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CELL CYCLE
DNA REPAIR
CELL DEATH
The 2001 Nobel Prize in Physiology or Medicine was awarded to Lee Hartwell, Paul Nurse, and Tim Hunt for their groundbreaking work on cell cycle regulation. Starting in the late 60s, Hartwell used budding yeast to identify mutants that blocked
specific stages of cell cycle progression. Nurse, working in fission yeast in the 70s, went on to isolate mutants that could also
speed up the cell cycle, thus focussing his attention on the original CDK kinase, cdc2. In the 80s, Hunt identified proteins in
sea urchin extracts, the levels of which varied through the cell cycle hence "cyclins". All three have continued to make
important advances in cell cycle research including the identification of checkpoints, mechanisms coupling cell morphology to
the cell cycle, and identification of additional classes of kinases, cyclins, and inhibitors.
THE PHASES OF THE CELL CYCLE
mitosis
cytokinesis
DNA replication
A CELL CYLE CONTROLLER SYSTEM
COORDINATES THE CELL CYCLE MACHINERY
CHECKPOINTS MONITOR PROGRESSION
TROUGH THE CELL CYCLE
Are cell proliferative factors present?
EXIT FROM THE CELL CYCLE-G0
G0
EXIT FROM THE CELL CYCLE-G0
Many times a cell will leave the cell cycle, temporarily or
permanently. It exits the cycle at G1 and enters a stage
designated G0 (G zero). A G0 cell is often called "quiescent", but
that is probably more a reflection of the interests of the
scientists studying the cell cycle than the cell itself. Many G0
cells are anything but quiescent. They are busy carrying out their
functions in the organism. e.g., secretion, attacking pathogens.
Often G0 cells are terminally differentiated: they will never
reenter the cell cycle but instead will carry out their function in
the organism until they die. For other cells, G0 can be followed by
reentry into the cell cycle. Most of the lymphocytes in human
blood are in G0. However, with proper stimulation, such as
encountering the appropriate antigen, they can be stimulated to
reenter the cell cycle (at G1) and proceed on to new rounds of
alternating S phases and mitosis. G0 represents not simply the
absence of signals for mitosis but an active repression of the
genes needed for mitosis. Cancer cells cannot enter G0 and are
destined to repeat the cell cycle indefinitely.
EXPERIMENTAL EVIDENCES OF CELL
CYCLE REGULATORS
Mixing nuclei together in the same cytoplasm (heterokaryon) to determine whether
they could influence one another.
CONCLUSION: THERE ARE DIFFUSIBLE FACTORS THAT CAN PROMOTE S OR
M PHASE. THE S PHASE PROMOTING FACTOR (SPF) ONLY WORKS ON G1
NUCLEI. THE M PHASE PROMOTING FACTOR (MPF) WORKS ON
EVERYTHING.
THE M PHASE PROMOTING FACTOR (MPF)
-IDENTIFICATION OF CDK
Xenopus laevis eggs: When a small sample taken from a meiosis -II
metaphase arrested secondary oocyte is injected into a meiosis-I G2 phase
primary oocyte, the G2-arrested cell will mature without progesterone and
reach to metaphase of meiosis-II, because the secondary oocyte contains
MPF. Cyclin dependent kinase (cdk) was isolated this way.
THE M PHASE PROMOTING FACTOR (MPF) IDENTIFICATION OF CYCLIN
Accumulation and degradation of cyclin B
Sea urchin embryos. Found when looking at total proteins in a
population undergoing synchronous division that some proteins go up
and down with the cell cycle: cyclin.
THE M PHASE PROMOTING FACTOR (MPF) IDENTIFICATION OF CYCLIN
Sea urchin adult/embryo.
THE M PHASE PROMOTING FACTOR (MPF):
CIKLIN+CDK
Cdk 1
Cyclin B
THE M PHASE PROMOTING FACTOR (MPF):
CIKLIN+CDK
MPF IS ACTIVE ONLY AT HIGH CYCLIN CONCENTRATIONS
MPF IS SUFFICIENT TO INDUCE THE
DRASTIC CHANGES OCCURING AT M-PHASE
 chromosome condensation
 breakdown of the nuclear envelope
 reorganization of the actin cytoskeleton
 reorganization of the microtubule cytoskeleton
 assembly of the mitotic spindle
 chromosome attachment to the kinetochore microtubules
CDKs/CYCLINS OF HIGHER
EUKARYOTES
Cyclin-dependent kinases
Cdk4
Cdk2
cyclins
M
D
G1
Cdk1
E
A
S
Start
Cell cycle phases
B(A)
G2
M G1
Control of the Cell Cycle
The passage of a cell through the cell cycle is controlled by proteins in the
cytoplasm. Among the main players in animal cells are:
CYCLINS
•a G1 cyclin (cyclin D)
•S-phase cyclins (cyclins E and A)
•mitotic cyclins (cyclins B and A)
Their levels in the cell rise and fall with the stages of the cell cycle.
