Cell Cycle Regulation. Adrian R. Black

Cell Cycle Regulation Adrian R. Black 845-3090 Adrian.black@roswellpark.org

Loss of Growth Control Block of Apoptosis Evasion of Senescence Angiogenesis Invasion

hases of the cell cycle Mitosis: Chromosome Segregation and Cytokenesis M

hases of the cell cycle Mitosis: Chromosome Segregation and Cytokenesis M Gap 2: reparation for Cell Division G 2 G 1 Gap 1: reparation for DNA Replication S Synthesis: DNA Replication

Cyclins regulate the cell cycle Expression Oscillates During the Cell Cycle Main Cell Cycle Regulatory Cyclins: cyclin D1-3, cyclin E, cyclin A and cyclin B

"Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms." Satyanarayana, A. and. Kaldis (2009). Oncogene 28(33): 2925-39. After a decade of extensive work on gene knockout mouse models of cell-cycle regulators, the classical model of cell-cycle regulation was seriously challenged. Several unexpected compensatory mechanisms were uncovered among cyclins and Cdks in these studies. The most astonishing observation is that Cdk2 is dispensable for the regulation of the mitotic cell cycle with both Cdk4 and Cdk1 covering for Cdk2's functions. Similar to yeast, it was recently discovered that Cdk1 alone can drive the mammalian cell cycle, indicating that the regulation of the mammalian cell cycle is highly conserved. Nevertheless, cellcycle-independent functions of Cdks and cyclins such as in DNA damage repair are still under investigation. Here we review the compensatory mechanisms among major cyclins and Cdks in mammalian cell-cycle regulation.

Cyclin Expression During Cell Cycle Cyclin E Cyclin A Cyclin B Cyclin D G 1 S G 2 M

hases of the cell cycle Mitosis: Chromosome Segregation and Cytokenesis Growth Factor Withdrawal M G 0 Growth Factor Addition G 2 G 1 eriod during which growth factors are required S Synthesis: DNA Replication Restriction oint oint at which growth factors no longer required

Cell cycle checkpoints Growth Factor Withdrawal G 0 Check of DNA integrity Spindle checkpoint M Growth Factor Addition G 2 G 1 S Check of DNA integrity and readiness for replication Check of DNA integrity

hases of the cell cycle Growth Factor Withdrawal Mitosis: Chromosome Segregation and Cytokenesis M G 0 Growth Factor Addition Gap 2: reparation for Cell Division G 2 G 1 eriod during which growth factors are required S Synthesis: DNA Replication Restriction oint oint at which growth factors no longer required

Cyclin Expression During Cell Cycle Cyclin E Cyclin A Cyclin B Cyclin D Growth Factors G 1 S G 2 M R

Cyclin D1 acts as a sensor of the growth environment for the cell cycle Ral RTK Ras Raf Mek Erk AK I3K Rac Akt 4EB1 mrna nuclear export ILK Integrins GSK3β mtor S6K β-catenin Wnt/ Frizzled AC Nuclear Export and roteasoma degradation Ub Ub Ub Translation cyclin D1 gene Transcription Ras KC

Cyclins Regulate Cyclin-Dependent Kinases Cyclin-dependent kinases (cdks) Multigene Family [cdk4, cdk6, cdk2, cdk1 (cdc2)] Serine/Threonine Kinases Require Cyclin Binding for Activity Regulated by hosphorylation Regulated by Cyclin-Dependent Kinase Inhibitors (ckis) Cyclins Regulatory Subunits of Cyclin/cdk Complexes Main Cell Cycle Regulatory Cyclins: cyclin D1-3, cyclin E, cyclin A and cyclin B Cyclin/cdk Associations: cyclin D/cdk4, cyclin D/cdk6, cyclin E/cdk2, cyclin A/cdk2, cyclin A/cdk1 and cyclin B/cdk1 Expression Oscillates During the Cell Cycle

