Mouse Models to Investigate Cell Cycle and Cancer Prof. Philipp Kaldis
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1 Mouse Models to Investigate Institute of Molecular and Cell Biology (IMCB) Cell Division and Cancer Laboratory, Proteos 3-09 Singapore Crystal structure of Cdk2/ cyclin A A.A Russo et al., (1996) Nat Struct Biol 3; T-loop, Unphos. T-loop, Phos. Phosphate group 3 1
2 Cyclin expression: periodicity cyclin A cyclin D cyclin E cyclin B G1 S G2 M
3 Cdk2 Binds to Cyclin E, A, B Activity peaks in S phase Major target of p27 Phosphorylates Rb and other targets Can replace yeast Cdk1 Essential for DNA replication Inactivation arrest cells in G1 Cdk2 is important (essential?) regulator of S phase 7 Cyril Berthet Eiman Aleem Cdk2 -/- knockout mice Current Biology Cdk2 knockout mice are viable! Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P (2003) Curr Biol 13(20): Ortega S et al. (2003) Nat Genet 35:
4 Atrophy in testis of Cdk2 -/- mice 3 weeks 11 weeks 11 weeks Cdk2 -/- Cdk2 +/+ H&E BrdU H&E 10 Absence of oocyte development in Cdk2 -/- mice H&E BrdU Cdk2 +/+ Cdk2 -/- 11 Expression of cell cycle regulators in mouse tissues 12 4
5 Kinase activity and Cdk/cyclin complexes in spleen extract 13 Growth defect in Cdk2 -/- mouse embryo fibroblasts 14 Cdk complexes and activity in mouse embryonic fibroblasts 15 5
6 Cdk2 -/- MEF analysis after starvation 16 Cdk activity in synchronized MEFs 17 Delayed immortalization in Cdk2 -/- MEFs 18 6
7 Summary Cdk2 is essential for meiosis but not mitosis Cdk2 -/- MEFs display growth defects Which genes/ proteins compensate Cdk2 function? 19 Cdk2 is not an essential gene in the mitotic cell cycle 20 The Retinoblastoma (Rb) protein pathway Cdk1, cyclin A, Cdc25, Cyclin E, Cdk6, etc. 21 7
8 Cyril Berthet Developmental Cell Cdk2 -/- Cdk4 -/- mutants die around E15 E13.5 E14.5 E15.5 WT DKO WT DKO WT DKO 23 Heart defects in Cdk2 -/- Cdk4 -/- embryos Cdk2 -/- Cdk4 -/- double knockout mice Cdk2-/-Cdk4-/- Wild type 24 8
9 In vivo proliferation in Cdk2 -/- Cdk4 -/- embryos DKO Wild type BrdU Liver 25 Hypophosphorylated Rb leads to decreasing expression of Cdk1 and cyclin A 26 Cdk2 -/- Cdk4 -/- MEFs display impaired proliferation P2 MEFs P4 MEFs 27 9
10 S phase entry defect in Cdk2 -/- Cdk4 -/- MEFs 28 Rb phosphorylation in Cdk2 -/- Cdk4 -/- MEFs IP/ Western blot analysis 29 E2F target gene expression in Cdk2 -/- Cdk4 -/- MEFs RT PCR analysis 30 10
11 Senescence in Cdk2 -/- Cdk4 -/- MEFs WT DKO P2 P4 SA-β-galatosidase staining 31 HPV-E7 neutralizes Rb and rescues Cdk2 -/- Cdk4 -/- phenotype 32 HPV-E7 mutants cannot rescue Cdk2 -/- Cdk4 -/- phenotype 33 11
12 Wild type Cdk2 -/- Cdk4 -/- Cdk1 expression is repressed in the absence of Cdk2 and Cdk4 34 Summary Cdk2 -/- Cdk4 -/- DKO die due to heart defects Rb is not phosphorylated and E2F mediated transcription is repressed Cdk2 and Cdk4 are essential genes, regulating the G1/S transition 35 Cdk2 and Cdk4 control the expression of Cdk1 through the Rb/E2F pathway 36 12
