1. p53 and Apoptosis: Master Guardian and Executioner
The Biology of Cancer
Chapter 9:
p53 and Apoptosis: Master Guardian and Executioner

2. Why p53? Need to eliminate malfunctioning cells
Must have a decision maker (p53)
If too much damage, initiates programmed cell suicide (Apoptosis)
P53 is dangerous for the cancer cell because its job is to kill cancer cell

3. Discovery of p53
Originally identified as an oncogene in association with Large T antigen in SV40
Over-expressed in murine tumors
Forms found in tumors are mutant, have to amino acid changes in functional domains
Does not conform to Knudson’s “two hit hypothesis” model

4. How can a mutant form foster tumor growth?
Yeast: Mutant alleles of gene can interfere with wild-type gene function
“Dominant-interfering” or “Dominant-Negative”
Wild-type P53 found to be a homo-tetramer (made from 4 identical units)
Single mutation produce normal and error copies of each unit in equal proportion

5. Figure 9.7a The Biology of Cancer (© Garland Science 2007)

6. Out of 16 tetramers, only one will function normally
Figure 9.7b The Biology of Cancer (© Garland Science 2007)

7. What happens when there is a mutation in p53?
Mutant form makes tetramer but it cannot function normally
P53 efficiency reduced to 1/16 th of normal
Advantage for tumors
Many tumors also have LOH at mutation locus for p53
They cannot tolerate even 1/16 efficiency of p53

8. Figure 9.4 The Biology of Cancer (© Garland Science 2007)

9. Variation in type of mutation seen in different genes in cancer
Figure 9.6a The Biology of Cancer (© Garland Science 2007)

10. Most p53 mutations are in the DNA binding domain
Figure 9.6b The Biology of Cancer (© Garland Science 2007)

11. P53 has a short half-life Cycloheximide blocks protein synthesis
When added to cells with wild-type p53 in-vitro, amount of p53 protein decayed with ½ life of 20 minutes
p53 has a “futile” cycle
Useful way to create a “fast switch” for a critical protein. Turns on immediately when its degradation is inhibited.

12. What activates p53? X-rays, UV radiation damage
DNA damaging chemo agents
Inhibitors of DNA synthesis
Microtubule disruption
Many carcinogens, drugs
Hypoxia
High Nitrous Oxide concentrations

13. More activation signals
Oncogene upregulation
Changes in methylation of chromosomes
Acidified growth medium
Reduction in ribonucleotides
Blocks in RNA/DNA synthesis

14. P53 causes “growth arrest” if it senses damage
Initiates a “Repair” program
If damage persists or repair fails, initiates cell suicide
Figure 9.8 The Biology of Cancer (© Garland Science 2007)

15. How p53 stops cell cycle X rays upregulate p53 which induces p21Cip1
p21 binds to Cyclin D, CDK4/6
X-rays do not induce
p21 when p53 mutated
Direct correlation seen in
p53 levels and apoptotic
rate for different amounts
of X-ray
Figure 9.9 The Biology of Cancer (© Garland Science 2007)

16. P53 protein is transcription factor for MDM2
p53/MDM2 “futile” cycle
P53 protein is transcription factor for MDM2
MDM2 ubiquitylates p53 for degradation in proteasome
Figure The Biology of Cancer (© Garland Science 2007)

17. P53 protein recognizes a specific DNA sequence:
Pu-Pu-Pu-C-A/t-T/a-G-Py-Py-Py repeated twice with a gap of 0-13
MDM2 binds p53 protein in a small N-terminus region
Grey area allows it to form a tetramer
Red: NLS or Nuclear Localization Signals + amino acid for DNA binding
“Proline rich” region contributes its apoptotic activity
Figure The Biology of Cancer (© Garland Science 2007)

18. p53 activation mechanism
Double stranded DNA breaks (X-rays)
ATM ATR Kinase  phosphorylation of p53 to stabilize it
DNA damaging agents
ATM CKII phosphorylate p53
Aberrent Growth signals
e.g. (pRb-E2F) disregulation: Mechanism ?
Hypoxia: Mechanisms?

