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Review
The P53 pathway: what questions remain to be
explored?
AJ Levine*
,1
,WHu
1
and Z Feng
1
1
Institute for Advanced Study and The Cancer Institute of New Jersey,
Princeton, NJ 08540, USA
* Corresponding author: AJ Levine, Institute for Advanced Study and The
Cancer Institute of New Jersey, Princeton, NJ 08540, USA.
Fax: þ 1-609-924-7592; E-mail: alevine@ias.edu
Received 22.12.05; revised 15.2.06; accepted 22.2.06; published online 24.3.06
Edited by A Braithwaite
Abstract
The p53 pathway is composed of hundreds of genes and their
products that respond to a wide variety of stress signals.
These responses to stress include apoptosis, cellular
senescence or cell cycle arrest. In addition the p53-regulated
genes produce proteins that communicate these stress
signals to adjacent cells, prevent and repair damaged DNA
and create feedback loops that enhance or attenuate p53
activity and communicate with other signal transduction
pathways. Many questions remain to be explored in our
understanding of how this network of genes plays a role in
protection from cancers, therapy and integrating the homeo-
static mechanisms of stress management and fidelity in a cell
and organism. The goal of this chapter is to elucidate some of
those questions and suggest new directions for this area of
research.
Cell Death and Differentiation (2006) 13, 1027–1036.
doi:10.1038/sj.cdd.4401910; published online 24 March 2006
Keywords: The p53 pathway; feedback loops; apoptosis; stress
responses; DNA damage
Abbreviations: MDM-2, murine double minute 2; COP-1, coat
protein complex 1
Introduction
The goals for this chapter
The p53 protein was first described in 1979
1–4
and since that
time there have been more than 35 000 papers published
on this topic. The description of this protein and its gene
has changed from a virus-associated tumor antigen to an
oncogene to a tumor suppressor gene.
5
The major functions
of the p53 protein have been elucidated and reasonable
explanations are in hand that describe why it is an important
tumor suppressor gene in humans and animals.
6
This
progress has been reported at 12 different meetings
dedicated solely to research with the p53 protein, which have
been held every other year starting in 1981, with the most
recent meeting being held in New Zealand (2004) and the next
one will be in New York City, USA (2006). As each meeting
results in hundreds of presentations and posters they provide
the latest concepts of how the p53 protein functions and what
its many roles are in the cell and the organism. From this a
consensus forms and new questions emerge that change the
concepts, ideas and directions of this area of research. That is
just what happened at the last meeting and the goal of this
chapter is then to help guide and formulate the questions that
remain to be explored over the next years. Some of these
questions address very specific observations and are quite
narrow, but important to answer. Other questions are global
and address large conceptual issues in the field. The format of
this chapter will be to describe the functioning of the p53
pathway and at each specific step to identify those narrow and
focused questions that require the attention of experimental-
ists and need answers. By asking these questions we may
uncover contradictions in the literature, point out results that
remain unclear or bring up issues that remain to be tested and
are unresolved. As we move through the descriptions of each
step in the p53 pathway and note these focused issues they
will collectively point to holes in our understanding and lead to
the bigger issues. In the final section of this chapter the larger
or global questions can be explored in the context of
understanding the details of the p53 network and its functions.
The p53 pathway may conveniently be divided up into five
parts (Figures 1 and 2); (1) The input signals that trigger or
induce the network into a functional state. (2) The upstream
mediators that detect and interpret those signals that initiate
the functional pathway and relay the inputs to the p53 protein
or molecules that most immediately (in minutes to an hour)
regulate the concentration and activity of the p53 protein. (3)
The core set of proteins, including the p53 protein itself, which
regulates p53 activity and function. (4) The downstream
events which are composed of a set of genes and their
proteins that are regulated by the p53 protein, most commonly
by transcriptional activation but in some cases by protein–
protein interactions. (5) The cellular outputs of these down-
stream events which include cell cycle arrest, cellular
senescence or apoptosis and often result in extensive
communication with other signal transduction pathways in
the cell. Employing this format we can identify the open
questions that remain to be explored. Then we can turn our
attention to more global questions about these processes in a
larger context of the cell and organism.
The P53 Pathway
The input signals that trigger or induce the p53
pathway
The p53 protein and its signal transduction pathway are
composed of a set of genes and their protein products that are
Cell Death and Differentiation (2006) 13, 1027–1036
&
2006 Nature Publishing Group All rights reserved 1350-9047/06
$
30.00
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designed to respond to a wide variety of intrinsic and extrinsic
stress signals. These stress signals all impact upon the
cellular homeostatic mechanisms that monitor and control the
fidelity of DNA replication, chromosome segregation and cell
division.
7
Among the stresses that activate the p53 protein are
damage to the integrity of DNA in a cell. There are many
physical and chemical causes of DNA damage including
gamma or UV irradiation, alkylation of bases, DNA cross-
linking, depurination of DNA, alteration of the deoxyribose
sugar moiety, reaction with oxidative free radicals and more.
DNA
Damage
Hypoxia
rNTP
Depletion
Oncogene
Activatio
n
Spindle
Damage
Nitric
Oxide
(NO)
Activation of p53
?
?
?
