CELL CYCLE

Lectures for 25-10-2015

Dr Saeb Aliwaini

– How cells make the decision to begin moving through the stagesof replication, and why some cells never make this journey

– How cells decide to die

– A multicellular organism needs to coordinate cell division acrossdifferent tissues & organs

– critical for normal growth, development & maintenance

Cell cycle

– critical for normal growth, development & maintenancecoordinate timing of cell division coordinate rates of cell division

– not all cells can have the same cell cycle

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• Frequency of cell division varies by cell type

– embryo

• cell cycle < 20 minute

– skin cells

• divide frequently throughout life

• 12-24 hours cycle

Frequency of cell division

G2

S G1

M

metaphaseprophase

anaphasetelophase

interphase (G1, S, G2 phases)mitosis (M)cytokinesis (C)

C

• 12-24 hours cycle

– liver cells

• retain ability to divide, but keep it in reserve

• divide once every year or two

– mature nerve cells & muscle cells

• do not divide at all after maturity

• permanently in G0

New cells arise from parental cells that complete the cell cycle

• Key Concepts :

– Cells divide by following carefully scripted program of molecular events collectively called the cell cycle.

– The cell cycle is subdivided into five phases named G1, S, – The cell cycle is subdivided into five phases named G1, S, G2, M, and G0. Cells not actively dividing reside in G1 or G0

phase.

– Progression through the cell cycle is under the control of proteins that form checkpoints to monitor whether the proper sequence of events is taking place. Cells halt at these checkpoints until they complete the necessary steps to continue.

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Overview of Cell Cycle Control

• Two irreversible points in cell cycle

– replication of genetic material

– separation of sister chromatids

• Checkpoints

– process is assessed & possibly halted

centromere

sister chromatids

single-strandedchromosomes

double-strandedchromosomes

Checkpoint control system

• Checkpoints

– cell cycle controlled by STOP & GO chemical signals at critical points

– signals indicate if key cellular processes have been processes have been completed correctly

Checkpoint control system

• 3 major checkpoints:– G1/S

• can DNA synthesis begin?

– G2/M• has DNA synthesis been completed • has DNA synthesis been completed

correctly?

• commitment to mitosis

– spindle checkpoint• are all chromosomes attached to

spindle?

• can sister chromatids separate correctly?

G1/S checkpoint

• G1/S checkpoint is most critical

– primary decision point

• “restriction point”

– if cell receives “GO” signal, it divides

internal signals: cell growth (size), cell nutrition • internal signals: cell growth (size), cell nutrition

• external signals: “growth factors”

– if cell does not receive signal, it exits cycle & switches to G0 phase

• non-dividing, working state

G0 phase

M

• G0 phase

– non-dividing, differentiated state

– most human cells in G0 phase

liver cells MMitosis

G1Gap 1

G0Resting

G2Gap 2

SSynthesis

liver cells

in G0, but can be “called back” to cell cycle by external cues

nerve & muscle cells

highly specialized

arrested in G0 & can never divide

• How do cells know when to divide?

– cell communication signals

• chemical signals in cytoplasm give cue

• signals usually mean proteins

– activators

Activation of cell division

– activators

– inhibitors

experimental evidence: Can you explain this?

“Point of no return”

Early experiments characterizing the activity of

Mitosis Promoting Factor.

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Mitosis Promoting Factor.

Proteins called cyclinsplay an important role inadvancing cells throughcheckpoints

Activation of cyclin-CDK complexes begins in G1 phase

Figure 13.04: Scientists discovered the first cyclins when they noted that high cyclin levels with the onset of mitosis in embryos. Cyclin levels drop sharply after this.

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Interaction of Cdk’s & different cyclins triggers the stages of the cell cycle

“Go-ahead” signals

• Protein signals that promote cell growth & division

– internal signals

• “promoting factors”

– external signals– external signals

• “growth factors”

• Primary mechanism of control

– phosphorylation

• kinase enzymes

• either activates or inactivates cell signals

Cell cycle signals

• Cell cycle controls

– cyclins• regulatory proteins

• levels cycle in the cell

– Cdk’s

inactivated Cdk

– Cdk’s• cyclin-dependent kinases

• phosphorylates cellular proteins– activates or inactivates proteins

– Cdk-cyclin complex• triggers passage through different stages of cell

cycle

activated Cdk

Control by cyclin/CDK complexes

Figure 13.05: Distinct cyclin-cdk complexes control progression through cell cycle checkpoints.

