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

Quiescence Resting cells Post- or pre-mitotic Potential to proliferate Naïve (Potential to differentiate) In G 0 of the cell cycle Metabolically active Not differentiating! Not proliferating! Growth arrested Differentiated cells Stem cells Resident pool of progenitor cells Proliferation Maintain cell number Differentiation Accomplish function

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

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

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

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

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

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

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 D-cyclin -CDK4 ↑↑↑ Sequests p27 KIP E2F ↑ Cyclin E ↑ Complexes with CDK2 but inhibited by p27 KIP CDK2-cyclin E Active G1 to S

DNA damage inhibits G1 to S transition until the DNA is repaired DNA damage (CDK inhibitor) Alternatively cells may undergo apoptosis Programmed cell death p53 stability ↑[p53] ↑[p21 CIP ] ↑ Binds G1 CDK- cyclin complex Cells stuck in G1 until DNA damage repaired [p53] ↓S-phase Normally p53 tumour suppressor is degraded when DNA is intact

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

Rb -/- NO! (Degrades p53) Knockout mice No inhibition of E2F G1 to SUncontrolled proliferation? Activates p19 ARF Binds to and inhibits MDM2 p53 ↑ P21 : p53 ↓Apoptosis E2F ↑↑↑ X There is a fine balance between proliferation and apoptosis

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

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

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

Apoptosis vs Necrosis Injury

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

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

Differentiation Differential expression or repression of genes to confer phenotype and function Progenitor cell Pre-Phenotype A Pre-Phenotype B Pre-Phenotype C Pre-Phenotype D Phenotype B Phenotype C Phenotype D Phenotype A ? 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

Mesenchymal stromal cell Pre- adipocyte Pre- osteoblast Pre- myoblast Pre- chondrocyte Adipocyte Osteoblast Myoblast Chondrocyte ? 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 Specialised function not conferred until terminal differentiation Inhibition of proliferation Dedifferentiation Transdifferentiation Terminal differentiation

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

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

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

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