p53 – The tumour suppressor gene

As“Guardian of the

genome”

Author: Shreesha V BhatProfile link: http://www.pharmainfo.net/shreeshabhat

Co author: Mr. Niraj Vyas(Faculty)Profile link: http://www.pharmainfo.net/nirajvyas4me

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

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.

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.

OBJECTIVE

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.

P53 : GATEKEEPER OF GENOME

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: 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.

ONCOGENES AND TUMOUR SUPPRESSOR GENES

p53 = CANCER

P53 MUTATIONS 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)

P53 AND CANCER

FREQUENCY OF P53 MUTATIONS IN VARIOUS CANCER

As the name says this gene is made up of mass of 53kilodaton having composed of 393 amino acids located in 17th chromosome.  

STRUCTURE

The tumor suppressor gene p53 is located at chromosomes region 17p13 and is one of the most frequently mutated gene in human cancers.

LOCATION

The N-terminus is a specialised «landing pad» for kinases transducing DNA damage signals. They are responsible for the activation of transcription factors leading to cell cycle arrest and apoptosis. The core domain

contains DNA binding region –activates gene expression upon binding. This is the most mutated region of p53.

Activation of p53 requires posttranslational modifications in C terminal.Negativaly regulates DNA binding activity.Acetylation causes structural changes in this domain that allows DNA binding activity

STRUCTURE OF P53: DOMAINS

TD: Transcriptional domainPR: Proline-rich domainASPP: Apoptotic-Stimulating Protein of p53OD: Oligomerization domainBR: Basic region Oncogene 23: 2809, 2004

ACTIVATION OF P53

In a normal cell

Activation of p53 is low (kept low by MDM2)

MDM2 binds to transactivation domain

MDM2 blocks interaction of p53 with transcription apparatus and prevents Phosphorylation

p53 is transported to the cytoplasm by MDM2

MDM2 recruits histone deacetylase HDAC1 (to c-terminal lysine residues) to make p53 available for ubiquitylation.

After DNA damage

Phosphorylation and then acetylation of p53 by

protein kinases and acetylases (MDM2 overridden)

Release of p53 from MDM2 Accumulation of p53 and its

functions.

ACTIVATION OF P53

Low level p53 in normal cells

DNA damage

Posttranslational modifications ( acetylation)

Mdm2 (inhibitory protein) causes proteasomal degradation of p53

Activated p53– no effect of Mdm2

P53 free to perform its function of cell cycle arrest, apoptosis,DNA repair.

ACTIVATION OF P53

ROLE OF ACETYLATIONIn 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.

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 .

MOUSE DOUBLE MINUTE 2(MDM2)

Regulators of p53

Cell cycle regulatory genes: p21/WAF1/Cip1,G

ADD45, cyclin G

Apoptosis genes : Bax, Puma, Perp,

Noxa,

Regulators of itself: Mdm2, Cop1,

Pirh2

P53 REGULATORS

REGULATION PATHWAY

1) Cell growth arrest

2) DNA repair

3) Apoptosis – programmed cell death

4) Gene marker

FUNCTIONS OF P53

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.

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”

FUNCTIONS

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 AND APOPTOSIS

PUMA: p53 upregulated modulator of apoptosis

Pro apoptotic genes

P53 is the transcription

factor that along with

release of cyt c also

activates pro apoptotic

genes.

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.

ROLE OF APOPTOSIS

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 AND CELL CYCLE ARREST

P53- the guardian genome

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 disaseFibroblasts 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.

DNA REPAIR

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.

P53 IN MICRO RNA PROCESSING : SLICING AND DICING

Gene therapy

A revolutionary step

THERAPIES TO RESTORE P53

P53 GENE THERAPY

•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 .

Vectors

Viral vectors Non viral vectors

VECTORS ENHANCE THE P53 GENE THERAPY

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.

RECENT DEVELOPMENTS IN GENE DELIVERY : USE OF FAT DROPLET

NANOPARTICLE

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.

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.

NANO DELIVERY

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.

conclusion

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.

ACKNOWLEDGEMENTS

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

REFERENCE