p53 and Control of the Cell Cycle

1. p53 and Control of the Cell Cycle

2. Learning Objectives • Conclude the role of p53 in preserving genome stability (Bloom’s Cognition Level 2, Understand) 2. Relate the functions of p53, MDM2, and Arf to triggering apoptosis and cell cycle arrest (Bloom’s Cognition Level 3, Apply) 3. Propose a mechanism for a cell’s response to a situation in terms of a familiar story (Bloom’s Cognition Level 6, Create)

3. A Little About p53… • Acts as a tumor suppressor gene • 2 Main Functions: • halts growth and division in cell cycle under aberrant conditions • induces apoptosis • Loss of p53 function leading cause in 30-50% of various types of cancers

4. Mutant Version of p53 • Knudson’s model of tumor suppressor genes: loss of one copy (haploinsufficiency) should reduce function of the gene by ~50%. • p53 does not follow this pattern: far more than 50% efficiency in haploinsufficient cells • Experiment: Mutant p53 cDNA introduced in rat embryo fibroblasts • Point-mutated p53 allele exerted dominant function: most of the tumor suppression was lost How does this make sense?

5. Mutant Version of p53 • Most mutant p53 alleles carry point mutations in reading frames that create missense codons instead of nonsense codons • How does mutant p53 foster tumor formation? • p53 is a dominant-negative allele • p53 exists as a homotetramer: assembly of four identical polypeptide subunits

6. Structure of p53 Because it acts as a tetramer, one mutant p53 can disrupt three healthy p53 proteins in the tetramer. Cells heterozygous for p53 can still make perfectly functional homotetramers, but only 1/16 of the possible combinations is healthy. In human tumor cells mutant at p53 locus, p53 locus found to have undergone loss of heterozygosity (LOH), in which wild type allele is discarded, yielding a cell with two mutant alleles.

7. p53 Protein Molecules Have Short Lifetimes • Nuclear localization suggests that p53 protein functions as a transcription factor (TF) • 3 mechanisms regulate its activity • Level of TF in nucleus modulated: can degrade it in proteasomes • Level of TF in nucleus held constant, but intrinsic activity regulated by some covalent modifications (phosphorylation, acetylation, methylation) • Level of collaborating TFs modulated • Treat cells with cycloheximide (protein synthesis inhibitor)- p53 disappeared with half life of 20 minutes • Conclude: p53 is a highly unstable protein

8. Variety of Signals Cause p53 Induction • Receives signals from a diverse array of surveillance systems • Increased expression of TF called E2F1, de-methylation of chromosomal DNA, exposure to nitrous oxide, blockage of RNA or DNA synthesis can increase p53 levels • Genotoxic (DNA damaging) agents and physiological signals increase p53 levels to act in cytostatic fashion (growth arrest or apoptosis)

9. Contd.. • Hypoxia (lowered oxygen tension), genomic damage, imbalances in signaling pathways governing cell proliferation are experienced by cancer cells • In response, functional p53 alarm system triggered to activate p53’s function • p53 acts as the “guardian of the genome”

10. Mdm2 and ARF Battle Over the Fate of p53 • Mdm 2 recognizes and binds the N-terminal domain of p53 • Blocks the ability of p53 to act as a transcription factor • Promotes p53 export from the nucleus

11. Mdm2 and ARF Battle Over the Fate of p53 • Mdm2 targets p53 for degradation • Adds a ubiquitin moiety • Upon export to the cytoplasm polyubiquitylation occurs • Negative feedback loop • p53 promotes Mdm2 gene expression • Mdm2 targets p53 for destruction

12. Mdm2 and ARF Battle Over the Fate of p53 • Phosphorylation of Mdm2 plays an important role • Multiple sites on Mdm2 can be phosphorylated • Phosphorylation can activate or inhibit Mdm2 function • Survival signals like Akt phosphorylate and activate Mdm2, leading to cell survival • ATM and ATR phosphorylate and inactivate Mdm2, at the p53 binding domain of Mdm2. The inhibition of Mdm2 permits p53 activation

13. Mdm2 and ARF Battle Over the Fate of p53 • Is p53 helpless in its defense from Mdm2? • No! ARF binds to Mdm2 • Prevents it from binding p53 • Sequesters it in nucleolus • p53 escapes ubiquitylation, can rise to high cellular levels

14. Mdm2 and ARF Battle Over the Fate of p53 • Need balance in the regulation of ARF, p53 and Mdm2 • Over/Under expression of Mdm2 leads to cellular disruption • Mouse embryo cells without Mdm2 expression do not proliferate • Over expression of Mdm2 can lead to excess proliferation

15. Mdm2 and ARF Battle Over the Fate of p53 • Mdm2 promoter critical in expression of Mdm2 • Ease of transcription factor binding increases expression of Mdm2 • More in next session….

