Ramanbhai Patel College of Pharmacy, Education Campus Changa, Anand- 388 421.

India

p53 – The tumour suppressor

gene
As
“Guardian of the genome”

Author: Shreesha V Bhat Co author: Mr. Niraj Vyas(Faculty)

Profile link: Profile link:
http://www.pharmainfo.net/shreeshabhat http://www.pharmainfo.net/nirajvyas4m
e
 INTRODUCTION
Cancer, today has turned the most devastating disease of all
times. The statistics itself reveals the true nature of the disease.

Various approaches have been used in the cancer treatment like

chemotherapy, radiation, surgery, etc.

But the need is for latest therapies like gene therapy which could
help to treat various types of cancer effectively.

   Human P53 gene is a tumour suppressor gene and its resulting

protein (p53 protein) is found in human cells.

Previous research has shown that in about 70% of all human

cancers, P53 gene is mutated.
 OBJECTIVE
The present work focuses on the tremendous potential of p53 gene
used as a suppressor gene to kill the tumour cells in cancer therapy.

The main objective is to study the role of p53 mutation in cancer, its
anatomy, its activation and regulatory pathways and its various functions.

Moreover to study the role of p53 in cell cycle arrest, apoptosis, DNA
repair and ultimately in cancer.

Lastly, the focus is on the p53 gene therapy in cancer and various
current researches related to the therapy.
P53 : GATEKEEPER OF GENOME

A gene which encodes a protein that regulates all growth and

is able to cause potential cancerous cells to destroy themselves.
The gene is an antioncogen.

“guardian of the genome”.

Transcription factor.
Key tetrameric protein in mammalian cell’s stress response
(DNA damage).
Regulates critical cellular function involving the G1 and G2
cell-cycle check points in response to DNA damage and
apoptosis induced by certain stimuli, such as DNA damaging
agents and hypoxia.
Inhibits and prevents tumor growth.
WHAT ARE TUMOUR SUPPRESSOR
GENES?

Tumor suppressor genes are

normal genes that slow down
cell division, repair DNA
mistakes, and tell cells when to
die (a process known as
apoptosis or programmed cell
death).

When tumor suppressor genes

don’t work properly, cells can
grow out of control, which can
lead to cancer. About 30 tumor
suppressor genes have been
identified, including p53,
BRCA1, BRCA2, APC, and RBI.
ONCOGENES AND TUMOUR
SUPPRESSOR GENES

Oncogenes:
1) Activation (TURNING
ON) leads to cancer.
2) Single mutation
enough to form
oncogene.

Tumour suppressor
genes:
1) Inactivation
( TURNING OFF) leads to
cancer.
2) Two mutations
required to inactivate TS
genes.
P53 MUTATIONS AND
CANCER

p53 = CANCER
P53 AND CANCER

Mutations in p53 or the pathway that

directly regulates it have been found in over
80% of human tumors..."
-         Lozano and Elledge (2000)
FREQUENCY OF P53 MUTATIONS IN
VARIOUS CANCER
 STRUCTURE
As the name says this gene is made up of mass of 53kilodaton
having composed of 393 amino acids located in 17th chromosome.  
 LOCATION
The tumor suppressor gene p53 is located at chromosomes
region 17p13 and is one of the most frequently mutated gene in
human cancers.
 STRUCTURE OF P53:
DOMAINS

The N-terminus is a specialised

«landing pad» for kinases Activation of p53
transducing DNA damage requires posttranslational
signals. They are responsible for modifications in C
the activation of transcription terminal.
factors leading to cell cycle Negativaly regulates
arrest and apoptosis. The core domain DNA binding activity.
contains DNA binding Acetylation causes
region –activates gene structural changes in this
expression upon domain that allows DNA
binding. This is the binding activity
most mutated region
of p53.
TD: Transcriptional domain
PR: Proline-rich domain
ASPP: Apoptotic-Stimulating Protein of p53
OD: Oligomerization domain
BR: Basic region Oncogene 23: 2809, 2004
 ACTIVATION OF P53
a l c ell After DNA dam
In a no rm a ge

 Activation of p53 is low (kept low by  Phosphorylation and

MDM2) then acetylation of p53 by
 MDM2 binds to transactivation protein kinases and acetylases
domain (MDM2 overridden)
 MDM2 blocks interaction of p53 with
 Release of p53 from MDM2
transcription apparatus and prevents
Phosphorylation  Accumulation of p53 and its
 p53 is transported to the cytoplasm functions.
by MDM2
 MDM2 recruits histone deacetylase
HDAC1 (to c-terminal lysine residues)
to make p53 available for
ubiquitylation.
 ACTIVATION OF P53

Low level p53 in DNA

normal cells damage

Mdm2 (inhibitory Posttranslational

protein) causes modifications
proteasomal Activated p53– no ( acetylation)
degradation of p53 effect of Mdm2

P53 free to perform its

function of cell cycle arrest,
apoptosis,DNA repair.
ACTIVATION OF P53
 ROLE OF ACETYLATION
In the «latent» (inactive) form, the protein is constitutively unstable and adopts a
conformation in which the extreme C-terminal domains hinder the interactions of the DNA-
binding domain with its target .

