1. Cell Cycle Checkpoints The Guardian Mechanisms of
the Genome
THEY ARE DISRUPTED
IN CANCER!
Figure 8.4 The Biology of Cancer (© Garland Science 2007)

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

3. 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

4. How do Cell Cycle Checkpoints Work?
Zhou and Elledge Nature 408, (2000)

5. Remember Inhibitory CDK Phosphorylation?
is a kinase is a phosphatase

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

7. Remember Kinetochore/Microtubule Attachments
During Metaphase?
Kinetochore
Microtubule

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

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

10. 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 R966 - R980

11. 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 Dec;18(6):

12. The Master Guardian of the Genome
p53
The Master Guardian of the Genome

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

14. 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

15. The Discovery of p53

16. SV40 large T protein binds to p53

17. 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!!

18. Cloning of the p53 gene, followed by successive experiments showed that it is actually a
tumor suppressor gene
Moshe Oren
Arnold Levine

19. Autosomal Dominant Li-Fraumeni syndrome
Inherited germ-line mutations in p53 cause predisposition for distinct cancers in variable ages

20. p53 Mutant Mice Develop Cancer

21. p53 is a transcription factor, active only as a homotetramer

22. 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

23. 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

24. 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%)

25. 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.

26. 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

27. 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)

28. 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

29. 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

30. p53 protein is ubiquitinated by the E3 ligase MDM2

31. Genetic Evidence that Mdm2 Inhibits p53
p53-/- mdm2-/-

32. Mdm2 is a p53 Target Gene
p53 control of Mdm2 transcription is a negative feedback loop

33. Some p53 mutants show over expression of inert p53 protein
p53 control of Mdm2 transcription is a negative feedback loop

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

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

36. p53 activates transcription of the CKI p21

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