Cancer P53 By Swati Seervi

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CANCER
[p53 - the guardian angel of genome]
[Abnormalities in the genes that regulate the cell death or apoptosis are
associated with many diseases. For example, damage to genes called
tumor suppressor genes, which produce proteins that normally inhibit
cell division, causes some types of cancer. Loss or alterations of a tumor
– suppressor genes called p53 on chromosome 17 is the most common
genetic change.]

Ms. Swati Seervi
Bachelor of Pharmacy (Final Year)
Jodhpur Pharmacy College
Guide Mr.Sanjay Sharma

Cancer p53 jodhpur pharmacy collage

1. INTRODUCTION

1.1 A Short history about cancer

Cancer has not been a new disease. The written descriptions about cancer can be found
on Egyptian papyrus dating back to roughly 1600 B.C. The Egyptians blamed this disease
on the gods and treated it with a cauterizing tool; they called it ―the fire drill‖. Then,
apparently the drill did not happen to work, as the corresponding writing on the papyrus
says ―there is no treatment‖

The Greek physician Hippocrates is believed to be the first person to use the word
―carcinos‖. Over the time this word became the now known ―cancer‖. When the first
autopsy was performed by Italian anatomist Giovanni Morgagni in 1771, the foundation
had been laid for the scientific study of cancer, thus the branch dealing with the study of
cancer is called oncology.

1.2 A brief introduction about cancer

Cancer (medical term: malignant neoplasm) is a class of diseases in which a group of cells
display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and
destruction of adjacent tissues), and sometimes metastasis (spread to other locations in
the body via lymph or blood). These three malignant properties of cancer differentiate
them from benign tumors, which are self-limited, and do not invade or metastasize. Most
cancers form tumor but some, like leukemia, do not.

Cancer affects people at all ages with the risk for most types increasing with age. Cancer
caused about 13% of all human deaths in 2007 (7.6 million).cancer is caused by
abnormalities in the genetic material of the transformed cells. These abnormalities may be
due to the effects of carcinogens, such as tobacco smoke, radiation chemicals, or
infectious agents. Other cancer promoting genetic abnormalities may randomly occur
through errors in DNA replication, or are inherited, and present in all cells by birth.

Genetic abnormalities found in cancer typically affect two general classes of genes. Cancer
– promoting oncogenes are typically activated in cancer cells, giving those cells new

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properties, such as hyperactive growth and division, protection against programmed cell
death, loss of respect for normal tissue boundaries and ability to become established in
diverse tissue environments. Tumor suppressor genes are then activated in cancer cells,
resulting in the loss of normal functions in those cells, such as accurate DNA replication,
control over the cell cycle, orientation and adhesion within tissues, and interaction with
protective cells of the immune system.

1.3 Introduction to various types of cancers

Cancer has been the most dreadful disease encompassing the world today. There are
many types of cancer existing in present following are its types.

1.3.1 Types of ovarian cancers - Germ cell tumors ,
 Rare tumors;
 Sex cord tumors;
 Epithelial tumors
1.3.2 Types of breast cancers - Ductal carcinoma
 Lobular carcinoma;
 Inflammatory breast cancer;
 Medullary carcinoma of the breast;
 Colloid carcinoma;
 Papillary carcinoma
1.3.3 Metaplastic carcinoma of breast
 Triple Negative Breast Cancer

1.4 Introduction about a tumor suppressor gene

A tumor suppressor gene, or anti-Oncogene, is a gene that protects a cell from one step
on the path to cancer. When this gene is mutated to cause a loss or reduction in its
function, the cell can progress to cancer, usually in combination with other genetic
changes.

1.4.1 Two-hit hypothesis

Unlike oncogenes, tumor suppressor genes generally follow the 'two-hit hypothesis',
which implies that both alleles that code for a particular gene must be affected
before an effect is manifested. This is due to the fact that if only one allele for the

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gene is damaged, the second can still produce the correct protein. In other words,
mutant tumor suppressor‘s alleles are usually recessive whereas mutant Oncogene
alleles are typically dominant. The two-hit hypothesis was first proposed by A.G.
Knudson for cases of retinoblastoma.[1] Knudson observed that the age of onset of
retinoblastoma followed 2nd order kinetics, implying that two independent genetic
events were necessary. He recognized that this was consistent with a recessive
mutation involving a single gene, but requiring biallelic mutation. Oncogene
mutations, in contrast, generally involve a single allele because they are gain of
function mutations. There are notable exceptions to the 'two-hit' rule for tumor
suppressors, such as certain mutations in the p53 gene product. p53 mutations can
function as a 'dominant negative', meaning that a mutated p53 protein can prevent
the function of normal protein from the un-mutated allele. Other tumor-suppressor
genes that are exceptions to the 'two-hit' rule are those which exhibit haploid
sufficiency. An example of this is the p27Kip1 cell-cycle inhibitor, in which mutation
of a single allele causes increased carcinogen susceptibility.

1.5 p53 as a tumor suppressor protein

A protein that is the product of a tumor suppressor gene, regulates cell growth and
proliferation, and prevents unrestrained cell division after chromosomal damage, as from
ultraviolet or ionizing radiation. The absence of p53 as a result of a gene mutation
increases the risk of developing various cancers.

A 53 kD nuclear phosphoprotein encoded by the proto-oncogene p53, on chromosome
17p13; in its wild form, p53 inhibits cell growth control and transformation; it activates
transcription of genes that suppress cell proliferation, acting as a tumor suppressor
protein; if p53 is physically lost or functionally inactive, cells can grow without restraint.

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2. HISTORY

2.1 The history of the tumor suppressor protein 53

p53 was identified in 1979 by Arnold Levine, David Lane and William Old, working at
Princeton University, Dundee University (UK) and Sloan-Kettering Memorial Hospital,
respectively. It had been hypothesized to exist before as the target of the SV40 virus, a
strain that induced development of tumors. Although it was initially presumed to be an
Oncogene, its character as a tumor suppressor gene was revealed in 1989.In 1993, p53
protein has been voted molecule of the year by the Science magazine. [12]

p53, also known as TP53 or tumor protein (EC: 2.7.1.37) is a gene that codes for a
protein that regulates the cell cycle and hence functions as a tumor suppression. It is very
important for cells in multi cellular 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 (Strachan and Read, 1999). The name is due to its molecular mass: it is
in the 53 kilodalton fraction of cell proteins.[13]

In 1979, scientists discovered a novel protein. This protein, which could bind to a
transforming protein (Large T antigen) from Simian Virus 40 (SV40), was more prevalent
in cells transformed (immortalized and made potentially tumorigenic) by this virus than in
normal cells. The protein and its corresponding gene were named p53, in reference to the
mass of the protein (53 kilodalton). The p53 gene is located on chromosome 17 at
position p13.

Although p53 was the second tumor suppressor to be discovered after Rb, scientists did
not understand its true role in the cell until ten years after its discovery. Because p53 was
present at increased levels in transformed cells, researchers first believed that it acted as
an Oncogene. This belief was supported by initial research. Scientists found that when the
p53 gene was transferred into cells, the cells underwent transformation. However,
researchers later discovered that the p53 gene that had been transferred was in fact a
mutant form of the gene. One normal function of the p53 gene is to prevent cell
transformation!

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3. DESCRIPTION OF THE GENE

3.1 Genetic Composition Of Cancer p53

The table 1 below shows the genetic composition of p53:

UniProt P04637 O70366
RefSeq (mRNA) NM_000546 NM_011640
RefSeq (protein) NP_000537 NP_035770
Location (UCSC) Chr17: 7.51 - 7.53 Mb Chr11: 69.4 - 69.41 Mb

The name p53 is in reference to its apparent molecular mass: it runs as a 53 kilodalton
(kDa) protein on SDS-PAGE. But based on calculations from its amino acid residues, p53's
mass is actually only 43.7kDa. This difference is due to the high number of proline
residues in the protein which slow its migration on SDS-PAGE, thus making it appear
heavier than it actually is.[5] This effect is observed with p53 from a variety of species,
including humans, rodents, frogs, and fish. [16]

3.2 Nomenclature

p53 is also known as:

 UniProt name: Cellular tumor antigen p53

 Antigen NY-CO-13

 Transformation-related protein 53 (TRP53)

 Tumor suppressor p53

3.3 Cancer p53 in different organisms

In humans, p53 is encoded by the TP53 gene located on the short arm of chromosome 17
(17p13.1). TP53 orthologs have been identified in most mammals for which complete
genome data are available.