Posttranslational modification like acetylation change in conformations

promote DNA binding activation of p53.

Several lysines in the C-terminus are covalently modified by acetylation, including lysine 320,
373 and 382.
Acetylation occurs in response to many forms of DNA-damage..
1. Acetylation may contribute to stabilise p53 by concealing lysines used as target sites for
ubiquitin, therefore inhibiting degradation.
2. Acetylation may induce conformational rearrangements of the C-terminus, increasing
DNA binding capacity.
3. Acetylation may play a role in the regulation of compartmentalisation of p53 between
nucleus and cytoplasm.
MOUSE DOUBLE MINUTE 2(MDM2)

The mdm2 gene encodes a zinc finger protein that negatively regulates p53
function by binding and masking the p53 transcriptional activation domain.
MDM2 protein inhibits p53 activity during normal cell growth.

By binding to p53, mdm2 not only earmarks the protein for degradation but
also conceals transcription activation domain and mediates p53 export from
the nucleus into the cytoplasm .

Furthermore, mdm2 expression is activated by p53, defining a feedback loop

in which mdm2 controls the level, extent and duration of p53 protein
activation

 The mdm2 protein is able to shuttle between the nucleus and the
cytoplasm and it is known to bind to the p53 protein in the N-terminal region.
Through binding to p53, mdm2 shuttles p53 out of the nucleus to the
cytoplasm for degradation .
 P53 REGULATORS
Apoptosis genes :
Bax, Puma, Perp,
Noxa,

Cell cycle
Regulators of
regulatory genes:
itself: Mdm2,
p21/WAF1/Cip1,G
Cop1, Pirh2
ADD45, cyclin G

Regulators
of p53
REGULATION PATHWAY
 FUNCTIONS OF P53
1) Cell growth arrest

2) DNA repair

3) Apoptosis – programmed cell death

4) Gene marker

p53 protein binds in sequence specific manner to sites (p53-

Response elements) in certain genes (p53-Target Genes) such
as WAF-1, BAX, MDM2 etc as a transcription factor.

Resulting regulatory protein checks the cell cycle and directly

initiates DNA-damage repair or cell destruction (Apoptosis)
based on the degree of DNA damage.
 FUNCTIONS
The p53-induced activation of target genes may result in
the induction of growth arrest either before DNA
replication in the G1 phase of the cell cycle or before
mitosis in the G2 phase.

The growth arrest enables the repair of damaged DNA.

 By programmed cell death, which is often referred to as

apoptosis according to its morphological appearance, the
cells damaged beyond repair are eliminated thus
preventing the fixation of DNA damage as mutations.

Because these processes ensure genomic integrity or

destroy the damaged cell, p53 has been called the
“guardian of the genome”
 P53 AND APOPTOSIS
P53 protein starts a pathway that
releases cyt c from mitochondria.

This cytochrome c complexes

with protein Apaf-1 and together
they activate capsase 9.

The effector caspases (e.g

caspase 3) start a pathway that
results in cleavage of cell
constituents : DNA, cytoskeletal
components, enzymes,etc.

Later phagocytosis of these

remaining components by
macrophages mark the end of
apoptosis.
 P53 is the
transcription
factor that
along with
release of cyt
c also
activates pro
apoptotic
genes.

Pro apoptotic
genes

PUMA: p53 upregulated modulator of apoptosis

 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. 
Possibilities include:

Control of malignant disease

Delay of premature senescence in neurodegenerative disease

Regulation of inflammatory disease

Treatment of autoimmune disorders

 Minimizing the area of infarct in ischemic disease, e.g. stroke,

MI.
 P53 and cell cycle arrest

P53
CHECKPOINT
 P53 AND CELL CYCLE
ARREST

P53 arrests the cell cycle primarily by up

regulating p21 (Cip1/Waf-1), which inactivates
CDK/cyclin.
Involvement of cyclins ensure successful
transitions from S phase to G1.
Since p21 (cyclin dependent kinase inhibitor-
p21) inhibits CDKs, it results in inhibition of
both G1 to S and G2 to mitotic transitions.

P21 is a kinase inhibitor

P53- the guardian
genome
 DNA REPAIR
DNA repair prevents the accumulation of mutations.