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Figure 13.02: Checkpoints control progression through the

cell cycle. Some of the major checkpoints are shown.

Cell cycle step 1: Signal transduction initiates cell cycle progression.

Figure 13.06: The family of mitogen activated protein kinases (MAPKs) and their

upstream regulatory proteins.

Figure 13.07: A simplified MAP kinase signaling pathway.

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Cell cycle step 2: Changes in gene expression are required for progression through the restriction point

• progression through the restriction point in mammalian cells requires activation of at least two cyclin/CDK complexes: cyclin D1/CDK4 (or CDK6) and cyclinE/CDK2

• expression of most CDKs does not vary much • expression of most CDKs does not vary much throughout the cycle, but without their corresponding cyclins, they are not functional

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Cell cycle step 3: Pro- and anti-growth signaling networks converge at the G1/S cyclin-CDK complexes

• Phosphorylation

• Binding by inhibitory kinases

• Subcellular location• Subcellular location

• Protein degradation

Figure 13.08: Summary of the cyclin/cdk activation-inactivation cycle.

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• The key function of G1-Cdk complexes in animal cells is to activate a group of gene regulatory factors called the E2F proteins, which bind to specific DNA sequences in the promoters of a wide variety of genes that encode proteins required for S-phase entry, including G1/S-cyclins, S-cyclins, and proteins involved in DNA including G1/S-cyclins, S-cyclins, and proteins involved in DNA synthesis and chromosome duplication.

• In the absence of mitogenic stimulation, E2F-dependent gene expressionis inhibited by an inter- action between E2F and members of the retinoblastoma protein (Rb) family.

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Cell cycle step 4: Active cyclin/CDKs phosphorylate pocket proteins, which activate E2Fs

Figure 13.09: The transcription factor E2F is inactivated by Rb

binding.

Figure 13.10: Examples of positive (green) and negative (red) feedback loops controlling E2F

function.

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How E2Fs enhance expression of some genes while suppressing expression of others remains

unclear

Figure 13.11: A model of how E2F transcription factors can suppress or activate gene transcription.

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Cell cycle step 5: The DNA replication machinery is activated by protein kinases

A key player is a large, multiprotein complex called the origin recognition complex (oRc), which binds to replication origins throughout the cell cycle. In late mitosis and early G1, the proteins cdc6 and other proteins bind to the

Figure 13.12: Assembly of the prereplication complex.

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proteins cdc6 and other proteins bind to the ORC at origins and help load a group of six related proteins called the Mcm pro-teins. The resulting large complex is the pre-RC, and the origin is now licensed for replication.

DNA replication occurs in S phase

• 3 key steps

Figure 13.13: Activation of the replication complex.

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Cell cycle step 6: DNA integrity is ensured by the G1/S, S/G2, and G2/M checkpoints

Figure 13.14: A current model for DNA repair.

Figure 13.15: Growth arrest induced by Chk1 and Chk2.

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Multicellular organisms contain a cell self-destruct program that keeps them healthy

• Key Concepts:

– Cells die either by traumatic injury (necrosis) or by a self-destruct program called apoptosis.

– Apoptosis begins through at least two molecular mechanisms, called intrinsic and extrinsic pathways.

– The family of proteins called caspases includes proteases that promote the degradation of organelles and cytosolicproteins during apoptosis.

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Cells die in 2 different ways: necrosis and apoptosis

Figure 13.22: Cellular damage can result in necrosis, as organelles swell and the plasma membrane ruptures.

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Apoptosis is a property of all animal cells and some plant cells

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Apoptosis is induced via at least 2 different pathways

Figure 13.24: Ligation of death receptors causes the recruitment of the adaptor

protein FADD to the intracellular region of the death receptor.

Figure 13.25: E2F1 lies at the heart of the growth-versus-death decision making

system.

