p53-MEDIATED GROWTH ARREST

& APOPTOSIS
p53, also known as TP53 or tumor protein is a gene that codes for a protein that
regulates the cell cycle and hence functions as tumor suppression. It is very important
for cells in multicellular organisms to suppress cancer. P53 has been described as "the
guardian of the genome", referring to its role in conserving stability by preventing
genome mutation. The name is due to its molecular mass: it is in the 53 kilodalton
fraction of cell proteins. The p53 protein is a transcription factor which plays a central
role in the regulation of cell cycle arrest and apoptosis following DNA damage.
The p53 gene contains eleven exons which encode for a 2.8 kb mRNA, translated
into a 53kD protein. Exon 1 is always non-coding. In human p53, a very large intron of 10
kb with unknown biological function is located between exon 1 and exon 2. The human
p53 gene is located on the seventeenth chromosome.

The exon are numbered in the diagram above. The pink region denotes the UTR, the blue region denotes
the coding region and the grey region denotes the internal exons within the introns.

p53 protein is a phosphoprotein made of 393 amino acids. It consists of four units
(or domains):
 A domain that activates transcription factors.
 A domain that recognizes specific DNA sequences (core domain).
 A domain that is responsible for the tetramerization of the protein.
 A domain that recognized damaged DNA, such as misaligned base pairs or single-
stranded DNA.

p53-MEDIATED GROWTH REGULATORY

FUNCTIONS
The p53 tumor suppressor protein plays a key role in the regulation of the cell cycle
and cell death. The p53 protein is also involved in cell differentiation, DNA repair,
senescence and angiogenesis. Wild-type (wt) p53 and intact signaling pathways are
essential for the prevention of cancer, consistent with a high tumor incidence observed
in p53 null mice and in p53-heterozygous Li-Fraumeni patients. It is estimated that
approximately one half of human cancers contain a mutation in p53. It is predicted that

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in the majority of the remaining tumors, the p53 signaling pathway is inactivated by up-
regulation of p53 inhibitors, such as Mdm2, or by down-regulation of p53 cooperators,
such as ARF. The increased predisposition to tumor development in the absence of p53
is due to the accumulation of genetic alterations and failure to eliminate these defective
cells.
Wt p53 is a labile protein with a short half-life. Accumulation and activation of the
protein can be triggered by a variety of stress signals including DNA damage, hypoxia,
nucleotide deprivation, viral infection, heat shock, and oncogenic activation. The specific
activity of p53 is further enhanced by post-translational modifications and by a variety of
positive and negative regulators. Activated p53 elicits cellular responses that ultimately
lead to growth arrest and/or programmed cell death (apoptosis).

1.1 - p53-MEDIATED CELL CYCLE ARREST

Activation of p53 may lead to growth arrest at both G1 and G2 phases of the cell
cycle. Arrest in G1 prevents replication of damaged DNA, while arrest in G2 prevents
improper segregation of chromosomes. p53 may also arrest DNA replication in S phase,
which is usually masked the prior G1 arrest. The ability of p53 to arrest cells at multiple
checkpoints is crucial for suppression of amplification of genetic alterations which
otherwise can lead to cancer. Inactivation of wt p53 results in a loss of the DNA damage-
induced G1/S checkpoint impaired G2 arrest. While the pathway leading to G1 arrest is
well established, the pathway leading to G2 arrest is beginning to emerge.

1.1.1 - p53-MEDIATED G1 ARREST

The p53-target gene, p21 (waf-1/cip-1), is the key player in G1 arrest. p21 inhibits
different complexes of cyclin/cyclin-dependent kinases (cdks) (Cyclin D-Cdk4/6 and
Cyclin A, E-Cdk2) that sequentially phosphorylate the retinoblastoma (pRb) protein, and
as a result release the S phase-promoting E2F-1 transcription factor. Cells deficient in
p21 show aberrant G1 arrest following radiation. Unlike p53 null mice, those lacking p21
develop normally, exhibit normal apoptotic response, and are not susceptible to
spontaneous malignancies. p53 also promotes G1 arrest by directly inhibiting the activity
of the CDK-activating kinase (CAK) complex CDK7/CyclinH1/Mat1, which activates cyclin
A-Cdk2 by phosphorylation. CAK is also a component of the TFIIH transcription factor
complex controlling the transcriptional activity of RNA polymerase II. Hence, binding of
p53 to CAK results in a strong reduction of CAK activity towards both Cdk2 and the C-
terminal repeat domain (CTD) of RNA polymerase II. In addition, p53 may activate pRb
via PC3 (TIS21, BTG2), a newly identified p53-target gene. The PC3 gene product
promotes accumulation of hypophosphorylated pRb by reducing Cyclin D1 protein level
and thereby inhibiting Cdk4 activity.