CYCLIN-DEPENDENT KINASES (CDKS)
•a G1 Cdk (Cdk4)
•an S-phase Cdk (Cdk2)
•an M-phase Cdk (Cdk1)
Their levels in the cell remain fairly stable, but each must bind the appropriate
cyclin (whose levels fluctuate) in order to be activated. They add phosphate
groups to a variety of protein substrates that control processes in the cell
cycle.
The anaphase-promoting complex (APC). (The APC is also called the cyclosome,
and the complex is often designated as the APC/C.) The APC/C
•triggers the events leading to destruction of the cohesins thus allowing
the sister chromatids to separate;
•degrades the mitotic cyclin B.
Steps in the cell cycle
A rising level of G1-cyclins bind to their Cdks and signal the cell to prepare the
chromosomes for replication.
A rising level of S-phase promoting factor (SPF) — which includes cyclin A bound to
Cdk2 — enters the nucleus and prepares the cell to duplicate its DNA (and its
centrosomes).
As DNA replication continues, cyclin E is destroyed, and the level of mitotic cyclins
begins to rise (in G2).
M-phase promoting factor (the complex of mitotic cyclins with the M-phase Cdk)
initiates
•assembly of the mitotic spindle
•breakdown of the nuclear envelope
•condensation of the chromosomes
These events take the cell to metaphase of mitosis.
At this point, the M-phase promoting factor activates the anaphase-promoting complex
(APC/C) which
•allows the sister chromatids at the metaphase plate to separate and move to the
poles (= anaphase), completing mitosis;
•destroys cyclin B. It does this by attaching it to the protein ubiquitin which
targets it for destruction by proteasomes.
•turns on synthesis of G1 cyclin for the next turn of the cycle;
•degrades geminin, a protein that has kept the freshly-synthesized DNA in S phase
from being re-replicated before mitosis. This is only one mechanism by which the
cell ensures that every portion of its genome is copied once — and only once —
during S phase.
MECHANISMS OF CDK REGULATION
MECHANISMS OF CDK REGULATION
PHOSPHORILATION-DEPHOSPHORILATION
MECHANISMS OF CDK REGULATION
INHIBITORY PROTEIN (P27/CKI) BINDING
INACTIVATES THE CYCLIN-CDK COMPLEX
MECHANISMS OF CDK REGULATION
UBIQUITIN-DEPENDENT
PROTEIN DEGRADATION
CELL CYCLE CHECKPOINTS
THE G1/S CELL CYCLE CHECKPOINT
DNA
Protein
kinase
activation
Stable,
Active p53
p53 foszforiláció
p53 binds to the promoter
of the p21 gene
p53 degradation
transcription
P21 mRNS
translation
P21 (CKI)
ACTIVE
INACTIVE
THE G1/S CELL CYCLE CHECKPOINT
The G1/S cell cycle checkpoint controls the passage of
eukaryotic cells from the first “gap” phase (G1) into the
DNA synthesis phase (S). Two cell cycle kinases,
CDK4/6-cyclin D and CDK2-cyclin E, and the
transcription complex that includes Rb and E2F are
pivotal in controlling this checkpoint. During G1-phase,
the Rb-HDAC repressor complex binds to the E2F-DP1
transcription factors, inhibiting downstream
transcription. Phosphorylation of Rb by CDK4/6 and
CDK2 dissociates the Rb-repressor complex, permitting
transcription of S-phase-promoting genes including
some that are required for DNA replication. Many
different stimuli exert checkpoint control including
TGFβ, DNA damage, contact inhibition, replicative
senescence and growth factor withdrawal. The first four
act by inducing members of the INK4 or Kip/Cip
families of cyclin dependent kinase inhibitors (CKIs).
