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CELL PROLIFERATION AND

APOPTOSIS

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Overview

• In this session we will deal with cell proliferation, apoptosis, repair, regeneration and how these relate to the actions of drugs.

• About 10 billion new cells are manufactured in the body daily through cell division-an output that must be counterbalanced by the elimination of a similar number of cells.

• We will first deal with the changes that occur within an individual cell when, after stimulation by growth factors, it gears up to divide into two daughter cells.

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Overview…

• We then consider the interaction of cells, growth factors and the extracellular matrix in cell proliferation

• We describe the phenomenon of apoptosis (the programmed series of events that lead to cell death), outlining the changes that occur in a cell that is preparing to die, and the intracellular pathways that lead to its demise.

• We consider how these processes relate to the repair of damaged tissue and the possibility of its regeneration.

• Lastly, we consider the pathophysiological significance of these events, and implications for the potential development of clinically useful drugs

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CELL PROLIFERATION:

• Cell proliferation (cell division) is involved in many physiological and pathological processes including:

Growth, Healing, repair, hypertrophy, hyperplasia and the development of tumours.

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• Angiogenesis (the development of new blood vessels) necessarily occurs during many of these processes.

• Proliferating cells go through what is termed the cell cycle, during which the cell replicates all its components and then bisects itself into two identical daughter cells.

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• Important components of the signaling pathways in proliferating cells are receptor tyrosine kinases or receptor-linked kinases and the mitogen-activated kinase (MAP kinase) cascade.

• In all cases, the pathways eventually lead to transcription of the genes that control the cell cycle.

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THE CELL CYCLE

• The cycle is an ordered series of events consisting of several sequential phases: G1; S; G2 and M.

• G1; preparation for DNA synthesis • S phase is the phase of DNA synthesis &

chromosome duplication.• G2: preparation for division• M is the phase of mitosis: division into two

daughter cells

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• G1 is the gap between the mitosis that gave rise to the cell and the S phase;

• During G1 the cell is preparing for DNA synthesis.• G2 is the gap between S phase and the mitosis that

will give rise to two daughter cells: during G2 the cell is preparing for DNA synthesis.

• In cells that are dividing continuosly, G1 S and G2

comprise interphase-the phase between one mitosis and the next

• Cell division requires the controlled timing of two critical events of the cell cycle: S phase ( DNA replication ) and M phase (mitosis).

• Entry into each of these phases is carefully regulated and this gives rise to two ‘check points’ (restriction points) in the cycle: one at the start of S and one at the start of M.

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Figure 5-1 The main phases of the cell cycle of dividing cells.

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• DNA damage results in the cycle being stopped at one or other of these. The integrity of the checkpoints is critical for the maintenance of genetic stability; and failure of the checkpoints to stop the cycle when it is appropriate to do so is a hallmark of cancer.

• In an adult; most cells do not constantly divide; most spend a varying amount of time in a quiescent phase outside the cycle as it were, in the phase termed G0 (‘G nought’ not the word ‘Go’).

• Neurones and skeletal muscle cells spend all their time in the G0; bone marrow cells and the lining cells of the gastro-intestinal tract divide daily.

• Quiescent cells can be activated into G1 by chemical stimuli associated with damage; for example, a quiescent skin cell can be stimulated by a wound into dividing and repairing the lesion.

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• The impetus for a cell to start off on the cell cycle ( i.e. to move from G0 into G1 ) can be provided by several stimuli, the most important being growth factor action. (Note: G-protein-coupled receptors can also stimulate cell proliferation)

• Growth factors stimulate the production of signal transducers of two types:

Positive regulators of the cell cycle that control the changes necessary for cell division.

Negative regulators that control the positive regulators.

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• The maintenance of normal cell numbers in tissues and organs requires that there be a balance between the positive regulatory forces and the negative regulatory forces.

• Apoptosis also has a role in the control of cell numbers.

