cells as compared with
their p53-negative counterparts (Fig.
8A and Supplemental Material,
Fig. 7). Inactivation of p53 by
GSE22 also decreased the Rac1 activity in HCT116 cells without affecting the
HCT116 p53/ cells (Fig.
8A). On the other hand, expression of p53 or p73
in the p53-deficient H1299 cells considerably increased the amount of active
Rac1 (Fig. 8, A and
B). Hence, the Rac1 activity correlates directly with the
level of p53 or p73
expression. These data agree with our previous
observations (1) of the
p53-mediated increase in lamellar activity at the cell edge, because Rac1 is
the essential inductor of membrane ruffling (reviewed in Refs.
28 and
29). Unlike changes in the
Rac1 activity, the expression of p53 did not affect the activities of Rho
(Fig. 8C) or Cdc42
(40) GTPases. The changes in
activity of Rac1 can constitute a part of the main mechanism by which p53 and
p73
modulate cell migration.
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One of the major regulators of Rac1 activity are guanine nucleotide exchange factors (GEFs). Over 30 different GEFs regulating the Rho family GTPases have been identified to date. Some of the GEFs, including representatives of the best characterized Vav family members, are activated by the second messengers generated by the enzymatic activity of PI3-kinase (reviewed in Refs. 28 and 41). Trying to learn more about possible pathways leading to the p53-mediated up-regulation of Rac1, we tested whether p53 expression could modulate PI3-kinase activity. We found that p53 expression did not alter the expression of p85 or p110 subunits of PI3-kinase (Fig. 9A). However, study of the PI3-kinase functional activity in p53-positive and p53-deficient serum-stimulated HCT116 cells showed that PI3-kinase precipitated from the p53-deficient cells had significantly lower phosphorylation activity than the enzyme from the parental HCT116 cells (Fig. 9B). Hence, we conclude that p53 may stimulate cell motility through the PI3-kinase/Rac1 pathway.
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The p53/p73-induced Stimulation of Cell
Migration Depends on Modulation of Intracellular Signaling Pathways Rather
Than on Enhanced Production of Secreted Motogenic/Chemoattractant
FactorsThere are two possible mechanisms by which p53 could
up-regulate the PI3-kinase/Rac1 pathway. First, the p53 expression can
increase production of secreted motogenic/chemoattractant factors. Second, p53
could act downstream by modulating some steps of intracellular signaling
involved in activation of PI3-kinase/Rac1 in response to motogenic factors.
The former supposition is based on the data that p53 can up-regulate
transcription of HB-EGF leading to activation the PI3-kinase/Akt pathway
(24). Some other genes
encoding motogenic factors proved to be direct transcriptional targets of p53
(17,
18,
21,
23,
24). Therefore, p53 may
enhance the expression of motogenic cytokines such as HB-EGF, hepatocyte
growth factor/SF, IGF1, etc. and consequently induces the activity of their
receptors whose phosphorylation mediates activation of the PI3-kinase
function.
To answer whether p53 or p73 affect cell migration through increased
production of secreted motogenic/chemoattractant factors, we studied the
changes in cell migration toward conditioned media obtained from the cells
with different p53 state. The conditioned medium obtained from the cells
expressing p53 or p73
showed only a slightly increased motogenic effect
as compared with the medium conditioned by the cells with inhibited p53
function (Fig. 10A).
In agreement with these data, we observed no changes in the migration activity
of HCT116 cells toward 10(1)tet/wt-p53 cells after stimulation of p53
expression by withdrawal of tetracycline
(Fig. 10B). This
result allowed us to propose that p53/p73
-mediated stimulation of cell
migration is mainly related to some changes in intracellular signaling rather
than to enhanced production of secreted motogenic factors.
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Next, we tested whether expression of p53 could modify cell response to motogenic factors, in particular to EGF. Stimulation with EGF of the p53-deficient HCT116/GSE22 cells resulted in significantly lower migration activity and proportion of the GTP-bound Rac1 as compared with the control p53-positive HCT116 cells (Fig. 11A). A similar result was obtained after addition of the serum that apparently contains a mixture of motogenic factors (Fig. 11A). Thus, p53-negative cells might have some defects in transduction signals from growth/motogenic factor receptors to PI3-kinase. This is consistent with the observation that the conditioned media additionally stimulates migration of p53-positive HCT116 cells, whereas it has almost no effect on the isogenic cells with the disrupted p53 gene (Fig. 10A).
