J. Physiol. Biochem., 60 (4), 287-308, 2004
p53: Twenty five years understanding
the mechanism of genome protection
M. Gomez-Lazaro, F. J. Fernandez-Gomez and J. Jordán
Centro Regional de Investigaciones Biomédicas, Facultad de Medicina, Universidad de
Castilla-La Mancha, Avda. Almansa, 02006 Albacete, Spain
(Received on November 10, 2004)
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ and J. JORDÁN. p53:
Twenty five years understanding the mechanism of genome protection (minireview).
J. Physiol. Biochem., 60 (4), 287-308, 2004.
This year the p53 protein, also known as “guardian of the genome”, turns twenty
five years old. During this period the p53 knowledge have changed from an initial
pro-oncogene activity to the tumorsupressor p53 function. p53 is activated upon
stress signals, such as gamma irradiation, UV, hypoxia, virus infection, and DNA
damage, leading to protection of cells by inducing target genes. The molecules activated by p53 induce cell cycle arrest, DNA repair to conserve the genome and apoptosis. The regulation of p53 functions is tightly controlled through several mechanisms including p53 transcription and translation, protein stability, post-translational modifications, and subcellular localization. In fact, mutations in p53 are the most
frequent molecular alterations detected in human tumours. Furthermore, in some
degenerative processes, fragmentation and oxidative damage in DNA take place, and
in these situations p53 is involved. So, p53 is considered a pharmacological target, p53
overexpression induces apoptosis in cancer and its expression blockage protects cells
against lethal insults.
Key words: Apoptosis, Necrosis, mdm2, Cancer, Tumor suppressor, Mutation.
Twenty-five years ago a 53KDa protein
with previously unknown properties was
discovered. This event was the culmination of two kind of studies: virologic and
serologic. In the virologic studies, SV40transformed cells were used. It was found
Correspondence to J. Jordán (Tel.: +34 967 599 000;
Fax: +34 967 599 327; e-mail: joaquin.jordan@uclm.
es).
that a 55kDa protein co-precipitated with
the large T antigen of simian virus 40
(SV40) (21, 82, 87, 95, 108). This protein
was also found overexpressed in several
murine SV40 transformed cells, and in
embryonic carcinoma cells (82, 95). On
the other hand, the serologic approach
showed how antisera prepared from
BALB/c Meth A sarcoma in syngeneic or
compatible F1 mice, recognized a protein
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M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
with an apparent molecular weight of 53
kDa in extracts of BALB/c cells (32). Animals suffering several types of tumors
exhibited an immune response specific for
this protein (82, 108, 132). Later studies
revealed the presence of antibodies against
this protein in 9% of sera from breast cancer patients (29) as well as in sera from
children with a wide variety of cancer (20,
63).
During the first decade after p53 was
discovered, this protein was suggested to
act as an oncogene. Indeed, p53 was implicated in positive regulation of cell growth
(129) and in cell transformation (43, 125).
In 1989, Levine and colleagues established
its real physiological function: the tumor
suppressor action, acting negatively to
block transformation (45). It then became
clear that mutant forms of p53 exhibited
oncogenic activities (43, 46, 59). In addition, p53 mutations were found very frequent in colon cancer (6). This was seen
later in most of the common types of
human cancers (61). Since these observations were done, a large amount of studies
have demonstrated that: i) inactivating
mutations and/or deletions of the p53
gene are a common event in different
types of human cancer (130, 139), ii) the
wild-type p53 protein acts as a transcription factor, while some p53 protein
mutant forms fail to exhibit this biochemical function (42, 44, 77) and iii) the target
gene products induced by wild-type p53
mediate its tumor-suppressor function
playing a direct role in modulating growth
arrest, apoptosis, membrane signalling,
protein degradation or oxidative stress
(127, 168, 170) (Table I). All these evidence established turning point in this
protein short life, as pointed out by the
growing number of publications referring
to it.
J. Physiol. Biochem., 60 (4), 2004
We now know that p53 function is
essential in the cellular response against
cell damage or genome mutation. Because
of this, in the beginning of the 90’s it was
called “The Guardian of the Genome”
(86).
p53 structure
In humans the p53 gene locus is located
in the shorter arm of chromosome 17
(17p1.3). Its mRNA is translated into a
393 amino acid (aa) protein that comprises several domains, including an acidic Nterminal region containing the transactivation domain, a core holding the
sequence-specific DNA-binding domain
and a complex C-terminal domain with
multiple functions.
– The N-terminal domain (aa 1–93)
holds a strong transcription activation signal (156), and is involved in transcriptional target gene activation, and is subdivided
into the transcription activation subdomain (aa 1–42, aa 43–63) and the prolinerich subdomain (aa 62–93). N-terminal
also contains the highly conserved BOX-I
transactivation domain that directs the
binding of p53 to the transcriptional
adapter protein p300 (5) .
– The central core domain (aa 102-292)
contains the specific DNA binding
region, and is the most preserved region of
the protein amongst vertebrates. The
stress-regulated transactivation function
of p53 is driven by its sequence-specific
DNA-binding domain and is co-ordinated by specific protein-protein interactions
that can in turn be modulated by covalent
and non-covalent modifications. The core
domain is organized into a large b -sandwich that forms a scaffold for a loop-b strand-helix motif and two loops that coordinate with zinc ion (25). The direct
contact with the DNA is through residues
TWENTY FIVE YEARS AROUND p53
289
Table I. Functions of p53 and targets genes.
Gene/protein
Synonims
Role
Cell cycle inhibition
p21WAF1
14-3-3s
CDK-interacting protein 1, MDA-6 Cyclin-dependent kinase inhibitor
KCIP-1, Protein 1054
Tethers cyclin B1-CDK1 complexes in the cytoplasm.
G2 cycle arrest
MyD118
Growth and apoptosis.
GADD45b
Activation of stress-responsive MTK1/MEKK4 MAPKKK
BTG2/TIS21 NGF-inducible anti-proliferative General transduction
protein PC3
Post-translational modifications.