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POSITIVE REGULATORS OF THE CELL CYCLE:

• The cell cycle is initiated when a growth factor acts on a quiescent cell provoking it to divide.

• One of the main actions of a growth factors is to stimulate production of the cell cycle regulators; which are coded for by the delayed response genes.

• The main components of the control system that determines progress through the cycle are two families of proteins:

cyclins and cyclin-dependent kinases (cdks ).

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• [ The name cyclin comes from the fact that these proteins undergo a cycle of synthesis and breakdown during each cell division ].

• The cdks phosphorylate various proteins ( e.g enzymes ) activating some and inhibiting others – to coordinate their activities.

• Sequential functioning of several different cdks activates the process that promote progress through the phase of the cycle.

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• Each cdks is inactive until it binds to a cyclin, the binding enabling the cdk to phosphorylate the protein(s) necessary for a particular step in the cycle. (Fig 5.2)

• It is the cyclin that determines which protein(s) are phosphorylated.

• After the phoshorylation event has taken place; the cyclin is degraded (Fig 5.2) by the ubiquitin/protease system.

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Figure 5-2 Schematic representation of the activation of a cyclin-dependent kinase. An inactive cdk. The inactive cdk is activated by

being bound to a cyclin; it can now phosphorylate a protein substrate (e.g. an enzyme). After the phosphorylating event, the cyclin is

degraded.

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• There are 8 main groups of cyclins. Those important in the control of the cell cycle are cyclins A, B, D and E.

• Each cyclin is associated with and activates particular cdk(s).

• Cyclin A activates cdks 1 and 2; cyclin B; cdk 1; cyclin D cdks 4 and 6 and cyclin E; cdk 2.

• Precise timing of each activity is essential and many cycle proteins are degraded after they have carried out their functions.

• The actions of the cyclin/cdk complexes in the cell cycle are depicted in Fig 5.3 (see next slide)

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Figure 5-3 Schematic diagram of the cell cycle, showing the role of the cyclin/cyclin-dependent

kinase complexes. The processes outlined in the cycle occur inside a cell such as the one shown in Figure 5.4. A quiescent cell

(in G0 phase), when stimulated to divide by growth factors, is propelled into G1 phase and prepares for DNA synthesis. Progress

through the cycle is determined by sequential action of the cyclin/cdk complexes-depicted here by coloured arrows, the arrows being given the names of the relevant cyclins: D, E, A and B. The

cdks (cyclin-dependent kinases) are given next to the relevant cyclins. The thickness of each arrow represents the intensity of

action of the cdk at that point in the cycle. The activity of the cdks is regulated by cdk inhibitors. If there is DNA damage, the products of

the tumour suppressor gene p53 stop the cycle at check point 1, allowing for repair. If repair fails, apoptosis (see fig. 5.5) is initiated. The state of the chromosomes is shown schematically in each G

phase-as a single pair in G1, and each duplicated and forming two daughter chromatids in G2. Some changes that occur during

mitosis (metaphase, anaphase) are shown in a subsidiary circle. After the mitotic division, the daughter cells may enter G1 or G0

phase. Rb, retinoblastoma gene.

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CELLS IN G0

• In quiescent G0 cells, cyclin D is present in low concentration and important regulatory protein- Rb protein is hypophosphorylated.

• (Note: the Rb protein is coded for by the Rb gene. The Rb gene is so named because mutations of this gene are associated with retinoblastoma tumours)

• Hypophosphorylated Rb holds the cell cycle in check at check point 1 by inhibiting the expression of several proteins critical for cell cycle progression.

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• The Rb protein accomplishes this by binding to the E2F transcription factors, which control the expression of the genes that code for cyclin E and A, for DNA polymerase, for thymidine kinase, for dihydrofolate reductase etc.- all essential for DNA replication during S phase.

• Growth factor action on a cell in G0 propels it into G1

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PHASE G1

• G1 is the phase in which the cell is preparing for S phase by synthesizing the messenger RNAs (mRNAs) and proteins needed for DNA replication.