Every time a cell prepares to divide into 2 new cells, it must

duplicate its DNA. This process is not perfect, and copying errors
sometimes occur.

Fortunately, cells have DNA repair genes, which make proteins

that proofread DNA. But if the genes responsible for the repair
are faulty, then the DNA can develop abnormalities that may lead
to cancer.

Thus p53 plays a pivotal role in DNA repair and thus combating
cancer.

P53 as a biomarker in alzheimer’s disase

Fibroblasts derived from AD patients expressed an altered conformational
status of p53 and were less sensitive to p53-dependent apoptosis
compared to fibroblasts from non-AD subjects.
Results from research show the potential of p53 as a biomarker in AD.
P53 IN MICRO RNA PROCESSING :
SLICING AND DICING
p53 helps slice long pieces of RNA into small regulatory
molecules called microRNAs. These microRNAs help control
production of proteins, including some involved in cell
proliferation, which can lead to cancer if unchecked.

The p53 could direct the production of long RNAs, called

primary transcripts, which eventually are broken up into
microRNAs. Previous work has shown that the protein turns on
production of a long RNA molecule that gets chopped into a
microRNA called miR-34.

Levels of both the hairpin-shaped intermediates and mature

microRNAs were lower in cells in which p53 was mutated.

Some of the affected microRNAs control production of proteins

involved in cell proliferation. Having too little of these microRNAs
could allow too much of these growth-promoting proteins to be
made, leading to uncontrolled growth and cancer.
 Gene
therapy

A revolutionary
step
THERAPIES TO RESTORE P53
P53 GENE THERAPY
 VECTORS ENHANCE THE P53 GENE
THERAPY
Vectors

Viral vectors Non viral vectors

•Viral vectors – lentivirus, adenovirus ONYX -015,herpes,retrovirus  act as

a carrier of p53 gene in cancer tumour which undergo apoptosis .

•Non viral vectors –gene gun , lipofaction naked plasmids , RNA , cationic
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 .
RECENT DEVELOPMENTS IN GENE DELIVERY
: USE OF FAT DROPLET NANOPARTICLE

The difficulties faced in the vector delivery system ( degradation

of the vector by immune system) can be overcome by the use of fat
drop nanoparticle.

This development is the first systemic, non-viral, tumor-targeted,

nanoparticle method designed to restore normal gene function to
tumor cells while completely bypassing normal tissue.
 NANO DELIVERY
In a work using animal models, functional p53 genes were delivered to
tumor cells and tumor metastases in 16 different types of cancer, including
prostate, pancreatic, melanoma, breast cancer and head and neck cancer.

 The presence of the replacement genes dramatically improved the efficacy

of conventional cancer therapy.

Thus the use of P53 delivery system eventually would allow physicians to
use a lower dose of therapies, achieving the same or enhanced therapeutic
results but sharply diminishing the side effects so troublesome in many
treatments.

The nanoparticle delivery system is designed in such a way that when a

normal P53 suppressor gene is reinstated, the nanoparticle – essentially
a little fat droplet wrapped around the gene – simply melts away,
unlike non-biodegradable delivery systems.
 conclusion
The cancer susceptibility genes p53
have been tested in ovarian & breast
cancer patients, , and have shown
some potential for this antitumor
strategy.
The p53 tumor suppressor and its
surrounding molecules are now the
focus of thousands of studies in
laboratories around the world.
These studies may one day lead to
new treatments for the most frequent
and life-threatening of cancers.
Truly, the the guardian of the
genome will hold the guard of “
GUARDIAN OF THE WHOLE HUMAN
MANKIND” in the near future.
ACKNOWLEDGEMENTS

I am thankful to my sister for her constant encouragement and support

during this preparation.

My sincere thanks to Mr. Niraj Vyas (lecturer, RPCP) for his everlasting
support and guidance.

I would also like to thank my institute and all the faculty members for
their love and support.

Last but not the least, I would like to thank my family and colleagues
without whom this presentation would not be possible.
 REFERENCE
1) Harris CC: The p53 tumor suppressor gene as a target for new anticancer
therapies.Adv Oncol 1998, 14:3-7.

2) Apoptosis. Its significance in cancer and cancer therapy. Kerr JF, Winterford CM,
Harmon BV.Department of Pathology, University of Queensland Medical School,
Herston, Australia .

3) Park BH, Vogelstein B. Tumor-suppressor genes. In: Kufe DW, Pollack RE,
Weichselbaum RR, et al, eds. Cancer Medicine. 6th ed. Hamilton, Ontario: BC Decker;
2003: 87–106.

4) L. Hjortsberg, J.M. Rubio-Nevado, D. Hamroun, C. Béroud, M. Claustre and T Soussi,.

The p53 Mutation handbook 2.0, available online; http://p53.free.fr