TGFβ also inhibits the transcription of cdc25A, a
phosphatase required for CDK activation. In response to
DNA damage-induced activation of the
ATM/ATR/Chk1/2 pathway, cdc25A is ubiquitinated and
targeted for degradation via the SCF ubiquitin ligase
complex. Targeted degradation of cdc25A in mitosis via
the APC ubiquitin ligase complex allows progression
through mitosis. Growth factor withdrawal activates
GSK-3β, which phosphorylates cyclin D, leading to its
rapid ubiquitination and proteosomal degradation.
Ubiquitin/proteasome-dependent degradation and
nuclear export are mechanisms commonly used to
rapidly reduce the concentration of cell cycle control
proteins. Some redundancy and tissue specific
requirements exist as shown by animal models.
p53
The p53 protein senses DNA damage and can halt progression of the cell cycle in
G1. Both copies of the p53 gene must be mutated for this to fail so mutations in
p53 are recessive, and p53 qualifies as a tumor suppressor gene. The p53 protein is
also a key player in apoptosis, forcing "bad" cells to commit suicide. So if the cell
has only mutant versions of the protein, it can live on — perhaps developing into a
cancer. More than half of all human cancers have p53 mutations and have no
functioning p53 protein. A genetically-engineered adenovirus can only replicate in
human cells lacking p53. Thus it infects, replicates, and ultimately kills many types
of cancer cells in vitro. Clinical trials are now proceeding to see if injections of this
virus can shrink a variety of types of cancers in human patients. In some way, p53
seems to evaluate the extent of damage to DNA, at least for damage by radiation.
At low levels of radiation, producing damage that can be repaired, p53 triggers
arrest of the cell cycle until the damage is repaired.
At high levels of radiation, producing hopelessly damaged DNA, p53 triggers
apoptosis. Possible mechanism:
•Serious damage, e.g., double strand breaks(DSBs), causes a linker histone (H1)
to be released from the chromatin.
•H1 leaves the nucleus and enters the cytosol where
•it triggers the release of cytochrome c from mitochondria leading to
•apoptosis.
ATM (ataxia telangiectasia mutated)
Symptoms of the disease ataxia telangiectasia. The ATM protein is involved in
detecting DNA damage, especially double-strand breaks; interrupting (with the aid
of p53) the cell cycle when damage is found; maintaining normal telomere length.
RB - the retinoblastoma gene
Retinoblastoma is a cancerous tumor of the retina. It occurs in two forms:
Familial retinoblastoma
Multiple tumors in the retinas of both eyes occurring in the first weeks of
infancy.
Sporadic retinoblastoma
A single tumor appears in one eye sometime in early childhood before the
retina is fully developed and mitosis in it ceases.
Familial retinoblastoma
Familial retinoblastoma occurs when the fetus inherits from both of its parents a
chromosome (number 13) that has its RB locus deleted (or otherwise mutated). The
normal Rb protein prevents mitosis.
Mechanism. The Rb protein prevents cells from entering S phase of the cell cycle.
It does this by binding to a transcription factor called E2F. This prevents E2F
from binding to the promoters of such proto-oncogenes as c-myc and c-fos.
Transcription of c-myc and c-fos is needed for mitosis so blocking the transcription
factor needed to turn on these genes prevents cell division.
Sporadic retinoblastoma
In this disease, both inherited RB genes are normal and a single cell must be so
unlucky as to suffer a somatic mutation (often a deletion) in both in order to
develop into a tumor. Such a double hit is an exceedingly improbable event, and so
only rarely will such a tumor occur. (In both forms of the disease, the patient's life
can be saved if the tumor(s) is detected soon enough and the affected eye(s)
CELL CYCLE CHECKPOINTS
THE G2/M CELL CYCLE CHECKPOINT
A complex of checkpoint proteins recognizes unreplicated or damaged DNA and
activates the protein kinase Chk1, which phosphorylates and inhibits the Cdc25
protein phosphatase. Inhibition of Cdc25 prevents dephosphorylation and
activation of Cdc2.
THE G2/M CELL CYCLE CHECKPOINT
cdk1
The G2/M DNA damage checkpoint prevents the cell
from entering mitosis (M-phase) if the genome is
damaged. The cdk1-cyclin B complex is pivotal in
regulating this transition. During G2-phase, cdk1 is
maintained in an inactive state by the kinases Wee1
and Myt1. As cells approach M phase, the
phosphatase cdc25 is activated by phosphorylation.
