p53The Master Guardian of the Genome

p53 gene mutations in human tumors

Lung (897) 56% Adrenal (31) 23% Colon (960) 50% Breast (1536) 22% Esophagus (279) 45% Endometrium (224) 22% Ovary (386) 44% Mesothelioma (23) 22% Pancreas (170) 44% Renal (102) 19% Skin (220) 44% Thyroid (299) 13% Gastric (314) 41% He matological (1916) 12% He ad & neck (524) 37% Carcinoid (13) 11% Bladder (308) 34% Melanoma (70) 9% Sarc oma (339) 31% Parathyroid (13) 8% Prostate (87) 30% Cevix (350) 7% He patoce llular (716) 29% Neuroblastoma (212) 1% Brain (456) 25% others (155) 0%

Tumor type (n) p53 mutation Tumor ty pe ( n)

p53 mutation

All tumors: 37%

Greenblatt et al. (1995) Cancer Res. 54:4855

50%

p53(low)

p53(high)

Cell cycle arrest Apoptosis

Genotoxic Stress(e.g. DNA damage)

The Basic Paradigm of p53 Function

INPUTS

OUTPUTS

Proliferative Stress(e.g. oncogenes)

The Discovery of p53

SV40 large T protein binds to p53

T

53 K

61 2 3 4 5

IPNRS anti-T

Hypothesis

Large T antigen and p53 are oncogenes

- p53, a proto-oncogene, is expressed in low concentrations in normal cells

- T antigen oncogenic activity leads to over-expression of p53 and the latter acts as an oncogene

WRONG!!

Cloning of the p53 gene, followed by successive

experiments showed that it is actually a

tumor suppressor geneMoshe

OrenArnold Levine

Autosomal Dominant Li-Fraumeni syndrome

Inherited germ-line mutations in p53 cause predisposition for distinct cancers in variable ages

p53 Mutant Mice Develop Cancer

p53 is a transcription factor, active only as a homotetramer

p53 acts only as a tetramerImagine a scenario:- One normal copy- One lof copy, encoding a mutated protein that can still bind to its partners

Does this mean that +/- heterozygotes do not need a second mutation for tumor progression?

Not quite, even 1/16 of p53 molecules have some activity

Missense mutations and not nonsense/frameshift are the common p53 mutations in cancer patients

p53 Mutations in Human Tumors are Found with HighFrequency In the DNA Binding Domain

In 143 families reported:point mutations (85%)deletions (9%)splice mutations (3.5%) insertions (2%)

Ribbon Model Space Filling Model

p53 Binds DNA

The most common mutation changes arginine 248, colored red here. Notice how it snakes into the minor groove of the DNA (shown in blue and green), forming a strong stabilizing interaction. When mutated to another amino acid, this interaction is lost. Other key sites of mutation are shown in pink, including arginine residues 175, 249, 273 and 282, and glycine 245.

p53(low)

p53(high)

Cell cycle arrest Apoptosis

Genotoxic Stress(e.g. DNA damage)

The Basic Paradigm of p53 Function

INPUTS

OUTPUTS

Proliferative Stress(e.g. oncogenes)

Low levels of p53 expression in normal cells

p53 protein levels increase upon exposure to UV (and many other agents)

Campbell et al. Biochemical Society Transactions (2001)

- p53 is a transcription factor, acting as a homotetramer

Summary

- Expressed when cells gone awry

- Two mutated copies in tumors, first is usually a dominant-negative mutation

- Acts as a tumor suppressor gene

In normal cells we find only low concentrations of the p53 protein

- p53 protein is actually synthesized all the time, but is degraded very fast via ubiquitin mediated proteolysis

p53 protein is ubiquitinated by the E3 ligase MDM2

Genetic Evidence that Mdm2 Inhibits p53

p53-/- mdm2-/-

p53-/- mdm2-/-

Mdm2 is a p53 Target Gene

p53 control of Mdm2 transcription is a negative feedback loop

Some p53 mutants show over expression of inert p53 protein

p53 control of Mdm2 transcription is a negative feedback loop

p53

DNA damage

Hyperproliferative stress

Many agents induce p53 activity

Grouped into two classes

Extensive DNA damage recruits the DNA damage response machinery

Two key players:The protein kinases ATM and ATR

ATM and ATR are recruited to distinct sites and phosphorylate downstream effectors