Anti-proliferative protein.
Transcription regulation mediated by ESR1
MIC-1
Colon cancer-associated
Growth factor inhibitor
protein Mic1
IGFBP3
None
Growth factor inhibitor
MDM2
Transformed 3T3 double
Inhibits p53 and p73- mediated cell cycle arrest
minute 2
and apoptosis. Ubiquitin ligase E3
p53-binding protein Mdm2
Nuclear export of p53 and targets for proteolysis
EGF-like TGF, ETGF, TGF type 1 Growth factor inhibitor
TGF-a
Cyclin B1 None
Cell cycle control (G2/M transition)
Cyclin D1 PRAD1 oncogene,
Cell cycle control (G1/S transition)
BCL-1 oncogene
Cyclin E
None
Cell cycle control (G1/S, start, transition)
Cyclin B2 None
Cell cycle control (G2/M transition)
Cyclin D2 None
Cell cycle control (G1/S transition)
Cyclin A
None
Cell cycle control (G2/M transition)
CDK4
PSK-J3
Cell cycle control.
CDK2
p33 protein kinase
Cell cycle control. Interacts with cyclins A, B3, D, or E.
Activity of CDK2 is maximal during S phase and G2
DCK1
Doublecortin-like and CAM
Calcium-signaling neuronal migration
kinase-like 1
None
Topological states of DNA by transient breakage and
TOPO-IIa
subsequent rejoining of DNA strands.
Double-strand breaks
PLK
PLK-1, STPK13
Cell division and G1 or S phase
PISSLRE
Serine/threonine, e-protein kinase Cdc2 related protein kinase
BRCA-1
None
DNA repair. E2-dependent ubiquitation.
Cdc6
CDC6-related protein, p62(cdc6), DNA replication
HsCDC6, HsCDC18
Checkpoint controls that ensure DNA replication
GOS2
None
Probably involved in translation
Apoptosis
Bax
Apaf-1
PUMA
P53AIP1
PIDD
None
None
None
None
None
J. Physiol. Biochem., 60 (4), 2004
Programmed cell death
Activation of pro-caspase-9
Mitochondrial cytochrome c release
Lost of YD m and apoptosis
Mitochondrial cytochrome c release
290
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
Table I. (Continuation).
Gene/protein
NOXA
KILLER/DR5
Bid
TRAF4
Fas/Apo1
BAK
Bcl-6
Wig1
Caspase 2
Caspase 3
Caspase 9
MIHB
MIHC
PDCD2
DNA repair
P53R2
PCNA
DDB2
LIG1
ERCC5
Synonims
None
TRAIL-R2
None
Cysteine-rich domain
associated with RING and
Traf domains protein 1,
Malignant 62
TNFR6, FASL receptor,
Apoptosis-mediating surface
antigen FAS, Apo-1 antigen,
CD95 antigen
Apoptosis regulator BAK,
BCL2-like 7 protein
Zinc finger protein 51
LAZ-3 protein, BCL-5,
Zinc finger and BTB domain
containing protein 27
Zinc finger protein WIG1
CASP-2, ICH-1 protease,
ICH-1L/1S
Apopain,
Cysteine protease CPP32,
Yama protein, SREBP, SCA-1
ICE-like apoptotic protease 6
ICE-LAP6,
Apoptotic protease Mch-6,
Apoptotic protease activating
factor 3
BIRC-2
Brush border myosin I,
BBM-I, BBMI,
Myosin I heavy chain, Myosin Ia
Zinc finger protein Rp-8
Zinc finger MYND domain
containing protein 7
None
None
Damage-specific DNA binding
protein 2, DDB p48 subunit,
DDBb, UV-damaged
DNA-binding protein 2
DNA ligase I
Polydeoxyribonucleotide
synthase [ATP]
DNA excision repair-related
gene, DNA-repair protein
complementing XP-G cells,
J. Physiol. Biochem., 60 (4), 2004
Role
Mitochondrial cytochrome c release
Receptor for the cytotoxic ligand TNFSF10/TRAIL
Pro-apoptotic
Activation of NF-kappa-B and JNK
Receptor for TNFSF6/FASL
Binding and antagonizing the repressor Bcl-2 or E1B
19k protein
Transcriptional regulator with an important role
in lymphomagenesis
TP53-dependent growth regulatory pathway
Activation cascade of caspases for apoptosis
Apoptosis execution
Binding to Apaf-1 leads to activation of the protease
cascade
Apoptosis inhibitor
Movement of organelles along actin filaments
DNA-binding protein with a regulatory function
DNA damaged repair
Auxiliary protein of DNA polymerase delta.
Control of eukaryotic DNA replication.
Repair of UV-damaged DNA. Binds to pyrimidine dimers
Seals nicks in double-stranded DNA
DNA excision repair
TWENTY FIVE YEARS AROUND p53
291
Table I. (Continuation).