Stress Signals
(input)
Mediators
Core Regulation
MDM2
p53
Nutrition
deprivation
E2F-1
P14/p19
ARF
ROS UV
γ-irradiation
REDD-1
ATR
CHK1
ATM
CHK2
AMPK
Heat/cold
shock
MDM-X
Kinases (ATM, ATR, CHK1, CHK2); Phosphatases (PP2A)
Acetyltransferases (p300, CBP, PCAF); Deacetylases (HDAC, SIRT-1)
Unbiquitin Ligases (UbcH5B/C); Deunbiquitinases (HAUSP)
Methylases (Set9); Sumoylases (PIAS-1, Ubc9, Topors)
Neddylation (NEDD8); Werner helicase (WRN); PML; HMG1
?
Figure 1 The input signals, mediators and core p53 functions of the p53 pathway. Several types of stress signals are detected by the cell and communicated to the p53
protein and its core constituents by the mediators. Several stress signals result in the degradation of the MDM-2 protein and the increase in the levels and activity of the
p53 protein. The MDM-2 protein levels are then restored via the p53-mediated transcription of the MDM-2 gene
Downstream Events of p53
Core Regulation
Inhibition of
angiogenesis
and metastasis
PAI
BAI-1
KAI
TSP1
Maspin
GD-AiF
A
p
o
p
tosis
IGF-BP3
PAG608
Siah-1
Scotin
PERP
Fas
PIDD
Killer/DR5
p53AIP
PIGs
Noxa
PUMA
Bax
Apaf-1
Cell cycle
arrest
G1-S
p21
14-3-3-σ
Reprimo
Gadd45
B99
G2-M
?
Cellular
senescenc
e
DNA Repair
and damage
prevention
p48
p53R2
PTEN
TSC2
IGF-BP3
Inhibition
of IGF-1
/mTOR
pathway
Siah-1
Wip1
Cyclin G
Cop-1
PIRH-2
deltaNp73
P53
negative
feedback
TSAP6
Exosome
mediated
secretion
Sestrins
Gadd45
MDM2
MDM2
p53
E2F-1
P14/p19
ARF
MDM-X
Cdc2
Cyclin B
Cdc25C
Cyclin E
Cdk2
Casp 8
Bid
Cyto C
Casp 9
Casp 3
Response
(output)
Figure 2 The core p53 functions, the downstream genes regulated by p53 and the outputs of the p53 pathway. The p53 protein is employed as a transcriptional
activator of p53-regulated genes. This results in three major outputs; cell cycle arrest, cellular senescence or apoptosis. Other p53-regulated gene functions
communicate with adjacent cells, repair the damaged DNA or set up positive and negative feedback loops that enhance or attenuate the functions of the p53 protein and
integrate these stress responses with other signal transduction pathways
The P53 pathway
AJ Levine et al
1028
Cell Death and Differentiation
Each of these types of DNA damage is different and each is
detected by a different set of proteins and then repaired by
different enzymes or activities that reverse the damage in
diverse ways. There are multiple DNA damage detection and
repair systems in the cell but every type of DNA damage
is reported to the p53 protein and its pathway.
8–10
The
duplication processes of DNA often make mistakes (i.e.
mismatch errors) that are in the main edited and repaired, but
when lesions remain they too can be detected by p53. In all of
these cases the p53 pathway functions to respond to errors (a
check on the fidelity of these processes) and eliminates those
cells with such mistakes. As cells divide and the telomeres
of chromosomes get shorter they reach a critical size that
can result in an enhanced rate of translocations or abnormal
recombination events. The presence of these shorter telo-
meres in a cell are reported to the p53 protein resulting in a
p53 response which in turn limits abnormal chromosome
translocations. In addition to DNA damage several cellular
deprivations activate the p53 response such as hypoxia,
glucose starvation or the depletion of ribonucleoside triphos-
phate pools in the cell. When ribosome biogenesis is stopped
or falls below a critical level the p53 protein is alerted and
activated. Agents that damage the cell spindle and result in
faulty chromosome segregation activate p53. Both heat and
cold shock as well as the altered or denatured protein
response in cells communicate with p53 by activating it. Nitric
oxide, which is often associated with infections and inflamma-
tion activate the p53 protein and its response. All of these
stresses will result in a loss of fidelity in the cellular duplication
process and as such their communication with the p53
pathway acts as a check point to prevent abnormal clones
of cells from arising. The mutational activation of some
oncogenes also result in the sensitization of the p53 response
and can often change the downstream output of the p53
pathway from cell cycle arrest to apoptosis. This very large
number of diverse cellular stress signals that all feed into a
central and single node to monitor and respond to those
stresses may be thought of as a poor design of a system that is
too vulnerable to the loss of a single central node.
7
Why don’t
different stress signals act upon different protein checkpoints?
Why is the pathway not duplicated or backed up so as to make
it more robust and less vulnerable to the mutational damage of
the p53 gene? The observation that demonstrates the unique
and central function of the p53 protein in this stress network is
that the p53 gene is mutated about 50% of the time in a wide
variety of cancers.
11
In a number of other cancers additional
genes in the network that encode proteins that act upon the
p53 functions are mutated. Thus, most cancers appear to
select for a loss of function of the p53 pathway.