Interaction of cyclins and cdks during the cell cycle Figure 8.8 The Biology of Cancer ( Garland Science 2007)

Cyclin Dependent-Kinase Inhibitors (ckis) INK Family p15 b, p16 a, p18 c, p19 d Bind cyclin D-associating cdks (cdk4 and cdk6) Block interaction of cdks with cyclins CI/KI Family p21 WAF1/CI1, p27 KI1, p57 KI2 Inhibit Cyclin A/cdk2 and Cyclin E/cdk2 Required for assembly of Cyclin D/cdk complexes Do not inhibit cyclin D/cdk complexes at physiological levels p21 binds CNA

Interaction of cdki s with cdks Figure 8.13a The Biology of Cancer ( Garland Science 2007)

Cip/Kip cdki s stimulate cyclin D/cdk association and activity Figure 8.17a The Biology of Cancer ( Garland Science 2007)

Cyclin D/cdk complexes sequester cip/ kip proteins form cyclin E/cdk2 Figure 8.17b The Biology of Cancer ( Garland Science 2007)

Cyclin D1 and TGF-β TGF-β Cyclin E Cdk2 CI/KI Cdk4/6 Cdk4/6 Cdk4/6 Cyclin E Cdk2 CI/KI Cyclin E CI/KI CI/KI Cyclin E CI/KI Cdk2 CI/KI Cdk4/6 Cdk4/6 Cyclin D Cyclin D

Cyclin D1 and TGF-β TGF-β Cyclin E Cdk2 Cyclin E Cdk2 CI/KI CI/KI Cyclin E Cdk4/6 Cyclin D Cyclin D CI/KI CI/KI Cyclin E CI/KI Cdk2 CI/KI Cyclin D Cdk4/6 Cdk4/6 Cyclin D Cdk4/6 Cyclin D Cdk4/6 Cyclin D Cyclin D Cyclin D Cyclin D

Cyclin D1 and TGF-β TGF-β Cyclin D Cyclin E Cdk2 CI/KI Cdk4/6 Cyclin D Cdk4/6 Cyclin D Cdk4/6 Cyclin D Cyclin E Cdk2 CI/KI Cyclin E CI/KI CI/KI Cyclin E CI/KI Cdk2 CI/KI Cyclin D Cdk4/6 Cyclin D Cyclin D Cdk4/6 Cyclin D Cyclin D

Cyclin D1 and TGF-β TGF-β Cyclin E Cdk2 Cyclin D Cdk4/6 Cyclin D Cdk4/6 CI/KI CI/KI Cyclin D Cdk4/6 CI/KI Cyclin D Cyclin E Cdk2 Cyclin E Cyclin E CI/KI Cdk2 Cyclin D Cyclin D Cyclin D CI/KI Cdk4/6 Cyclin D Cdk4/6 Cyclin D CI/KI

How do cyclin/cdk complexes regulate the cell cycle?

The E2F-pRB pathway Disrupted in most if not all cancers A major regulator of cell cycle progression Involved in regulation of apoptosis

E2F-pRB pathway Major layers: E2F Family of Transcription Factors Retinoblastoma Family of roteins (ocket roteins) Cyclins and Cyclin Dependent Kinases (cdks) Cyclin Dependent Kinase Inhibitors (ckis)

E2F-Regulated Genes DNA Synthesis Genes Growth Regulatory Genes dihydrofolate reductase (DHFR) DNA polymerase α thymidylate synthase (TS) thymidine kinase (TK) proliferating cell nuclear antigen (CNA) licensing factors (e.g. cdc6, mcm s) c-myc B-myb prb p107 cyclin E cyclin A E2F1 E2F2 cdk1 p21 WAF1/CI1 p27 KI1 p19 ARF