Gene/protein
RPA1
XRCC9
RFC4
Synonims
Xeroderma pigmentosum group
G complementing protein,
DNA excision repair protein
ERCC-5
RF-A,
Replication factor-A protein 1,
Single-stranded DNA-binding
protein
FACG protein,
DNA-repair protein XRCC9
Replication factor
C 37 kDa subunit,
A1 37 kDa subunit,
RF-C 37 kDa subunit, RFC37
Angiogenesis and metastasis inhibitors
Maspin
Protease inhibitor 5
KAI1
Metastasis suppressor homolog
Senescence
Ras
Raf
Raf-1, C-RAF, cRaf
P14/ARF None
MAPK
Extracellular signalregulated kinase 2
Mitogen-activated protein kinase 2,
p42-MAPK, ERT1
E2F1
RBBP-3,
PRB-binding protein E2F-1,
RBAP-1
P16
P16INK4A tumor suppressor
protein
PML
None
Transcription
ATF3
None
Signal transduction
DUSP5
Dual specificity protein
phosphatase hVH3
DGKA
Diglyceride kinase,
DGK-alpha, DAG kinase alpha,
80 kDa diacylglycerol kinase
CDC25C
M-phase inducer phosphatase 3
Oxidative stress
PIG3
Quinone oxidoreductase
homolog
J. Physiol. Biochem., 60 (4), 2004
Role
Absolutely required for simian virus 40 DNA
replication in vitro
Post-replication repair
Elongation of the multiprimed DNA template
Serine protease inhibitor
Metastasis suppressor protein
Transduction of mitogenic signals
Transduction of mitogenic signals
Regulator of MDM2
Phosphorylates microtubule-associated protein-2
(MAP2). Myelin basic protein (MBP) and Elk-1
Control of cell-cycle progression from G1 to S phase
Cyclin-dependent kinase inhibitor
Cell proliferation and apoptosis
Represses transcription
Displays phosphatase activity
Converts diacylglycerol into phosphatidate and
attenuates protein kinase C activity
Inducer in mitotic control
Oxidative stress
292
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
Table I. (Continuation).
Gene/protein
Synonims
Role
Protein metabolism
LOXL1
Lysyl oxidase-like protein 1
LOL
Cell-cell signaling
EDN2
ET-2,
Preproendothelin-2,
PPET2
Protocadherin-1 None
R148 and R273, while other residues are
necessary for the integrity of the structure.
– The C-terminus (aa 293–393) is a flexible region, which has a basic end and a
tetramerization domain that ensures
assembly of p53 into conformationally
active tetramers. It is subdivided in the
tetramerization domain (326–353) and the
regulatory domain (CTRD; 363–393).
Flanking the p53 tetramerization domain,
there is a negative regulatory domain
whose post-translational modification
may play an important role in modulating
the specific activity of p53 in vivo. One
function for this regulatory C-terminal
domain is to maintain p53 protein in a
latent state for specific DNA binding, but
also: nuclear import, nuclear export, nonspecific DNA binding and homooligomerization. In the p53 latent form, this
region is tucked back inside the protein
central region preventing the binding to
its DNA target sequence.
p53 family
p53 protein belongs to a family of transcription factors composed by at least two
other members: p73 and p63 (also known
as KET, p40, p51 or p73L). All of them
J. Physiol. Biochem., 60 (4), 2004
Lysyl oxidase family
Endothelins are endothelium-derived vasoconstrictor
peptides
Cell to cell adhesion and communication,
Extracellular matrix
share substantial sequence homology, and
p63 and p73 can, at least when overproduced, activate p53-responsive promoters
and induce apoptosis. They contain very
large introns (98) and while split in their
primary sequence, present divergences in
their C-terminal regions and also in exon
number, functions, regulation and tissue
distribution. On the basis of phylogenetic
analysis, it has been postulated that the
p53 gene may have evolved from an ancestral gene similar to p73/p63 (75, 166). The
fact that no other p53 family members
have been identified within the Drosophila or the Caenorhabditis elegans genome
suggests that the p63/p73 subfamily
evolved after the division of the arthropod
and vertebrate lineages (121).
The N-domain is poorly conserved,
while the DNA binding domain presents
the highest homologies. Therefore, the
essential residues for p53 protein folding
are conserved among its family, for this
reason p73 and p63 can bind to p53 DNA
binding sequences. Strikingly, p63 shows
the highest preservation of primary amino
acid sequence in the N-terminal domain
between human and mouse with 99%
identity (166). This high conservation of
the transactivation domain is possibly
related to the fact that p63 plays a unique
role among the p53 family members with
TWENTY FIVE YEARS AROUND p53
respect to normal murine and human
development.
In addition, gene processing of p53 is
different as well. While p63 and p73 present different proteic isoforms, two p53
mRNA isoforms (normal splice, NS, and
alternative splice, AS) can be detected in
the mouse. ASp53, but not NSp53, fails to
bind non-specifically to single-stranded
nucleic acids and is constitutively active
for specific DNA binding (8). These proteins are generated at different times during the cell cycle and also at different levels between different tissues. With respect
to p63, there are at least three different
protein isoforms, differing in the C-terminal end (a , b and g forms) that may vary
also at the transactivation domain because
of the cryptic promoter located at intron 3
that generates additional transcripts
(D Np63a , D Np63b and D Np63g ) (166).
In the case of p73 there are six different
variants of the protein (a , b , g , d and e
forms) (89). On the other hand, the
expression pattern is also different, therefore, while p53 is ubiquitously expressed,
p73 is located in the epidermis, sinuses,
inner ear and brain and p63 in epidermis
proliferating basal cells , cervix, urothelium and prostate.
There are also differences in the
upstream signalling pathways involved in
the activation of these genes. Both cellular
and viral oncoproteins can discriminate
between p53 and the rest of its family
(105). p53 activators like dactinomicine
and UV radiation are not able to activate
p73, although both proteins are accumulated in response to DNA damage.
However, the greatest difference
between these proteins is their mutational
prevalence in human cancer. While p53
mutations are found in a high percentage
of cancers mutations on p63 and p73 are
unusual. Besides, and in contrast to what
J. Physiol. Biochem., 60 (4), 2004
293
happens in p53-/- mice (64) the absence of
p63 or p73 is not involve in tumor development in animal models. Instead, the
absence of one of these two proteins is
responsible for developmental abnormalities.
In summary, p53, p63 and p73 appear
to have overlapping but also distinct functions: p53 regulates the stress response for
suppressing tumors; p63 is essential for
ectoderm development; and p73 might
regulate both the stress response and
development.
p53 protein level regulation
p53 is a short-lived protein with a halflife of ~5-20 min in most cell types studied. The levels of this protein are under
severe control (123, 131). Stressful situations including genotoxic (irradiation,
chemical carcinogens, cytotoxic drugs,
cyclobutan pirimidin dimmers) (47, 56,
76, 94, 119), non-genotoxic (telomere erosion, hypoxia, hyperthermia, depletion of
ribonucelotides, nitric oxide), and oncogenic activation of growth signalling cascades are all responsible for the stabilization and activation of the p53 protein, by
increasing the half-life of protein by several fold (102, 128), yielding to an increase
in protein levels. These increments are
mainly a consequence of post-translational mechanisms, although small changes at
the transcriptional level are also involved.