12
The most
common answer to why evolution has designed the network to
have a single central node that receives inputs from a wide
variety of stress signals is that one entity or protein can act as
the most efficient integrator of information about the different
types of stress that act upon the cell. Multiple proteins
receiving multiple input signals would have to have a much
more complicated communication system to integrate infor-
mation about the environmental stresses. Because all of
these stresses lead to a lack of fidelity in the duplication
process, and that could lead to disease, the p53 protein is a
central player in the go-no go decision of a cell to grow or
reproduce. The question remains; is this explanation a correct
interpretation of the network architecture or is there a lot more
to learn so as to answer this enigma?
What other stress signals can induce a p53 response?
What more remains to be elucidated in the type or nature of
the input signals? Can psychological stress, in the form of
depression or nervous system activity activate the p53
protein? Do some hormonal changes result in the activation
of the p53 protein? What nutritional changes, in addition to
glucose deprivation,
13,14
can induce a p53 response? It will be
important to fill in those inputs to the p53 pathway over the
next few years. In some cases (i.e. psychological stress) it will
be a real challenge to design experiments to prove these
points. Most of the experiments in the past that test for the
activation of the p53 protein and therefore define an input
signal for this pathway have been carried out in cell culture. In
order to better define environmental stress we will need
to carry out experiments (like testing psychological stress)
in the whole organism taking into account cell type and
tissue specificity as well as the variable of time (age) and
developmental stage.
Orthologues of the p53 gene have been identified in worms,
flies, mice and humans and it will clearly be instructive to
elucidate a comparative genomic and network analysis and
follow changes in p53 function over evolutionary time scales.
Similarly two homologues of the p53 gene, p63 and p73, have
been identified
15,16
and it will be important to complete the
characterizations of their functions, their possible role in
diseases and their impact upon the p53 gene and its protein.
17
Do p63 or p73 respond to stress signals and if so which ones?
P73 is phosphorylated by the Abl kinase after DNA damage
and p73 can induce cellular apoptosis. There is a great deal to
be learned about these functions in the cell and organism.
The upstream mediators of the P53 response
In response to an input stress signal the p53 protein is
activated. We can define activation experimentally as an
increase in the concentration of the p53 protein and an
increased activity of a p53 protein for the transcription of a set
of genes that have a p53 DNA response element
18
and are
transcriptionally regulated by the p53 protein. The levels of the
p53 protein are predominantly regulated by it’s proteolytic turn
over. The p53 protein has a short half-life of 6–20 min in
several cell types and the ubiquitin ligase that confers this
short half-life is the MDM-2 protein. Recently, two other
ubiquitin ligases were shown to act upon the p53 protein,
COP-1 and PIRH-2.
19,20
Why is this redundancy employed?
What are the roles of these enzymes for the many diverse
stress inputs, in different tissue or cell types or at the different
times in development of an organism? After some types of
DNA damage (gamma radiation) the MDM-2 protein is auto-
poly-ubiquitinated resulting in its degradation and an asso-
ciated increase in p53 levels and activity.
21
While this
observation helps to explain why the p53 protein has a longer
half-life after some types of DNA damage, this mechanism is
not observed in all types of DNA damage or stress signals.
Indeed given the large number of stress signals employed as
an input to the pathway we know almost nothing about how
these diverse inputs are communicated to the p53 protein.
The P53 pathway
AJ Levine et al
1029
Cell Death and Differentiation
That is a big challenge for future experiments. We do know
that the p53 and MDM-2 proteins are extensively modified
after a stress signal.
22
The p53 protein is phosphorylated on a
large number of serine and threonine residues by many
different protein kinases. It is acetylated by histone acetyl-
transferases, methylated by methylases, ubiquitinated, sum-
molated and neddylated (at epsilon amino groups of lysine at
the carboxy-terminus of the p53 protein).
23–25
In some cases it
is clear that this is part of the process that mediates the
response from DNA damage to the p53 protein. For example,
gamma radiation activates the ATM protein kinase that then
phosphorylates MDM-2 and p53 (probably through a CHK
kinase) as well as participating in the DNA repair process. In
spite of these clear observations, mutations in many of the
p53 protein serine and threonine residues that then block
p53 phosphorylation events still result in fairly normal p53
activation and function. Similarly, the use of Nutlin (a drug
that blocks p53-MDM-2 binding) activates p53 normally with
no phosphorylation events.
26
Changes of the lysines in p53
protein, that are normally acetylated or modified by various
peptides, to arginine residues that cannot be modified resulted
in normal p53 responses in mice with these mutant proteins.
These types of experiments indicate that the protein
modifications of the p53 protein after a stress input are not
essential for the activation of the p53 protein (or are heavily
backed up by other functions not eliminated in these
experiments). This brings up the question; what are the
functions, if any, of the protein modifications in p53 or the
MDM-2 protein after exposure to a stress? Most people
believe there is a functional role for these modifications. First
of all the extent and nature of these protein alterations of the
p53 protein differs with different types of inputs. Thus, these
protein modifications form a chemical code that could inform
the p53 protein and the cell about the type of stress that is
occurring. Second the p53-regulated transcriptional output
after different stresses is different, and these protein
modifications could well dictate which p53-responsive genes
are transcribed by the cell based upon the specific protein
modifications of the p53 protein. Do p53 protein modifications
determine the genes which are transcriptionally regulated by
that p53 protein? These ideas need to be tested. P53 protein
interactions with other cellular proteins (which may be initiated
by protein modifications) impact upon gene selection and
the nature of the downstream events.