The E2F Family of Transcription Factors Cyclin A binding DNA binding Dimerization Marked box ocket protein binding Transactivation Cell cycle regulation E2F- 1 E2F- 2 E2F- 3 rotein expression limited to late G 1 and S-phase. E2F- 4 E2F- 5 Expressed throughout. Nuclear during G 0 /G 1, otherwise cytoplasmic. E2F- 6 Repressor D- 1 D- 2 E2F- 7 Expressed throughout the cell cycle. Repressor

ocket roteins Founding member is the Retinoblastoma Gene roduct (prb) mutated in familial retinoblastoma Other members are p107 and p130, named for molecular weight Bind to transactivation domain of E2F s with dual effect a) Blocks transactivation function b) Recruitment of histone deacetylase 1 (HDAC1), causing general repression of promoters Regulated by phosphorylation a) Hypophosphorylated forms bind E2F s and HDAC1 b) Hyperphosphorylated forms do not bind E2F s or HDAC1 c) hosphorylated by cyclin/cdk complexes

ocket proteins bind E2F on DNA and actively repress transcription Deacetylation hdac ocket rotein E2F X

Cyclin/cdks phosphorylate and inactivate pocket proteins Cyclin Cdk Cyclin Cdk

The E2F-Retinoblastoma athway is Targeted by Viral Oncoproteins Adenovirus E1A SV40 large TAg Human apillomavirus E7 E2F prb family E2F S phase Viral Oncoproteins prb family

Cyclin D/cdk and cyclin E/cdk sequentially phosphorylate pocket proteins

Figure 8.21b The Biology of Cancer ( Garland Science 2007)

ocket rotein Interactions with the E2F Family of Transcription Factors Cyclin A binding DNA binding Dimerization Marked box pocket protein binding Transactivation E2F- 1 prb p107 p130 E2F- 2 E2F- 3 E2F- 4 E2F- 5 E2F- 6

Components of the E2F-pRB pathway disrupted in cancer RB. mutated or deleted 130.... mutated (rare?) yclin D1 overexpressed (amplification/transcriptional) yclin E (rare).. overexpressed dk4 overexpressed mutated to disrupt p16 a binding 16 a... deleted or transcriptionally repressed 27 KI1 downregulated

Some levels of regulation that could impinge on the control of E2F activity by p53 roteasome CB/ p300 Mdm2 p19 p53 p21 Cyclin D/E cdk ocket rotein E2F roliferation Cyclin A cdk Apoptosis Black and Azizkhan-Clifford, 1999

Cyclin Expression During Cell Cycle Cyclin E Cyclin A Cyclin B Cyclin D Growth Factors G 0 G 1 S G 2 M R

hases of the cell cycle Mitosis: Chromosome Segregation and Cytokinesis M G 0 Check of DNA integrity Gap 2: reparation for Cell Division G 2 G 1 Gap 1: reparation for DNA Replication S Synthesis: DNA Replication Restriction oint: Check of DNA integrity and readiness for replication

Ras Regulation of Cyclin D1 Expression Synchronized Cyclin D1 Growth Factors Ras Ras G 0 G 1 S G2 M R Cycling Cyclin D1 Ras G 1 S G2 M

M Early G1 Growth Factors G 0 hdac Deacetylation ocket rotein E2F Cyclin D Cdk4/6 X CI/KI Cdk4/6 Cyclin D S G 1 hdac ocket rotein E2F Cyclin Cdk4/6D Cdk4/6 X CI/KI Acetylation and remodeling

Early G1 M Growth Factors G 0 Deacetylation hdac ocket rotein E2F Cyclin D Cdk4/6 X CI/KI Cdk4/6 Cyclin D G 1 hdac ocket rotein Cyclin Cdk4/6D Cdk4/6 CI/KI S E2F cyclin E Acetylation and remodeling