• During G1, the concentration of cyclin D increases and the cyclin D/cdk complex phosphorylates and activates the necessary proteins.

• In mid- G1, the cyclin D/cdk complex phosphorylates the Rb protein, releasing transcription factor E2F;

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• This then activates the genes for the components specified above that are essential for the next phase – DNA synthesis- namely cyclins E and A, DNA polymerase and so on.

• The action of cyclin E/cdk complex is necessary for transmission from G0 to S phase, i.e past check point 1.

• Once past check point 1, into the S-phase, the processes that have been set in motion cannot be reversed and the cell is committed to continue with DNA replication an mitosis

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S PHASE:

• Cyclin E/cdk and cyclin A/cdk regulates progress through S phase, phosphorylating and thus activating proteins/ enzymes involved in DNA synthesis.

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G2 PHASE:

• In G2 phase; the cell, which now has doubled the number of chromosomes; must duplicate all other cellular components for allocation to the two daughter cells.

• Synthesis of the necessary mRNAs and protein occurs.

• Cyclin A/cdk and cyclin B/cdk complexes are active during G2 phase and are necessary for entry into M phase, i.e. for passing check point 2.

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• The presence of cyclin B/cdk complexes in the nucleus is required for mitosis to commence.

• Unlike cyclins C, D, and E, which are short lived, cyclin A and B remain stable throughout interphase but undergo proteolysis by a ubiquitin-dependent pathway during mitosis.

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MITOSIS:

• Revise processes involved in mitosis

• Mitosis is a continuous process but can be considered to consist of 4 stages.

• Prophase: The duplicated chromosomes ( which have up to this point formed a tangled mass filling the nucleus ) condense, each now consisting of 2 daughter chromatids, ( the original chromosome and a copy ). These are released into the cytoplasm as the nuclear membrane disintegrates.

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• Metaphase:The chromosomes are aligned at the equator.

• Anaphase: A special device, the mitotic apparatus, captures the chromosomes and draws them to opposite poles of dividing cell.

• Telophase: A nuclear membrane forms round each set of chromosomes.

• Finally the cytoplasm divides between the two forming daughter cells. Each daughter cell will be in G0 phase and will remain there unless stimulated into G1 phase.

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NEGATIVE REGULATORS OF CELL CYCLE:

• One of the main negative regulators is the Rb protein that holds the cycle in check while it is hypophosphorylated.

• Another negative regulatory mechanism is the action of inhibitors of the cdks. These bind to and inhibit the action of the complexes, the main action being at check point 1.

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There are two families of inhibitors-:

• The CIP family (cdk inhibitory proteins; also termed KIP or kinase inhibitory proteins-proteins p21, p27, and p57

• The Ink family ( inhibitors of kinases )-proteins p16, p19 and p15.

• The action of p21 serves as an example of the role of a cyclin/cdk inhibitor.

• Protein p21 is under control of p53 gene- a particularly important negative regulator that operates at check point 1-which is relevant in carcinogenesis.

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INHIBITION OF THE CYCLE AT CHECK POINT 1:

• The p53 gene has been called the ‘ guardian of the genome’ . It codes for a protein transcription factor- the p53 protein. In normal health cells, the steady-state concentration of p53 protein is low.

• But when there is DNA damage, the protein accumulates and activates the transcription of several genes, one of which codes for p21.

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• Protein p21 inactivates cyclin/cdk complexes, thus preventing Rb phosphorylation, which means the cycle is arrested at check point 1. This allows for DNA repair. If the repair is successful, the cycle proceeds past check point 1 into S phase. If the repair is unsuccessful; the p53 gene triggers apoptosis- cell suicide.

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INHIBITION OF THE CYCLE AT CHECK POINT 2:

• There is evidence that DNA damage can result in the cycle being stopped at check point 2 but the mechanisms involved are less clear than those at check point 1.

