Cell Cycle Checkpoints The Guardian Mechanisms of the

Cell Cycle Checkpoints The Guardian Mechanisms of the Genome THEY ARE DISRUPTED IN CANCER! Figure 8. 4 The Biology of Cancer (© Garland Science 2007)

Entering Mitosis Before the Completion of S phase is Bad for Chromosomes and Therefore Bad for Cells

Caffeine Abrogates the Replication Checkpoint The point: the combination of cellular stress and an inactive Checkpoint is very detrimental to cells. Molecular Biology of the Cell © 1994 by Bruce Alberts et al

How do Cell Cycle Checkpoints Work? Zhou and Elledge Nature 408, 433 - 439 (2000)

Remember Inhibitory CDK Phosphorylation? is a kinase is a phosphatase

G 2 Arrest Induced by DNA Damage Signal Sensor Transducer Effector Cytoplasm Nucleus

Remember Kinetochore/Microtubule Attachments During Metaphase? Microtubule Kinetochore

Remember the Metaphase to Anaphase Transition? Lara-Gonzalez et al. Current Biology Volume 22, Issue 22 2012 R 966 - R 980

Mitotic Defects Leading to Aneuploidy Weaver BA, Cleveland DW. Does aneuploidy cause cancer? Curr Opin Cell Biol. 2006 Dec; 18(6): 658 -667.

The Spindle Assembly Checkpoint Prevents Anaphase Errors Very robust: e. g. A single unattached kinteochore will activate the SAC Lara-Gonzalez et al. Current Biology Volume 22, Issue 22 2012 R 966 - R 980

Spindle Assembly Checkpoint Genes that are Mutated and/or Misregulated in Human Cancers Weaver BA, Cleveland DW. Does aneuploidy cause cancer? Curr Opin Cell Biol. 2006 Dec; 18(6): 658 -667.

p 53 The Master Guardian of the Genome

p 53 gene mutations in human tumors 50% Greenblatt et al. (1995) Cancer Res. 54: 4855

The Basic Paradigm of p 53 Function Genotoxic Stress (e. g. DNA damage) p 53 (low) Proliferative Stress (e. g. oncogenes) INPUTS p 53 (high) Cell cycle arrest Apoptosis OUTPUTS

The Discovery of p 53

SV 40 large T protein binds to p 53

Hypothesis Large T antigen and p 53 are oncogenes - p 53, a proto-oncogene, is expressed in low concentrations in normal cells - T antigen oncogenic activity leads to overexpression of p 53 and the latter acts as an oncogene WRONG!!

Moshe Oren Arnold Levine Cloning of the p 53 gene, followed by successive experiments showed that it is actually a tumor suppressor gene

Autosomal Dominant Li-Fraumeni syndrome Inherited germ-line mutations in p 53 cause predisposition for distinct cancers in variable ages

p 53 Mutant Mice Develop Cancer

p 53 is a transcription factor, active only as a homotetramer

p 53 acts only as a tetramer Imagine 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 p 53 molecules have some activity Missense mutations and not nonsense/frameshift are the common p 53 mutations in cancer patients

p 53 Mutations in Human Tumors are Found with High Frequency In the DNA Binding Domain In 143 families reported: point mutations (85%) deletions (9%) splice mutations (3. 5%) insertions (2%)

p 53 Binds DNA Ribbon Model Space Filling Model The most common mutation changes arginine 248, colored 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.

The Basic Paradigm of p 53 Function Genotoxic Stress (e. g. DNA damage) p 53 (low) Proliferative Stress (e. g. oncogenes) INPUTS p 53 (high) Cell cycle arrest Apoptosis OUTPUTS

Low levels of p 53 expression in normal cells Campbell et al. Biochemical Society Transactions (2001) p 53 protein levels increase upon exposure to UV (and many other agents)

Summary - p 53 is a transcription factor, acting as a homotetramer - 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 p 53 protein - p 53 protein is actually synthesized all the time, but is degraded very fast via ubiquitin mediated proteolysis

p 53 protein is ubiquitinated by the E 3 ligase MDM 2

Genetic Evidence that Mdm 2 Inhibits p 53 mdm 2 -/- p 53 -/- mdm 2 -/-

Mdm 2 is a p 53 Target Gene p 53 control of Mdm 2 transcription is a negative feedback loop

Some p 53 mutants show over expression of inert p 53 protein p 53 control of Mdm 2 transcription is a negative feedback loop

Many agents induce p 53 activity Grouped into two classes DNA damage Hyperproliferative stress p 53

What about outputs? DNA damage Hyperproliferative stress p 53 Cell cycle arrest Apoptosis

p 53 activates transcription of the CKI p 21 (p 16) (p 21)

What about outputs? DNA damage Hyperproliferative stress p 53 Cell cycle arrest Apoptosis