27,28
Just what protein
modifications of p53 and MDM-2 accomplish in a cell remains
a big question? In particular, the protein modifications and
protein–protein interactions (with p19 ARF, cyclin G, etc.) of
MDM-2 remain to be explored in more detail.
One of the most interesting methods of activating p53
results from the mutational inactivation of a tumor suppressor
gene like the retinoblastoma protein (which activates the
E2F-1 transcription factor) or the APC tumor suppressor
(which activates the beta catinin-TCF4 transcription factor) or
the mutational activation of oncogenes like myc or Ras. The
activities of the transcription factors that result from mutations
in these genes can transcribe the ARF gene and the ARF
protein then binds to the MDM-2 protein and inhibits its activity
as a ubiquitin ligase. This raises the p53 levels in a cell and
helps to bring about a p53 response or output that protects the
organism from the development of cancers.
29,30
This is an
excellent example of how diverse signal transduction path-
ways in a cell communicate and create feedback loops that
contribute to cellular homeostasis. Just who the other players
or modifiers of this circuit are remains to be explored? There
are some suggestions that ARF has additional functions and
may be regulated in several ways, and these questions
remain a high priority for the field.
The core control of P53
Once activated the p53 protein transcribes a number of
genes. Among these is the MDM-2 gene (and the COP-1 and
PIRH-2 genes) which is the major negative regulator of p53 in
the cell.
12
The transcriptional regulation of MDM-2 by p53 is a
slow step (hours) but once the MDM-2 protein is produced it
binds to p53 (inhibiting its activity as a transcription factor) and
ubiquitinates it enhancing its proteolytic breakdown. This
occurs as a rapid step. This auto-regulatory loop produces an
MDM-2-p53 oscillator composed of changing protein levels of
p53 and MDM-2, out of phase with one another, in the cell.
31,32
When p53 levels rise this increases MDM-2 levels which in
turn lower p53 levels, resulting in less MDM-2, etc. These
oscillations have been observed in vivo to be variable from
cell to cell but they can last a long time and the number of
oscillations is roughly proportional to the input dose of
radiation.
32
Does transcriptional activation of the downstream
genes depend upon this oscillator or does it optimize the
output of the pathway? Just what the functions of such
oscillations are, if any, and the factors that can modify these
oscillations remain to be determined? Do COP-1 and PIRH-2
participate in this process? The reason that the field has
focused upon MDM-2 as the major regulator of p53 is that it
was found first
33,34
and the gene knockout of MDM-2 in mice is
lethal early in development, just after implantation.
35
This
could be due to an hypoxia response of the p53 protein in the
absence of MDM-2, so that p53 activity is uncontrolled. This
idea needs to be tested. The double knockout mouse, with no
MDM-2 or p53 genes, is viable proving that MDM-2 acts to
block p53-mediated cell death early in development (is this
due to hypoxia?). This also demonstrates that PIRH-2 and
COP-1 do not back up MDM-2, at least early in development.
Now similar experiments should be done with COP-1 and
PIRH-2 knockouts.
The gene products that regulate MDM-2 (and p53) levels
and activities will need to be better characterized. The MDM-X
(or MDM-4) gene was identified because it is related to the
MDM-2 gene in its DNA and protein sequence. A knockout of
the MDM-X gene in mice is lethal and a MDM-X and p53
double knockout is viable.
36
Thus, MDM-X is as important as
MDM-2 in regulating p53 in vivo. It now appears that MDM-X
can negatively regulate p53 directly and positively regulates
MDM-2. After a stress signal, the poly-ubiquitination of MDM-
2 results in the degradation of MDM-X and MDM-2, increasing
p53 levels and activity. Just how this is accomplished (via
protein modification of MDM-X or 2, or protein–protein
interaction) remains to be studied. The activities that modify
MDM-X and MDM-2 (cyclin G-PP2A, ARF and its activators
and HAUSP) act only on MDM-2 and remain to be explored
in more detail. High levels of p53 appear to repress the
transcription of the ARF gene and the p53 knockout mouse
The P53 pathway
AJ Levine et al
1030
Cell Death and Differentiation
(no p53 protein) has much higher levels of ARF mRNA and
protein than a wild-type mouse. How the p53 transcription
factor acts to repress transcription remains a mystery and is in
need of study.
Several studies have suggested additional levels of the
control of the p53 protein, at the transcriptional and at
the translational level, which should now form the basis for
future research. In addition, the p53 and MDM2 proteins can
shuttle between the nucleus and the cytoplasm which
undoubtedly changes their activity and regulation.
37,38
Just
what factors control this cellular localization and therefore can
impact upon p53 function remain to be explored. The AKT-1
kinase can phosphorylate the MDM-2 protein increasing its
activity and sending it into the nucleus of the cell.
39,40
Cyclin
G, a p53-regulated gene product, combines with the catalytic
subunit of the PP2A phosphatase to remove a phosphate
residue from the MDM-2 protein and increase its activity.
41
These two modifiers of the MDM-2 protein form feedback
loops that connect the core p53 activity to other signal
transduction pathways as well as regulate the core functions
of the network. Indeed there are at least 10 negative or
positive feedback loops that start with a p53-regulated gene
product and result in increasing or decreasing the activity of
p53.
12
These feedback loops connect the core p53 activities
to other signal transduction pathways. When a positive
feedback loop is activated it results in apoptosis and cell
death, a negative feedback loop attenuates the p53 pathway.