Mid-Late G1 ocket rotein Cyclin E Cdk2 E2F cyclin E G 1 ocket rotein Cyclin Cdk4/6E Cdk2 Cyclin Cdk4/6D Cdk4/6 Cyclin E Cdk2 CI/KI CI/KI E2F Transcription of S-hase Genes, cyclin E and E2F1-3

ositive Feedback Loop for E2F activation Cdk4/6 CI/KI CI/KI Cyclin Cdk4/6D Cdk4/6 Cyclin Cdk4/6E ocket rotein hdac p27 cdk2 E2F X Degradation

Mid-Late G1 ocket rotein Cyclin E Cdk2 E2F cyclin E G 1 ocket rotein E2F Cyclin Cdk4/6E Cdk2 Other substrates (DNA replication) Cyclin Cdk4/6D Cdk4/6 Cyclin E Cdk2 Transcription of S-hase Genes and E2F1-3 CI/KI CI/KI

M E2F 4/5 G 2 G 1 Cyclin AE Cdk2 cytoplasm S E2F 1-3 Transcription of S-hase Genes ocket rotein Cyclin A Cdk2 E2F 4/5 E2F 1-3 S phase

M G 2 Cdk1 ocket rotein Cyclin A Cdk2 S Early-mid G 2

Mitotic ocket rotein X Cyclin B Cdk1 cdc25 Cyclin A Cdk2 G 2 M S G Late G 2

M phase hdac Deacetylation ocket rotein E2F X Transcription of S-phase Genes hdac ocket rotein 1 M E2F cytoplasm G 2 S

Early G1 hdac Deacetylation ocket rotein E2F X Transcription of S-phase Genes G 1 S

Transition from G 0 p27 kip1 p27 kip1 p21 waf1 p21 waf1 prb E2F 4/5 prb p130 E2F 4/5 p107 p130 p107 cyclin D cyclin D quiescent cells (G 0 ) cycling cells

Transition into G 0 p27 kip1 p27 kip1 p21 waf1 p21 waf1 p21 waf1 prb p130 p107 prb E2F 4/5 p130 E2F 4/5 p107 cyclin D cyclin D cycling cells quiescent cells (G 0 )

ositive Feedback Loop for E2F activation Cdk4/6 CI/KI CI/KI Cyclin Cdk4/6D Cdk4/6 Cyclin Cdk4/6E ocket rotein hdac p27 cdk2 E2F X Degradation

E2F1 and Apoptosis Overexpression of E2F1 induces apoptosis Apoptotic effects of E2F1 blocked by prb Induces p53-dependent and -independent apoptosis p53-dependent a) E2F upregulates p53 levels (through ARF) b) enhances apoptotic function of p53 (e.g. by competition for p300) p53-independent a) enhanced by p300 b) blocks antiapoptotic pathways (e.g. NFkB activity, IA expression) c) inhibition of E2F1 phophorylation in S-phase enhances apoptosis d) May involve p73

Negative Feedback Downregulation of E2F activity. Cyclin Cdk4/6D Cdk4/6 Cyclin Cdk4/6E CI/KI ocket rotein Cyclin Cdk4/6A cdk2 hdac cdk2 E2F p27 p21 Antiapoptotic signals p19 ARF Mdm2 p53 Apoptosis X p53 p19 ARF Mdm2 Degradation

otential Effects of Overactivation of E2F Activity? Cyclin Cdk4/6D cdk4/6 Cyclin Cdk4/6E CI/KI ocket rotein Cyclin Cdk4/6A cdk2 hdac cdk2 E2F E2F1 p21 p27 Apoptosis Antiapoptotic signals Apoptosis X p53 p19 ARF Mdm2 p19 ARF Mdm2 p53 Degradation

The E2F-Retinoblastoma athway is Targeted by Viral Oncoproteins Adenovirus E1A SV40 large TAg Human apillomavirus E7 E2F prb family E2F S phase Viral Oncoproteins prb family

The p53 athway is Targeted by Viral Oncoproteins p53 Apoptosis

The p53 athway is Targeted by Viral Oncoproteins Adenovirus E1A SV40 large TAg Human apillomavirus E6 Viral Oncoproteins p53 p53 Apoptosis

