Cell Fate

1. Cell Fate Quiescence Proliferation Apoptosis Differentiation Senescence Necrosis Cell states are mutually exclusive

2. Quiescence Resting cells Post- or pre-mitotic Metabolically active Growth arrested • Naïve (Potential to differentiate) Differentiated cells Not differentiating! Potential to proliferate Not proliferating! In G0 of the cell cycle Stem cells Differentiation Proliferation Accomplish function Resident pool of progenitor cells Maintain cell number

3. (Cell Division /Mitosis) Proliferation Cellular division of non-terminally differentiated cells Mitotic stimulation by hormones, growth factors and cytokines Stimulation of Immediate Early Genes (transcription factors c-fos, c-myc, c-jun) Protein products of IEGs regulate transcription of late genes Needed to maintain cell population Change expression of proteins in the cell cycle Functional tissue Regulation of cell cycle / mitosis

4. The cell cycle (a one-way street) Stages cells pass through during cell division G0 (Quiescence) G0 Senescence Terminal differentiation G2-M Restriction Checkpoint 2 Gap Gap DNA synthesis RNA transcription low G1-S Restriction Checkpoint 1

5. Cell cycle is regulated by cyclins and CDKs Degradation of structural components and cyclins Cyclin are the regulatory subunit of the Cyclin- Dependent Kinases (CDKs) Production of protein complexes for M-phase Activity of CDKs regulated by the availability of the corresponding cyclins Production of protein complexes for S-phase Degradation of cell cycle inhibitors (ubiquitination) Phosphorylation of complexes for chromatin synthesis

6. G1-S restriction Primary regulation point for the control of the cell cycle Growth factors Immediate early Genes Delayed response genes Cyclin E, D-cyclins, E2F-1, CDK2/4 Needed to pass G1-S restriction E2F Group of transcription factors Required for transcription of genes for G1-S transition (DNA replication)

7. Normally (Growth Factor deprived) X E2F inhibited by retinoblastoma (Rb) G1 to S Rb Binds activation domain of E2F T.F. Disrupts transcription Binds histonedeacetylase (HDAC) Folds DNA into nucleosome (DNA/histone complex) Hinders TF access Growth factors [Cyclin] ↑ G1 CDK/cyclin complex formed Rb-P Dissociates from E2F Active kinase Transcription of genes for G1 to S

8. The cell cycle is regulated by the fine balance of cyclin expression CDK2/cyclin E complex is essential for G1 to S transition

9. The cell cycle is regulated by the fine balance of cyclin expression CDK2/cyclin E complex is essential for G1 to S transition GFs [D-cyclins] ↑ Complex with CDK 4/6 More cyclin-D P-Rb E2F ↑ D-cyclin -CDK4 ↑↑↑ Cyclin E ↑ Complexes with CDK2 but inhibited by p27KIP Sequests p27KIP CDK2-cyclin E Active G1 to S

10. DNA damage inhibits G1 to S transition until the DNA is repaired Normally p53 tumour suppressor is degraded when DNA is intact p53 stability ↑ [p53] ↑ [p21CIP] ↑ DNA damage (CDK inhibitor) Cells stuck in G1 until DNA damage repaired Binds G1 CDK-cyclin complex S-phase [p53] ↓ Alternatively cells may undergo apoptosis Programmed cell death

11. Knockout mice Indicates the in vivo function of the protein No inhibition of G1 CDK/cyclin complex Into S-phase with DNA damage p21CIP-/- NO! Apoptosis No proliferation Tumouragenesis? Ratio p21 : p53 important p21CIP is anti-apoptotic!! Growth arrest P21 : p53 ↑ Apoptosis P21 : p53 ↓ Fine tuning of expression of cyclins, cell cycle inhibitors and tumour suppressors to allow mitogenesis

12. Knockout mice X Rb-/- No inhibition of E2F G1 to S Uncontrolled proliferation? NO! E2F ↑↑↑ Activates p19ARF Binds to and inhibits MDM2 (Degrades p53) Apoptosis P21 : p53 ↓ p53 ↑ There is a fine balance between proliferation and apoptosis