ATR is recruited to single - stranded DNA

- ATR-dependent phosphorylation of the Rad9 adaptor protein is needed for activating Chk1

- Activated Chk1 is released to phosphorylate its effectors

ATR

Rad9

Chk1 (inactive)

Chk1 (active)

Phosphorylation of p53 (by ATM/ATR and/or Chk2) makes it unable to bind MDM2

Phosphorylated p53 acts as a transcription factor

DNA damage response activates p53 by stabilizing the protein via phosphorylation (and additional mechanisms)

Additional inhibitory phosphorylation of MDM2

p53

DNA damage

Hyperproliferative stress

E2Fs

e.g. high activity of E2Fs

Hyperproliferative stress response is mediated through the ARF protein

- E2Fs induce transcription of the ARF gene

- ARF binds to and sequesters MDM2- p53 is stabilized

Over activity of oncogenes stimulates apoptosis through ARF

ARF stands for Alternative Reading Frame

- Found in the same locus of the p16 gene

- Uses an alternative promoter

p53 and Ink4a are the two most frequently mutated genes in human tumors

LocusChromosome location type of alterations

estimated frequency of alterations

p53

INK4a

17p13

9p21

nucleotide substitution

homozygous deletion nucleotide substitution small deletion/insertion promoter methylation

~ 50%

~ 40%

- p53 is a transcription factor, acting as a homotetramer

Summary

- Transcribed constitutively, but has a very short half life

- DNA damage and a stalled replication fork induce p53 phosphorylation and activation

- Hyperproliferative stress (e.g. oncogenic signaling, hypoxia) activates p53 via ARF

- Ubiquitinated by the E3 ligase MDM2

p53

DNA damage

Cell cycle arrest Apoptosis

What about outputs?

Hyperproliferative stress

(p16)

(p21)

p53 activates transcription of the CKI p21

p53

DNA damage

Cell cycle arrest Apoptosis

What about outputs?

Hyperproliferative stress

Programmed cell death: aka Apoptosis

Specific cells are dying in a programmed manner

http://mathematica.edublogs.org/files/2011/03/C-elegans-2kbu264.JPG

Worms are transparent

C. elegans

One can follow the individual lineage of each of the cells

<1000 cells

http://www.imsc.res.in/~sitabhra/research/neural/celegans/

Deciphering the cell lineage of C. elegans

Cell death is also a “cell fate”

Use genetics to identify mutants

Epistasis analysis and biochemical studies allow the scientists to build a pathway

Genetic control of programmed cell death

CED-4No death signal

CED-4 is inactive

Death signal

CED-9

Activates CED-3 caspase for the killing

CED-9CED-4

ced-9 Cell deathced-3ced-4

Gain of function mutations in bcl-2 were associated with human cancers (bcl= b cell

lymphoma resulting from a translocation event)

Our body uses the same apoptotic mechanisms used in PCD, for killing “bad” or unwanted cells

http://herkules.oulu.fi/isbn9514266676/html/i267425.html

Lineage and programmed

cell deathBob Horvitz John Sulston Sydney Brenner

Physiology and Medicine 2002

Nobel Prize

- Pro-apoptotic signals open up the channels, allowing cyt C to be released to the cytoplasm

A simplified representation of apoptosis

NoxaProapoptotic(BH3-only)

Proapoptotic(multidomain)

Bax

Bcl-2

Cytochrome C

Apaf

Procaspase

Caspase

Mitochondria

All the regulators are from the same protein family (containing BH domains)

Antiapoptotic

http://www.weizmann.ac.il/home/ligivol/apoptosis_project/apoptotic_pathways.html

p53 triggers apoptosis