Cdc25 then activates cdk1, establishing a feedback
amplification loop that efficiently drives the cell into
mitosis. DNA damage activates the DNAPK/ATM/ATR kinases, initiating two parallel
cascades that inactivate cdk1-cyclin B. The first
cascade rapidly inhibits progression into mitosis: the
Chk kinases phosphorylate and inactivate cdc25,
which can no longer activate cdk1. The second
cascade is slower. Phosphorylation of p53 dissociates
it from MDM2, activating its DNA binding activity.
Acetylation by p300/PCAF further activates its
transcriptional activity. The genes that are turned on
by p53 constitute effectors of this second cascade.
They include 14-3-3, which binds to the
phosphorylated cdk1-cyclin B complex and exports
it from the nucleus; GADD45, which apparently
binds to and dissociates the cdk1-cyclin B complex;
and p21/Cip1, an inhibitor of a subset of the cyclindependent kinases including cdk1.
CELL CYCLE CHECKPOINTS
THE META/ANAPHASE TRANSITION
THE META/ANAPHASE TRANSITION
THE META/ANAPHASE TRANSITIONSPINDLE ASSEMBLY CHECKPOINT
What if There Were No Checkpoint?
THE META/ANAPHASE TRANSITIONSPINDLE ASSEMBLY CHECKPOINT
Unattached Kinetochores Cause a Checkpoint Delay
THE META/ANAPHASE TRANSITIONSPINDLE ASSEMBLY CHECKPOINT
APC is the target of the spindle assembly checkpoint
THE META/ANAPHASE TRANSITIONSPINDLE ASSEMBLY CHECKPOINT
Mad2 cycles through kinetochore and inhibits cdc20
MAD AND CANCER
MAD (="mitotic arrest deficient") genes (there are two) encode proteins that
bind to each kinetochore until a spindle fiber (one microtubule will do) attaches
to it. If there is any failure to attach, MAD remains and blocks entry into
anaphase.
Mutations in MAD produce a defective protein and failure of the checkpoint.
The cell finishes mitosis but produces daughter cells with too many or too few
chromosomes (aneuploidy). Aneuploidy is one of the hallmarks of cancer cells
suggesting that failure of the spindle checkpoint is a major step in the
conversion of a normal cell into a cancerous one.
Infection with the human T cell leukemia virus-1 (HTLV-1) leads to a cancer
(ATL = "adult T cell leukemia") in about 5% of its victims. HTLV-1 encodes a
protein, called Tax, that binds to MAD protein causing failure of the spindle
checkpoint. The leukemic cells in these patients show many chromosome
abnormalities including aneuploidy.
CELL CYCLE CONTROL MECHANISMS
DNA
damage
KEY PLAYERS AND
EVENTS
CHECKPOINTS
Inappropiate
environment
mitogen
stimulation
non
replicated
DNA
Unattached
kinetochores
DNA
damage
p53
G1-Cdk
G1/S-Cdk
Hct1
CKI
S-Cdk
Cdc25
APC
M-Cdk
Synthesis of G1/S
cyclin
DNA replication
Synthesis of S cyclin
G1
S
G2
M
G1
MUTATION
DNA REPAIR
SPONTANEOUS MUTATIONS
The spontaneous mutation rate is low and different in
different
organisms.
bacteria: 10-10-10-6/gene/generation
The diploid nature of higher eukaryotes allows tolerance of higher
mutation rates than in prokaryotes, provides greater genetic
heterogeneity and thus evolutionary adaptability.
Drosophila: 10-4-10-5/gene/generation
Mouse: 10-5/gene/generation
Human: 4x10-6-10-4/gene/generation
Many of the recessive mutations are lethal: incompatible with life in
homozygous condition. It is estimated that an average human is
heterozygous for 3-5 recessive lethal mutations.
SPONTANEOUS MUTATIONS - TAUTOMERIC SHIFTS
because of seldom and
short lived tautomeric
shifts, adenine and
cytosine attain the rare
imino, guanine and
thymine the rare enol
form.
tautomeric shifts lead to unusual base pair formation (so-called
mismatches)
SPONTANEOUS MUTATIONS - TAUTOMERIC SHIFTS
In their rare forms A* pairs with C, T* with G, G* with
T and C* with A
SPONTANEOUS MUTATIONS - TAUTOMERIC SHIFTS
If tautomeric shift happens at the time of replication,
BASE PAIR SUBSTITUTION happens in the DNA
SPONTANEOUS MUTATIONS - TAUTOMERIC SHIFTS
The base pair exchange is a MUTATION: heritable
change in the genetic material.