Apoptotic Factors Altered in Cancer p53 p19 ARF (p14 ARF in Humans) Others e.g. NFκB survivin (caspase inhibitor)

Effect of p19 ARF Mutation on Feedback Downregulation of E2F activity.? Cyclin Cdk4/6D cdk4/6 Cyclin Cdk4/6E CI/KI ocket rotein Cyclin Cdk4/6A cdk2 hdac cdk2 E2F E2F1 p21 p27 Apoptosis Antiapoptotic signals Apoptosis Degradation X X p53 p19 ARF Mdm2 p19 ARF Mdm2 p53 Degradation

Effect of p53 Mutation on Feedback Downregulation of E2F activity.? Cyclin Cdk4/6D cdk4/6 Cyclin Cdk4/6E ocket rotein Cyclin Cdk4/6A cdk2 hdac cdk2 E2F E2F1 p21 p27 Apoptosis Antiapoptotic signals Apoptosis X p53 p19 ARF Mdm2 p19 ARF Mdm2 p53 Degradation

E2F-regulation is cell type-dependent He et al., (2000). E2F is Reqired To revent Inappropriate S-hase Entry of Mammian Cells. Mol Cell Biol 20: 363-371

hases of the cell cycle Mitosis: Chromosome Segregation and Cytokenesis Growth Factor Withdrawal M G 0 Growth Factor Addition Gap 2: reparation for Cell Division G 2 G 1 Gap 1: reparation for DNA Replication (2 N DNA Content) (4 N DNA Content) S Synthesis: DNA Replication

Flow cytometric analysis of cell cycle S distribution G 1 /G 0 G 2 /M Harvest cells as single cell suspension Number of cells Stain with DNA-intercolating fluorescent dye Laser Detector Fluorescence (DNA Content) Collect and analyze data Run cells through FACScan

Flow cytometric analysis of the cell cycle S G 1 /G 0 G 2 /M Number of cells DNA Content

Effects of MA on Cell Cycle in IEC18 cells MA, 100 nm U 6 h 12 h 16 h 24 h

Effects of MA on Cell Cycle in IEC18 cells U 16 6 h12 h h 6 h12 16 24 hh h MA, 100 nm U 6 h 12 h 16 h 24 h

Detection of Apoptosis by Flow Cytometry Sub G 1 Apoptotic S G 1 /G 0 G 2 /M Number of cells DNA Content

REFERENCES Sherr, C. J. and J. D. Weber (2000). The ARF/p53 pathway. Curr Opin Genet Dev 10(1): 94-9. Hanahan, D. and R. A. Weinberg (2000). The hallmarks of cancer. Cell 100(1): 57-70. Stacey, D.W. (2003). Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Current Opinion in Cell Biology 15:158 163 Seville, L. L., N. Shah, A. D. Westwell and W. C. Chan (2005). "Modulation of prb/e2f functions in the regulation of cell cycle and in cancer." Curr Cancer Drug Targets 5(3): 159-70. Musgrove, E. A. (2006). "Cyclins: roles in mitogenic signaling and oncogenic transformation." Growth Factors 24(1): 13-9. Malumbres, M. and Barbacid, M. (2009). Cell cycle, CDKs and cancer: a changing paradigm. Nature Reviews: Cancer 9: 153-166. Gopinathan, L., C. Ratnacaram and. Kaldis (2011). Established and Novel Cdk/Cyclin Complexes Regulating the Cell Cycle and Development. In Cell Cycle in Development. J. Z. Kubiak, Springer Berlin / Heidelberg. 53: 365-389. Novak, B., O. Kapuy, M. R. Domingo-Sananes and J. J. Tyson (2010). "Regulated protein kinases and phosphatases in cell cycle decisions." Current Opinion in Cell Biology 22(6): 801-808.