Do these loops act in all cell types? How are they regulated or
triggered? What is the impact of the activation of another
signal transduction pathway upon the core p53 regulation?
A good example of this is the starvation of a cell for glucose.
This engages the mTOR pathway. The absence of glucose
activates the LKB kinase and the AMP kinase. The AMP
kinase phosphorylates the TSC-2 protein in a TSC-1/2
complex. TSC-1/2 is a GAP that inactivates a G-protein,
RHEB, and RHEB activity controls the kinase activity of
mTOR. Thus, glucose starvation inactivates mTOR which
leads to the activation of autophagy, a catabolic process
that breaks down proteins, fats and carbohydrates into their
momomeric components, supplying endogenous nutrients to
the starved cell. While this proceeds the AMP-kinase also
phosphorylates serine-15 on the p53 protein.
13,14
In normal
cells this does not activate the p53 protein but in transformed
cells this is a proapoptotic event. P53 serine-15 phosphory-
lation is a precursor for other phosphorylation events for the
p53 protein.
22
When glucose becomes available and mTOR
reactivates, mTOR activates a subunit of PP2A which can
then remove the phosphate residue from p53 serine-15.
42
This restores p53 to its original state and attenuates the
feedback loop.
As there are two distinct transcriptional start sites for the
p53 mRNA and several alternative splicing patterns of this
mRNA there are nine different isoforms of the p53 protein that
have been detected. The amino-terminus of the p53 protein
has two distinct transcriptional activator regions of the protein
and one or both can be present. The carboxy-terminus of the
p53 protein has a basic domain which can be spliced out or
replaced by another amino-acid sequence. All combinations
of these alternative protein isoforms have been observed
43
but many questions remain. Are these forms found preferen-
tially in different cell or tissue types or at different stages of
development? What are the functions of each alternative
domain and the resultant protein? Does a protein with its
DNA-binding domain intact but without a transactivation
domain act as a repressor? How do the three alternative
forms of the carboxy-terminus alter the functions of the p53
protein? Recently, one of these isoforms has been shown to
have a distinct and critical function in the development of the
gut of zebra fish.
44
There is now clearly more to learn.
The downstream events in the p53 pathway
Once p53 is activated in response to a stress signal it
gains the ability (and increased concentration) to bind to
p53-responsive DNA sequence elements in the genome.
The consensus DNA sequence for p53 binding is
RRRCWWGYYY, where R is a purine, W is A or T and Y is
a pyrimidine. A p53-responsive element is composed of two
of these 10 base paired sequences, separated by a spacer of
0–21 base pairs, and the sequences are often located 5
0
to the
gene or in the first or second intron of the gene regulated by
p53.
45
It is a challenge to identify all of the p53-responsive
sequences in a genome because degenerate sequences
also function in a p53-dependent fashion. An algorithm has
been described
18
that has successfully identified novel p53-
responsive genes.
13,46
It has become clear that different types
of stress signals as inputs result in different genes being
transcribed under p53 control.
47
In addition stress signals
received by different cell or tissue types also produce different
transcriptional programs regulated by the p53 protein. We
do not understand what mediates these different regulatory
responses. Both p53 protein modifications and protein–
protein interactions could play a role as well as differences
in the DNA sequences of response elements. In addition,
there are large differences in both the amounts of mRNA and
proteins produced by different p53-regulated genes and in the
kinetics of synthesis after a p53-activation event. Why this is
so remains unclear. There appear to be some p53-regulated
genes that are transcribed in response to many different types
of stress signals and in all tissues responding to the stress
(p21, cyclin G, MDM-2, GADD-45) and others that are either
stress- or tissue specific (PTEN, TSC-2). What regulates
these differences remains unclear? The functions of the p53-
response genes fall into several categories. A set of genes are
clearly involved in cell cycle arrest (p21, 14-3-3 sigma, GADD-
45). A second set of p53-regulated genes are involved in
apoptosis. These can be divided into the intrinsic and extrinsic
apoptotic pathways. In the extrinsic pathway p53 regulates
Fas production (a secreted protein), as well as killer/DR5, the
trail receptor and a membrane protein. These proteins, along
with PIDD, activate caspase 8 and Bid to release cytochrome
c which acts with APAF-1 (a p53-regulated gene) to activate
caspase 9 and then 3 resulting in apoptosis. The intrinsic
pathway is populated with many p53-regulated genes of which
bax, noxa and puma may work in different cell types to
promote cytochrome c release. Several other p53-regulated
genes have been implicated in the enhancement of apoptosis
(Perp, scotin, some PIGS, p53 AIP) but their mechanism of
action remain to be elucidated. Elucidating the details of how
p53 initiates apoptosis employing its transcriptional program
The P53 pathway
AJ Levine et al
1031
Cell Death and Differentiation
is a high priority for the field. More recently several groups
48,49
have provided good evidence that the p53 protein itself can
move out of the nucleus and act upon the mitochondria or
proteins in the mitochondria (BCl-2, BCL-XL) to promote
cytochome c release and apoptosis. A large challenge
remains to prove these mechanisms are functional in an
animal and augment or replace, under some circumstances,
the transcriptional-mediated apoptosis. This is presently a
confusing and challenging area of research and needs to be
resolved with a consensus being formed in the field. The third
output of the p53 response is senescence of cells. There are
some suggestions that senescence is as important as
apoptosis in mediating the tumor suppressor functions of
p53. In spite of this we have no idea which p53-regulated
genes contribute to senescence and what gene products
populate the pathway to cellular senescence. As normal cells
divide and age in culture and telomeres shorten, p53 levels
rise, but what transcriptional program this triggers remains
to be elucidated? Mice with an overactive p53 protein, where
the threshold for stress responses is lowered, die prematurely
and have compromised stem cell capabilities (senescence of
the stem cells).