• Inhibition of the accumulation of cyclin B/cdk complex in the nucleus seems to be a factor.

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INTERACTIONS BETWEEN CELLS, GROWTH FACTORS AND EXTRACELLULAR MATRIX:

• During cell proliferation, there is integrated interplay between growth factors, cells, the extracellular matrix (ECM), and the matrix metalloproteinases (MMPs).

• The extracellular matrix supplies the supporting frame work for the cells of the body and is secreted by cells themselves.

• Matrix expression is regulated by the action on the cell of growth factors and cytokines.

• The activation status of some growth factors is, in turn, determined by the matrix.

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• Since they are sequestered by interaction with matrix components released by enzymes ( e.g. metalloproteinases ) secreted by the cells.

• It is clear that the action of growth factors- which act through receptor tyrosine kinases or receptor-coupled kinases initiating the cell cycle is fundamental part of these processes.

• There are numerous growth factors; important examples being fibroblast growth factor (FGF), epidermal growth factor (EGF ) , platelet-dependent growth factor ( VEGF ) and transformating growth factor- ( TGF-).

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• The main components of the extracellular matrix are-:• Fibre forming elements e.g. collagen species and

elastin. Collagens:These are main proteins of extracellular matrix.

• Non-fibre-forming, e.g proteoglycans, glucoproteins and adhesive proteins

• Proteoglycans: These have a growth regulating role, in part by functioning as a reservoir of sequestrated growth factors. Some proteoglycans are associated with the cell surface, where they bind cells to the matrix.

• Adhesive proteins (e.g. Fibronectin ): These link the various elements of the matrix together

and also form links between the cells and the matrix through integrins on the cells.

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• Integrins are transmembrane receptors with alpha and -subunits.

• On interaction with elements of the extracellular matrix (ECM) , cooperate with growth factor signalling pathways (this is necessary for the optimum cell division) and also mediate cytoskeletal adjustments within the cell)

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ANGIOGENESIS:

• Angiogenesis, which normally accompanies cell proliferation, Is the formation of new capillaries from existing blood vessels,

• an important stimulus being vascular endothelial growth factor (VEGF ).

• The sequence of events is as follows:1. VEGF induces nitric oxide and also the

expression of proteases ( e.g metalloproteinases ).

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• Nitric oxide causes local vasodilatation and the protease degrade the local basement membrane and local matrix and

• also mobilize further growth factors from matrix.

2. Endothelial cells migrate out forming a solid capillary sprout.

3. The endothelial cells behind the leading cells are activated by growth factors and start to divide.

4. A lumen forms in the sprout

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5. Local fibroblasts; activated by growth factors; proliferate and lay down matrix around capillary sprout.

6. A process of maturation occurs in which there is stabilization of the endothelial layer through cell to cell binding by adherence proteins and integrin binding of the cells to the matrix.

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APOPTOSIS AND CELL REMOVAL:

• Apoptosis is cell suicide by a built in self-destruct mechanism; it consists of a genetically programmed sequence of biochemical events.

• It is therefore, unlike necrosis, which is disorganized disintegration of damage cells resulting in products that trigger the inflammatory response.

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• Apoptosis is the mechanism which each day un-obtrusively removes 10 billion cells from the adult human body.

• It is involved in the shedding of intestinal lining, the regression of mammary gland cells after lactation and the death of time expired neutrophils.

• It is the basis for development of self-tolerance in the immune system and is implicated in the pathophysiology of cancer; autoimmune disease, neurodegenarative conditions, cardiovascular disease and the acquired immunodeficiency syndrome (AIDS ).

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• It plays an important role in embryogenesis, helping to shape organs during development by eliminating cells that have become redundant

• It has a role in the monitoring of cancerous change because it acts as a first line defense against mutations- purging cells with abnormal DNA that could become malignant.

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MORPHOLOGICAL CHANGES IN APOPTOSIS:

• As cell dies it rounds up, the chromatin in the nucleus condenses into dense masses and cytoplasm shrinks.

