Cancer and the Cell Cycle

1. Cancer and the Cell Cycle

2. Outline of the lecture • What is cancer? • Review of the cell cycle and regulation of cell growth • Which types of genes when mutated can  cancer? • Roles for screening for mutations in specific genes • Tumor suppressor p53 • Have you figured it out?

3. What is cancer? • Cancer = uncontrolled proliferation of cells within the body  tumor. • Tumor = clone of cells resulting from series of sequential genetic mutations  loss of growth control. • Cancer is also known as malignancy. • Development of cancer = oncogenesis • Study or treatment of cancer = oncology

4. Cancer is a multi-step process.

5. How does this happen? In the following slides: = a non- dividing cell 1, 2, 3 = successive mutations, each contributing in some way to an increased rate of cell division or decreased rate of cell death.

6. 1 Non-dividing cells

7. 1 1 Non-dividing cells 2

8. 1 1 1 1 1 2 2 2 3 Non-dividing cells 2

9. Non-dividing cells 1 2 1 2 1 1 1 2 1 2 1 123 123 123 123 123 123 123 123 123 This process continues, with each successive mutation leading to a faster rate of cell division, slower rate of cell death, and eventually loss of cell adhesion.

10. Review of the Eukaryotic Cell Cycle

11. The cell cycle • The cell cycle has four phases: M, during which the cell divides; G1, during which the cell grows larger; S , during which DNA synthesis occurs; and G2, during which the cell continues to grow and prepare for mitosis. The cycle is regulated at several points. • The restrictive point in late G1 phase is a time when the decision is made whether to continue the cycle or to to exit the cycle in a nondividing state called G0. • Cells in G0 may differentiate and assume specialized functions. • A cell can remain in G0 indefinitely, or it may re-enter the cell cycle in response to signals from a variety of growth factors.

12. The cell cycle • Once the cell passes the restriction point in G1, the cycle will continue until it is arrested at one of several checkpoints in response to some problem that needs to be corrected. • Progression is halted in late G1 and late G2 if DNA damage has occurred. The checkpoints allow time for the damaged DNA to be repaired before the cycle resumes. • The checkpoint in G2 also responds to the presence of unreplicated DNA and prevents mitosis from occurring until all of the DNA has been copied. • A checkpoint in late M phase halts the cell cycle until all of the chromosomes are properly aligned.

13. The Cell Cycle • Proteins from several different families interact to regulate progression through the cell cycle. • Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CKIs) all interact either to block or unblock phases of the cycle. • Cyclins, and Cdks act together as a dimer, functioning as the regulatory and catalytic subunits, respectively. • Cyclins are degraded at the end of their functional period, thus inactivating their Cdk partner in the dimer. • The assembly of the dimers is regulated by other proteins.

14. The cell cycle • In the event that a cell enters an S phase with damaged DNA, apoptosis may be triggered to prevent the mutant cell from reproducing itself.

15. Seven levels of regulation of cell growth An unrepaired mutation in a gene for a DNA-repair protein, a cell-cycle control protein, or an anti-apoptosis protein can increase the likelihood of a cancer developing.

16. An Example of Cell Cycle Regulation by a Serum Growth Factor • Cyclin D is made following the binding of the serum growth factor to its receptor and the ensuing cascade of phosphorylations.

17. An Example of Cell Cycle Regulation by a Serum Growth Factor

18. An Example of Cell Cycle Regulation by a Serum Growth Factor • Cyclin D associates with either Cdk4 or Cdk6. • P16 may block the assembly. • After assembly the Cdk becomes phosphorylated. • This may be blocked by either p21 or p27 • The target of the active dimer is Rb which is bound to a transcription factor called E2F. • The Rb/E2F dimer blocks transcription of genes needed to enter the S phase. • Phosphorylation of Rb results in its dissociation from E2F.

19. An Example of Cell Cycle Regulation by a Serum Growth Factor • This results in activation of S phase genes. • In addition to its ability to block the association of cyclin D with a Cdk, P16 can also directly block the phosphorylation of Rb.

20. Phosphorylation of Rb

21. An Example of Cell Cycle Regulation by a Serum Growth Factor • P16, p21, and p27 are regulated by p53 (more on this later) which blocks the cell cycle in the G1 phase if there is DNA damage. • P53, Rb, p21, p16, and p27 are called tumor supressors because their normal function is to prevent the growth of cells with damaged DNA.

22. An Example of Cell Cycle Regulation by a Serum Growth Factor • P53 also responds to unrepaired DNA damage by triggering apoptosis of the injured cell. • It interacts with a member of the Bcl-2 family of proteins which, in turn activate special enzymes called caspases. • Caspases initiate a protease cascade that results in digestion of the DNA. • This ultimately leads to cell death.

