1. Chapter 8: pRb and Control of the Cell Cycle Clock From: Biology of Cancer: Robert Weinberg: Copyright © Garland Science 2007

2. pRb and the Cell cycle Cell-cell signaling decides cell fate Normal cells will not divide without growth signals TGF-B can stop growth in presence of growth signals Cells can divide OR Cells can become quiescent (G 0 phase) (reversible) Cells can become post-mitotic – an irreversible state of non-division

3. Signaling and Growth Surface receptors collect signals and funnel them into the cell Integration of signals by proteins help cells decide to divide/not-divide There must be a master cellular program deciding cell fate (quiescence, division, post-mitotic) Master Program Called Cell Cycle Clock

4. Figure 8.1 The Biology of Cancer (© Garland Science 2007)

5. Cancer Cells Always proliferate Oncogenes keep cell cycle on, regardless of lack of growth signals Tumor suppressor genes can be hijacked by cancer cells to change circuitry and keep cell cycle on

6. Normal Cell Cycle Cells in culture with growth medium will keep growing and dividing In vivo, a newly divided cell has to decide whether to divide again or not Cell division requires circuits for –Growth in size –Duplication of chromosomes –Correct chromosome complements in divided cell

7. Normal Cell Cycle After a new cell is made, RNA and protein synthesis begins immediately But chromosome duplication is delayed by 12-15 hours (G 1 or Gap 1 phase) In G 1, cell is deciding what to do G 1 is followed by S phase (6-8 hours) Then by G 2 phase (Gap 2) of 3-5 hours Then by M phase (~1-2 hours)  division

8. M-Phase Prophase – chromosomes condense, become visible, mitotic spindle forms Metaphase – chromosomes align along plane that bisects cell, nuclear membrane disappears, mitotic spindle migrates to poles Anaphase – chromosome halves pulled apart to poles by mitotic splindle Telophase – Chromatids decondense, nuclear wall forms around them Cell splits into 2 daughter cells – mitosis complete

9. Figure 8.3b The Biology of Cancer (© Garland Science 2007) Mitosis DemoDemo

10. Checkpoints MUST exist Necessary for fidelity of cell division Decision on when to enter cycle phases Accurate duplication of chromosomes Division of correct complement to daughter cells

11. Where are these checkpoints? G 1  S phase checkpoint –Integrate growth signals and decide its time to divide –Check that genome is undamaged S  G 2 phase checkpoint –Accurate DNA replication (no DNA damage) G 2  M phase checkpoint –DNA replication completed Anaphase checkpoint in M –Chromosomes attached to splindle Other checkpoints must exist –Decatenation checkpoint in late G phase (no tangles in Chr)

12. Figure 8.4 The Biology of Cancer (© Garland Science 2007)

13. R (Restriction) checkpoint Cells make growth decision during specific interval in G 1 phase –In culture, if cells are < 80-90% in G 1 phase, inhibition of growth serum will restrict entry into S phase –In last 10% of G 1 phase, restriction of serum has no effect. Cell committed to S phase entry –TGF-B is able to halt cell cycle only in early G 1

14. Figure 8.6 The Biology of Cancer (© Garland Science 2007)

15. R Checkpoint This is a GO/NO-GO decision made by the cell in late G 1 phase More important than other checkpoints –Once in S phase, cells seem oblivious to external signaling –They activate fixed schedule S  G 2  M Cancers must subvert R checkpoint

16. Cyclin Dependent Kinases Embryonic frog and sea urchin cells have a cyclical expression of Cyclin-B correlated with cell cycle

17. Cyklin Dependent Kinases Play a major role in R checkpoint Act only in the presence of cyclins which are subunits (form a dimer-complex) Phosphorylated at Serine/threonine instead of tyrosine (as in receptors – eg EGF) 40% identity amongst themselves (evolved by gene duplication from progenitor gene) 430 distinct known CDKs in humans Enormous enzymatic potential –Cyclin A + CDK2 increases activity by 400,000 X

18. When are they active? G 1 phase –Kinase CDK4/CDK6 –Cyclin D1, D2, D3 Early S phase –Kinase CDK2 –Cyclin E1, E2 Late S phase –Kinase CDC2 (CDK1) –Cyclin A1, A2 G 2 phase –Kinase CDC2 (CDK1) –Cyclin B1, B2