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Targets of pro- and anti-apoptotic transcription factors are members

of bcl-2 family

Figure 13.26: The Bcl-2 family proteins share up to four Bcl-2 homology domains

(BH) and can be antiapoptotic or proapoptotic.

Figure 13.27: The Bcl-2 family of proteins compete with members of the

antiapoptotoic group to access the apoptotic group in an elaborate hierarchy.

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Mitochondrial Outer Membrane Permeabilization (MOMP)

Figure 13.28: Signals for the induction of apoptosis trigger changes in the Bcl-2 family proteins, which function to inhibit or promote apoptosis. Activation of caspase 9 by the apoptososme. Insert, three views of apoptosome

structure as determined by electron microscopy.

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Apoptosis triggers the activation of special proteases: the caspases

Figure 13.29: Different types of vertebrate caspases are shown schematically.

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BALANCED SALT SOLUTIONS (BSS)

composed of inorganic salts and may include sodiumbicarbonate and, in some cases, glucose.

BSS forms the basis of many complete media, and commercialsuppliers will provide Eagle’s MEM with Hanks’s salts

Dr Saeb Aliwaini

• Hanks’s salts requires sealed flasks with a gas phase of air,whereas Earle’s salts imply a higher bicarbonateconcentration compatible with growth in 5% CO2.

• Short incubations up to about 4 h (usually with glucosepresent)

BALANCED SALT SOLUTIONS (BSS)

present)

• PBS without Ca2+ and Mg2+ is known as PBS Solution A

• HEPES (10–20 mM) is currently the most effective buffer inthe pH 7.2 to pH 7.8 range

Dr Saeb Aliwaini

COMPLETE MEDIA

• It is usually made up of unstable constituents, such asglutamine

• And various supplements, such as serum, growthfactors, or hormones, may be added just before use.

• Antibiotics were originally introduced into culture media toreduce the frequency of contamination. However, the useof laminar-flow hoods, coupled with strict aseptic technique,makes antibiotics unnecessary. Indeed, antibiotics have anumber of significant disadvantages:

• They have antimetabolic effects that can cross-react withmammalian cells.

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SERUM

• Serum contains growth factors, which promote cellproliferation, and adhesion factors and antitrypsin activity,which promote cell attachment.

• Minerals, lipids, and hormones.

• Protein• Protein

• The functions of many proteins in vitro remain obscure

• Albumin which may be important as a carrier of lipids,minerals, and globulins

• Fibronectin

• α2-macroglobulin

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Serum

• Growth Factors:

• Platelet derived growth factor (PDGF) : polypeptides withmitogenic activity and is probably the major growth factor inserum.

• TGF-β, may inhibit growth or promote differentiation inepithelial cellsepithelial cells

• fibroblast growth factors (FGFs)

• epidermal growth factor (EGF)

• Endothelial cell growth factors such as vascular endothelialgrowth factor (VEGF) and angiogenin

• insulin-like growth factors IGF-I and IGF-II

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PDGF

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Transforming growth factor-beta

• Transforming growth factor(TGF)-beta mediates G1 cellcycle arrest by inducing oractivating cdk inhibitors,and by inhibiting factorsand by inhibiting factorsrequired for cdk activation.Mechanisms that lead tocell cycle arrest by TGF-beta are reviewed.

Dr Saeb Aliwaini

Dr Saeb Aliwaini

Vascular endothelial growth factor promotes cell-cycle transition from G0 to G1phase in subcultured endothelial cells of diabetic rat thoracic aorta

An overview of studies from ourlaboratory on the hormone-responsiveMCF7, ZR75 and T47D human breastcancer cell lines indicates thatestrogens, via ERα, trigger a concertedphenotypic program that allows thesecells to replicate and divide. Amongnumerous estrogen-induced proteins,intracellular transcription factors (fos,intracellular transcription factors (fos,jun, c myc…) and cyclins (D and E) actas intracrine mitogens to trigger theentry of cells into an active G1 phase ofthe cycle (Fig. 1). As reported byseveral authors at this meeting, theydirectly affect the regulation of the cellcycle

Dr Saeb Aliwaini

plating hela

Dr Saeb Aliwaini