13. G2 to M restriction G2 to M transition also finely controlled Cyclin B synthesis and phosphorylation Controls breakdown of nuclear lamina Multiple checks that prevent uncontrolled proliferation (tumourigenesis)

14. Apoptosis (programmed cell death) Apoptosis = Falling off Ordered set of events Blebbing Nuclear fragmentation Cellular fragmentation into apoptotic bodies Chromatin condensation and fragmentation Phagocytosis Important during embryogenesis Development of free and independent digits Massive death of interdigital mesenchymal cells Syndactyly

15. ~ 50% neurones undergo apoptosis before adulthood (selection) Brain development Immune system Majority of lymphocytes undergo apoptosis during negative selection of antigen receptor Man Turnover of several billion cells / day! Nutrient deprivation External activation Stimulated by cell stress Radiation Common pathway Heat Viral infection Cytokines Hypoxia GCs

16. Apoptosis vs Necrosis Injury

17. Apoptotic pathways Intrinsic pathway Death-inducing signal complex Extrinsic pathway Loss of membrane integrity ATP synthesis stops Change in redox potential Caspase = Cysteine proteases Components of the apoptotic pathway are always present in the cell Waiting for stimulus Anti-apoptotic pathway keeps cells viable

18. Insufficient apoptosis Cancer Activating mutations in BCL-2 Many oncogenes sensitise the cells to apoptosis Also requires apoptosis to be turned off Autoimmunity (Failure to eliminate autoimmune antibodies) Persistent infections (Failure to eliminate infected cells) Excessive apoptosis Neurodegeneration Alzheimer’s disease, Parkinson’s disease, Huntington’s disease Autoimmunity (Uncontrolled apoptosis in specific organs) AIDS (depletion of T lymphocytes) Ischemia (stroke, myocardial infarction)

19. Differentiation Differential expression or repression of genes to confer phenotype and function All diploid cells contain the full complement of genetic material Whole organism from a single cell Inhibition of proliferation Specialised function not conferred until terminal differentiation ? Pre-Phenotype A Phenotype A Pre-Phenotype B Progenitor cell Phenotype B Phenotype C Pre-Phenotype C Phenotype D Pre-Phenotype D

20. Differentiation Differential expression or repression of genes to confer phenotype and function All diploid cells contain the full complement of genetic material Whole organism from a single cell Inhibition of proliferation Specialised function not conferred until terminal differentiation ? Pre- adipocyte Adipocyte Pre- osteoblast Mesenchymal stromal cell Osteoblast Myoblast Pre- myoblast Chondrocyte Pre- chondrocyte Dedifferentiation Terminal differentiation Transdifferentiation

21. Expression of specific markers indicates state of differentiation Adipose-derived stromal cells Adipocyte Lipid droplets Cell-cell contact is essential! Differentiation medium Coordinated and Synchronized expression of genes Terminal differentiation

22. Senescence (aging) Characterised by inability of cells to proliferate Cells are viable but function is compromised Larger, fatter, express βgalactosidase Limits the number of cell divisions and possibly somatic mutations An alternative to apoptosis to prevent spread of cancer Progressive telomere shortening Alters RNA splicing in proteins such as progerin (lamin A) which compromise functionality (protective, non-coding DNA at end of chromosome) Many cancers are immortal Up-regulation of telomerase (90% tumours) Adds non-coding DNA onto the telomeres No telomere shortening Stem cells Also upregulate telomerase Proliferation needed to supply tissue

23. Aging Gompetz-Makeham law of mortality Mortality rate increases with age Man at 80 years Genetically determined Mouse at 3 years Old enough for reproductive success! Longevity Differences in Antioxidant enzymes DNA repair Free radical production Hormone signalling over age Damage and repair Stress Chronic activation of the HPA axis Aging ↑

24. Cell Fate  Quiescence  Proliferation  Apoptosis  Differentiation  Senescence