19. P53 stabilized by phosphorylation
Phosphorylation done by kinases like ATM, Chk1, Chk2 which are activated by DNA damage
They alter the p53 domain recognized by MDM2
ATM can also phosphorylate MDM2 to deactivate it
ATM  p53
ATM --| MDM2
MDM2 --| p53
Figure The Biology of Cancer (© Garland Science 2007)

20. MDM2 is a “funny” oncogene
Acts by “antagonizing” a tumor suppressor
Prevents entry into cell cycle arrest, senescence, cell suicide
MDM2 is under control of other proteins:
E.g. Ras Raf  MapK  ETS/AP-1 which through FOS + Jun transcription factors can increase MDM2 levels
SNP 309 in MDM2 intron is cancer promoting

21. P14ARF ARF = Alternate Reading Frame
Transcriptional promotor 13 kb upstream of gene for p16INK4A deletes exon 1 and an alternate reading frame in Exon 2
Result = ARF mRNA
Figure The Biology of Cancer (© Garland Science 2007)

22. ARF is a tumor suppressor
Shuts down MDM2 by sequestering it in nucleolus
ARF + p53 monitor intracellular signalling and cell cycle
pRb --| E2F  ARF --| MDM2 --| p53  death
p14ARF has an E2F recognition sequence
Elimination of ARF removes check on E2F levels

23. Figure 9.15b The Biology of Cancer (© Garland Science 2007)

24. Table 9.2 The Biology of Cancer (© Garland Science 2007)

25. P53 is not involved in “routine” cell death e.g. in morphogenesis
Mouse hand morphogenesis. Black dots are cells undergoing apoptosis
Figure The Biology of Cancer (© Garland Science 2007)

26. Li-Fraumini Syndrome
Predisposition to Glioblastomas, Leukemias, Breast, Lung, Pancreatic tumors
Mandelian inheritance of trait
Traced to scattered mutations in p53 gene on Chr17-p13
Figure The Biology of Cancer (© Garland Science 2007)

27. The bcl-2 Oncogene
B-Cell Lymphoma Gene seen to be upregulated in tumors
Mutation is reciprocal translocaton in Chr14/Chr18
Mutant bcl-2 inserted in mouse germline and expressed in lymphocyte precursor has no effect on survival
Myc upregulation + bcl-2 mutation killed mice in < 2 months
Myc upregulation is also potent killer but not as
potent as Myc + bcl-2
Figure 9.22b The Biology of Cancer (© Garland Science 2007)

28. Mechanism of action of bcl-2
Mutant bcl-2 prolongs survival of lymphocytes in-vitro
In vivo, prolongs lymphocyte life but cells are not proliferating  no tumors induced
Bcl-2 seems to act in the “opposite way” from p53, causing a halt in “death” program

29. Figure 9.27c The Biology of Cancer (© Garland Science 2007)

30. Apoptosis and bcl-2 bcl-2 is anti-apoptotic
Found localized in outer membrane of mtDNA !
Apoptosis is triggered by depolarization of outer membrane of mtDNA and release of Cytochrome C
Cytochrome C associates with other proteins to initiate cell death

31. Apoptosis = “Programmed Cell Death”
Figure The Biology of Cancer (© Garland Science 2007)

32. Two apoptotic pathways
P53 induced – driving genes
like Bax which open
mtDNA channels
Extracellular, initiated by
signals from
transmembrane
“death receptors”

33. There is a third pathway
T-cell and NK cell mediated
Immune system
Killer cells attach a protease (like a bomb) to cell outer membrane. When internalized it cleaves and activates procaspase 3,8,9  Cell death

34. Convergence of Intrinsic and Extrinsic Apoptotic pathways
Figure The Biology of Cancer (© Garland Science 2007)

35. P53 can also use the extrinsic pathway by
inducing a gene for the Fas “death” receptor
inducing IGFBP-3 which binds to extracellular IGF-1,2 ligands
to reduce effect of external anti-apoptotic signals
Figure The Biology of Cancer (© Garland Science 2007)

36. Anti-Apoptotic Strategies of Cancer Cells
Point mutations in p53 to disable pathways
Deletion or promotor methylation of ARF
Overexpression of MDM2
Mis-localization of p53 (sequestering in cytoplasm)
Hijacking of apoptotic pathway (Melanoma)
Inactivating Bax (Colon Cancer)
Disregulating bcl-2 (Follicular Lymphoma)
Hyperactivation of PI3K  Aft  PkB pathway
Activation of NF-kB pathway

37. Anti-apoptotic Pathway:
Figure The Biology of Cancer (© Garland Science 2007)

38. Table 9.5 The Biology of Cancer (© Garland Science 2007)

39. Different post transcriptional modifications of p53 protein
Figure The Biology of Cancer (© Garland Science 2007)

40. Autophagy or Cell Self-Cannibalism
A survival mechanism. Is it a mechanism for therapy?
Beclin-1 induces autophagy and is down-regulated by tumors
Figure The Biology of Cancer (© Garland Science 2007)

41. The p53 circuit board
Figure The Biology of Cancer (© Garland Science 2007)