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In humans, the two most common polymorphisms occur involve the substitution of an
Arginine base for a Proline base. This polymorphism arises out of a SNP mutation on the
72 codon, where a guanine bases is replace by a cytosine. [17]

For these mammals, the gene is located on different chromosomes:

 Chimp and orangutan, chromosome 17

 Macaque, chromosome 16

 Mouse, chromosome 11

 Rat, chromosome 10

 Dog, chromosome 5

 Cow, chromosome 19

 Pig, chromosome 12

 Horse, chromosome 11

 Opossum, chromosome

3.4 Cancer p53 as an Oncogene

p53 protein was first identified in 1979 as a transformation-related protein [21] and
a cellular protein which accumulates in the nuclei of cancer cells and binds tightly
to the simian virus 40 (SV402) large T antigen [22]. The gene encoding p53 was
initially found to have weak oncogenic activity as the p53 protein was observed to be over
expressed in mouse and human tumor cells [23]. However, almost 10 years later,
researchers discovered that it was a missense mutant of p53 which had originally
been considered as wild-type p53 (wt p53) in that previous study, and that the
oncogenic properties resulted from a p53 mutation [24,25] ,which was later called
―gain of oncogenic function‖ [26]. By the early 1990s, data from the first p53
knock- out mice provided inarguable evidence in support of the potent tumor
suppressor action of wt p53[27]. In subsequent studies, p53 became widely
recognized as a tumor suppressor, and the p53 gene became probably the most
common site for genetic alterations in human cancers[28,29]. Subsequent research
with wt p53 clearly demonstrated that p53 was a biological consequences of p53

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activity include cell-cycle regulation, induction of apoptosis, development,
differentiation, gene amplification, DNA recombination, chromosomal segregation, and
cellular senescence [30].
Presently, p53 is known to play a key role in practically all types of human
cancers, and the mutation or loss of the p53 gene can be identified in more than
50% of all human cancer cases worldwide. This significant involvement in oncogenes is
extended far beyond the simple role in viral transformation p53 was suspected of playing
in earlier investigation.p53 belongs to an unique probe are structurally and functionally
related to each other, p53 seems to have evolved in higher organisms to prevent tumor
development, whereas p63and p73 have clear roles in normal developmental biology [31-
33]. Because p53 plays a pivotal role in regulation of the cell cycle and induction of
apoptosis, there has been enthusiasm about its potential for therapeutic application. It is
not surprising that the prominent position p53 plays in tumor development has spurred
extensive research into both its basic biologic and clinical aspects. To better understand
the relationship between p53 antineoplastic activities and its structure and function, this
review focuses on describing biochemical modifications of p53 and p53 mutations.

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4. STRUCTURE OF THE GENE

4.1 Description about the structure of cancer p53

Human p53 is 393 amino acids long and has seven domains .These domains
with respect to residues are as follows:

 N-terminal transcription-activation domain (TAD), also known as activation
domain 1 (AD1) which activates transcription factors: residues 1-42.

 Activation domain 2 (AD2) important for apoptotic activity: residues 43-63.

 Proline Central DNA-binding core domain (DBD) . Contains one zinc atom and
several arginine amino acids: residues 100-300.

 Nuclear localization signaling domain, residues 316-325.

 Homo-oligomerization domain (OD): residues 307-355. Tetramarisation is
essential for the activity of p53 in vivo.

 C-terminal involved in down regulation of DNA binding of the central domain:
residues 356-393.[7]

 A tandem of nine-amino-acid transactivation domains (9aaTAD) was
identified in the AD1 and AD2 regions of transcription factor p53.[8 ] [9]

The 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 tetramarisation of the protein.
 A domain that recognized damaged DNA, such as misaligned base pairs
or single-stranded DNA.

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Figure 1 : The structure of the core domain of the p53 protein
(light blue) bound to DNA dark blue).The six most frequently
Mutated amino acids in human cancer are show in yellow
– all are residues important for p53 binding to DNA –Red ball:
Zinc atom, {reproduced from cho,Y,et al : (1994) science
1265, 346-355, with kind permission.

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4.2 Structure with description about the mutation hot spots in p53

Figure 2

Figure 2: Schematic representation of the p53 structure. p53 contains 393 amino acids,
consisting of three functional domains, i.e. an N terminal activation domain, DNA binding
domain and C-terminal tetramarisation domain. The N-terminal domain includes
transactivation sub domain and a PXXP region that is a Proline-rich fragment. The central
DNA binding domain is required for sequence-specific DNA binding and amino acid
residues within this domain are frequently mutated in human cancer cells and tumor
tissues. TheArg175, Gly245, Arg248, Arg249, Arg273, and Arg282 are reported to be
mutation hot spots in various human cancers. The C-terminal region is considered to
perform a regulatory function. Residues on this basic C-terminal domain undergo
posttranslational modifications including phosphorylation and acetylation. Numbers
indicate residue number. NLS, nuclear localization signal sequence; NES, nuclear export
signal sequence.[35]

Human p53 is a nuclear phosphoprotein of MW 53 kDa, encoded by a 20-kb gene
containing 11 exons and 10 introns [16], which is located on the small arm of
chromosome 17 [17]. This gene belongs to a highly conserved gene family containing at
least two members, p63 and p73.

Wild-type p53 protein contains 393 amino acids and is composed of several structural
and functional domains : a N-terminus containing an amino-terminal domain (residues 1-

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42) and a Proline-rich region with multiple copies of the PXXP sequence (residues 61-94,
where X is any amino acid), a central core domain (residues 102-292), and a C terminal
region (residues 301-393) containing an oligomerization domain (residues 324-355), a
strongly basic carboxyl terminal regulatory domain (residues 363-393), a nuclear
localization signal sequence and 3 nuclear export signal sequence [18-20]. The amino-
terminal domain is required for transactivation activity and interacts with various
transcription factors including acetyltransferases and MDM2 (murinedouble minute 2,
which in humans is identified as Hdm2) [21,22]. The Proline-rich region plays a role in
p53 stability regulated by MDM2, wherein p53 becomes more susceptible to degradation
by MDM2 if this region is deleted [23]. The central core of this protein is made up
primarily of the DNA-binding domain required for sequence-specific Unbinding (the
consensus sequence contains two copies of the 10-bp motif 5‘-PuPuPuC (A/T)-(T/A)
GPyPyPy-3‘, separated by0-13bp) [24]. The basic C-terminus of p53 also functions as a
negative regulatory domain [20], and has also been implicated in induction of cell death
[25]. According to the allosteric model, in which C-terminal tail of p53 was
considered as a negative regulator and may regulate the ability of its core DNA
binding domain to lock the DNA binding domain as an latent conformation.

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Figure 3 – Structural Organization of p53 Core domains occupies four core-nodes of the
EM m light orange). Atomic coordinates of the oligomerization domain and two α-helices
representing terminus were fitted in to the N/C node of the 3D map. Zn [37]

Figure 4: Schematic representations of the corresponding monomer interactions.
N/C nodes are represented as blue/magenta joints, the linkers representing N-terminus
are in blue, and those for C terminus are in magenta. The core nodes are shown as

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spheres colored in relation to their corresponding core domains. Grey linkers represent
core node to N/C node contacts. [37]

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5. MECHANISM OF WORKING FOR CANCER p53

5.1 A brief about working of cancer p53

It plays an important role in cell cycle control and apoptosis. Defective p53 could allow
abnormal cells to proliferate, resulting in cancer. As many as 50% of all human tumors
contain p53 mutants.

In normal cells, the p53 protein level is low. DNA damage and other stress signals may
trigger the increase of p53 proteins, which have three major functions: growth arrest,
DNA repair and apoptosis (cell death). The growth arrest stops the progression of cell
cycle, preventing replication of damaged DNA. During the growth arrest, p53 may
activate the transcription of proteins involved in DNA repair. Apoptosis is the "last resort"
to avoid proliferation of cells containing abnormal DNA.

The cellular concentration of p53 must be tightly regulated. While it can suppress tumors,
high level of p53 may accelerate the aging process by excessive apoptosis. The major
regulator of p53 is Mdm2, which can trigger the degradation of p53 by the ubiquitin
system. [38]

5.2 Target Genes of p53

P53 is a transcriptional activator, regulating the expression of Mdm2 (for its own
regulation) and the genes involved in growth arrest, DNA repair and apoptosis. Some
important examples are listed below.

 Growth arrest: p21, Gadd45, and 14-3-3.
 DNA repair: p53R2.
 Apoptosis: Bax, Apaf-1, PUMA and NoxA. [39]

5.3 A brief about several mechanisms of cancer p53

In its anti-cancer role, p53 can work through several postulated mechanisms which are
as follows:

 It can activate DNA repair proteins when DNA has sustained damage.

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 It can induce growth arrest by holding the cell cycle at the G1/S regulation point on
DNA damage recognition (if it holds the cell here for long enough, the DNA repair
proteins will have time to fix the damage and the cell will be allowed to continue
the cell cycle.)
 It can initiate apoptosis, the programmed cell death, if the DNA damage proves to
be irreparable.

Figure 5: p53 pathway

In a normal cell p53 is inactivated by its negative regulator, mdm2. Upon DNA damage or
other stress, various pathways will lead to the dissociation of the p53 and
mdm2complex.Once activated; p53 will either induce a cell cycle arrest to allow repair and
survival of the cell or apoptosis to discard the damage cell. How p53 makes this choice is
currently unknown.

Activated p53 binds DNA and activates expression of several genes including WAF1/CIP1
encoding for p21. p21 (WAF1) binds to the G1-S/CDK (CDK2) and S/CDK complexes
(molecules important for the G1/S transition in the cell cycle) inhibiting their activity. P53
has many anticancer mechanisms, and plays a role in apoptosis, genetic stability, and
inhibition of angiogenesis.

When p21 (WAF1) is complexed with cdk2 the cell cannot pass through to the next stage
of cell division. Mutant p53 can no longer bind DNA in an effective way, and as a

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consequence the p21 protein is not made available to act as the 'stop signal' for cell
division. Thus cells divide uncontrollably, and form tumors.

Recent research has also linked the p53 and RB1 pathways, via p14ARF, raising the
possibility that the pathways may regulate each other.

P53 by regulating LIV has been shown to facilitate implantation in the mouse model and
possibly in humans.