In eukaryotes the frequency of base pair
substitutions is 10-3/kb/replication.
It is estimated that eight base pair mutations occur
during one round of replication of the human
genome.
SPONTANEOUS MUTATIONS–BP. ADDITION & DELETION
Mispairing of the bases in the complementary DNA strands
leads to deletion or addition of nucleotides during replication
from one and into the another of the DNA molecules. As a
result, FRAME SHIFT MUTATIONS orginate.
SPONTANEOUS MUTATIONS–BP. ADDITION & DELETION
Mispairing of the complementary DNA strands may lead to
the formation of extrahelical loops. Most of the extrahelical
loops form following single strand breaks (that arise due to
radiation, replication, recombination, reparation of DNA as
well as due to structurally altered DNA.). Mispairing may lead
to deletion of base pairs from one or addition to the another
DNA strand following replication. As a result of base pair
deletions and additions the frame of the genetic information
is shifted and hence the originated mutations are called
frame shift mutations.
The so-called intercalating agents stabilize the extrahelical
loops, and increase the probability of base pair deletions and
additions, i.e. frame shift mutations.
INDUCED MUTATIONS
physical : ionizing radiation, UV
chemical: chemical mutagens
biological : transposons,
retroposons
direct mutagenes:
induce mutations by acting directly
on the DNA.
indirect mutagenes :
become chemically modified for
excretion form the body, and the
intermediates possess mutagenic
activities
The formation of mutagenic substance from the
nonreactive benzopyrene.
INDUCED MUTATIONS
The UV indirectly causes base substitutions. Pyrimidine, mostly
thymine dimers form upon UV radiation: the adjacent thymine bases
of the same DNA strand become covalently bound. The thymine
dimers cannot act as template during replication and consequently
nucleotides are added randomly opposite to the thymine dimers and
hence base substitutions result.
THE MUTATIONS DISCUSSED SO FAR,
WHETHER SPONTANEOUS OR INDUCED,
ARE RESTRICTED TO VERY SHORT
STRETCH OF THE DNA AND ARE HENCE
CALLED POINT MUTATIONS OR GENE
MUTATIONS.
THE CONSEQUENCES OF MUTATIONS
BASE PAIR SUBSTITUTIONS

Base substitutions do not have usually consequences in case of the
SILENT mutations that happen in the third base of the genetic
codes. Degeneracy of the genetic code implies the free exchange of
many of the third bases.
 In case of the QUIET mutations, base substitution is followed by
replacement of an amino acid with another one of the same type
(e.g. hydrophilic to hydrophilic). The quiet mutations do not usually
change significantly the function of the encoded protein. In fact,
genetic codes of the amino acids of similar character are rather
similar.
 Base pair substitutions at the second position are of the most
dramatic consequences. In case of any MISSENSE mutation, a
single base pair substitution leads to a single amino acid substitution
in the encoded protein. For example in sickle cell anemia a single base
pair substitution (5'GAA3' to 5'GUA3') results in a Glu to Val change at
position #6 of the ß-hemoglobin molecules. The Glu to Val change is
inherited as an autosomal recessive mutation and causes severe anemia.
THE CONSEQUENCES OF MUTATIONS
BASE PAIR SUBSTITUTIONS
 The NONSENSE mutations change an amino acid coding code to
one of the three stop codons. As the consequences of nonsense
mutations, translation of the mRNA molecules is prematurely
terminated. The forming shorter than normal proteins are usually
non functional and are quickly degraded.
 The CHAIN-ELONGATION mutations convert a STOP code into
sense code that encodes for the incorporation of an amino acid
into the synthesized protein molecule. E.g. UAG (STOP) to UAC
(Tyr). The longer than normal protein molecule is most of the
time nonfunctional, sometimes possess reduced activities. Several
types of anemia are caused by chain elongation mutations.