50
These mice are more resistant to developing
cancers initiated with carcinogens. These mice also die at
younger ages than wild-type mice with a normal p53 protein.
We need to design new experimental approaches to explore
these questions of cancer resistance and longevity in more
detail. These experiments also introduce the question; what
are the functions, if any, of the p53 protein in regulating
longevity in animals and cells? The next few years will see
these questions addressed in worms, flies, mice and even
humans.
While cell cycle arrest, apoptosis and senescence are
traditionally thought of as the major outputs of the p53
pathway several other p53-responsive genes are beginning
to define additional functions of the p53 pathway. The p48
protein and p53R2 subunit of ribonucleotide reductase are
p53-responsive genes that aid in DNA repair. The sestrins are
a set of p53-regulated genes that counter the presence of
reactive oxygen species in the cell.
51
Clearly some p53
pathway functions help to protect the cell from exogenous or
endogenous stresses while others enhance the cellular repair
process. A second p53-regulated function is to communicate
with cells in its environment that a cell has DNA damage or
senses a stress signal. A number of p53-responsive or p53-
regulated genes produce secreted proteins. These proteins
fall into several functional categories. The IGF-BP3 binds the
IGF-1 hormone and prevents it from activating a growth
response signal transduction pathway (IGF-1, IGF-1R, PI3 K,
PDK, AKT-1, forkhead transcription factors).
12,52
In this case
p53 is negatively regulating cell growth, mitogen signaling and
preventing cell division in surrounding cells after a stress
signal. The p53-regulated and -secreted genes PAI and
maspin are protease inhibitors that result in alterations to the
extracellular matrix and cell surface.
53,54
Similarly the p53-
regulated thrombospondin gene produces a secreted protein
that alters the cellular matrix and is anti-angiogenic.
55
Secreted proteins permit communication between cells. More
recently the p53-regulated TSAP-6 gene was shown to
enhance the rate of exosome production from cells under-
going a p53 response to stress.
56
Exosomes also can
communicate with other cells both in the immune system
and cells in its own vicinity. Cells in stress will want to
coordinate their responses with the surrounding cell layer
in vivo and this is a new field opening up to future research
efforts. Finally, a large number of p53-responsive genes (p21,
WIP-1, SIAH-1, PTEN, TSC-2, IGF-BP-3, cyclin G, p73delta
N, MDM-2, COP-1 and PIRH-2) initiate positive or negative
feedback loops with the p53 protein and the core gene
products of the pathway.
12
This results in the integration of a
stress signal with other pathways in the cell that regulate cell
growth, division or cell death. These p53-regulated gene
products either reinforce or inhibit the p53 response and
network. This then results in a coordination of cellular
processes that need to be explored in future experiments.
The cellular outputs of the downstream events
This brief review has discussed at least six different outputs
of the p53 pathway which fall into two distinct categories,
primary response and secondary responses. The three
primary responses to a stress input signal by the p53 pathway
are; either cell cycle arrest, cellular senescence or apoptosis.
The cell cycle arrest may be reversible or in other cases
irreversible (which might be related to senescence). At this
time we do not know all of the gene products (p53 regulated or
not) that bring about cell cycle arrest. While p21 clearly plays
some role in a G-1 arrest, it is not the entire story (based upon
knockout experiments).
57,58
There is some controversy about
whether p53 mediates a G-2 arrest, when it does so and how?
There are several examples of a role for p53 in blocking the
reinitiation of a second S-phase in cells that can not enter
cytokinesis because of treatments with spindle poisons.
59
This is one of the ways in which p53 prevents karyotypic
instability in cells, which is a major function or output of the p53
pathway. Whether the p53 pathway has a method to ‘count’
the number of chromosomes that segregate into a daughter
cell, and if this number is not correct it kills the cell, or it
monitors segregation in another fashion remains an important
line of enquiry? There is good evidence that the p53 protein
and its functions regulate or monitor the number of centro-
somes produced at each cell cycle. The p53 knockout mouse
produces cells with abnormal numbers of centrosomes.
60
The way in which the p53 pathway brings about cellular
senescence is completely unknown. Just which p53-regulated
genes initiate this state remain unclear. Indeed, there are very
few reliable biomarkers for the senescent state and we do
not know what mediates this process of irreversible division
leaving the cell in a metabolically active state? There are good
reasons to suspect that senescence occurs in vivo as well
as in cell culture (cells from older animals undergo fewer
divisions in culture) and plays a role in cancer responses.