The post-translational modifications that
p53 can suffer are phosphorylations,
dephosphorylation, acetylation, sumoylation and changes in redox state (Table II).
The phosphorylation of residues in the
C-terminal and N-terminal regions modulates p53 function. The enzymes with
kinase activity that can phosphorylate p53
are: casein kinase I (Ser6 y Ser 9) and II
(Ser392), DNA-PK (Ser15 y Ser37), ATM
294
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
Table II. Post-transcriptional p53 modifications.
Position
Modifying
enzymes
Stress
signal
Function
Phosphorylation
Ser-6
Unknown
IR, UV
Enhanced acetylation of Lys382
Ser-9
Unknown
CK1
IR, UV
IR
Enhanced acetylation of Lys382
Signal amplification
Ser-15
ATM, ATR
DNA-PK
p38
IR, UV
IR, UV
UV
P300 binding
Enhanced sequence specific transactivation
Enhanced sequence specific transactivation
and stability (Apoptosis)
Ser-20
ChK1, ChK2
IR, UV
Reduced Mdm2-binding
Ser-33
CAK
p38
NK
UV
JNK
IR, UV
DNA binding
Enhanced sequence specific transactivation
and stability (Apoptosis)
Enhanced stability
Ser-37
ATR
DNA-PK
IR, UV
IR, UV
Acetylation
Enhanced sequence specific transactivation
Ser-46
p38
UV
Enhanced sequence specific transactivation
and stability (Apoptosis)
Unknown
HIPK2
IR
UV
Activation of p53-dependent transcription
and apoptotic pathways
Ser-149
CSN
Ser-315
CKI
CDK
Ser-371
CyclinA-cdk2
NK
Enhanced sequence specific transactivation
Ser-376
PKC
CAK
CyclinA-cdk2
UV
NK
NK
Enhanced sequence specific transactivation
DNA binding
Enhanced sequence specific transactivation
Ser-378
CAK
CyclinA-cdk2
PKC
NK
NK
IR
DNA binding
Enhanced sequence specific transactivation
Enhanced sequence specific transactivation
Ser-392
CKII
P38
UV
UV
Tetramerization
Enhanced sequence specific transactivation
and stability (Apoptosis)
Thr-18
CKI
PKR
IR, UV
IFN, dsRNA
Reduced Mdm2-binding
Enhanced sequence specific transactivation
Thr-55
TAF1
Unstressed cells
Cell G1 progression and apoptosis
JNK
ROS
DNA-damage
Apoptosis
Enhanced p53 transactivation potential
Thr-81
Targets p53 to degradation by the ubiquitin system
IR, UV
NK
Reduced Mdm2-binding
DNA binding
Thr-150
CSN
IFN
Targets p53 to degradation by the ubiquitin system
Thr-155
CSN
IFN
Targets p53 to degradation by the ubiquitin system
J. Physiol. Biochem., 60 (4), 2004
TWENTY FIVE YEARS AROUND p53
295
Table II. (Continuation).
Position
Modifying
enzymes
Stress
Function
signal
Dephosphorylation
Ser-315
Unknown
IR
Cdc14 phosphatase
Increase binding to 14-3-3
NK Nuclear localization
Lys-320
PCAF
IR, UV
DNA binding
Lys-373
P300/CBP
p33(ING1b)
IR, UV
NK
DNA binding
Apoptosis
Lys-382
p33(ING1b)
P300/CBP
NK
IR, UV
Apoptosis
DNA binding
Acetylation
Sumoylation
Lys-386
E1 and hUbc9 UV
Stabilization
Ribosylation
PARP
IR
Enhanced sequence specific transactivation and stability
dsRNA, double stranded RNA; IR, ionizing radiation; IFN, interferon; NK, not known; UV, ultraviolet.
and ATR (Ser15), CDK-activating kinase
(CAK; Ser 33), cdc2/cdk2 (Ser315) and
PKC (Ser378) (97, 111). In addition to all
these proteins that regulate the stability
and function of p53 through phosphorylation, a functionally distinct group of
proteins, is now emerging. This new
group of proteins appears to operate as
cofactors stimulating one or more of the
wild-type properties of p53. One such
family, which is possibly involved in
breast cancer, is the apoptosis stimulating
protein of p53 (ASPP) (134).
p53 redox regulation involves two clusters of cysteine residues in the central
domain of the protein, one of them (aa
121, 132, 138 and 272) being located near
the loop-sheet-helix region of p53 that
contacts with the consensus DNA
sequences. Oxidized p53 loses its
sequence-specific DNA-binding capabilities (56), and divalent metal cations,
whose binding to p53 is dependent on the
J. Physiol. Biochem., 60 (4), 2004
reduced state of certain cysteine residues,
are critical for p53 function (33, 56, 155).
Furthermore, nitric oxide and thioredoxin
reductase affect p53 conformation and/or
transcriptional activity (17, 126). The
demonstration that the redox/repair protein, Ref-1, activates the DNA-binding
and transactivation activities of p53 (68)
further stresses the potential functional
importance of the redox state of p53.
Like other proteins, p53 is degraded.
The major degradation pathway of p53 is
the ubiquitin-26S proteosome (100), but it
can also be hydrolysed by cysteine proteases such as calpains (83). Because p53
degradation takes place entirely in the
cytoplasm, a return of p53 from the nucleus to the cytoplasm is necessary. This
return occurs via the CRN1 pathway.