When p53 expression is stimulated by sunlight, it begins the chain of events leading to
tanning.

The p53 protein senses DNA damage and can halt progression of the cell cycle in G1 (by
blocking the activity of Cdk2). Both copies of the p53 gene must be mutated for this to fail
so mutations in p53 are recessive, and p53 qualifies as a tumor suppressor gene. [40]

The p53 protein is also a key player in apoptosis, forcing "bad" cells to commit suicide.
So, if the cell has only mutant versions of the protein, it can live on — perhaps developing
into a cancer. More than half of all human cancers do, in fact, harbor p53 mutations and
have no functioning p53 protein. [41]

5.4 activation of p53 by two categories of protein kinases

The protein kinases that are known to target this transcriptional activation domain of p53
can be roughly divided into two groups. A first group of protein kinases belongs to the
MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of
stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. A
second group of protein kinases (ATR, ATM, CHK1 and CHK2, DNA-PK, CAK) is implicated
in the genome integrity checkpoint, a molecular cascade that detects and responds to
several forms of DNA damage caused by genotoxic stress. Oncogenes also stimulate p53
activation, mediated by the protein p14ARF.

5.5 Mdm 2: its significance in mechanism of action of p53

In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A
protein called Mdm2 (also called HDM2 in humans) binds to p53, preventing its action
and transports it from the nucleus to the cytosol. Also Mdm2 acts as ubiquitin ligase and

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covalently attaches ubiquitin to p53 and thus marks p53 for degradation
by the proteasome. However, ubiquitylation of p53 is reversible. A ubiquitin
specific protease, USP7 (or HAUSP), can cleave ubiquitin off p53, thereby
protecting it from proteasome-dependent degradation. This is one means by
which p53 is stabilized in response to oncogenic insults.

Phosphorylation of the N-terminal end of p53 by the above-mentioned protein kinases
disrupts Mdm2-binding. Other proteins, such as Pin1, are then recruited to p53 and
induce a conformational change in p53 which prevents Mdm2- binding even more.
Phosphorylation also allows for binding of transcriptional co activators, like p300 or
PCAF, which then acetylates the carboxy-terminal end of p53, exposing the DNA binding
domain of p53, allowing it to activate or repress specific genes. Deacetylate enzymes,
such as Sirt1 and Sirt7, can deacetylate p53, leading to an inhibition of apoptosis. [19]
Some oncogenes can also stimulate the transcription of proteins which bind to MDM2
and inhibit its activity.

As mentioned above, p53 is mainly regulated by Mdm2. The regulation mechanism is
illustrated in the following figure 6.

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Figure 6.0 Regulation of p53

From figure 6 we come to know about the following points:

 Expression of Mdm2 is activated by p53.
 Binding of p53 by Mdm2 can trigger the degradation of p53 via the ubiquitin
system.
 Phosphorylation of p53 at Ser15, Thr18 or Ser20 will disrupt its binding with
Mdm2. In normal cells, these three residues are not phosphorylated, and p53
is maintained at low level by Mdm2.

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 DNA damage may activate protein kinase (such as ATM, DNA-
PK, or CHK2) to phosphorylate p53 at one of these three residues, thereby
increasing p53 level. Since Mdm2 expression is activated by
p53, the increase of p53 also increases Mdm2, but they have no
effect while p53 is phosphorylated. After the DNA damage is
repaired, the ATM kinase is no longer active. p53 will be quickly
dephosphorylated and destroyed by the accumulated Mdm2. [42]

5.6 The regulation of p53’s level in cell

Under normal circumstances, p53 is maintained at very low concentrations within the cells
and exists mainly in an inactive latent form [43]. During the cell cycle progression, the
low basal level of wt p53 has to be precisely controlled [44].

In normally growing cells, the half-life of p53 is limited to Minutes, whereas cellular
stress or exposure to DNA damaging agents prolongs it to hours [45] . Increased
levels of the p53 protein are primarily regulated through lengthening of its half-life.
The level of p53 and its activities in the cell depend on the cell‘s situation
and extracellular stimuli. The figure 7 shows the p53 pathway and associated genes in
apoptosis.

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Figure 7: p53-associated genes and pathways involved in apoptotic cell death. p53
induces apoptosis mainly via two pathways: extrinsic and intrinsic pathways. The p53-
associated extrinsic pathway is mainly executed by activating caspase 8 to induce
apoptosis, whereas the p53-associated intrinsic pathway is almost executed by influencing
mitochondrial proteins, by which activate caspase 9 to induce apoptosis. In addition p53
may directly activate Apaf-1 to induce apoptosis

5.7 Genes involved in regulating p53 level in cell and its activity

The regulation of p53 level and activity involves a complex network of a multitude of
cellular proteins as shown in figure 7 , including HPV16E6[46] , WT-1 [94], E1B/E4[47]
, SV40 ,T-antigen[49,50] ,MDM2[51,52] , JNK[53] , Pirh2[54,55], and PARP-1[62,57].
Moreover, p53 can switch between a latent and an active form in its function as a
transcription factor. The binding of SV40 T antigen, WT1 or E1B/E4 with p53 increases its
stability, whereas the association of E6 or MDM2 with p53 accelerates its degradation.
MDM2 is an important related protein, which is the product of a p53 inducible gene. The

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importance of MDM2 in the regulation of p53 levels is demonstrated by the fact that
disruption of the MDM2 gene is lethal in early embryos, whereas the concurrent
inactivation of the p53 gene rescues the animal from a lethal consequence [58]. MDM2
inhibits p53 activity by blocking its transcriptional activity, favoring its nuclear export and
stimulating its degradation. MDM2 protein has been found to play an additional role in
blocking the interaction of p53 with the transcriptional apparatus by binding to and
shielding the transactivation domain ofp53 within its N-terminus[52,59] .MDM2 protein
possesses intrinsic E3 ubiquitin ligase activity and mediates both the ubiquitylation and
proteasome dependent degradation of p53[60] . Ubiquitinated p53 is exported to the
cytoplasm, thereby moving it away from its site of action and promoting its rapid
degradation by the proteosome [51]. MDM2 can also recruit the histones deacetylase1
(HDAC1) to deacetylate key lysine residues in the C Terminus of p53, thus making them
available for ubiquitination. The MDM2 gene itself also contains ap53-dependentpromoter
and is transcriptionally regulated by p53 following challenge of the cell by various stresses
[61]. In this fashion, the p53protein regulates the MDM2 gene at the level transcription
and the MDM2 protein regulates the p53 protein at the level of its activity, and an
autoregulatory feedback loop is established that regulates both the activity of the p53
protein and the expression of the MDM2 gene. In this sequence of events, it is wtp53 that
is targeted by MDM2 for degradation, whereas p53 is outside of this negative feedback
loop and accumulates to high levels in cancer cells. It is becoming evident that a number
of mechanisms exist to abolish MDM2-mediated degradation of p53, thereby allowing the
maintenance of a p53 response initiated by various genotoxic stimuli [20, 30]. Under
stress conditions, distinct signaling pathways can be activated to prevent p53 from
ubiquitylation and degradation through post translational modifications and abolishment
of MDM2 activity. Several regulators of p53 have been identified recently, such as the
positive regulator PML (promyelocytic leukemia protein) [66, 67] and the negative
regulators YY1 (Yin Yang1) [68], survivin [64] and PLD (phospholipase D)[65]. All these
regulators appear to influence p53 through MDM2, and they all appear to affect the
transcriptional activity of p53.

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6. ROLE OF P53 IN CANCER TREATMENT

6.1 Role of P53 and MDM2 in Treatment Response of Human Germ Cell Tumors

PURPOSE: Testicular germ cell tumors (TGCTs) of adolescents and adults are very
sensitive to systemic treatment. The exquisite chemo sensitivity of these cancers has
been attributed to a high level of wild-type P53.

RESULTS: Immunohistochemistry demonstrated absence of staining for P53 in 36%, 41%,
and 17% of the unselected, responding, and nonresponding TGCTs, respectively. Of the
positive TGCTs, most tumors, i.e., 49%, 41%, and 33%, showed 1% to 10% positive
nuclei. This overall low level of P53 was confirmed by Western blotting. Mutation analysis
revealed only one silent P53 mutation in one of the responding patients. All embryonal
carcinomas were homogeneously positive for MDM2, encoded by the full length mRNA,
while a heterogeneous pattern was found for the other histological components.
Amplification of MDM2 was detected in one out of 12 embryonal carcinomas.

CONCLUSION: The results of this abstract show that a high level of P53 does not relate
directly to treatment sensitivity of these tumors, and only inactivation of P53 is not a very
exact cause in the development of cisplatin resistance.

6.2 Adenovirus-based p53 gene therapy in ovarian cancer

Mutations of the p53 tumor suppressor gene are the most common molecular genetic
abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53
as a molecular target for gene therapy in ovarian cancer, an adenovirus vector containing
the wild-type p53 gene was constructed. The ability of this adenovirus construct (Ad-CMV-
p53) to express p53 protein was examined by Western blot analysis in the H358 lung
cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the
adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus

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containing the beta-galactosidase reporter gene under the control of the CMV promoter
(Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53
mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for
beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell
growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the
effect of these agents on the growth of 2774 cells was determined using an in vitro
growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-
CMV-p53 showed expression of wild-type p53 protein.