As such this is clearly an important line of research for the
future. It appears that the majority of the p53-regulated genes
play a role in promoting apoptosis. This pathway seems to
contain examples of redundant functions so experiments
where genes are knocked out are less informative. It also
appears that tissue specific p53-regulated gene products
play a role here. The transcriptional program that initiates
apoptosis triggers both the intrinsic and extrinsic apoptotic
pathways. The proposed p53 transcription-independent
The P53 pathway
AJ Levine et al
1032
Cell Death and Differentiation
pathway to apoptosis needs additional evidence and several
lines of proof that it is employed in vivo, and not only in cell
culture under abnormal circumstances. It is an important
potential mechanism and proof of its validity should be held to a
high standard. An important question that remains in examining
the output of the p53 pathway is how a cell which has received a
stress signal chooses which of these three outputs (apoptosis,
cell cycle arrest or cellular senescence) to implement? We do
know that normal fibroblasts taken from an animal and
irradiated undergo G-1 arrest while their transformed counter-
parts undergo apoptosis. Normal irradiated T cells undergo
apoptosis, so both the state of cellular differentiation and the
input of other signal transduction pathways influence this
decision. In other experimental studies, hormones or other
mediators which turn on signal transduction pathways altered
the p53 output response. Now we need to understand the rules
which regulate the p53-mediated output.
The secondary outputs of the p53 pathway come from p53-
regulated gene products that either (1) prevent DNA damage
(sestrins) or aid in DNA repair, (2) mediate communication
between the cell under stress and its neighbors, the
extracellular matrix shared by these cells or even other more
distant cells in the body, and (3) create intracellular or
extracellular p53 feedback loops that modulate p53 activity
and the pathway and simultaneously communicate with other
networks in the cell.
12
Prevention of DNA damage by
removing free radicals or other chemical insults needs to be
explored in more detail. This concept brings up the question
of whether the p53 pathway is solely inductive, responding to
stress and then preventing more damage, or is also active
before a stress signal and is therefore truly preventing the
initiation of DNA damage? The p53 functions that aid in DNA
repair suggest that the cell cycle arrest that is preventing
errors is reversible and the cell lives to divide again. The
nature of the communication between a cell undergoing a p53
response (apoptosis, senescence or cell cycle arrest) and its
neighbors is a poorly explored area of research. The soluble
secreted p53-responsive proteins can change the extracel-
lular matrix (maspin, PAI-1), block hormonal signaling (IGF-
BP3) to another pathway, and alter angiogenesis in the vicinity
of the alarmed cells (thrombospondin). In addition p53
activation can enhance the rate of exosome production by
the damaged cell by inducing the p53-regulated TSAP-6
gene.
56,61
Exosomes can communicate with adjacent cells
by cell fusion and with T cells by presenting antigens to the
immune system. Could p53-mediated apoptosis enhance
immunization of the host in this fashion? The activation of p53
(and its inactivation) was first detected in virus-infected
cells.
1,2
Could the p53 pathway be playing an important role
in infectious diseases and immunity? Is it possible that a
cancer cell undergoing a p53 response is also immunizing its
host to cancer antigens and preventing or slowing the disease
in this fashion? If this was the case then cancer cells with
mutant p53 would fail to immunize their host and selection for
a mutant p53 could be due to an immunoselective process.
These are provocative ideas that will need to be tested in the
future. The negative- and positive-feedback loops that are
initiated by p53-regulated genes and then act upon p53
activity and functions have been reviewed elsewhere
12
and in
this chapter. They serve two functions; (1) to modulate up or
down p53 activity; and (2) to communicate with other signal
transduction pathways in the cell. For example, after a
commitment to apoptosis, a PTEN-mediated feedback loop
modulates up p53 activity (a positive feedback) and shuts
down the IGF-1 and mTOR pathways (growth and cell division
pathways). This activates other processes such as autophagy
which provides nutrients for adjacent cells and exosome
production. Both autophagy and exosome production are
functions of the lysosomal compartment and pathway. Could
this indicate some additional fundamental roles of this cellular
compartment in the p53 pathway and its control of cellular
processes?
Questions about the p53 pathway in a larger
context
Several observations have demonstrated that the p53 gene
and its protein play a central role in the origins of cancers: (1)
the role of the p53 protein and its gene in viral and cellular
transformation, (2) the cancers in the p53 gene knockout
mice, (3) cancers observed with the Li-Fraumeni families and
(4) the high incidence of p53 gene mutations in many human
cancers. However, just what does p53 do to prevent cancers
from arising in murine and human populations? The presence
of hundreds of genes in the p53 pathway and genetic
polymorphisms in those genes suggests that individuals
may have more or less efficient p53 pathways. The p53 gene
itself has a codon 72 polymorphism (proline–arginine change)
and the arginine allele appears to be more efficient in
apoptosis in cell culture.
62
The observation that the ratio of
proline to arginine alleles in individuals changes as the latitude
changes from the equator to the north pole already suggests
that selection is playing a role upon these alleles.
63
There are
several studies that suggest that one or the other allele could
influence the occurrence or progression of cancer.
64,65
At
present however the results are confusing and a better
understanding of the role of these two alleles in the pathway
will be important. Recently, a polymorphism in the promoter of
the human MDM-2 gene has been identified (SNP309).
66
The
minor allele enhances the levels of the MDM-2 protein in cells,
lowers the levels of p53 after a stress response and lowers the
frequency of apoptosis in these cells. This same allele lowers
the age of onset of the development of several types of
cancer.
66
More recently, a separate study demonstrated that
the minor allele of SNP309 increased the frequency of
esophageal cancer in individuals and that the codon 72
polymorphism further increased the odds ratio of developing
this cancer (in an additive fashion) and that smoking further
increased this odds ratio (in a more than additive fashion).