Three systems involve in the signalling for
p53 degradation have been described: the
signalosome COP9 (CSN) that phosphorylates p53 at Thr155 and the neighbour-
296
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
ing residues (9); the JNK kinase that phosphorylates p53 at Thr81; and the MDM2
protein (58), a phosphoprotein with E3
ubiquitin protein ligase activity, which is
able to interact with the p53 amino-terminal end (67). The most important of these
is the one mediated by the oncogenic
MDM2 protein (15, 99), which can guide
p53 to the cytoplasm where it is degraded
(62, 146). The ability of p53 to bind and
upregulate the transcription of MDM2
gene (7), further increases the significance
of this pathway. Therefore, the rise of p53
protein levels results in the increase in
MDM2, which in turns guides p53 to its
proteolitic degradation. In this context,
MDM2 could be responsible for the low
levels of p53 seen in cells that are not
under stress conditions (122). In some situations, MDM2 transcription is induced
later than other p53 target genes, which
favours the presence of a time period that
allows p53 to exert its activity before its
degradation takes place (165). This idea is
supported by the observation that in situations where MDM2 protein function is
cancelled, such as in MDM2-/- mice, an
upregulation of p53 takes place. This is
lethal in embryonic stages, but can be
reversed if p53 is inactivated (72). The
p14 ARF protein (murine p19ARF) modulates p53 half-life by downregulation of
the MDM2 action on p53. p14ARF action
involves the kidnapping of MDM2 in the
nucleolus, so as to prevent the MDM2p53 interaction in the nucleoplasm (160)
or even the inhibition of MDM2 activity
(67). Recent studies of the ubiquitin pathway have resulted in the identification of
two new ubiquitin ligases E3 for p53,
the constitutively photomorphogenic 1
(Cop1) and Pirh2 proteins. Thus, negative
regulation of p53 seems to occur through
at least four E3 or E3-like proteins
(COP1, Pirh2, Mdm2, Mdm4) (37) and
J. Physiol. Biochem., 60 (4), 2004
three of these, COP1, Pirh2 and Mdm2,
are p53-induced genes. This creates a very
complex and responsive control circuit
able to fine-tune the p53 response. Another new gene is the deubiquitinating
enzyme herpesvirus-associated ubiquitinspecific protease (HAUSP, also called
USP7) that in human interacts with p53
protein, and removes the ubiquitin from
ubiquitinated p53. Thus, human HAUSP
stabilizes the status of p53 and induces
p53-dependent cell growth repression and
apoptosis (91).
The role of phosphorylation in modulating p53 function becomes apparent during senescence, quiescence and after exposure to DNA damaging agents, where
steady-state phosphorylation of p53
increases at Ser15 (161). Phosphorylation
at Ser15 increases binding to CBP (85) and
p300 (39), and simultaneously decreases
binding to MDM2 (85). Two phosphoaceptor sites recently identified are: at
Thr18 in the BOX-I domain, which is
modified in human breast cancer (28),
induced during senescence (161) or transiently following ionizing radiation (133).
The second site at Ser20 is modified constitutively in normal human fibroblasts
and oxidant stresses can result in de-phosphorylation at this site (27). In addition,
an intact Ser20 residue is required for
effective p53 activity (151) and the ionizing irradiation-induced form of p53 protein is phosphorylated at Ser20 by a
Chk2-dependent pathway (137, 138).
Acetylation processes are also involved
in the p53 and MDM2 regulation. p53
acetylation not only controls its transcriptional activity but also inhibits its MDM2mediated ubiquitination, while MDM2
acetylation prevents its activity. In the latter mechanism two proteins are involved:
the CREB binding protein (CBP) and, in
a less extent, p300 (159). Phosphorylation
297
TWENTY FIVE YEARS AROUND p53
of p53 in the transactivation domain at
Ser15 activates p53 by an ATM-dependent
pathway. Adjacent phosphorylation at
Thr18 or Ser20 by CHK2 activates p53 by
stabilizing the binding of p300 to p53 (36).
CBP/p300-mediated acetyl-transferase
activity is critical to play its role in
catalysing p53 acetylation and activating
p53-mediated function during stress
response. In fact p300/CBP-mediated
acetylation of p53 is negatively regulated
by MDM2 (66). Interestingly, two additional regulators have also been identified
in the p53 acetylation pathway:
PID/MTA2 and Sir2alpha. PID/MTA2
induces p53 deacetylation by recruiting
the histone deacetylase complex 1
(HDAC1), and Sir2alpha, a NAD-dependent histone deacetylase, attenuates p53
transcriptional activity through deacetylation. Both enzymes are critical for p53dependent stress response (54).
p53 is also subjected to modification by
conjugation of SUMO-1 (22). Sumoylation of p53 by the ubiquitin-like protein,
SUMO-1/sentrin/PIC1, has been shown
to stimulate its transcriptional activation
activity.
Besides the interactions referred above
in which p53 acts as a substrate, p53 protein interacts with other cellular and viral
proteins. These include the adenovirus
E1B-55kDa (which inhibits the transcriptional activity of p53) (14, 120), the two
subunits of the TFIID complex (important for transmitting activation signals
between p53 and the initiation complex of
the RNA polymerase) (88, 96), TBP
(involved in both activation and repression of transcription) (24), the single
stranded DNA binding protein RP-A and
the p62 subunit of the transcription/repair
factor TFIIH (40), and BRCA1 a transcriptional coactivator for p53.
J. Physiol. Biochem., 60 (4), 2004
p53 functions
From a mechanistical point of view, the
p53 protein acts as a cellular stress sensor
(49) and the rise in p53 levels causes the
cell to undergo either one of two possible
fates: a) arrest in the G1 phase of the cell
cycle or, b) apoptosis or genenetically
programmed cell death (88). G1 arrest is
part of a checkpoint response whereby
cells with sustained DNA damage pause
in G1 so as to allow for DNA repair
before cell cycle progresses. By this mechanism, the propagation of potentially
oncogenic mutations is limited. The p53dependent apoptotic pathway is also
induced by DNA damage in certain cell
types, as well as in cells undergoing inappropriate proliferation.
