Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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
Dr. Saeb Aliwaini2
• 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.
Dr. Saeb Aliwaini 4
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.
Dr. Saeb Aliwaini 11
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.
Dr. Saeb Aliwaini 12
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.
Dr. Saeb Aliwaini 15
Dr. Saeb Aliwaini 16
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.
Dr. Saeb Aliwaini 17
Dr. Saeb Aliwaini 18
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
Dr. Saeb Aliwaini 19
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.
Dr. Saeb Aliwaini 20
• 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.
Dr. Saeb Aliwaini 21
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.
Dr. Saeb Aliwaini 22
Dr. Saeb Aliwaini 23
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.
Dr. Saeb Aliwaini 24
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.
Dr. Saeb Aliwaini 25
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.
Dr. Saeb Aliwaini 26
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.
Dr. Saeb Aliwaini 27
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.
Dr. Saeb Aliwaini 28
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.
Dr. Saeb Aliwaini 29
Apoptosis is a property of all animal cells and some plant cells
Dr. Saeb Aliwaini 30
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.
Dr. Saeb Aliwaini 31
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.
Dr. Saeb Aliwaini 32
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.
Dr. Saeb Aliwaini 33
Apoptosis triggers the activation of special proteases: the caspases
Figure 13.29: Different types of vertebrate caspases are shown schematically.
Dr. Saeb Aliwaini 34
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.
Dr Saeb Aliwaini
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
Dr Saeb Aliwaini
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
Dr Saeb Aliwaini
PDGF
Dr Saeb Aliwaini
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