1. Strategy (ies) in Cell Cycle Research Aleš Hampl DNA Replication Nuclear division Cytokinesis Sequentially occur in every cell division Cell cycle that contains G1, S, G2, and M phases =

3. Aspect no. 1 - Approaches Little bit of not that long history ( ~3 decades ) 80ties of the last century = beginnings of cell cycle research 1971 - first description of the cytoplasmic activity called „MPF“ in meiotically maturing frog oocytes (Masui and Markert, J. Exp. Zool.) MPF activity G2-arrested oocyte oocyte at MI oocyte at interphase MII-arrested egg zygote at interhase progesteron sperm penetration „ MPF assay“ GVBD

5. 1980, 1981 – it was shown the major role of the product of cdc2 gene in controlling the cell cycle in the fission yeast Schizosaccharomyces pombe (Nurse and Thuriax, Genetics ; Nurse and Bisset, Nature) Temperature sensitive mutants of the cdc2 gene become arrested either in G1 before S-phase or in G2 before mitosis, when S. pombe is incubated at nonpermissive temperature. S. pombe cdc2 gene has its homolog CDC28 in Saccharomyces cerevisiae. Important lesson learned from yeast :

7. Vertebrates also have it: 1987 – human homolog of cdc2 gene was identified by functional complementation of the S. pombe cdc2 mutation (Lee and Nurse, Nature) 1987 – identification of p34 protein in human cells that is homologous to p34 cdc2 protein of S.pombe and p36 CDC28 of S. cerevisiae (Draetta et al., Cell) H uman p34 protein was immunoprecipitated from [ 35 S ] methionine labeled HeLa cells using monoclonal antibody raised against p34 cdc2 crossreacting with p34 CDC28 .

8. Link between p 34 cdc2 and MPF becomes established: 1988 – MPF isolated (by affinity chromatography using p13 suc1 ) from frog oocytes is composed from two major components, proteins of molecular weights 34 kD and 42 kD, with p34 most likely being homolog of p34 cdc2 (Dunphy et al., Cell, July 29 ) 1988 – MFP purified from frog oocytes contains 32 kD protein that is recognized by antibody against p34 cdc2 ; 32 kD protein is associated with 42 kD protein thus creating a complex that expresses protein kinase activity (Gautier et al., Cell, July 29 ) 1988 – when purified from starfish oocytes, „M phase-specific“ histone H1 kinase (H1K) activity coelutes with 34 kD protein that crossreacts with antibody against p34 cdc2 ; MPF and H1K appear to be the same entity ( Arion et al., Cell)

10. 1983 – described for the first time as the protein that undergoes repeated destruction in cleaving sea urchin eggs (Evans et al., Cell) 1989 – cyclin synthesis is necessary for entry into mitosis in frog eggs (Minshull, Blow, and Hunt ; Cell) 1989 – destruction of cyclin is required for exit from mitosis in early frog embryos (Murray, Solomon, and Kirschner ; Nature) Cyclin comes to play: Example: fluctuation of cyclins A and B in early clam embryos minutes after fertilization cyclin A cyclin B Westendorf et al., J. Cell Biol., 1989

12. Things are not that simple in higher eukaryotes: PARADIGM „ cell cycle as a cdc2 cycle“ appealing for its simplicity further studies have complicated the issue SEEMED BUT 1991 – new human p34 protein kinase (CDK2 – c ell d ivision k inase) identified by complementation of a cdc28 mutation in S. cerevisiae (Elledge, Spottswood, EMBO J.) CDK2 has 66 % amino acid sequence identity with the CDC2Hs identified by Lee and Nurse (1987) by complementation of a cdc2 mutation in S. pombe FIRST

14. Current status (simplified in graphics)

15. Aspect no. 2 – Important phenomena Uncontrolled hyperproliferation And the ways of approaching their cell cycle-related molecular basis Restricted proliferation Proliferative diseases (cancer, psoriasis, rheumatoid arthritis, …) Failure to replenish diseased/injured cell type or tissue P henomenon: P henotype: P rofit: Finding biomarkers Designing drugs Induction of „stem cell“ proliferation Understanding of the molecular mechanisms that regulate cell proliferation is being converted into designing novel „smart“ diagnostical and therapeutical strategies.

16. A simple logic of looking at cell cycle regulators: Positive Negative Regulators of cell cycle: Members: CDKs, C yclins Inhibitors of CDKs Down-regulated & I nactivated Up-regulated & A ctivated Up-regulated & A ctivated Down-regulated & I nactivated In hyperproliferating cells: In nonproliferating cells:

20. What did we learn until now ? & Important take home lessons:

21. Protein Function Defect(s) Tumour type(s) p16 Cdk4/6 inhibitor Deletion ~95% T-cell ALL Point mutation ~75% Familial melanomas Silencing ~75% Pancreatic carcinomas ~52% Oesophageous SCC 12 - 30% NSC lung carcinomas ~30% Gliomas ~20% Bladder carcinomas Sporadic melanomas ~95% Tumour cell lines p18 Cdk6 inhibitor Deletion Cell ALL Point mutation Breast carcinoma cell lines p21 Cdk inhibitor Underexpression Cell lines which lack p53 p27 Cdk2 inhibitor Degradation Breast carcinomas Colorectal carcinomas cdk1 Catalytic subunit Overexpression Breast carcinomas Prostate carcinomas Colorectal carcinomas Gastric carcinomas Various defects to cell cycle regulators occur in human malignancies: (adopted from: Webster, K.R., Exp. Opin. Invest. Drugs, 1998)

22. Protein Function Defect(s) Tumour type(s) cdk2 Catalytic subunit Overexpression Colorectal carcinomas cdk4 Catalytic subunit Amplification Sarcomas, gliomas Point mutation Familial melanoma Overexpression Familial adenomatous polyposis Cyclin A Activator of cdk1/2 Stabilisation Hepatocellular carcinoma Cyclin B1 Activator of cdk1 Overexpression 88% Colorectal carcinomas Cyclin D1 Activator of cdk4/6 Amplification 40 - 80% Breast carcinomas Overexpression ~70% Familial adenomatous polyposis 50% B-cell lymphomas ~47% NSC lung carcinomas ~35% Head and neck SCC 25 - 50% Oesophagus SCC ~25% Bladder carcinoma Cyclin E Activator of cdk2 Amplification ~90% Colorectal carcinomas Overexpression 30 - 80% Breast carcinomas ~70% Prostate carcinomas ~18% Ovarian carcinomas Gastric carcinomas

24. Prognostic value of expression of cell cycle regulators is not completely understood p27 is not a good predictor for outcome of carcinoma of the bladder ! p27 – inhibitor of CDKs Is it a good predictor in bladder cancer ?

25. p27 Growth of mouse embryonal carcinoma cell lines differing in the amount of p27 p27 A.1 1.3 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 0 2 4 4 8 7 2 9 6 total protein per dish (mg) A . 1 1 . 3 hours Growth EC lines A .1 X.1 1.1 1.3 1.5 MAPK

27. Thank you for your attention Questions and comments at : a hampl @med.muni.cz