Originally, it was postulated that p53
was not essential for a normal cell function. This idea was based in the observation that in some cell types its latent form
in physiological conditions is almost
undetectable by Western Blot or inmunocytochemiscal techniques. Furthermore,
first studies in mice with a mutant form of
p53 showed normal embryogenesis (35).
However, later studies revealed that a
small number of p53-deficient embryos
displayed developmental irregularities
such as abnormal neural tube closure (3,
116).
p53 induces cell cycle arrest in the G1/S
transition step through a mechanism
involving genes such as p21WAF1/CIP1,
retinoblastoma, E2F, PCNA or GADD45
(Fig. 1A). The p21WAF1/CIP1 gene is a
direct transcriptional target for the p53
protein. It is widely accepted that p21 is a
cyclin-dependent kinase (cdks) inhibitor
that can influence cell cycle progression
by controlling the activity of cdks. This
kinase can act on retinoblastoma tumour
suppressor protein which, in a hypophos-
298
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
Fig. 1. Functions of p53: The protein p53 is activated by several types of stress signals, such as DNA damage,
hypoxia or nucleotide depletion. As a consequence of p53 activation, either cell cycle arrest or apoptosis programmes are activated. A. Cell cycle arrest: p53 activation turns on the transcription of p21CIP1, that inhibits
cyclin-dependent kinases, causing hypophosphorylation of retinoblastoma, preventing the release of E2F and
blocking the G1-S transition. B. Apoptosis: If the cell is unable to restore DNA damage, p53 might upregulate
p53 target genes such as Bax, Noxa or PUMA which induce the release of mitochondrial apoptogenic factors.
phorylated state, associates with E2F
transcription factors to prevent the activation of genes required for progression into
S phase (41).
Once the cell cycle arrest has been
induced, p53 contributes to DNA damage
restoration processes (51) in cooperation
J. Physiol. Biochem., 60 (4), 2004
with p21WAF1/CIP1 and GADD45 pathway (that forms complexes with PCNA
protein) (52). Moreover, GADD45 regulates the transcription of the nucleotide
reductase R2 subunit necessary for DNA
synthesis and repair (145), and also regulates the transcription of genes directly
TWENTY FIVE YEARS AROUND p53
involved in the nucleotide excision repair
of DNA.
However, when the cell is unable to
restore the DNA damage, p53 induces the
activation of the apoptotic signalling pathways, with or without de novo transcription (Fig. 1B). Among the apoptotic genes
controlled by p53 are members of the Bcl2 family (including PUMA, NOXA, bax),
the p53-induced Gene family (PIG) and
death receptor genes expression (CD95,
DR5 or PIDD) (4, 93).
The Bcl-2 family includes proteins with
pro- and anti– apoptotical function.
PUMA (p53 upregulated modulator of
apoptosis) is an essential mediator of p53dependent and –independent apoptosis.
In vivo (69) PUMA is quickly induced by
p53, due to the existence of a p53 binding
sequence in PUMA first intron. PUMA
mediates p53-induced cell death through
the cytochrome c/Apaf-1-dependent
pathway, and the inhibition of the expression of its antisense reduces the apoptotic
response to p53 (114). NOXA encodes a
Bcl-2 homology 3 (BH3)-only member of
the Bcl-2 protein family. When ectopically expressed, NOXA undergoes BH3
motif-dependent localization to mitochondria and interacts with anti-apoptotic Bcl-2 family members (such as Bcl-2
and Bcl-xL), but not with pro-apoptotic
proteins like bax. Bax protein is also
induced by p53. Under normal physiological conditions Bax is located in the
cytosol, but when an apoptotical signal is
received bax translocates to the outer
mitochondrial membrane (34, 57, 70),
where it interacts with the transitional
permeability pore. Bax ablation generates
a partial or complete resistance against
p53 –mediated apoptotic stimuli (169).
The p53-inducible gene 3 (PIG3) was
originally discovered in a serial analysis of
gene expression designed to identify genes
J. Physiol. Biochem., 60 (4), 2004
299
induced by p53 before the onset of apoptosis (127). There are some evidences that
suggest that the PIG3 protein is involved
in the generation of ROS (127), which are
important downstream mediators of the
p53-dependent apoptotic response (71,
92). First, PIG3 expression precedes the
appearance of ROS in p53-induced apoptosis (127). Second, PIG3 has sequence
similarity with numerous NAD(P)H
quinone oxidoreductases (127). Third,
certain p53 mutants capable of inducing
cell cycle arrest but not apoptosis retain
the ability to activate target genes such as
p21WIF1, but not PIG3 (19, 154). However, PIG3 expression alone is insufficient to
cause apoptosis (127).
p53 regulates the membrane expression
of the death receptors such as CD95/
Fas/APO-1 and TRAIL receptor 2/
KILLER/DR5 in a transcriptional or
non- transcriptional manner (124, 163).
Nevertheless, p53 not only acts by
means of the transcriptional activation of
its target genes (136, 167), but also
through its ability to interact with other
proteins in order to modify their function
and to induce apoptosis in the presence of
transcriptional and protein synthesis
inhibitory drugs (16, 115). For example, in
the absence of other proteins, p53 directly
activates Bax to permeabilize the mitochondrion and engage the apoptotic program in tumor cells. In these cells a fraction of p53 protein localizes to the mitochondria (104, 109, 110). Indeed, p53
directly interacts with the antiapoptotic
proteins bcl-xl and bcl-2, allowing bax
and bac liberation, which causes changes
in the mitochondrial membrane permeability resulting in cell death (109).
Further, ASPP1 and ASPP2 (apoptosis
stimulating of p53 proteins 1 and 2) stimulate the apoptotic function of p53 and
also induce apoptosis independently of
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M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
p53 by binding to p63 and p73. Theses
proteins are the first common activators
of all p53 family members yet identified
(12), and selectively induce the expression
of pro-apototic genes such as Bax, PIG2
and PUMA but not mdm2 or p21.
p53 can function as a regulator of the
senescence growth arrest as well (18). p53
contributes to the reversible growth factor-dependent arrest of quiescence (G0).