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 Induction of G1 cell cycle arrest by p53. The activation of p21 is central for mediating G1 arrest by
inhibition of multiple cyclin/CDK complexes. The induction of PC3 and inhibition of CAK activity contribute
to this effect. As a result, pRb is not phosphorylated, and it inactivates E2F-1 through recruitment of
histone deacetylase (HDAC1).

1.1.2 - p53-MEDIATED G2 ARREST

Contribution of p53 to G2 cell cycle arrest. p53 triggers several parallel pathways that block the formation
of the mitotic cyclin B/Cdc2 complex and inhibit its activity. The activation of Chk1 by ATM is also
important for this effect. Defects in one of these pathways cause premature entry into M phase. This
activity of p53 is essential for maintaining the G2 arrest.

Activation of p53 can promote and maintain G2 arrest. This depends on functional
pRb128 and is mediated by several target genes. Acting in concert, p53 and its target
genes efficiently inhibit the cyclin B1/Cdc2 activity, which is essential for cells to enter
mitosis. p21 inhibits the activity of the cyclin B1/Cdc2 complex. GADD45 binds Cdc2 and
disrupts its ability to complex with cyclin B. The importance of GADD45 is manifested in
GADD45-/- mice which exhibit both genetic instability, failure of G2 arrest, and
centrosome amplification. The 14-3-3–σ protein sequesters and inhibits the

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phosphorylated form of Cdc25C. The Cdc25C phosphatase dephosphorylates and
thereby activates the cyclin B/Cdc2 complex. Also, 14-3-3–σ sequesters Cdc2 in the
cytoplasm, preventing it from translocating into the nucleus in the late G2. These effects
of p21, GADD45 and 14-3-3–σ are further compounded by the transcriptional repression
of cdc2 and cyclin B1 by p53. Although the role of p53 in triggering G2 arrest is still
unclear, its ability to induce the expression of p21 and 14-3-3–σ is essential for
sustaining this arrest.

1.2 - P53-MEDIATED APOPTOSIS

Apoptosis is a genetically controlled mechanism of cell death that is essential for
the elimination of unwanted cells during normal development and for the maintenance
of tissue homeostasis. One of the major apoptosis signaling pathways involves the p53
tumor suppressor. Tumor protein p53 is a nuclear transcription factor that regulates the
expression of a wide variety of genes involved in apoptosis. p53 stimulates a wide
network of signals that act through two major apoptotic pathways: Extrinsic Pathways
and Intrinsic Pathways.

1.2.1 - THE EXTRINSIC APOPTOSIS PATHWAY

The Extrinsic pathway involves engagement of particular `death receptors that
belong to the tumor TNFR (Tumor Necrosis Factor Receptor) family and, through the
formation of the DISC (Death-Inducing-Signaling-Complex) leads to the recruitment of
initiator caspases 8 and 10 through the adaptor Fas-associated death domain (FADD)
which activate effector caspases 3, 6, and 7, leading to apoptosis.Most common death
receptors involved in extrinsic apoptosis Fas, DR5 (Death Receptor-5) and PERP.
The cell-surface receptor Fas (CD95/Apo-1), a member of the TNFR family of
receptors, is a key component of the extrinsic death pathway. Fas is activated by binding
of its ligand, FasL. p53 induces Fas mRNA expression. This induction occurs in response
to Gamma-irradiation, and it appears to be strictly tissue specific. In addition to
stimulating Fas transcription, overexpressed p53 may enhance levels of Fas at the cell
surface by promoting trafficking of the Fas receptor from the Golgi.
The second member of this receptor family that is induced by p53 is DR5/Killer, the
death-domain-containing receptor for TRAIL (TNF-Related Apoptosis-Inducing Ligand).
DR5 is induced by p53 in response to DNA damage and in turn promotes cell death
through Caspase8. DR5 induction is cell type specific.
Another apoptotic gene, PERP, is induced in response to DNA-damage. PERP is a
putative tetraspan transmembrane protein that represents a new member of the PMP-
22/Gas family of proteins implicated in cell growth regulation. The mechanism by which
PERP contributes to p53-mediated apoptosis is yet to be defined.

