Transcript PharmacoLecture 7 - pharmacology1lecnotes

Cellular mechanism: Cell
proliferation and apoptosis
Objective/ learning out come:
Introduction.
 The cell cycle.
 Positive
regulators of the cell cycle.
 Negative
regulators of the cell cycle.
 Angiogenesis.
 Apoptosis
and cell removal.
Therapeutic implications
Introduction:
Cell proliferation is involved in many physiological and
pathological processes including growth, healing,
repair, hypertrophy, hyperplasia and development of
tumours.
Proliferating cells go through cell cycle, during
which the cell replicates all its components and then
bisects itself into two identical daughter cells.
Receptor tyrosine kinase and mitogen -activated
kinase cascade are very important and these
leads to transcription of the genes that control the
cycle.
The cell cycle:
The cell cycle is an ordered series of events consisting
of several sequential phase: G1,S,G2 and M
 M is the phase of mitosis.
 S phase is the phase of DNA synthesis.
 G1 is the gap between the mitosis that gave rise to the
cell and S phase; during G1the 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 the mitotic division into two daughter cells.
S phase (DNA replication) and M phase (Mitosis) are two very
critical event in cell division. Therefore entry into each of
these phases is carefully regulated by two check points
known as (restriction points) in the cycle.
DNA damage results in the cycle being stopped at one or
other of these, therefore the integrity of the check points is
critical for the maintenance of genetic stability.
Positive regulators of the cell cycle
The cell cycle is initiated when a growth factor acts on a
quiescent cell provoking it to divide. They achieve these
through the stimulation of the various cell cycle regulators.
Cyclins and cyclin - dependence kinase (cdks) are two
major proteins that determine the progress of cell cycle.
The cdks phosphorylate various proteins (e.g. enzymes)
activating some and inhibiting others to coordinate their
activities.
Each cdk 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.
It is the cyclin that determines which protein(s) are
phosphorylated.
.There
are eight main groups of cyclins, for control of
cell cycle cyclin A,B,D and E are very important. 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; cyclin E cdk 2.
The activity of these cyclin/cdk complexes is
modulated by various negative regulatory forces.
 cells in G0- cyclin D is present in low concentration.
Helps in phosphorylation of Rb proteins.
 phase G1- prepare for S-phase by synthesizing
RNAs (mRNAs) and proteins needed for DNA
replication. At this phase the concentration of cyclin
D increases and the cyclin D/cdk complex
phosphorylates and activates the necessary
proteins.
S-phase- Cyclin E/cdk and cyclin A/cdk regulate progress
through S-phase, phosphorylating and thus activating
proteins/enzymes involved in DNA synthesis.
G2 phase – the chromosomes in cell is doubled, cellular
components are duplicated synthesis of mRNAs and
proteins occur.
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. 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, cyclins A and B
remain stable throughout interphase but undergo proteolysis by
a ubiquitin - dependent pathway during mitosis.
Mitosis
Mitosis is a continuous process but can be considered to
consists of four stages: prophase, metaphase, anaphase
and telophase.
Negative regulators of the cell cycle
One of the main negative regulator of cell cycle is the Rb
protein, which holds the cycle in check while it is
hypophosphorylated.
Another negative regulatory mechanism is the action of
the inhibitors of cdks. These bind to and inhibit the action
of the complexes, their main action is on the check point
1.
There are two families of inhibitors:
 the CIP family – (cdk inhibitory proteins); also termed
KIP or kinase inhibitory proteins): p21, p27 and p57
 the Ink family ( inhibitors of kinase) : p16, p19,p15.
 The action of p21 serves as an example of the role
of a cyclin/cdk inhibitor. Protein p21 is under the control
of p53 gene- a particularly important negative regulator
that operates at check point 1.
Inhibition of the cycle at check point 1
 p53 gene guardian of genome, it codes for a
protein transcription factor-p53 protein.
 normal healthy person concentration of p53 is
low,
 but when there is DNA damage, the protein
accumulates and activates the transcription of several
genes one of which codes for p21.