23. Apoptosis

24. Which types of genes when mutated can  cancer? • Oncogenes = genes whose products turn DNA synthesis ON • Tumor suppressors/anti-oncogenes = genes whose products turn DNA synthesis OFF • Genes whose products contribute to genomic stability • Genes whose products contribute to cell longevity

25. Which types of genes when mutated can  cancer? • Oncogenes (turn DNA synthesis ON) • In progression towards cancer, a gene for a protein that normally stimulates DNA synthesis (proto-oncogene) is either • consitutively expressed at high levels or • mutated such that protein product is constitutively active, i.e., can not be inactivated • Classes I-IV from Slide # 15 generally give rise to dominantly active oncogenes. • Examples: see next slide.

26. Oncogenes

27. Which types of genes when mutated can  cancer? • Tumor suppressors/anti-oncogenes (turn DNA synthesis OFF) • In the progression towards cancer, a gene for a protein that normally inhibits DNA synthesis is either • permanently inactivated or • mutated such that the protein product is inactive • Mutations in Class VI, cell-cycle control proteins, from Slide #15. • Examples: • APC inhibits Wnt gene product from activating myc • Rb inhibits activation of transcription of DNA synthesis genes

28. Which types of genes when mutated can  cancer? • Contributors to genomic stability • Some tumor suppressors turn DNA synthesis off when DNA is damaged. • The progression toward cancer occurs when a gene for a protein which contributes to DNA repair is • permanently inactivated or • mutated such that protein product is inactive • Mutations in repair genes increase likelihood of mutations in proto-oncogenes and tumor suppressors. • Examples: • p53 gene product induces genes for DNA repair • MDM2 gene product destabilizes p53 • MutS and MutL gene products repair UV or chemically damaged DNA

29. Which types of genes when mutated can  cancer? • Contributors to cell longevity (anti-apoptosis genes) • Progression toward cancer can occur when an anti-apoptosis gene is • constitutively expressed or • mutated such that protein product is constitutively active • Allows survival of cells with oncogenic mutations • Example: Bcl2

30. Roles for screening for mutations in specific genes • To determine • Type of cancer • Familial predispositions • Progression of the cancer

31. What is p53? • A protein of ~53 kilodaltons • A nuclear phosphoprotein       

32. What is p53? • Transcriptional regulator • Binds to 12 bp recognition sequence in the promoters (regulatory regions) of the genes it regulates • Activates transcription by interacting with RNA polymerase complex  

33. What is p53? • Acts as a tetramer • Individual molecules associate at tetramerization region • Oligomerization of mutated p53 with wt p53  inactive p53 complex 

34. What is p53? • Binding of damaged DNA fragments to p53 causes p53 to be stabilized and accumulate in the cell. • When damaged DNA is not present, p53 is turned over rapidly and does not accumulate because • the protein MDM2 binds to the transcription-activation region of p53 and targets p53 for degradation by a proteosome.  (TAD)  Note: MDM2 binds when the TAD is NOT phosphorylated.

35. p53 as a transcriptional regulator • If DNA damage is detected by binding of DNA fragments to the non-specific DNA binding region of p53, p53 stops DNA synthesis until the damage is repaired. • If DNA damage is detected, then • p53 is phosphorylated by a protein known as ATM • MDM2 is released from being bound to the transcriptional activation domain of p53 and • p53 is able to act as a transcriptional activator and turn on genes for • cyclin dependent kinase inhibitor p21, which • stops or prevents DNA synthesis • DNA repair • Example: GADD45 • If DNA damage is extensive and can not be repaired, p53 induces genes for apoptosis (programmed cell death).

36. p53 as a transcriptional regulator • p53 activates the gene for MDM2 • MDM2 • targets p53 for degradation and prevents inappropriate build up • prevents transcriptional activation by p53 • So, it’s a negative feedback loop! • p53 also turns expression of some genes off.

37. How does p53 inhibit DNA synthesis? Let’s work backwards. • E2F transcription factor turns on transcription of genes for DNA synthesis. • E2F can’t turn on genes if it is bound to Rb, a tumor suppressor. • Rb can’t bind E2F if it is heavily phosphorylated. • Rb is phosphorylated by cylin-dependent kinases (CDKs).

38. How does p53 inhibit DNA synthesis? • Cylin dependent kinases can be inhibited by cyclin dependent kinase inhibitors (CDKIs). If CDKs are inhibited • Rb won’t be phosphorylated • E2F will be bound by Rb • DNA synthesis gene will not be transcribed • And remember . . . . P53 induces expression of CDKI p21, a cyclin dependent kinase inhibitor! • Check out the next slide for a visual of these pathways.

39. Phosphorylation of Rb

40. Figure legend on next slide.

42. p53 Mutations - where are they?

43. This magnification of mutations in the DNA binding region of p53 gives more information regarding how the mutation affects p53. Note particularly that some mutations cause p53 to be misfolded (denatured) and others do not.

44. Have you figured it out? For our assay, the samples are cell extracts from two mouse cell lines, BC3H1 and C2C12. One line is wild type for p53; one is mutant. One accumulates detectable levels of p53; one doesn’t. Based on this lecture and your assay results, have you figured out which cell line does what?