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1.2.2 - THE INTRINSIC APOPTOSIS PATHWAY
The intrinsic pathway is triggered by the p53 tumor-suppressor in response to DNA
damage and other types of severe cell stress. Conventional anticancer therapies, such as
chemotherapy and radiotherapy, activate this pathway via p53.
The Intrinsic apoptotic pathway is dominated by the Bcl2 family of proteins, which
governs the release of Cytochrome-C from the mitochondria. The Bcl2 family comprises
anti-apoptotic and pro-apoptotic members. Pro-survival proteins members are most
structurally similar to Bcl2, such as BclXL; pro-apoptotic proteins are BAX and BAK. The
Bcl2 family genes are p53 targets, including BAX, Noxa, PUMA (p53-Upregulated
Modulator of Apoptosis) and the most recently identified, BID.
In response to stress activation, BAX forms a homodimer and releases CytoC from
the mitochondria. CytoC, APAF1 (Apoptotic Protease-Activating Factor-1) and
Procaspase9 then form a complex termed the apoptosome, in which Caspase9 is
activated and promotes activation of Caspase3, Caspase6 and Caspase7, that after
cleavage of vital death substrates induce the final demise of the cell. The requirement
for BAX in p53-mediated apoptosis appears to be cell-type dependent.
The PUMA gene is also directly induced by p53 in response to DNA damage, through
p53-responsive elements within the first intron of PUMA. In humans, PUMA encodes
two BH3-domain-containing proteins, PUMA-Alpha and PUMA-Beta. BAX is absolutely
required for PUMA-mediated apoptosis. BAX participates in the death response as an
indirect target of p53 through PUMA.
Another p53 target gene, Noxa encodes a BH3-only protein and hence is likely to
contribute to p53-mediated apoptosis in a similar manner to PUMA and BAX, although
this is yet to be demonstrated.
An important p53 transcription target gene was identified as p53AIP1 (p53-
regulated apoptosis-inducing protein 1), which is located in the mitochondrial
membrane and is directly involved in p53-dependent mitochondrial apoptosis. Recent
evidence has suggested the existence of a transcription-independent pathway of p53-
mediated apoptosis. A fraction of the p53 molecules that accumulate in damaged cells
translocates to mitochondria, and this translocation is sufficient for p53 to induce
permeabilization of the outer mitochondrial membrane through formation of complexes
with the protective proteins BclXL and Bcl2, resulting in the release of CytoC into the
cytosol. p53 has been shown to transcriptionally repress the antiapoptotic genes Bcl2,
BclXL and Survivin.
Although Extrinsic and Intrinsic pathways are two separate pathways, recent studies
link the Extrinsic and Intrinsic pathways. The pro-apoptotic BID connects the activation
of the extrinsic and intrinsic pathway. Activation of BID involves cleavage of cytoplasmic
BID by Caspase8, which undergoes post-translational myristoylation. Myristoylated BID
translocates to the mitochondria, inserts into the membrane and activates BAX and BAK
to initiate mitochondrial events leading to apoptosome formation. The BID gene is
transcriptionally regulated by p53 in response to Gamma-irradiation through response

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elements in the first intron of the human gene. p53 therefore appears to promote the
convergence of the intrinsic and extrinsic pathways through BID regulation. Akt also play
an important role in regulating p53 pathway under growth/survival conditions and
under stress signals. Under normal conditions, activation of Akt can begin with several
events, mainly the binding of a ligand to a Receptor in the cell membrane. Most
common ligands activating Akt include growth factors, cytokine and hormones. Growth
factors bind to RTK (Receptor Tyrosine Kinase) and cause autophosphorylation of
tyrosine residues on the intracellular domain of the receptor.
Mutations in p53 are associated with genomic instability and increased
susceptibility to cancer. It is the most frequently mutated protein in all cancer with an
estimated 60% of all cancers having mutated forms that affect its growth suppressing
activities.

A model for p53-mediated apoptosis. This model depicts the involvement of p53 in the extrinsic and
intrinsic apoptotic pathways. p53 target genes are shown in red. The convergence of the two pathways
through Bid is shown.

CONCLUSION
The ability of p53 to kill defective cells with genomic instability, or with aberrant
oncogenic events, is probably the most critical function of p53 as a tumor suppressor.
The role of p53 in the G1 checkpoint, and in the maintenance of the G2 checkpoint,
minimizes the accumulation of genetic abnormalities. This can be seen as 50% of cancers
have missense point mutations in the p53 gene; these mutations impair its anti-cancer
gene inducing effects. Restoring its function would be a major step in curing many
cancers.

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