67
Clearly this is one way to relate the participation of the p53
pathway to the development of cancers, the progression
of cancers and the treatment of cancers. There will be more
p53 pathway polymorphisms, more environmental variables
tested and more studies that will uncover the functions of the
p53 network in humans.
Several lines of evidence suggest that the p53 pathway
contains a sexual dimorphism that distinguishes gender
responses. The p53 gene knockout mice have a sex ratio
distortion, the severity of which depends upon the genetic
The P53 pathway
AJ Levine et al
1033
Cell Death and Differentiation
background of the inbred strain.
68–70
This is due to the fact
that many female mice are born with an exencephalic
condition, where the brain case never closes and the brain
is outside of the head.
69
This is seen only in females. Whether
the cranium fails to close or be developed or the brain is too
large (failure of neuronal apoptosis in development?) remains
unclear and will be important to explore. The genetic back-
ground determines the penetrance of this phenotype so some
female mice are born. Female heterozygous p53 knockout
mice develop more osteogenic sarcomas than do their
male counterparts indicating that sexual dimorphism is
operative.
71,72
In humans with Li-Fraumeni Syndrome (also
heterozygotes) the females develop cancers at a higher
frequency and earlier in life
73
and this is the case even if breast
and prostate cancers are not considered. The MDM-2 gene,
can under certain circumstances be regulated by the estrogen
receptor.
74
The IGF-1 pathway (which interfaces with the
p53 pathway via the activation of MDM-2 by AKT-1) is also
influenced by sexual hormones and sensory inputs.
39,40
The
SIR-2 or SIRT-1 gene product, an NAD-dependent histone
deacetylase, acts to remove acetyl groups from both the
forkhead and the p53 proteins and can modulate the activity
of the IGF-1 pathway.
75,76
Certain types of mutations in the
IGF-1 pathway and SIR2/SIRT-1 play a central role in
extending the longevity of an organism (yeast, worms, flies
and mice).
77,78
Will the p53 pathway play a role in determining
longevity in worms, flies, mice and humans? Longevity is also
sexually dimorphic in some of these species. In mice (2–3
years life span) and dogs (8–16 years life span) and humans
(75 years life span) most cancers arise in the last quarter
of their life spans rather than a specific number of years
after birth. In fact the frequency of cancer in these species
increases exponentially (over 1- to 10 000-fold) during the last
quarter of their life span. Thus evolutionary selection has fixed
in each of these species the age of sexual maturity, the age of
producing offspring, the age of developing the majority of
cancers in the population, and in relation to that, the longevity
of the species (there would be no species if you died before
reproducing). The longevity of a species may be set in part by
alterations in the IGF-1 pathway and the age of onset of
cancers by tumor suppressor genes like p53 and their
pathways. The reproductive age is fixed by a related and
interactive set of genes and their networks. Future experi-
ments will explore these variables and their relationships.
In this evolutionary context, how did a gene function like p53
arise and what does (did) it do for the animal? Worms and flies
are born with a body plan that is largely (except for the germ
line) postmitotic. No more divisions occur in an adult animal.
In these cases the major job of p53 is the surveillance of
the germ line for environmental insults that would result in
poor offspring. The body plan of adult vertebrates, however,
contains many tissues that continue to divide throughout life
and renew themselves. Here p53 has been employed in the
surveillance of somatic cells to prevent cancers from arising in
the organism. The exploration of the functions of the p53
pathway throughout evolution and how it has adapted to novel
situations and organisms will be a profitable avenue of
research.
We now know enough about the p53 pathway to move in
some new directions. One of the input signals that activates
p53 is nitric oxide which is produced and employed for
signaling after inflammatory responses. Indeed inflammatory
syndromes have been shown to cause cells in the vicinity of
lymphocyte invasions to undergo a p53 response.
79,80
Communication may occur in the other direction with p53-
regulated exosomes signaling to T cells and immunizing the
organism with parts of damaged cells. These ideas suggest
the exploration of whether p53 plays a role in the recovery
from infectious diseases, inflammation or the initiation of
autoimmunity or the establishment of tolerance? The apopto-
tic functions of the p53 pathway may normally be employed by
the immune system for optimal function. These same
apoptotic pathways could play a role in neurodegenerative
diseases. There is a growing body of evidence for a p53-
regulated program cell death in neurons, glial cells or
Schwann cells. What role does the p53 pathway and its
response to environmental insults play in the origins or
propagation of neurodegenerative diseases? With an increas-
ing age of the host does the p53 pathway become more active
(is there a pathological condition that makes it more active?)
or does it decay in its efficiency? Could it be the case that the
large and rapid increase in cancers in a population during the
last quarter of our life span is due to accumulated mutations
in oncogenes and tumor suppressor genes in a cell and a
declining efficiency of the p53 response which increases the
rate of these fixed mutations in that cell? These are more
complicated questions to answer and more difficult experi-
ments to perform but the answers will open up new avenues of
research and understanding.
One thing we can be sure of, some of the answers to the
questions posed in this chapter will be answered. Other
questions will be rephrased or changed completely before it is
productive to test these ideas. However, all of these questions
will be discussed, probed and eventually explored. We have
uncovered and explored a process central to life, how a cell
responds to stress or perturbations in its environment.
Understanding these homeostatic mechanisms central to all
life processes ensures a productive future of questioning.
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The P53 pathway
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