Conclusion

When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.)
of 10 PFU/cell, > 90% of cells shows beta-galactosidase activity, demonstrating that these
cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells
infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The
ability of the adenovirus vector to mediate high-level expression of infected genes and the
inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could
be further developed into a therapeutic agent for ovarian cancer. [75]

6.3 Functional inactivation and structural mutation in p53 causes liver cancer

Structural mutations in the p53 gene are seen in virtually every form of human cancer. To
determine whether such mutations are important for initiating tumorigenesis, the
hepatocellular carcinoma have been studied, in which most cases are associated with
chronic hepatitis B virus infections. Using a transgenic mouse model where expression of
a single HBV gene product, the HBx protein, induces progressive changes in the liver,
showed that tumour development correlates precisely with p53 binding to HBx in the
cytoplasm and complete blockage of p53 entry into the nucleus.

Conclusion

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Analysis of tumour cell DNA shows no evidence for p53 mutation, except in advanced
tumors where a small proportion of cells may have acquired specific base substitutions.
The results suggest that genetic changes in p53 are late events which may contribute to
tumour progression [76-82].

6.4 Introduction of wild-type p53 in a human ovarian cancer cell line not
expressing endogenous p53

Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant
p53 at 37°C and a wild-type like p53 at 32°C, we transfected a human ovarian cancer cell
line (SKOV3) which does not express endogenous p53. Among the different clones
obtained, we selected three clones. Two were obtained from simultaneous transfectlon of
p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was
obtained from transfectlon experiments utilizing the neomycin resistance gene only (SKN).

Conclusion

Introduction of mutant p53 did not alter the morphology or growth characteristics of this
ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change
in morphology and growth rate was observed in SK23a and SK9 cells that are associated
with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32°C did
not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells
did not show evidence of apoptosis when measured up to 72 hours of maintenance at
32°C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at
32°C were not blocked in G1, but they were accumulated in G2-M. This accumulation was
transient and could be due either to a blockade or to a delay in the G2 progression. No
down-regulation of c-myc was observed in p53 expressing clones when shifted to the
permissive temperature. In these conditions gadd45 mRNA expression was highly
stimulated in SK9 and SK23a cells but not In SKN cells. In both clones Gas1 mRNA was
not detected either at 37°C or 32°C. This system represents a new and useful model for
studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53
(SK23a and SK9 kept at 37°C) or wild type p53 (SK23a and SK9 kept at 32°C) on the
mechanism of response of cancer cells to DNA damaging agents. [83]

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6.5 p53 alterations in recurrent squamous cell cancer of the head and neck
refractory to radiotherapy

Abstract

The aim of the study was to determine the incidence of p53 alterations by mutation,
deletion or inactivation by mdm2 or human papillomavirus (HPV) infection in recurrent
squamous cell cancer of the head and neck (SCCHN) refractory to radiotherapy. Twenty-
two tumours were studied. The p53 status of each tumour was analysed by sequencing of
exons 4–9 and by immunohistochemistry. Mdm2 expression was assessed by
immunohistochemistry and HPV infection was assessed by polymerase chain reaction of
tumour DNA for HPV 16, 18 and 33. Fifteen (68%) of the 22 tumors studied had p53
mutations, while seven had wild-type p53 sequence. p53 immunohistochemistry
correlated with the type of mutation. HPV DNA was detected in 8 (36%) tumors and all
were of serotype HPV 16. Of these, five were in tumors with mutant p53 and three were
in tumors with wild-type p53.

Conclusion

Mdm2 over expression was detected in 11 (50%) tumors. Of these, seven were in tumors
with mutant p53 and four were in tumors with wild-type p53. Overall, 21 of the 22 tumors
had p53 alterations either by mutation, deletion or inactivation by mdm2 or HPV. In this
study, the overall incidence of p53 inactivation in recurrent head and neck cancer was
very high at 95%. The main mechanism of inactivation was gene mutation or deletion
which occurred in 15 of the 22 tumors studied. In addition, six of the seven tumors with
wild-type p53 sequence had either HPV 16 DNA, over expression of mdm2 or both which
suggested that these tumors had p53 inactivation by these mechanisms. This high
incidence of p53 dysfunction is one factor which could account for the poor response of
these tumors to radiotherapy and chemotherapy. Therefore, new therapies for recurrent
SCCHN which either act in a p53 independent pathway, or which restore p53 function may
be beneficial in this disease. [84]

6.6 Endocrine-Related Cancer

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Background

The scientists Lane and Harris have reviewed that the tumour suppressor gene p53 and its
protein control critical cellular functions are involved in apoptosis and in the control of the
cell cycle. The encoded protein consists of 393 amino acids giving a molecular mass of 53
000 Daltons. Kirsch and Kastan reviewed that the N terminal part of the protein is
involved in transcription control, the middle portion is responsible for the DNA binding and
the carboxy-terminal third of the protein facilitates the tetramarisation of the protein,
which is claimed to be required for its function. Garber et al and Serrano et al in 1997
found that the p53 gene can be activated via the ataxia telangectasia gene (ATM) by
carcinogens, cytostatics, radiation, ultraviolet light, and hypoxia or by an Oncogene. When
cells acquire irreparable damage, the reparable damage the cell cycle is retarded via p53-
initiated downstream activationof the cyclin-dependent kinases. This results in inhibition
of the cyclins, together with interaction with the retinoblastoma gene product aiming at
controlling the cell cycle at specific checkpoints.The normal p53 function can be
inactivated by somatic and germ line mutations, binding to the Oncogene murinedouble
minute (MDM2) and binding to different viraloncoproteins (humanpapilloma virus protein
E6, SV40large T antigen, hepatitis B viral X protein, adenovirus protein E1A) which was
reviewed in Harris 1996. Clarke et al. 1993, Lowe et al.1993, 1994, O‘Connor et al. 1993,
Fan et al. 1994, Lim etal. 1994demonstrated that somatic mutation of the p53 gene is, so
far, the most common geneticabnormality described in humancancer.In pre-clinical model
systems it was demonstrated thattumours with wild-type p53 status responded better
tocertain ontological therapeutic modalitiesthan tumourswith an altered p53 status .
Clarke et al.1993 say that despite these straight forward findings it is also obvious that
radiation and certain cytostaticsmay alsoinduce apoptosis via a p53-independent pathway
which was reviewed in Beck & Dalton 1997. However, the issue is complex, sincemutant
p53 has been claimedto interfere ‗with the p53-independent pathways of apoptosis‘ (Li et
al. 1998). Jost et al. 1997, Kaghad et al.1997, Osada et al.1998, Trink et al demonstrate
that p40, p51 and p73 have more or less sequence homology with p53 .These genes are
now grouped togetherin the p53family. How they are activated and may replace
andfunction whenp53 is mutated is not known. These aspectsmay be important both for
p53dependent and-independent apoptosis. [85]

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6.7 Clinical studies of p53 in treatment and benefit of breast cancer patients

Abstract

This review article focuses on discussing p53 inrelation to its predictive potential. So far,
no firm conclusions can be made based on the articlesstudied. This may be, in part,
because many studies have usedless than optimal techniques fordetermination of the p53
status, together with the fact that the studies lacked power to detect an Endocrine-
Related Cancer (1999) 6 51-59potential difference in outcome from specific therapy in
relation to p53 status.Bergh: p53 treatment in breast cancer patients aspect of p53. In
comparative studies, the use ofimmunohistochemistry and another protein measurement
will result in false negative and false positive results.

Conclusion

P53 has critical functions for the control of the cell cycle and apoptosis. Cytostatics,
tamoxifen and radiation may induce apoptosis via p53-dependent and –independent
pathways. The clinical data with reference to the potential predictive value of p53 are still
conflicting, which is due partly to suboptimal methods for determination of the p53status,
and partly to too small studies combined withselected patient materials. The data so far
indicate that CMF-based regimens and tamoxifen may be suboptimalfor patients with
mutant p53 andpostoperativeradiotherapy may be extra beneficial for breast cancerswith
mutant p53. For the future, larger studies onpopulation-based cohorts using
optimalmethods for p53measurements are warranted, and ideally the studies should have
patient populations. [86]

6.8 Boehringer Ingelheim and Priaxon announce a collaboration to research and
develop novel treatments for cancer

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The two Companies to advance mdm2/p53 inhibitors, which hold potential to address
various tumor types: Munich, Ingelheim/Germany, 18 January 2010 - Boehringer
Ingelheim and Priaxon entered into a worldwide collaboration to research and develop
mdm2/p53 inhibitors for the treatment of cancer. Priaxon is providing its innovative and
proprietary small molecule drug discovery expertise which is particularly suited to
investigate inhibition of protein-protein interactions. P53 is a human tumor suppressor
protein. It has been shown that in tumors with wild-type p53, the restoration of p53
tumor-suppressive functions can be achieved by blocking a cellular interaction of mdm2 1
and p53. This may reactivate the ―genome guardian‖ function of p53 and is therefore an
interesting approach for treating under the terms of the collaboration and license
agreement, Boehringer Ingelheim will lead development and commercialisation of the
potential mdm2/p53 inhibitor products to capitalise on its global marketing and sales
expertise. Boehringer Ingelheim will pay significant up-front and near-term payments to
Priaxon including research funding to support further discovery efforts. In addition,
Priaxon will be eligible to receive from Boehringer Ingelheim EUR 86 million in milestone
payments upon achievement of certain development, regulatory and commercial
milestones as well as royalties on potential future net sales of products.