19. Figure 8.10 The Biology of Cancer (© Garland Science 2007)

20. What dergrades them so fast? Ubiquitylation followed by degradation in proteosome

21. Cyclin D’s are different Controlled by extracellular signaling (mitogens) They inform cell about external conditions Three different types D1, D2, D3 for flexibility of response and refinement of input sensing Most often/easily targeted by carcinogens, viruses, oncogenes Probably newer than other CDKs (since the evolution of complexity) and so more error prone

22. Figure 8.11b The Biology of Cancer (© Garland Science 2007) Factors affecting Cyclin D 1 levels

23. Each cyclin pairs with a specific Cyclin Dependent Kinase

24. Figure 8.12 The Biology of Cancer (© Garland Science 2007)

25. CDK inhibitors p21, p27

26. Early G 1 : Cyclin D-CDK4/6 complexes accumulate, bind and sequester p27 and p21 including those associated with E-CDK2 complexes Late G 1 : Sufficient numbers of p21, p27 are bound to D-CDK4/6 to liberate enough E-CDK2 complexes to transit R point

27. How does TGF-B work? Increases p15 levels which block formation of D-CDK4/6 complexes and inhibits those already formed Cell cannot advance through R checkpoint If Cell is already past R, TGF-B has no effect since D-CDK4/6 complex is irrelevant

28. pRb gene controls R Checkpoint Defective pRb gene isolated in 1986 in retinoblastomas Encodes a nuclear phospho-protein ~ 105 kD Called pRb or RB pRb is unphosphorylated in G 0 pRb is hypo-phosphorylated in early G 1 pRb is hyper-phosphorylated in late G 1 and rest of cell cycle After exit through mitosis, phosphate groups on pRb are stripped off by PP1 (protein phosphatase 1)

29. How was it identified as important? Several DNA virus (eg. SV40) oncoproteins target pRb by binding to it to sequester it or inactivate it They only bind to (seem to care only about) hypo-phosphorylated state of pRb This means that only the hypo-P state of pRb is relevant for tumors

30. How does pRb get Phosphorylated? D-CDK4/6 initiate phosphorylation of pRb E-CDC2 complex complete the phosphorylation of pRb Puzzle: pRb has homologues p107 and p130 which also have similar phosphorylation states and histories. Why are they not involved in tumorogenesis?

31. Figure 8.22 The Biology of Cancer (© Garland Science 2007)

32. Figure 8.19 The Biology of Cancer (© Garland Science 2007)

33. So why is hypo-P of pRb important? Unphosphorylated pRb Binds and sequesters E2F Hypo-phosphorylated pRb Releases some E2F Hyper-phosphorylated pRb Releases a lot of E2F

34. Figure 8.23a The Biology of Cancer (© Garland Science 2007)

35. What does E2F do? E2F is bound to promotor region of genes that initiate transcription When E2F is bound to pRB, it attracts histone- deacytylases – which remove acetyl groups from histones and make them inaccessible to transcription machinery (inhibits transcription) When E2F is not bound to pRB, it attracts histone-acytelases which add acetyl groups to histones and make them accessible to transcription machinery (promotes transcription)

36. Figure 8.24a The Biology of Cancer (© Garland Science 2007) Transcription control by chromatin modification induced by by Pocket Proteins pRb, p107, p130

37. Many cancers disable pRb Retinoblastomas, osteosarcomas, small cell lung carcinomas: pRb lost by mutation Cervical CA: HPV protein E7 binds to pRb and makes it inactive Breast Cancer : Overexpression of Cyclin D1 by gene amplification Melanoma : methylation of p16 gene promotor etc etc……

38. Myc and Subtle Dis-Regulation of pRb Myc overexpression –increases Cyclin D2  phosphorylation of pRb –Overexpression of CDK4  same effect –Sequesters p27, drives E-CDK2 complexes and makes cell enter S phase –Degrades Cull protein which degrades p27, induces expression of genes which encode E2Fwhich drives transcription Myc Disregulation Resets Cell-Cycle Regulatory Dials  Causes proliferation !!

39. Eukaryotic Cell Cycle and Pathway

40. Table 8.3 The Biology of Cancer (© Garland Science 2007)

41. Table 8.4 The Biology of Cancer (© Garland Science 2007)

42. Figure 8.38 The Biology of Cancer (© Garland Science 2007) Cyclin E levels and Breast Cancer Progression: Plot shows survival in women presenting with stage III disease (large primary tumors and LNs but no distant metastasis)

43. Next week Read Chapter 9 We will talk about the p53 gene, guardian and master executioner !