 Protein p21 inactivates cyclin/cdk complexes,
thus preventing Rb phosphorylation, which means
arresting the cycle 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.
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 mechanism 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.
Angiogenesis
Angiogenesis, which normally accompanies cell proliferation,
is the formation of new capillaries from existing small blood
vessels.
Angiogenic stimuli, in the context of cell proliferation, includes
the action of various growth factors and cytokines, in particular
VEGF.
The sequence of events is as follows:
The basement membrane is degrade locally by proteases
Endothelial cells migrate out forming a sprout
Endothelial cells following the leading cells proliferate under
the influence of VEGF
Matrix is laid down around the new capillary.
Apoptosis and Cell Removal
Apoptosis is cell suicide by a built-in self destruct mechanism;
it consists of a generally programmed sequence of
biochemical events.
It is, therefore, unlike necrosis, which is a disorganized
disintegration of damaged cells resulting in products that
trigger the inflammatory response.
There are two main pathways to activation of the effectors
caspases : the death receptor pathway and the
mitochondrial pathway.
The death receptor pathway involves stimulation of
members of the tumor necrosis factor receptor (TNFR)
family; and the main initiator caspase is caspase 8
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.
This, in turn, complexes with protein Apaf-1 and together
they activate initiator caspase 9.
In undamaged cells, survival factors (cytokines, hormones,
cell-to-cell contact factors) continuously activate antiapoptotic mechanisms. Withdrawal of survival factor
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,
cytoskeleton components, enzymes, etc. This reduces the
cell to a cluster of membrane-bound entities that are
eventually phagocytosed by macrophages.
Pathophysiological Implications of Apoptosis
Cell proliferation and apoptosis are involved in many
physiological and pathological processes:
 the growth of tissues and organ
 the replenishment of lost or time-expired cells such as
leucocytes, gut epithelium, uterine endometrium, etc.
 healing and repair after injury or inflammation
 the hyperplasia (increase in cell number) associated with
chronic inflammatory, hypersensitivity and autoimmune
disease.
 the growth, invasion and metastasis of tumor
Therapeutic Implication
Targets For New Drug Development
Angiogenesis has a critical role in numerous bodily
processes, some physiological (e.g. growth, repair) some
pathological (e.g. tumor growth, chronic inflammatory
conditions)
Angiogenesis Inhibitors
Angiogenesis Inhibitors are being sought for use in
pathological angiogenesis and there are currently 30
compounds in clinical trial. The approaches being used
include:
Interference with endothelial cell growth, for example by
the use of monoclonal antibodies that prevent the
interaction of VEGF and FGF with their receptors
Interference with the necessary adherence of endothelial
cells in the endothelial sprout to the matrix; an antiintergrin monoclonal antibody has shown promise
Interference with the necessary degradation of the matrix
round the developing endothelial sprout; inhibitors of
metalloproteinases are under test.
Angiogenesis Stimulators
Angiogenesis Stimulators are being investigated for use in
various ischaemic condition, for example coronary disease,
limb ischaemia, and gastrointestinal ulcers associated with
insufficient local perfusion.
The main compound under investigation is VEGF. In pilot
studies, naked DNA encoding the gene for VEGF has been
injected directly into the relevant tissue along with a viral
promoter.
Apoptotic Mechanisms
Defective apoptosis is a factor in several diseases, and
compounds that modify it are under investigation
Examples of defective apoptosis include cancer cell
proliferation, resistance to cancer chemotherapy and
ineffective eradication of virus – infected cells.
Examples of over-exuberant apoptosis of T cells in human
immunodeficiency virus (HIV) infection, allograft rejection,
loss of neurons in neurodegenerative diseases, and loss of
chondrocytes in osteoarthritis
Several anti – apoptosis compounds are in clinical trials
for neurodegenerative and inflammatory diseases and
numerous pro – apoptosis compounds are in clinical trials
for cancer. Various approaches to apoptosis – based
therapies are being explored.