About mdm2/p53 Inhibition

The human p53 tumor suppressor protein has been one of the most investigated proteins
in cancer research due to the fact that loss of p53 function through mutation and/or
deregulation is involved in about 50% of all human cancers. The role of p53 in controlling
the cell cycle and monitoring the integrity of the genome has made it known as the
―guardian of the genome‖. Besides the functional loss of p53 through mutation, it can also
be inactivated by the over expression or amplification of MDM2 (murine double minute 2),
which is the case in many p53 wild-type tumors. Thus, disruption of the MDM2–p53
interaction is considered a novel therapeutic strategy for cancer cells that still are
endowed with wild-type p53, and a variety of small molecule drug like compounds haveto
the p53 binding site of MDM2.

About Priaxon

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Priaxon is an emerging pharmaceutical company building a pipeline of novel drug
candidates in different therapeutic fields, but mainly focusing on protein-protein
interactions in oncology and other diseases. The goal is to discover and develop
candidates for validated but hard-to-drug targets using two orthogonal drug discovery
platforms.

About Boehringer Ingelheim

The Boehringer Ingelheim group is one of the world‘s 20 leading pharmaceutical
companies. Headquartered in Ingelheim, Germany, it operates globally with 138 affiliates
in 47 countries and 41,300 employees. Since it was founded in 1885, the independent,
family-owned company has been committed to researching, developing, manufacturing
and marketing novel products of high therapeveterinary medicine.

In 2008, Boehringer Ingelheim posted net sales of 11.6 billion euro while spending one
fifth of net sales in its largest business segment Prescription Medicines on research and
development. Mdm2 is an important negative regulator of the p53 tumor suppressor. It
is the name of a gene as well as the protein encoded by that gene. [87]

6.10 Regulation of cancer stem cells by p53

The hypothesis those cancer stem cells are responsible for the chemoresistant and
metastatic phenotypes of many breast cancers has gained support using cell sorting
strategies to enrich the tumor-initiating population of cells. The mechanisms regulating
the cancer stem cell pool, however, are less clear.

Two recent publications suggest that loss of p53 permits expansion of presumptive cancer
stem cells in mouse mammary tumors and in human breast celllines.These results add
restriction of cancer stem cells as a new tumor suppressor activity attributed to p53.

The recent identification and characterization of stem cells in a variety of adult tissues
has led interest in there of stem cells in cancers.Cancer stem cells are hypothesized to be

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a small population of cells within a tumor that are capable of self-renewal and that can
undergo differentiation to generate the phenotypic heterogeneity observed in
tumors.Contemporary methods to study cancer stem cells have often used cell surface
markers to enrich the subset of cells capable of initiating a tumor upon transplantation
into an appropriate host. Molecular pays that limit expansion of the tumor-initiating cell
population could be targeted to eradicate tumors.Using mammary tumors arising
spontaneously from transplants of BALB/c-Trp53-/- mammary epithelium, Zhang and
coworkers show that cells expressing markers of mouse mammary stem cells (lin-
/CD29hi/CD24hi) had a greater tumor-initiating frequency. This observation was
consistent among tumors with heterogeneous expression of markers for the luminal
epithelium and the basal epithelium. The lin-/CD29hi/CD24hi population shared additional
features of mammary stem cells, including radiation resistance and the formation of
secondary mammospheres.
But how might loss of p53 lead to formation or expansion of the tumor-initiating pool?
Using a unique culture model of luminal breast epithelial cells (BPEC-T), Godar and
coworkers demonstrate that p53 binds to the promoter of CD44, a commonly used marker
of cancer stem cells, and represses CD44 expression. Constitutive expression of CD44
blocked p53-dependent apoptosis and rendered cells resistant to doxorubicin. Conversely,
suppression of CD44 expression restricted tumor-initiating cells.

Conclusion
These results link the loss of p53 function to increased expression of CD44, which
promotes expansion of tumor-initiating cells purified in tumors. The p53 protein appears
to play a similar role in embryonic stem cells, where p53 represses expression of Nanog –
which limits the pool of pluripotent cells. In contrast, loss of p53 extends the repopulating
activity of tissue-specific stem cells. Disruption of BRCA1 also allows expansion of breast
stem cells. The restriction of stem cells may therefore be a fundamental pathway for
tumor suppression.

While expansion of the tumor-initiating cell population in p53-deficient mammary
epithelial cells is consistent in both mouse mammary and human breast epithelial cells,
the role of CD44 is not. Although loss of p53 expression resulted in increased levels of
CD44 protein in BPEC-T cells and in basal mammary epithelium of Trp53-/- mice , there
was no enrichment for tumor-initiating cells within the CD44+/CD24-population in

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BALB/c-Trp53-/- mammary tumors . This apparent discrepancy points to heterogeneity in
the expression of markers among cancer stem cells. In mammary tumors from
Brca1ΔExon11/Trp53+/- mice, two discrete tumor-initiating populations were identified
that express CD44+/CD24-or CD133+. As coexpression of CD44 and CD133 was not
detected in these pools of cells, it appears that CD44 is not essential for sustaining the
pool of cancer stem cells.
Indeed, p53 represses expression of more than 20 target genes that may contribute to
maintenance of the pool of tumor-initiating cells. Genes such as Nanog may have direct
actions in supporting self-renewal of cancer stem cells, allowing the pool to expand. Loss
of p53 would also allow increased expression of the multidrug-resistance gene (ABCB1 or
MDR1) that renders cells resistant to chemotherapies. Similarly, both increased
proliferation and decreased apoptosis would be expected to result from de-repression of
CDC25C and BIRC5/Survivin when p53 function is disrupted. CD44 may therefore be only
one mechanism by which p53 may act to restrict the tumor-initiating population of cancer
cells.

It is clear that that p53 plays a pivotal role in tumor suppression. Mutation and loss of
function of p53 are among the most common alterations in epithelial cancers., and gene
expression signatures associated with dysfunctional p53 have been shown to predict
patient survival .The p53 protein regulates a variety of pathways (cell cycle arrest,
apoptosis, DNA repair, senescence and autophagy) that can contribute to suppression of
tumors. The publications by Zhang and colleagues and by Godar and colleagues now add
suppression of cancer stem cells as an additional activity by which p53 can inhibit tumors.
On the one hand, loss of p53 may promote genetic instability – resulting in plasticity of
phenotypes due to random mutations and clonal evolution.

.

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Figure 8: Loss of p53 function and effects on tumor heterogeneity. In normal epithelia,
p53 represses expression of potential oncogenes (for example, CD44, NANOG, BIRC5,
CDC25C) as well as transcriptionally activating tumor suppressor pathways [10]. Loss of
p53 (more ...)

In this model, the behavior of the p53-deficient cancer cells would be stochastic and
would require therapeutics targeting multiple oncogenic pathways. If the apparent
phenotypic plasticity of p53-deficient breast tumors is due to the expansion of the cancer
stem cell pool, however, therapies targeting the self-renewal pathways may be extremely
effective. Loss of p53 function in breast tumors is strongly correlated with the basal-like
gene expression signatures. This suggests that either these tumors originate from breast
stem cells or that loss of p53 allows cancer cells to acquire characteristics of stem cells.
These results favor the possibility that p53 deficiency allows expansion of cancer stem
cells and that the expression profiles of tumor-initiating cells will identify effective
therapeutic targets. [88]

6.9 Role in disease

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Figure 9:
.

If the TP53 gene is damaged, tumor suppression is severely reduced. People who inherit
only one functional copy of the TP53 gene will most likely develop tumors in early
adulthood, a disease known as Li-Fraumeni syndrome. The TP53 gene can also be
damaged in cells by mutagens (chemicals, radiation, or viruses), increasing the likelihood
that the cell will begin decontrolled division. More than 50 percent of human tumors
contain a mutation or deletion of the TP53 gene. Increasing the amount of p53, which
may initially seem a good way to treat tumors or prevent them from spreading, is in
actuality not a usable method of treatment, since it can cause premature aging. However,
restoring endogenous p53 function holds a lot of promise. Loss of p53 creates genomic
instability that most often results in the aneuploidy phenotype.

Certain pathogens can also affect the p53 protein that the TP53 gene expresses. One such
example, the Human papillomavirus (HPV), encodes a protein, E6, which binds the p53
protein and inactivates it. This, in synergy with the inactivation of another cell cycle
regulator, p105RB, allows for repeated cell division manifested in the clinical disease of
warts. Infection by oncogenic HPV types, especially HPV16, can also lead to progression
from a benign wart to low or high-grade cervical dysplasia which is reversible forms of
precancerous lesions. Persistent infection over the years causes irreversible changes
leading to Carcinoma in situ and eventually invasive cervical cancer. This results from the

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effects of HPV genes, particularly those encoding E6 and E7, which are the two viral
oncoproteins that are preferentially retained and expressed in cervical cancers by
integration of the viral DNA into the host genome.

In healthy humans, the p53 protein is continually produced and degraded in the cell. The
degradation of the p53 protein is, as mentioned, associated with MDM2 binding. In a
negative feedback loop MDM2 is itself induced by the p53 protein. However mutant p53
proteins often don't induce MDM2, and are thus able to accumulate at very high
concentrations. Worse, mutant p53 protein itself can inhibit normal p53 protein levels.[89]

6.10 P53 Gene Therapy: A Potential Panacea To Cancer

The extensive work focuses on gene therapy as a novel method to cure cancer [ovarian
&breast], especially p53 gene having tremendous potential used as a suppressor gene to
kill the tumor cells, their types, roles and use of p53.