Proliferating, senescent and quiescent
cells exhibit different p53 phosphorylation patterns (161), suggesting that p53
acts differently in these growth states. p53
binding and transactivating activities are
enhanced in quiescent cells and the abrogation of p53 function delays the entry
into G0 following growth factor withdrawal (65).
There is an important question that still
remains to be answered is: How does the
cell choose between cell cycle arrest and
apoptosis? Different answers to this question have been proposed: i) a preferential
gene induction model, in which p53
induces pro-apoptotic genes after death
stimulation, while pro-apoptotic genes are
not activated in p53 cell cycle arrest
induction; ii) a model in which p53 always
induces the same set of genes and the p53mediated apoptosis requires an additional
and independent signal and finally, a
model in which; iii) the transcriptional
activation of the different target genes is
not the same and this transcription
depends on the p53 activation rate. Therefore in response to DNA damage the
phosphorylation is achieved for Chk2,
ATM or ADN-PK kinases in the following residues: Ser6, Ser9, Ser15, Ser29 and
Ser37. This phosphorylation stimulates
the expression of the genes related with
restoration or cell cycle arrest, while
phosphorylation mediated by HIPK2 or
J. Physiol. Biochem., 60 (4), 2004
p38 kinases (a kinase that phosphorylates
p53 in Ser46) induces apoptotic genes.
On the other hand, p53 function could
be related to its localization. The latent
form of p53 protein presents a diffuse cell
localization and, in some cases is placed in
the cytoplasm bound to proteins such as
Parc (for p53-associated parkin-like cytoplasmic protein) (117). Once activated or
stabilized, p53 translocates to the nucleus
(81), where acts as a multi-functional transcription factor (157). This nuclear localization is essential for its tumor suppressor function. Recently, it has been shown
that p53 is located in the mitochondrion
during cell death processes.
p53 in pathological situations
Since p53 integrates numerous signals
that control cell life and death its disruption has severe consequences. Because of
the central role of p53 in the regulation of
cell cycle, mutations in the p53 gene are
the most frequently observed genetic
lesions in human cancers. Therefore, in
cancer cells p53 function is impaired
either because of mutation of the gene or
following binding of viral or cellular
oncogene-derived proteins. This causes
p53 modification and genetic alterations
accumulate rapidly leading to tumor formation.
Depending on the location of the modification alterations in p53 gene have different effects on the activity of the gene.
Thus, mutations may occur in regulatory
regions that control often, and when, the
gene is transcribed. Likewise, a mutation
in the promoter region can result in a
decrease or absence of p53 in the cell.
Mutations in one allele assert a dominantnegative effect over the remaining wildtype allele, and a pernicious effect on the
p53 function (13). Some mutations retain
TWENTY FIVE YEARS AROUND p53
wild-type activities toward a subset of
promoters, whereas others are pro-oncogenic (140). Inactivating mutations in p53
at over 200 different aa positions within
this core DNA-binding domain have been
detected in human cancers. Biochemical
and biophysical characterization of these
p53 mutant forms have led to the suggestion of the existence of at least three distinct classes of mutants with unique biochemical defects in tetramerization, conformational regulation and intrinsic folding and stability. Tumor-specific point
mutations occur in many forms of human
cancer and as many as 50% of cancers
containing a p53 mutation, affecting p53DNA interactions in the 90% resulting in
a partial or complete loss of transactivation functions. 20% of mutations are concentrated at five ‘hotspot’ codons which
are located in the DNA-binding “core”
domain. These loci presnt five clusters of
highly conserved residues found in
sequence analysis comparisons of p53
sequences from different species (158).
Inherited p53 mutations are rare. However, germ line mutations have been found
in some families with Li-Fraumeni syndrome (101, 142). This results in an inherited predisposition to a wide spectrum of
cancers including breast cancer, osteosarcomas, soft tissue sarcoma, melanoma,
adenocortical carcinomas, and leukaemias,
all of which appear at an early stage.
Today we have more evidence that biological variations between mutants may
have clinical importance and this is the
reason why individual patients respond in
a different way to cytotoxic therapy. For
example, breast cancer patients with
mutations within the DNA binding
domain appear to have increased resistance to doxorubicin but not to taxol (1,
48).
J. Physiol. Biochem., 60 (4), 2004
301
In some degenerative processes fragmentation and oxidative damage of DNA
takes place. In these situations, which
include atherosclerotic lesions (107) and
neurodegenerative disorders (73, 144,
149), p53 is involved. Recent studies using
mice lacking both p53 and apolipoprotein
E (ApoE) (p53-/-apoE-/- mice) indicate
that p53 is important for atherogenesis;
p53-/-apoE-/- mice developed larger atherosclerotic lesions as compared to apoE-/mice (55). In vitro studies show that p53
facilitates apoptosis of smooth muscle
cells isolated from human atherosclerotic
lesions (10, 11) and that p53 is involved in
oxidized LDL-induced apoptosis of
human macrophages (78).
p53 levels are high in the frontal and
temporal lobes in Alzheimer´s disease
patients (79) and immunohistochemical
studies show p53 colocalization within
apoptotic cells in cortical neurons and
white glial cells (31). Further, b -amyloid
peptide [25-35] induces cell death in neuronal cultures by mechanisms that involve
p53 (153).
p53 protein plays a role also in Parkinson’s disease. In experimental models 6hydroxydopamine (164) and MPTP (103,
147) act as an activator of p53, and the
inhibition of p53 reduces the degeneration
process in dopaminergic neurons (38,
148). Indeed, this disorder is accompanied
by widespread occurrence of intracytoplasmic Lewy bodies, formed from fibrillary a -synuclein and hyperphosphorylated neurofilament proteins (50). The
involvement of sinuclein in the neurodegenerative processes is a source of controversy. Thus, the a -sinuclein is able to
block p53 expression and its transcriptional activity, but it is not helpful against
toxicity induced by 6-OHDA (2). However, the b -synuclein reduces cell death
induced by 6-OHDA and staurosporine.
