1. CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase Kinetochore attachment Repair of DNA damage G1 G1 DNA damage checkpoint Cell Cycle Checkpoints p53  p21 S DNA damage checkpoint ATR  Chk1 ATM  Chk2

2. Geminin Cdt1

3. HCT116 contGem4 Phospho Chk1 Phospho Chk2 Chk1 Chk2 Loading control Geminin Loss of geminin leads to re-replication and activation of Chk1 and Chk2

4. 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.

5. S and M have to alternate: if not, genomic instability S M G1G1 G2G2 1)elevated activity of cdks 2)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

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

12. , Cdk9, cyclin T

18. 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 ofCdc25A

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

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

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

27. Ubiquitinylation by an E3 ubiquitin ligase: SCF in G1 and S APC in M Proteasome recognizes polyubiquitinylated substrate and degrades it K K 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 Regulated proteolysis is an important component of cell-cycle regulation

28. CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase Kinetochore attachment Repair of DNA damage G1 G1 DNA damage checkpoint Cell Cycle Checkpoints p53  p21 S DNA damage checkpoint ATR  Chk1 ATM  Chk2

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

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

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

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

34. 1/v 0 (pmol/min) -1 1/[CDC6(wt)] (µM) -1 1/v0 (pmol/min)-1 Cy peptide Competitively Inhibits Cyclin E/cdk2 and Cyclin A/cdk2 K i = 7.5 ± 0.5 µM K i = 117.5 ± 11.6 µM Cyclin E/cdk2Cyclin A/cdk2

35. 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. A new class of cdk inhibitors

36. 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 Effect of statins on prostate cancer cells

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

38. Mevastatin induces p21 and inhibits cdk2

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

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

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

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

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

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

47. 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?