It focuses on the mechanism of p53 apoptosis [programmed cell death]. It suggests
advanced therapies in combination of chemotherapy with gene therapy. It also shed light
on various current research viral & non viral vectors used in p53 gene therapy. The figure
11 shows role of p53 in apoptosis.

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Figure 10:

Role of apoptosis

In each of these diverse areas implicates immense potential manipulation of apoptosis to
treat disease. Research is already underway to harness apoptosis as a therapeutic tool in
modern medicine.

As chemotherapy show disadvantage of regrowth of cell, metastasis after surgery
treatment, chemoresistance to tumor, gene therapy has been a revolutionary step
towards cancer treatment. Genetic correction strategies are presently being developed
and tested in animal models for human malignancies and in early patient trials. The
cancer susceptibility genes p53 have been tested in ovarian & breast cancer patients, ,
and have shown some potential for this antitumor strategy. P53 gene therapy may be

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effective even against tumors that lack p53 mutations, because p53 may function as a
growth inhibitor in a variety of gene transfer settings.

Key problems at present include the degradation of vector by the immune system and a
need for higher levels of gene transduction. Solutions will require the development of
improved vectors, improved vector delivery systems, and the fine-tuning of human gene
therapy in appropriate models of human cancer. [90]

6.11 Role of p53 Gene in Metabolism Regulation in Patients with Li-Fraumeni
Syndrome

Purpose

This study will examine metabolic and biological factors in people with Li-Fraumeni
syndrome, a rare hereditary disorder that greatly increases a person's susceptibility to
cancer. Patients have a mutation in the p53 tumor suppressor gene, which normally helps
control cell growth. This gene may control metabolism as well as cancer susceptibility, and
the study findings may help improve our understanding of not only cancer but also other
conditions, such as cardiovascular function.[91]

6.12 The role of p53 in treatment responses of lung cancer

Abstract
Resistance to radio- and chemotherapy is a major problem in treatment responses of lung
cancer. In this disease, biological markers, that can be predictive of response to treatment
for guiding clinical practice, still need to be validated. Radiotherapy and most

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chemotherapeutic agents directly target DNA and in response to such therapies, p53
functions as a coordinator of the DNA repair process, cell cycle arrest, and apoptosis. In
fact, it participates in the main DNA repair systems operative in cells, including NHEJ,
HRR, NER, BER, and MMR. Given the high p53 mutation frequency in lung cancer which
likely impairs some of the p53-mediated functions, a role of p53 as a predictive marker for
treatment responses has been suggested. In this review, we summarize the conflicting
results coming from preclinical and clinical studies on the role of p53 as a predictive
marker of responses to chemotherapy or radiotherapy in lung cancer. [92]

6.13 The Association of p53 with Specific Cancers

Germline (inherited) mutations of the p53 gene are associated with the rare inherited
cancers classified under the Li-Fraumeni syndrome (LFS). Somatic p53 genetic mutations
have been shown to be involved in tumors of the anus, bone, bladder, brain, breast,
colon, cervix, esophagus, stomach, liver, lung, lymphoid system, ovary, prostate and skin.
A brief look at the p53 gene's association with certain cancers is presented below.

Lung cancer

Benzo[a]pyrene is a mutagen found in cigarette smoke. This chemical binds to DNA and
ultimately can cause G (guanine) to T (thymine) substitutions in DNA. Other chemicals in
cigarette smoke have been shown to produce C (cytosine) to A (adenine) changes. When
these occur in the p53 gene, the mutations can cripple the p53 protein, disrupting
its tumor-suppressing function.

Liver cancer

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Two major causes of liver cancer are infection with the Hepatitis-B virus and exposure to
aflatoxin, a mutagen produced by a mold that grows on improperly stored grains and food
crops, specifically wet corn. Aflatoxin, like benzopyrene, may change the gene that codes
for p53, thereby disrupting the tumor-suppressing ability of p53.The Hepatitis-B virus
works to inactivate p53 in a different way; it produces a protein that has the ability to
bind p53 and prevent it from interacting effectively with its target genes.

Skin cancer

The ultraviolet (UV) rays in sunlight can cause damage to DNA. If the DNA in a skin cell is
damaged beyond repair, the p53 protein can induce cell death. However, if the UV light
causes a mutation in the p53 gene rendering the protein nonfunctional, the damaged cell
may reproduce and potentially lead to the formation of a cancerous growth.

Cervical cancer

Human papillomavirus (HPV) is a sexually transmitted virus that can infect cervical cells.
Once inside the cell, the virus produces a protein that binds to p53 and causes the p53
protein to be degraded. The result of this degradation is a decrease in available p53
protein and a loss of functinal p53 activity.

Breast cancer

In many breast cancers the p53 gene appears to be normal. However, it has been shown
that in some cases the protein MDM2 is enhanced in the cells and binds to the p53
protein, inhibiting its antitumor activity. This allows for the growth of malignant breast
cells and inhibits the p53 induced apoptotic pathway.[93]

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7. POTENTIAL THERAPEUTIC USE AND SIGNIFICANCE OF CANCER p53

7.1 Trifluorothymidine Induces Cell Death Independently of p53

Abstract

Trifluorothymidine (TFT), a potent anticancer agent, inhibits thymidylate synthase (TS)
and is incorporated into the DNA, both events resulting in cell death. Cell death induction
related to DNA damage often involves activation of p53. The role of p53 was determined
in TFT cytotoxicity and cell death induction, using, respectively, the sulforhodamine B-
assay and FACS analysis, in a panel of cell lines with either wild type, inactive, or
mutated p53. Neither TFT cytotoxicity nor cell death induction changed with TFT exposure
in cell lines with wt, inactive or mutated p53. Conclusion: sensitivity to TFT is not
dependent on the expression of wt p53.

In-vitro introduction of p53 in to p53-deficient cells has been shown to cause rapid death
of cancer cells or prevention of further division. The rationale for developing therapeutics
targeting p53 is that "the most effective way of destroying a network is to attack its most
connected nodes". P53 is extremely well connected (in network terminology it is a hub)
and knocking it out cripples the normal functioning of the cell. 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 (Vogelstein et al 2000).

Various strategies have been proposed to restore p53 function in cancer cells like the one
given by Blagosklonny in 2002. A number of groups have found molecules which appear
to restore proper tumour suppressor activity of p53 in vitro. These work by altering the
conformation of mutant conformation of p53 back to an active form. So far, no molecules
have shown to induce biological responses, but some may be lead compounds for more
biologically active agents. A promising target for anti-cancer drugs is the molecular
chaperone Hsp90, which interacts with p53 in vivo.

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Adenoviruses rely on their host cells to replicate; they do this by secreting proteins which
compel the host to replicate the viral DNA. Adenoviruses have been implicated in cancer-
causing diseases, but in a twist it is now modified viruses which are being used in cancer
therapy. ONYX-015 (dl1520, CI-1042) is a modified adenovirus which selectively
replicates in p53-deficient cancer cells but not normal cells says Bischoff in 1996. It is
modified from a virus that expresses the early region protein, E1B, which binds to and
inactivates p53. P53 suppression is necessary for the virus to replicate. In the modified
version of the virus E1B has been deleted. It was hoped that the viruses would select
tumour cells, replicate and spread to other surrounding malignant tissue thus increasing
distribution and efficacy. The cells which the adenovirus replicates in are lysed and so the
tumour dies.

Preclinical trials using the ONYX-015 virus on mice were promising however clinical trials
have been less so. No objective responses have been seen except when the virus was
used in combination with chemotherapy says McCormick in 2001. This may be due to the
discovery that E1B has been found to have other functions vital to the virus. Additionally
its specificity has been undermined by findings showing that the virus is able replicate in
some cells with wild-type p53. The failure of the virus to produce clinical benefits may in
large part be due to extensive fibrotic tissue hindering virus distribution around the
tumour says McCormick in 2001. [95]

7.2 Therapeutic applications of p53

The insights which have been provided by p53 researchover the years have been for
improvement of diagnostic techniques, accuracy of prognosis, and treatment of
cancer.Since about half of all human tumors have an abnormal p53 which can occur early
in carcinogenesis, and since post translational modifications of p53 can reflect the type
and magnitude of cellular stress [114], p53 can be a useful biomarkerin carcinogenesis.
Indeed, p53 has been used as a molecular signature to study both target tissues and
surrogate fluids such as blood in high-risk cancer populations [173]. Mutated p53 protein
accumulation and posttranslational modification endpoints could also prove useful