16. Activity 1 (5 min) What characters in the movie Batman: The Dark Knight best represent p53, Mdm2, and ARF, and why? (Use handout to record your answers) http://www.youtube.com/watch?v=FXMB5KNYgMw&feature=related

17. Activity 1 – Results • p53: Batman – Guards the genome, protects Gotham • Mdm2: Joker – Inhibits p53, creates chaos by inactivating Batman • ARF: Gordon—Binds mdm2, prevents it from binding p53; helps Batman by jailing the Joker

18. Why Is Batman p53? • Guardian of Gotham city (genome) • Acts to prevent crime (cell cycle arrest), and when necessary destroy evil (apoptosis) • Unstable depiction of heroism

19. Weapons!!!...Batman (p53) Can Be ACTIVATED! Once p53 is phosphorylated (Lucius Fox = p53 kinase called Chk2), Batman is ready to GO

20. Batman (p53) Is Signaled!! • Increase in p53 levels (Batman fighting), triggers p21Cip1 protein, a CDK inhibitor to halt cell cycle advance (allow time to fight crime) • The weapons Lucius Fox supplies Batman halt crime from happening in Gotham City

21. Who Is Mdm2 in the Dark Knight? • The Joker • Why? • Goal is to disrupt Gotham City by creating chaos • Occupies Batman so he can’t protect the city • Mdm2 prevents p53 from functioning

22. Who Is ARF in the Dark Knight? • Helps Batman • Attempts to catch the Joker • Brings the Joker to jail Officer / Commissioner Gordon

23. Activity 2 (5-10 min) Complete the handout given at the start of class (you may discuss with a partner) to match molecules/cellular activities with characters in the Batman movie

24. Activity 2 – Results • Two Face: Mdm2 Promoter – Over expression allows Joker to create more chaos • Batman’s weapons: p21cip1 (CDK inhibitor)— Batman’s weapons needed to fight crime • Gotham: Genome—Fighting over control between Batman and the Joker • Bat Signal: ATM/ATR kinases— Signals Batman when crime is occurring • Gotham Police Department: Proteins that cause apoptosis, thereby stopping DNA damage. • Lucius Fox: Chk2 kinase—gives Batman his suit to activate his capabilities to stop crime • Crime alert system: Cell cycle checkpoints—Crime taking place in Gotham city initiates Batman to take action

25. Functional p53 Triggers Apoptosis • Apoptosis is a more drastic response than halting the cell cycle • Plasma membrane herniates, forming blebs • Nucleus collapses • Cell breaks up into fragments • Neighboring cells ingest the fragments to recycle chemical matter

26. What Triggers Apoptosis? • Hypoxia • DNA damage (many causes) • Oncogene signaling Apoptosis is triggered by various stressors. Since so many things could go wrong, virtually all human cancer cells should have anti-apoptotic strategies to survive

27. Cancer Cell Strategies for Avoiding Apoptosis • Inactivation of the p53 pathway • Mutated gene • Altered promoter • Inhibition of protein • Overexpression of Mdm2 • Altered promoter • Interaction with p53

28. Mutation of p53 • Li-Fraumeni syndrome was discovered in the early 1980s • Found a group of families that showed increased susceptibility to various cancers • In 1990 found that in many of these cases, there was a mutation in chromosome 17, where the p53 gene is located • In 70% of multicancer families, mutations in the alleles of p53 found to be genetically inherited • Seemed reasonable that mutations in p53 gene should predispose a person to development of various types of tumors

29. Mutation of p53 • p53 gene is altered in almost half of all human cancer cell genomes • Have found a variety of point mutations scattered across the p53 reading frame

30. The Dark Knight • What happens to Batman in the middle of the movie? • How does this relate to loss of p53 function?

31. Overexpression of Mdm2 • A small percentage of human tumors will overexpress Mdm2 • Due to a mutation in the promoter region • Excess Mdm2 proteins bind to p53 proteins and prevent functional activation of apoptosis

32. The Dark Knight Describe Mdm2 overexpression in terms of the Joker. The Joker has a group of men working with him to fight Batman (p53) and stop its function of protecting Gotham (genome)

33. Hyperactivation of Akt/PKB Pathway • Activated by tyrosine kinase receptors and Ras • PI3K is activated, increasing PIP3 and activation of Akt/PKB • Akt/PKB can phosphorylate pro-apoptotic proteins, inhibiting them

34. Hyperactivation of Akt/PKB Pathway • Akt/PKB can also cause Mdm2 to inhibit p53 • Akt/PKB kinase can phosphorylate Mdm2 at a site that sends it to the cytoplasm to target p53 for degradation

35. Advantages Gained by Tumor Cells • When p53 is mutated, there is limited apoptosis and tumor cells can survive during hypoxia while the blood supply is made • p53 triggers apoptosis in response to DNA damage – cells with damaged DNA have better survival rate, increased gene alterations and accelerated tumor development Chaos!

