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

G2 Arrest Induced by DNA Damage Signal Sensor Transducer Effector Cytoplasm Nucleus

Remember Kinetochore/Microtubule Attachments During Metaphase? Kinetochore Microtubule

Remember the Metaphase to Anaphase Transition? Lara-Gonzalez et al. Current Biology Volume 22, Issue 22 2012 R966 - R980

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 R966 - R980

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.

The Master Guardian of the Genome p53 The Master Guardian of the Genome

Greenblatt et al. (1995) Cancer Res. 54:4855 p53 gene mutations in human tumors 50% Greenblatt et al. (1995) Cancer Res. 54:4855

p53 (high) The Basic Paradigm of p53 Function INPUTS OUTPUTS Genotoxic Stress (e.g. DNA damage) Proliferative Stress (e.g. oncogenes) INPUTS p53 (high) p53 (low) Apoptosis OUTPUTS Cell cycle arrest

The Discovery of p53

SV40 large T protein binds to p53

Large T antigen and p53 are oncogenes 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 gene Moshe Oren Arnold 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 tetramer Imagine a scenario: - One normal copy - One lof copy, encoding a mutated protein that can still bind to its partners

Not quite, even 1/16 of p53 molecules have some activity 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 High Frequency In the DNA Binding Domain In 143 families reported: point mutations (85%) deletions (9%) splice mutations (3.5%) insertions (2%)

p53 Binds DNA Ribbon Model Space Filling Model 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 (high) The Basic Paradigm of p53 Function INPUTS OUTPUTS Genotoxic Stress (e.g. DNA damage) Proliferative Stress (e.g. oncogenes) INPUTS p53 (high) p53 (low) Apoptosis OUTPUTS Cell cycle arrest

Low levels of p53 expression in normal cells Campbell et al. Biochemical Society Transactions (2001) p53 protein levels increase upon exposure to UV (and many other agents)

Summary - p53 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 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-/-

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

Hyperproliferative stress Many agents induce p53 activity Grouped into two classes DNA damage Hyperproliferative stress p53

Hyperproliferative stress What about outputs? DNA damage Hyperproliferative stress p53 Cell cycle arrest Apoptosis

p53 activates transcription of the CKI p21

Hyperproliferative stress What about outputs? DNA damage Hyperproliferative stress p53 Cell cycle arrest Apoptosis