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instudying the efficacy of chemopreventive agents [38].As p53 plays a key role in the
cellular response to stress; it serves as a major barrier to tumorigenesis. This obstacle
has to be removed in order for tumor development to proceedand restoration of wt p53
function is thus a potential key in anticancer therapies. Since MDM2 is an
importantnegative regulator of p53, MDM2 hyperactivity may inhibitthe function of p53
and lead to the development of a widevariety of cancers. For example, 30% of human
sarcomasshow no p53 mutations, but have an overexpressed MDM2gene [174]. It is
believed that inhibiting the E3 activity ofMDM2 and blocking the interaction of p53 with
MDM2 are potential effective strategies for killing certain tumor cellsselectively by
restoring the function of wt p53 [41]. Therefore, many studies have focused on the p53-
MDM2 interactionas the basis of a drug development strategy. A series ofsmall molecule
inhibitors have been developed, and some ofthese can bind to MDM2 and block its
interaction with p53, including peptides that have been shown to elevate the levelsof p53
protein and its transcriptional activity and triggerp53-dependent apoptosis in tumor cells
[175,176]. Class ofsmall molecules named nutlins have been identified to block
p53/MDM2 interaction in vitro and in vivo. Treatment of tumor cells with nutlins results in
induction of p53 and its target genes and triggering of apoptosis. Recently, a novelseries
of benzodiazepinedione antagonists of the p53/MDM2interaction have been discovered
which increase the transcriptionof p53 target genes and decrease proliferation oftumor
cells expressing wt p53 [177]. One study suggests that antisense oligodeoxynucleotides
targeted against MDM2and p21Waf1/Cip1 could be employed in a potential therapeutic
strategy sensitizing tumor cells to certain antineoplastic agents [178]. One of the major
concerns about blocking thep53/MDM2 interaction for use in treatment of cancer was the
idea that activation of p53 might be toxic to normal tissues.However, certain data suggest
that the mechanisms governing p53 activity in tumor cells and normal cells are quite
different, so the different effects of p53 in reactivating different molecules in tumor cells
and normal cells might providea molecular basis for a therapy without the need for tumor
targeting [179].Another factor in p53 inactivation is the presence of the human papilloma
virus (HPV). In cervical carcinomas, p53 istargeted by HPV encoded E6 protein, which
potentiates p53degradation and inactivates its function in 90% of cervical cancers [180].
Drugs that inhibit E6 should promote p53reactivation and thus have selective therapeutic
effect. ItE6 expression, stabilize p53 and induce apoptosis in a model system of cultured
cells [181]. Interestingly, both drugs can inhibit MDM2-mediated inactivation of p53,

Page 41 of 62


possiblyvia inhibition of p53 ubiquitylation (leptomycin B) or by Decreasing MDM2 gene
transcription (actinomycin D) [182].

Because the apoptotic function of p53 is critical for tumorsuppression, induction of
apoptotic pathways through p53-induced apoptotic targets may be an attractive strategy
for anti-cancer treatment. Furthermore, the p53 apoptotic targets,unlike p53, are rarely
mutated in human cancers [30].Some of the p53 apoptotic targets, such as bax,
Puma,p53AIP1, Noxa and others could potentially be used as targets transfer of bax can
act synergistically with chemotherapy to induce apoptosis in tumors[184]. A recent study
has demonstrated that siRNA targeting of survivin, a negative regulator of apoptosis
which is downregulated by p53, could be potentially useful for increasing sensitivity to
anticancer drugs, especially in drug-resistant cells with mutated p53 [185]. However, the
effects of p53-dependent apoptosis are not always favorable for clinical use, and so the
inhibitors of p53-mediated apoptosis might be used to Significance of PML and p53
protein as molecular prognostic markers of gallbladder carcinomas detection and
prognostic prediction, but may also serve as potential therapeutic targets.Inorder to
identify reliable molecular markers for prognostic prediction in gall bladder carcinoma
(GBC), we evaluated the immunohistochemical expression of 15proteins, namely p53,
p27, p16, RB, Smad4, PTEN, FHIT, GSTP1, MGMT, E-cadherin,nm23, CD44, TIMP3,
S100A4, and promyelocytic leukemia (PML) in 138 cases of protein. Over expression of
p53 and S100A4, and loss of p27, p16, RB, Smad4, FHIT, Ecadherin and PML expression
were associated with poor survival. In particular, PML andp53 showed considerable
potential as independent prognostic markers. Patients with normal PMLand p53 expression
displayed favorable outcomes, compared to those showingabnormal expression of either
or both proteins (49%vs. 23% in a 5-year survival rate; 60 monthsvs. 11 months in
median survival, respectively;P=0.009). Thus, PML and p53 are potential candidates for
development as clinically applicable molecular prognostic markers of GBC, and may be
effective therapeutic targets for the disease in the future. [96]

7.3 Clinical significance of p53 alterations in surgically treated prostate cancers

Abstract

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Despite the high number of previous studies, the role of p53 alterations in prostate cancer
is not clearly defined. To address the role of p53 alterations in prostate cancer biology, a
total of 2514 cancers treated by radical prostatectomy were successfully analyzed by
immunohistochemistry in a tissue microarray format. Overall a low rate of p53-positive
tumors was found (2.5%). A significant underestimation of p53-positive cases was
excluded by subsequent large section analyses and direct sequencing of the p53 gene in
subsets of our patients. Large section analysis of 23 cases considered negative on the
tissue microarray yielded only one weakly p53-positive tumor. Only 4 out of 64 (6.4%)
high-grade tumors, that were considered negative for p53 by immunohistochemistry,
presented exon 5–8 mutations.

Conclusion

These data suggest a high sensitivity of our immunohistochemistry approach and confirm
the overall low frequency of p53 alterations in clinically localized prostate cancer. A
positive p53 immunostaining was strongly associated with presence of exon 5–8
mutations (P<0.0001), advanced pT-stage (P<0.0001), high Gleason grade (P<0.0001),
positive surgical margins (P=0.03) and early biochemical tumor recurrence (P<0.0001). A
higher rate of positive p53 immunostaining was detected in late-stage diseases including
metastatic prostate cancer (P=0.0152) and hormone-refractory tumors (P=0.0003).
Moreover, p53 expression was identified as an independent predictor of biochemical tumor
recurrence in the subgroup of low- and intermediate-grade cancers. In summary, the
results of this study show that p53 mutations characterize a small biologically aggressive
subgroup of prostate cancers with a high risk of progression after prostatectomy. The rate
of p53 alterations increases with prostate cancer progression. [97]

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8.Future trends of p53 in curing various cancers

8.1 Double-edged swords as cancer therapeutics: simultaneously targeting p53
and NF-κB pathways

The p53 and nuclear factor-κB (NF-κB) pathways play crucial roles in human cancer, in
which inactivation of p53 and hyperactivation of NF-κB is a common occurrence.
Activation of p53 and inhibition of NF-κB promotes apoptosis. Although drugs are being
designed to selectively activate p53 or inhibit NF-κB, there is no concerted effort yet to
deliberately make drugs that can simultaneously do both. Recent results suggest that a
surprising selection of small molecules have this desirable dual activity. In this Review we
describe the principles behind such dual activities, describe the current candidate
molecules and suggest mechanisms and approaches to their further development.[99]

8.2 Novel Regulatory Mechanism Identified for Key Tumor Suppressor p53:

(Philadelphia – November 15, 2006) – Collaborating scientists from The Wistar Institute in
Philadelphia and The Vienna Biocenter in Austria identified a novel mechanism involved in
normal repression of the p53 protein, perhaps the single most important molecule for the
control of cancer in humans.

The new molecular pathway described in the study suggests intriguing approaches to
diagnosing or intervening in the progression of many types of cancer. A report on the
team‘s findings will be published online November 15 in the journal Nature.

Shelley L .Berber says that ―The p53 protein is vital for controlling cancer throughout the
body,‖ the Hilary Koprowski Professor said that at The Wistar Institute and senior author
on the study. ―The new mechanism we describe, driven by a previously unknown enzyme,
represses the p53 protein when its activity is not needed.

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―What we‘re looking at now is the possibility that this enzyme, if over-expressed or over-
active, might interfere with p53‘s normal tumor suppressor function and perhaps cause
cancer. If that‘s the case, then we could develop drugs to inhibit the enzyme that would
have the effect of freeing p53 to do its job of suppressing cancer. Unusually high levels of
the newly identified enzyme might also be useful as a diagnostic marker for cancer.‖

Responsible for tumor suppression throughout the body, the p53 protein has been found
to be mutated and dysfunctional in more than half of human cancers. When working
properly, p53 acts by binding to DNA to activate genes that direct cells with damaged DNA
to cease dividing until the damage can be repaired. Cells with such damage include cancer
cells, since all cancers track to genetic flaws of one kind or another, whether inherited or
acquired. If repairs cannot be made, p53 commands the cells with damaged DNA to self-
destruct so they are no longer a danger to the body.

This powerful ability of the p53 protein to shut down cell division and induce cell death
points to why the availability of a repressive mechanism such as the one outlined in the
new study might be crucial for cellular survival.

Conclusion

In their study, the scientists identified an enzyme called Smyd2 that adds a methyl group
to the p53 protein at a specific site, with the result being that p53 cannot bind to DNA
and, therefore, cannot act. The ability to bind to DNA is critical for p53‘s function,‖ says
Jing Huang, Ph.D., one of the study‘s two lead authors. ―What we found was that
methylation at the site we identified prevents p53 from binding to DNA, which also
explains why it‘s a repressive modification.Berger and Huang note that this is one of only
a small number of studies to identify methylation as playing a role in regulating the
activity of proteins that are not histones. Histones are relatively small proteins around
which DNA is coiled to create structures called nucleosomes. Compact strings of
nucleosomes, then, form into chromatin, the substructure of chromosomes.With histones,
methylation is well recognized as a regulatory mechanism, but the fact that other proteins
are also be modified in the same way is a relatively new observation. Berger believes that
scientists will likely find this type of regulatory mechanism at work in many other protein
systems over the next few years.

Page 45 of 62


Interestingly, only one other study has shown a role for methylation in regulating p53. In
that study, a methyl group added to a specific site on p53 called K372 was shown to
activate the tumor-suppressor molecule rather than repress it. The site identified in the
current study, dubbed K370, is adjacent to that first site. An additional finding of note is
that the two sites interact closely.