302
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ AND J. JORDÁN
In these conditions a dramatic drop in p53
levels has been observed. This probably,
by means of a post-transcriptional process
in which MDM2 and p38 might be
involved (30).
Other systems where apoptosis is
induced in a p53-dependent manner are:
the methamphetamine-induced apoptosis
in the dopaminergic neurons (60), oligodendrocytes from patients with multiple
sclerosis (162); patients with amyotrophic
lateral sclerosis (106) and excitotoxic
models (112).
p53 as pharmacological target
Pharmacologists have made some
attempts to target the p53 pathway. Major
advances have been made in the oncology
field where gene therapy represents a new
means for cancer therapy. This therapeutical approach is mainly focused on gene
replacement of defective p53 individuals.
Furthermore, it should be feasible to
restore some functional activity of p53
mutants by enhancing the stability of the
protein or by supplying additional DNA
contacts (13, 118, 140). Modified adenoviruses provide a potent vector for delivering p53 into cancer cells. The insertion
of p53 can arrest cancer cell growth and
induce apoptosis and tumor regression in
advanced cancers such as squamous cell
carcinoma of the head and neck, and nonsmall cell lung cancer. Reduction of 50%
in head and neck cancers has been
achieved with adenoviral-p53 treatments
(26). In nasopharyngeal carcinoma p53
gene therapy combined with ionizing
radiation appears to interact in a synergistic manner (90) and promising results have
been reported on prostate cancer according to preliminary clinical data. Therefore,
the p53 therapy may provide an alternative treatment to surgical radiotherapy
J. Physiol. Biochem., 60 (4), 2004
treatments for these patient population.
Although still in a developmental phase,
other pharmacological research lines propose to use mimetic peptides of the active
p53 regions, in order to obtain antitumoral effects.
A promising approach to activate p53 is
the inhibition of the p53-MDM2 interaction with compounds including nutlin,
chalcones and chlorofusin. Nutlin
behaves as a p53-MDM2 interaction
blocker, interfering in protein-protein
interactions that yield in MDM2 failure to
guide p53 degradation and causing an
increase in p53 protein levels (152). Chalcones (1,3-diphenyl-2-propen-1-ones) are
also MDM2 inhibitors that bind to a subsite of the p53 binding cleft of MDM2 and
disrupt the MDM2/p53 protein complex,
releasing p53 from both the p53/MDM2
and DNA-bound p53/MDM2 complexes
(143). Cells treated with these compounds
suffered cell cycle arrest and apoptosis.
The fungal metabolite, chlorofusin also
antagonizes the p53/MDM2 interaction
(143).
On the other hand, cancer chemopreventive selenium compounds in the form
of selenomethionine (SeMet) activate p53
by a redox mechanism that requires the
redox factor. SeMet induces sequencespecific DNA binding and transactivation
by p53 and shows promising results in the
prevention of prostate and other human
cancers (135, 141).
Finally, since p53 up-regulation has
been described as a common feature of
several degenerative disorders, p53
inhibitors can be used in these pathologies. The blockade of p53 expression by
means of the use of antisense oligonucleotide prevents neuronal cultures from
excitotoxic processes almost completely
(74, 84, 150). Recent studies identify the
tetrahydrobenzothiazole
analogue,
303
TWENTY FIVE YEARS AROUND p53
pifithrin-alpha, as a p53 inhibitor, which
is effective in protecting neuronal cells
against a variety of lethal insults and
reducing the side effects of anticancer
drugs (80, 171). Furthermore, the neuroprotective actions of lithium and antioxidant drugs like OPC-14117 in excitotoxic
processes can be due to a modulator effect
in both mRNA expression levels and p53
protein (23, 113).
Acknowledgements
This work has been supported by SAF200204721 from CICYT and SEF-Almirall Prodesfarma
to J.J. M. G-L and F.J. F-G are fellows from JCCM.
We thank Prof. E. Nava for helpful discussion and
critical reading of the manuscript.
M. GOMEZ-LAZARO, F. J. FERNANDEZ-GOMEZ y J. JORDÁN. p53: Veinticinco años acerca del mecanismo de protección del
genoma (minirrevisión). J. Physiol. Biochem.,
60 (4), 287-308, 2004.
En este año, la proteína p53, también conocida como “el guardián del genoma”, cumple
veinticinco años. Durante este periodo, el
conocimiento sobre las funciones desempeñadas por p53 ha ido cambiando desde una actividad pro-oncogénica hasta su función oncosupresora. Esta proteína se activa en respuesta
a estímulos de estrés como radiaciones gamma
y ultravioleta, hipoxia, infección vírica y daño
en el ADN, protegiendo a la célula mediante
acción sobre sus genes diana. Las moléculas
activadas por p53 inducen parada en ciclo celular y reparación del ADN con el fin de conservar el genoma o de inducir apoptosis. La regulación de las funciones de p53 está controlada
estrechamente a través de varios mecanismos
que incluyen la transcripción, traducción, estabilidad de la proteína por modificaciones posttranscripcionales y su localización subcelular.
Una de las alteraciones moleculares detectadas
con más frecuencia en los tumores humanos
son las mutaciones en p53. Mas aún, en algunos
procesos degenerativos, donde tiene lugar la
J. Physiol. Biochem., 60 (4), 2004
fragmentación y el daño oxidativo en el ADN,
la p53 está implicada. Así, se considera a la p53
como una posible diana farmacológica, ya que
su sobreexpresión induce apoptosis en células
cancerosas y el bloqueo de su expresión protege a las células contra daños letales.
Key words: Apoptosis, Necrosis, mdm2, Cáncer,
Supresor tumoral, Mutación.
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