13 The p27 kip1 network TGFβ, Cell cycle exit (GO) Active kinase Ubiquitin mediated degradation 37 Eiman Aleem Cdk2 -/- p27 -/- double knockout mice Nature Cell Biology Cdk2 -/- mice vs. wt Smaller body size Sterility of both males and females None None None MEFs: lower proliferation, 4h delay in S phase entry p27 kip1-/- mice vs. wt Increased body size Female sterility, disordered estrus, impaired luteal cell differentiation Pituitary tumors Retinal dysplasia Thymic hyperplasia, increased T cell proliferation, hematopoietic progenitors MEFs: normal cell cycle kinetics 39 13
14 Cdk2 -/- p27 -/- mice 40 Ovary tumors in Cdk2 -/- p27 -/- mice Wild type Cdk2 -/- p27 -/- p27 -/- Cdk2 -/- Wild type Cdk2 -/- p27 -/- 41 Pituitary tumors Wild type Cdk2 -/- p27 -/- p27 -/- Cdk2 -/- H&E 42 14
15 Thymus: normal levels of cell death Wild type Cdk2 -/- p27 -/- p27 -/- Cdk2 -/- TUNEL 21d H&E 21d 43 Thymus: high levels of S phase and mitosis Wild type Cdk2 -/- p27 -/- p27 -/- Cdk2 -/- PO 4 -H3 21d Br rdu 42d 44 Cdk activity in the absence of Cdk2 and p
16 Cdk1 binds to p27 and cyclin E 46 Proliferation of mouse embryonic fibroblasts 47 Silencing of Cdk1 in Cdk2 -/- MEFs WT + shcdk1 WT Cdk2-/- + shcdk
17 Summary Cdk2 -/- p27 -/- P27 regulates Cdk2 and Cdk1 Cdk1 interacts with cyclin E and forms active complexes Cdk1/cyclin E complexes most likely promote S phase and compensate for Cdk2 49 Cdk1 binds to p27 and cyclin E Wild type S phase G2 M phase Cdk2 -/- p27 -/- Cdk1/cyclin E complexes can drive S phase 50 Cdk1 compensates for Cdk2 functions in S phase 51 17
18 Cdk1 translocates to the nucleus prematurely in the absence of Cdk2 52 Satya Ande DNA damage response in Cdk2 -/- knockout mice Molecular Biology of the Cell Response to γ-irradiation in MEFs γ-irradiation 54 18
19 Response to γ-irradiation in MEFs γ-irradiation 55 In vivo response to γ-irradiation: partial hepatectomy model Cdk2 +/+ and Cdk2 -/- mice subjected to 70% partial hepatectomy (surgery) Control γ-irradiationi 56 In vivo response to γ-irradiation: molecular outcome γ-irradiation 57 19
20 Cdk1 and p21 can interact because they co-localize 58 Proliferation of MEFs after irradiation 59 Cdk2 -/- mice are sensitive to γ-irradiation 60 20
21 DNA damage: comet assay Initial damage Recovery/ repair 61 DNA damage: γ-h2ax staining 62 Disturbed kinetics of DNA damage foci in Cdk2 -/- MEFs 6 hr 6 hr 24 hr 24 hr 30 hr 30 hr 63 21
22 Wild type Cdk2 -/- DNA damage DNA damage Cdk2 ATM ATR ATM ATR p53 p21 p53 p21 Cdk2 Cdk1 G1 S G2 M 64 DNA damage in the absence of Cdk2 Cdk2 -/- mice are sensitive to γ-irradiation Cdk1 compensates for Cdk2 and is inhibited by p21 DNA damage foci formation is disturbed DNA damage repair is delayed Cdk1 is less efficient in DNA damage repair 65 Acknowledgements Kasim Diril Eiman Aleem Shuhei Kotoshiba Cyril Berthet Xinde Zheng Padmakumar VC Stefan Lim Weimin Li Shuhui Lim Satya Ande Daniel Chew Kristy McDowell June Wang Mary Beth Hilton Lino Tessarollo Vincenzo Coppola Eileen Southon Shyam Sharan Nancy Jenkins Neal Copeland Hiroaki Kiyokawa Northwestern Jonathan Keller Kim Klarmann Hyung Chan Suh Ben Asefa John Gooya 66 22
23 67 23
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