Huang says that there‘s important crosstalk between the two sites, but only in one
direction. If the previously identified site is already methylated, the site we found cannot
be methylated. But the reverse is not the case.

The two lead authors on the Nature study are Jing Huang, Ph.D., at Wistar and Laura
Perez-Burgos, Ph.D., at The Vienna Biocenter. The additional Wistar co-authors are
Brandon J. Placek, Ph.D., Jean A. Dorsey, and senior author Shelley L. Berger, Ph.D. The
additional co-authors at The Vienna Biocenter are Roopsha Sengupta, Mario Richter,
Stefan Kubicek, and Thomas Jenuwein, a senior scientist who led the collaboration at The
Vienna Biocenter.

Funding for the research was provided to the Berger laboratory by the National Institutes
of Health and the Commonwealth Universal Research Enhancement Program of the
Pennsylvania Department of Health. Support for the Jenuwein laboratory was provided by
Boehringer Ingelheim, the European Union, and the Austrian Ministry of Education,
Science, and Culture.

The Wistar Institute is an international leader in biomedical research, with special
expertise in cancer research and vaccine development. Founded in 1892 as the first
independent nonprofit biomedical research institute in the country, Wistar has long held
the prestigious Cancer Center designation from the National Cancer Institute. Discoveries
at Wistar have led to the creation of the rubella vaccine that eradicated the disease in the
U.S., rabies vaccines used worldwide, and a new rotavirus vaccine approved in 2006.
Wistar scientists have also identified many cancer genes and developed monoclonal
antibodies and other important research tools. Today, Wistar is home to eminent
melanoma researchers and pioneering scientists working on experimental vaccines against
flu, HIV, and other diseases. The Institute works actively to transfer its inventions to the
commercial sector to ensure that research advances move from the laboratory to the
clinic as quickly as possible. [100]

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8.3 p53 at the cross roads of cancer and ageing

ABSTRACT

The p53 tumour suppressor plays an undisputed role in cancer. p53's tumour suppressive
activity stems from its ability to respond to a variety of stresses to trigger cell cycle
arrest, apoptosis or senescence, thereby protecting against malignant transformation. An
increasing body of evidence suggests that p53 also drives organismal ageing. Although
genetic models with altered p53 function display age-related phenotypes and thus provide
in vivo evidence that p53 contributes to the ageing process, p53's role in organismal
ageing remains controversial. Anti-cancer therapies that target p53 and reactivate or
enhance its activity are considered good alternatives for treating various neoplasms.
Therefore, it is important to determine whether these clinical approaches compromise
tissue homeostasis and contribute to ageing. This review presents a number of models
with altered p53 function and discusses how these models implicate p53 as part of a
molecular network that integrates tumour suppression and ageing. Copyright © 2007
Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons,
Ltd.[101]

8.4 p53 statuses may be predictive of outcome in breast cancer

The association between p53 alterations and clinical outcome in breast cancer has been
the subject of numerous investigations. The possibility that p53 status influences
biological behaviour was raised in an early study in which the presence of p53 mutations
in aggressive breast cancer was demonstrated [37], and the majority of studies support
an association between worse survival and the presence of p53 mutations. This
association was confirmed in a comprehensive meta-analysis of the effect of somatic p53
mutations on prognosis in breast cancer [8].

Potential correlations between the type of p53 mutation and the clinical phenotype in
breast cancer have been described [38]. In this study, it was shown that mutations
affecting amino acids critical for DNA binding were associated with very aggressive
cancers, whereas null mutations and other missense mutations were associated with an
Page 47 of 62


indeterminate clinical phenotype. A recent study suggests that p53 mutation may be an
important molecular genetics correlate of breast cancer progression [39]. In a further
study of primary breast carcinomas, expression of the angiogenic vascular endothelial
growth factor was shown to correlate with poor prognosis and with mutation in p53 [40].

The serpin family member maspin is an inhibitor of angiogenesis, invasion and metastasis.
A step-wise decrease in the expression of maspin in the sequence DCIS > invasive cancer
> lymph node metastasis has been described, strongly supporting an important role in
breast cancer progression [41]. Maspin is directly transcriptionally induced by wild-type
p53, thus providing an interesting connection between p53 and progression in ductal
breast carcinomas [42]. It will clearly be of interest to determine how expression of
maspin relates to p53 status in breast cancer.

Studies of the effect of p53 mutations on chemo sensitivity of human tumours have
produced conflicting results. In breast cancer, there is evidence that specific mutations
correlate with primary resistance to doxorubicin and that the presence of such mutations
may be predictive of early relapse [43]. This hypothesis was further supported by a later
study from the same group [44]. In another study, cancers with p53 mutation were more
likely to respond to paclitaxel [45].

A number of recent reports have described the detection of tumour-specific DNA in plasma
from patients with breast carcinomas. p53 mutations can be detected in peripheral blood
in a significant proportion of patients whose primary tumours contain mutations.
Furthermore, the presence of p53 mutations in plasma DNA is strongly correlated with
various clinicopathological parameters and is a significant prognostic factor [46]. p53
autoantibodies are also detectable in patients with breast cancer. These were reported to
occur in 15% of patients but the presence of such antibodies had no relationship to
disease status [47].[102]

8.5 Suggested therapies on combination and improvement of p53 therapy

 cisplatin based chemotherapy
 Avoidance of estrogen foodstuff – eg. soy
Page 48 of 62


 Consumption of more alkaline foodstuff – as cancer survives and multiplies in acidic
environment.
 Use of herbal medicine like – tulsi [osmium sanctum], Granoda lucidum has been a
revolutionary fungal mushroom having high anti neoplastic activity .
 Use of antioxidant vitamins A .D .E etc as cancer needs oxygen for its survival .
 Use of stem therapy is also tried on experimental basis .
 Monoclonal antibody therapy.
 Human pappillavirus vaccines.
 Vectors enhance the p53 cancer gene therapy
 Viral vectors – lentivirus, adenovirus ONYX -015, herpes, retrovirus act as a carrier
of p53 gene in cancer tumor which undergo apoptosis .
 Non viral vectors –gene gun, lipofaction nakedplasmids, RNA, c ationic
liposome peptide DNA complex are carrier of this gene therapy.
 now the use of virus with combination of liposomes complex is used on animal
xenograft models[103]

8.6 p53 and stem cells: new developments and new concerns.

As the guardian of the genome, the tumor suppressor p53 prevents the accumulation of
genetic mutations by inducing cell cycle arrest, apoptosis or senescence of somatic cells
after genotoxic and oncogenic stresses. Recent studies have identified the roles of p53 in
suppressing pluripotency and cellular dedifferentiation. In this context, p53 suppresses
the self-renewal of embryonic stem cells after DNA damage and blocks the
reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). If the
inactivation of p53 pathway is a prerequisite for successful reprogramming, these findings
raise concerns for the genomic stability and tumorigenecity of iPSCs and their derivatives.
Elucidation of the roles of p53 as a barrier to pluripotency and cellular dedifferentiation
might also reveal the mechanisms by which p53 coordinates tumor suppression and
aging. [104]

Page 49 of 62


8.7 Inhibition of p53 Transcriptional Activity: A Potential Target for Future

Development of Primary Demyelination

Oligodendrogliopathy, microglial infiltration, and lack of remyelination are detected in the
brains of patients with multiple sclerosis and are accompanied by high levels of the
transcription factor p53. In this study, we used the cuprizone model of demyelination,
characterized by oligodendrogliopathy and microglial infiltration, to define the effect of
p53 inhibition. Myelin preservation, decreased microglial recruitment, and gene expression
were observed in mice lacking p53 or receiving systemic administration of the p53
inhibitor pifithrin- , compared with untreated controls. Decreased levels of
lypopolysaccharide-induced gene expression were also observed in vitro, in p53–/–
primary microglial cultures or in pifithrin- -treated microglial BV2 cells. An additional
beneficial effect of lack or inhibition of p53 was observed in Sox2+ multipotential
progenitors of the subventricular zone that responded with increased proliferation and
oligodendrogliogenesis. Based on these results, we propose transient inhibition of p53 as
a potential therapeutic target for demyelinating conditions primarily characterized by
oligodendrogliopathy.

Multiple sclerosis (MS) is a demyelinating disorder characterized by heterogeneity of
clinical and neuropathological signs and disease susceptibility (Lucchinetti et al., 2000 ).
At least two broad categories have been defined based on neuropathological findings: one
is characterized by immune-mediated demyelination occurring at perivenous locations,
whereas the other one is characterized primarily by myelin loss consequent to
oligodendrogliopathy and is associated with extensive microglial infiltration (Barnett and
Prineas, 2004 ). An additional difference between these two neuropathological findings is
the presence of remyelination attempts that are frequently detected in the immune-
mediated forms and are less prominent in cases with extensive oligodendrogliopathy
(Lucchinetti et al., 2000 ). Very little is known regarding the causes of oligodendroglial
death in MS patients; however, increased levels of the pro-apoptotic transcription factor
p53 and of its downstream genes have been detected in cases characterized by
oligodendrogliopathy, microglial infiltration, and relative lack of remyelination (Kuhlmann
et al., 2002 ; Aboul-Enein et al., 2003 ; Wosik et al., 2003 ; Stadelmann et al., 2005 ).

Page 50 of 62

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