• This is followed by blebbing of plasma membrane and finally transformation of the cell into cluster of membrane-bound entities, which are rapidly phagocytosed by macrophages

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MAJOR PLAYERS IN APOPTOSIS:

• The major players are the caspases- a family of cysteine proteases present in inactive form.

• They do not perform generalized proteolysis; they undertake delicate protein surgery, selectively cleaving a specific set of target proteins ( enzymes; structural components , etc. ) inactivating some and activating others.

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• A cascade of about 9 different caspases take part in bringing about apoptosis, some functioning as initiators that transmit the initial apoptotic signals and some being responsible for the final effector phase of cell death.

• The caspases are not the only executors of apoptotic change.

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• Various pathways that result in apoptosis without the action of caspase fraternity have recently been described.

• One involves a protein termed AIF (apoptotic initiating factor), which is released from mitochondria, enters the nucleus and triggers cell suicide.

• NB:not all caspases are death-mediating enzymes; some have a role in the processing and activating of cytokines e.g caspase 8 is active in processing the inflammatorycytokines IL-1 and IL-18

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PATHWAY TO APOPTOSIS:

• There are two pathways to activation of effector caspases: the death receptor pathway and the mitochondrial pathway

• The death receptor pathway involves stimulation of members of tumour necrosis factor ( TNFR ) family; and the main initiator caspase is caspase 8.

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• The mitochondrial pathway is activated by internal factors such as DNA damage, which results in transcription of gene p53.

• The p53 protein activates a subpathway that results in release from mitochondrion of cytochrome c and the end result is activation of initiator caspase 9.

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• In undamaged cells, survival factors ( cytokines, hormones, cell to cell contact factors ) continuously activates anti-apoptotic mechanism. Withdrawal of survival factors stimulation causes cell death through the mitochondrial pathway.

• The effector caspases ( e.g. caspase 3 ) start a pathway that results in cleavage of cell constituents: DNA; cytoskeletal components, enzymes ; etc. This reduces the cell to a cluster of membrane- bound entities that are eventually phagocytosed by macrophages.

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TARGETS FOR NEW DRUG DEVELOPMENT:

• Angiogenesis has a critical role in numerous bodily processes, some physiological ( e.g. growth, repair) some pathological (e.g. tumour growth, chronic inflammatory conditions)

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• ANGIOGENESIS INHIBITORS

• these are being sought for use in pathological angiogenesis and there are currently 30 compounds in clinical trial. The approaches being used include:

1. Interference with endothelial cell growth, for example by the use of monoclonal antibodies that prevent the interaction of VEGF (vascular endothelial growth factor) and FGF (fibroblast growth factor) with their receptors.

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2. Interference with the necessary adherence of endothelial cells in the endothelial sprout to the matrix; an anti-integrin monoclonal antibody has shown promise.

3. Interference with the necessary degradation of matrix round the developing endothelial sprout; inhibitors of metalloproteinases are under test

• It should be noted that though anti-angiogenesis drugs may be helpful in some conditions ( e.g cancer ) they could be harmful in others ( e.g heart disease ).

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ANGIOGENESIS STIMULATORS:

• Angiogenesis stimulators are also being investigated for use in various ischaemic conditions; for example coronary disease, limb ischaemia, and gastro-intestinal ulcers associated with insufficient local perfusion.

• The main compound under investigation is VEGF.

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APOPTOTIC MECHANISMS:

• Example of defective apoptosis include cancer cell proliferation, resistance to cancer chemotherapy and ineffective eradication of virus-infected cells.

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• Examples of over-exuberant apoptosis include depletion of T cells in human deficiency virus ( HIV ) infection; allograft rejection, loss of neurones in neurodegenerative disease and loss of chrondocytes in osteoarthritis

• Several anti-apoptosis compounds are in clinical trials for neurodegenerative and inflammatory disease.

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• PLEASE READ THOROUGHLY & DISCUSS!