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It should be noted that the EGF-dependent pathway does not seem to be the only and, probably, the major mechanism through which p53 affects cell motility. In fact, treatment with a specific inhibitor of EGF receptor (AG1478) only partially inhibited the migration and did not eliminate the difference in migration level of p53-positive and p53-negative HCT116 cells. Meanwhile, wortmannin, a specific inhibitor of PI3-kinase, almost completely inhibited migration of all tested cells (Fig. 11B). Moreover, treatment of peritoneal macrophages that may not express the EGF receptor (42, 43) with the AG1478 inhibitor did not affect migration of either p53-positive or p53-deficient cells (Fig. 11B). This allows us to suggest that different p53 targets are probably responsible for stimulation of cell migration in distinct cell contexts.
Analysis of gene expression profiles has revealed a number of p53- or
p73-regulated genes involved in positive control of cell migration,
such as Eps8, Ha-Ras-1, ACK, GNA13, inositol-1,3,4-triphosphate
5/6-kinase, smooth muscle actin, etc.
(9,
1719,
24,
38). Notably, the set of
p53/p73
-up-regulated genes is cell type-specific. So it is likely that
several different p53/p73 target genes are involved in positive control of
cell migration. On the other hand, p53 and p73
can cause some cell
type-specific changes in gene expression resulting in inhibition of directed
cell migration such as repression of fibronectin in fibroblasts
(1,
45), repression of
metalloproteinase-2 in melanocytes
(46), or yet unidentified
modulation of downstream targets of Cdc42 in mouse embryo fibroblasts
(40). Additional studies are
required for identification and verification of particular p53/p73-dependent
genes that are involved in regulation of cell migration in each cell
context.
The Role of the p53/p73-regulated Cell Migration in Vivo The question arises what is the physiological significance of the p53/p73-mediated modulation of cell migration. We assumed that such regulation might stimulate remodeling of tissues and, in particular, could facilitate wound healing. This idea is based on two observations. First, the p53-deficient cells showed decreased migration into the in vitro wound scratched on the surface of the plate with growing culture (Fig. 1). Second, the obtained data (data not shown) and the published data (47, 48) demonstrated that the re-epithelialization of skin wounds was accompanied by enhanced levels of p53 protein in the basal layers of the epidermis. However, measuring the kinetics of skin wound closing showed only marginal difference with a tendency of delayed wound healing in the p53 knockout mice (Fig. 12). The absence of a clear effect of p53 gene knockout could be explained by compensatory mechanisms. For example, the lack of p53 could lead to elevated transcriptional activity of the p53 family members, because p53 proved to up-regulate expression of the dominant-negative form of p73 (49, 50). Thus, the decrease in cell migration due to p53 deficiency could be compensated by stimulation of motility through increased functional activity of its family members, particularly p73. Hence, involvement of p53 in tissue remodeling and wound healing remains in question.
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Involvement of the p53/p73-mediated regulation of cell motility in other
physiological processes can be proposed. It can take place during inflammatory
response directing certain type of cells to the sites of infection, or during
embryogenesis to establish tissue patterns and to drive certain morphogenetic
pathways. The role of p53 and p73 in inflammation response is still poorly
studied. We found that the p53-positive peritoneal macrophages showed
significantly higher motility (Fig.
4B) and thus could have increased phagocyte activity as
well. Notably, it was shown that p73 knockout mice have severe defects in
inflammation response (15). In
addition, the p53 family members, particularly p73, are implicated in the
control of cell migration during neurogenesis. Deficiency in p73 leads to a
severe distortion of hippocampal formation suggesting that p73 function is
important for proper differentiation and migration of neurons
(15,
16). The p53 can be also
implicated in neurogenesis, as a certain proportion of p53-deficient mice show
defects in neuronal tube closure
(44,
51). Moreover, p53 regulates a
set of genes involved in neural crest cell migration and neural tube closure
(19). We can speculate that
the ability of p53 to modulate cell migration is a feature of its evolutional
ancestors that, similar to p63 and p73, played an important role in
ontogenesis. This feature could be attributed to a constitutive function of
p53 in the absence of stress, which explains why stress activation is not
important for p53-mediated increase in cell motility. Hence, our finding
demonstrates a novel activity of p53 and p73 that might be related to
obscure functions of the p53 family under normal physiological conditions.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains Figs. 1-7.
|| Both senior authors contributed equally to this work.
** To whom correspondence may be addressed. Tel.: 216-444-9540; E-mail: chumakp{at}ccf.org.
To whom correspondence may be addressed. Tel.: 7-095-324-1739; E-mail:
bkopnin{at}yahoo.com.
1 The abbreviations used are: HB-EGF, heparin-binding EGF-like growth factor;
EGF, epidermal growth factor; PI3, phosphatidylinositol 3; GFP, green
fluorescent protein; GSE22, genetic suppressor element 22; FBS, fetal bovine
serum; EMS, ethyl methanesulfonate; GEF, guanine-nucleotide-exchange
factor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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