S and M have to alternate: if not, genomic instability

1. S G1 G2 M S and M have to alternate: if not, genomic instability • elevated activity of cdks • elevated level of geminin • 3) assembly of pre-RC can only occur in a window in G1 (Cdc6 exported, Cdt1 degraded, Mcm2-7 phosphorylated in S) • 4) If despite this re-replication occurs: checkpoint pathways stop the cell-cycle

6. P (S/T)PX(K/R) (S/T)PX(K/R) (S/T)PX(K/R) Cdks phosphorylate substrates on S/T cdk2 Cyclin

8. , Cdk9, cyclin T

14. Cancers increase activators of cyclin-cdk • Cyclin D is amplified or over-expressed by translocations in parathyroid adenomas, in esophageal cancers, in breast cancers (30-60%) • Cyclin E is amplified or over-expressed in breast cancers • Cdc25A is over-expressed in 30-60% of breast cancers • Myc oncogene (8q24:14q32 translocation in Burkitt's lymphoma; amplified in lung cancers) transcriptionally activates the production of Cdc25A

15. ATM mutations (in Ataxia-telangiectasia patients) predispose • to cancers

19. Arrested before X mitosis DNA replication interrupted DNA replication Checkpoint M G2 S DNA replication Normal Preparation for mitosis

22. Cdc25C M G2 S ATR Kinase activated Cdc25C Phosphatase Phosphorylated X Chk1 kinase phosphorylated 14-3-3s binds to phosphoCdc25C and inhibits it DNA replication interrupted

23. Cdt1 Geminin

24. Loss of geminin leads to re-replication and activation of Chk1 and Chk2 HCT116 cont Gem4 Loading control Geminin Phospho Chk1 Chk1 Phospho Chk2 Chk2

25. Depletion of geminin activates G2/M checkpoint, resulting in sequestration of Cdc25C outside the nucleus (red on right panel: cytoplasmic Cdc25C). Rereplication by depletion of geminin activates the G2/M checkpoint.

26. Regulated proteolysis is an important component of cell-cycle regulation K K Ubiquitinylation by an E3 ubiquitin ligase: SCF in G1 and S APC in M Proteasome recognizes polyubiquitinylated substrate and degrades it Examples of substrates degraded in this manner: G1: Cdk inhibitor, p27 S: Cdt1 M : securin, a molecule that inhibits the protease that separates daughter chromosomes cyclin A, cyclin B

27. Sister chromatid separation Cell Cycle Checkpoints G1 S G2 Metaphase Anaphase CDK2 CDK1 APC ATRChk1 p53  p21 ATMChk2 Chromosome condensation Repair of DNA damage DNA replication; Repair of damage G1 DNA damage checkpoint DNA replication checkpoint Kinetochore attachment S DNA damage checkpoint Spindle checkpoint

29. p21 uses Cy motif to interact with cyclin-cdk2 K p21 cdk2 Cy Cyclin Chen, MCB 2002

30. Crystal structure of cdk inhibitor p27N in complex with cyclin A/Cdk2 Pavletich. Nature 1996

31. - (X)n - 40 A n = 2, 6, 12, or 18 wildtype = 16 Effect of Linker Length on Substrate Phosphorylation Linkers shorter than 40 A should be ineffective

32. Cy peptides inhibit Cyclin-Cdk2 Chen et al. 1996, MCB

33. 1/v0 (pmol/min)-1 1/v0 (pmol/min)-1 1/[CDC6(wt)] (µM)-1 1/[CDC6(wt)] (µM)-1 Cy peptide Competitively Inhibits Cyclin E/cdk2 and Cyclin A/cdk2 Cyclin E/cdk2 Cyclin A/cdk2 Ki = 7.5 ± 0.5 µM Ki = 117.5 ± 11.6 µM

34. A new class of cdk inhibitors • Existing cdk inhibitors are all ATP mimetic chemicals that competitively inhibit the binding of ATP to the cdk2 • Cy mimetic chemicals will be a new class of cdk inhibitors : • specific for sub-classes of substrates • specific for a given cyclin that might be de-regulated in a cancer • could synergise with ATP mimetic chemicals.

35. Effect of statins on prostate cancer cells Statins are widely used (FDA approved!) chemicals that inhibit HMG CoA reductase and reduce the levels of cholesterol: Fluvastatin (Lescol) - Novartis Atorvastatin (Lipitor) - Pfizer Simvastatin (Zocor) - Merck Pravastatin (Pravachol) - Bristol Myers Squibb Lovastatin (Mevacor) - Merck They also have anti-proliferative effect on epithelial cells

36. Mevastatin blocks prostate cancer cell PC3 at G1-S

37. Mevastatin induces p21 and inhibits cdk2

38. RNAi in flies and worms Input ds RNA Dicer siRNA (21-23 nt) UU UU RISC Homologous RNA transcripts Degraded RNA

39. RNAi in mammalian cells 21 nt RNA duplex UU 5’ UU 5’ Oligofectamine

40. RNAi of p21 prevents the induction of p21 by mevastatin

41. RNAi of p21 does not prevent the G1-S block and Rb dephosphorylation induced by mevastatin

44. Mevastatin inhibits the activating phosphorylation of cyclin E/cdk2 on T160

45. …but Mevastatin does NOT inhibit the putative mammalian CAK: cyclin H/Cdk7

46. Summary of the mechanism by which statins inhibit the cell-cycle in prostate cancer cells • Mevastatin blocks the cell-cycle at G1-S transition • Rb is de-phosphorylated, cyclin D1/cdk4 unaffected, cyclin E/cdk2 inhibited and cyclin A downregulated • p21 is induced, but not necessary for cyclin E/cdk2 inhibition • T160 phosphorylation is inhibited, but the conventional CAK cyclin H/cdk7 is active • T160-P phosphatase activity is not increased • Do statins affect a new (undiscovered) CAK?

47. Unsolicited advice to future scientists • Always remember: you are in charge • You are not doing this for money or for nice lifestyle • Scientific research is the most exciting enterprise that the human race has devised: you are part of a grand tradition • Be an optimist: no one else will be • There is no substitute for work at the bench AND for keeping on top of the Literature