36. Activity 3 – Role Play! (30 min) Time to put together everything we have learned today and characterize the Dark Knight in terms of proteins involved in cancer pathways.

37. Here are your movie characters: Batman Lucius Fox Gordon Harvey Dent/ Joker Two Face

38. Activity 3 Scenario A: Commissioner Gordon arrests the Joker and brings him to the jail. How does this affect Batman’s next action and significance to Gotham City? Scenario B: Detective Gordon is thought to be dead. How does his disappearance impact the conflict between the Joker and Batman in Gotham City? What would change if Two-Face joins the Joker?

39. Learning Objectives • Conclude the role of p53 in preserving genome stability (Bloom’s Cognition Level 2, Understand) 2. Relate the functions of p53, MDM2, and Arf to triggering apoptosis and cell cycle arrest (Bloom’s Cognition Level 3, Apply) 3. Propose a mechanism for a cell’s response to a situation in terms of a familiar story (Bloom’s Cognition Level 6, Create)

40. Questions??

41. For next time • Read research article: Post SM, Quintás-Cardama A, Pant V, Iwakuma T, Hamir A, Jackson JG, Maccio DR, Bond GL, Johnson DG, Levine AJ, Lozano G. A high-frequency regulatory polymorphism in the p53 pathway accelerates tumor development. Cancer Cell. 2010 Sep 14;18(3):220-30. • Pay close attention to the six figures in the article, and be prepared to explain them or bring specific questions about them, for discussion.

42. A High-Frequency Regulatory Polymorphism in the p53 Pathway Accelerates Tumor Development

43. Learning Objectives • Recall the functions of Mdm2 and p53 and how the regulation of each affects apoptosis and cell cycle arrest(Bloom’s Cognition Level1, Remember) 2. Graph the expected results when the conditions of a given experiment are altered (Bloom’s Cognition Level 3, Apply) 3. Invent an analogy to present biological data in a more familiar context (Bloom’s Cognition Level 6, Create)

44. Review: Why Is Batman p53? • Guardian of Gotham city (genome) • Acts to prevent crime (cell cycle arrest), and when necessary destroy evil (apoptosis) • Unstable depiction of heroism

45. Activity 1 (10 minutes) What are some other functions of p53? What is its relationship with Mdm2? What can trigger p53 inactivation in the p53/Mdm2 pathway?

46. New Material! Introduction • Mdm2 amplified in >30% of sarcomas, over-expressed in human cancers with wild type p53 • Mdm2 is a proto-oncogene, encodes E3 ubiquitin ligase that negatively regulates p53 protein stability and transcriptional activity • Mdm2 gene has two promoters. P1 controls “basal” expression and P2 is regulated by many TFs including p53. • Single Nucleotide Polymorphism (T-to-G) in P2 (SNP309G) yields homozygotes (G/G) or heterozygotes (T/G) • G/G mutation enhances binding of transcriptional activator Sp1 to P2 resulting in increase in Mdm2 transcription

47. Single Nucleotide Polymorphism • A single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population • 2 alleles differ in one location • Common: 1 of every 300 nucleotides, on average • Most often occur between genes, have no effect

48. Increased levels of Mdm2, decreased levels of p53 -> increased cancer risk! • Mdm2-SNP309G associated with an increased cancer risk in human tumors that express wild type p53, but not in cells with mutant p53 • To understand mechanisms regulating this pathway during tumorigenesis, most research focuses on mouse models that genetically delete p53, over express genes that regulate p53, or produce mutant proteins that mimic human mutations • This study used naturally occurring polymorphism in Mdm2 promoter to generate two humanized Mdm2 (SNP309G and SNP309T) alleles to examine polymorphoism on tumor development

49. Figure 1A: Generation of Mice Containing Either the Humanized Mdm2-SNP309G (mutant) or Mdm2-SNP309T (control) Allele • - Generated the humanized allele constructs by replacing mouse intron 1 containing P2 promoter with human intron 1 with G or T polymorphism • Black boxes are Mdm2 exons • Mdm2 translation begins at exon 3, downstream from construct

50. Figure 1B - Southern Blot Analysis revealed correct targeting of both constructs at Mdm2 locus - Southern Blot Analysis using probe verified single copy integration and indicated absence of other insertions