THE CONSEQUENCES OF MUTATIONS
FRAME SHIFT MUTATIONS
The frame shift mutations scramble the amino acid
sequence of the encoded protein form the site of the
mutation towards the C-terminal. Frequently
polypeptides form that are shorter or longer than
normal. These are the worst amongst the point
mutations.
CHROMOSOME MUTATIONS
STRUCTURAL CHANGES DUE TO CHROMOSOME BREAKS
The ionizing radiations (and also several of the chemical
and biological mutagens) can cause breakages of the DNA
with several further consequences.
In the INVERSIONS a sequence of bases is reversed.
The TRANSLOCATIONS move large DNA segments into
unusual positions. (In reciproc translocations chromosome
segments are exchanged.)
CHROMOSOME MUTATIONS
STRUCTURAL CHANGES DUE TO CHROMOSOME BREAKS
 DEFICIENCIES originate by removal of large
numbers of bases. (The DELETIONS eliminate one
or only a few base pairs.)
Large numbers of already existing sequences are
added in the DUPLICATIONS.
The so-called chromosome rearrangements frequently have deleterious
effects. A typical example is the Cri-du-chat syndrome that develops due
to genetic imbalance, an altered gene-dosage as the consequence of a
translocation of a piece of the 5th to the 13th chromosome
CHROMOSOME MUTATIONS
MUTATIONS DUE TO NUMERICAL CHROMOSOME
ABERRATIONS
The amount of DNA in the cells/organisms can change due
to gain or loss of entire chromosomes.
The change of chromosome number is the consequence of
chromosome loss and nondisjunction.
MUTATIONS IN THE GERM LINE AND SOMA
Naturally, both germ-line and somatic cells are
susceptible to mutations and, of course, mutations
occur in both cell types. Mutations in somatic cells
cause only altered somatic functions (including
cancer) and, fortunately, are not propagated for
the offspring. Mutations in the germ line cells,
however, may be inherited to the offspring,
increasing the so-called genetic load of the species.
DNA REPAIR
DNA in the living cell is subject to many chemical alterations.
If the genetic information encoded in the DNA is to remain
uncorrupted, any chemical changes must be corrected.
A failure to repair DNA produces a mutation.
The recent publication of the human genome has already
revealed 130 genes whose products participate in DNA repair.
REPAIRING DAMAGED BASES
Damaged or inappropriate bases can be repaired by several
mechanisms:
DIRECT CHEMICAL REVERSAL OF THE DAMAGE
EXCISION REPAIR, in which the damaged base or bases
are removed and then replaced with the correct ones in a
localized burst of DNA synthesis. There are three modes of
excision repair, each of which employs specialized sets of
enzymes.
•BASE (NUCLEOSIDE) EXCISION REPAIR (BER)
•NUCLEOTIDE EXCISION REPAIR (NER)
•MISMATCH REPAIR (MMR)
XERODERMA PIGMENTOSUM (XP)
XP is a rare
inherited disease
of humans which,
among other
things, predisposes
the patient to
pigmented lesions
on areas of the
skin exposed to
the sun and an
elevated incidence
of skin cancer. It
turns out that XP
can be caused by
mutations in any
one of several
genes — all of
which have roles to
play in NER.
REPAIRING STRAND BREAKS
Ionizing radiation and certain chemicals can produce both
single-strand breaks and double-strand breaks in the DNA
backbone.
SINGLE-STRAND BREAKS
Breaks in a single strand of the DNA molecule are repaired
using the same enzyme systems that are used in BaseExcision Repair (BER).
REPAIRING STRAND BREAKS
DOUBLE-STRAND BREAKS (DSBs)
There are two mechanisms by which the cell attempts to repair
a complete break in a DNA molecule:
1.DIRECT JOINING OF THE BROKEN ENDS
This requires proteins that recognize and bind to the exposed
ends and bring them together for ligating. They would prefer to
see some complementary nucleotides but can proceed without
them so this type of joining is also called Nonhomologous EndJoining (NHEJ). Errors in direct joining may be a cause of the
various translocations that are associated with cancers.
Examples: Burkitt's lymphoma, the Philadelphia chromosome in
chronic myelogenous leukemia (CML), B-cell leukemia.
REPAIRING STRAND BREAKS
2.HOMOLOGOUS RECOMBINATION
Here the broken ends are repaired using the information on the
intact sister chromatid (available in G2 after chromosome
duplication), or on the homologous chromosome (in G1; that is,
before each chromosome has been duplicated). This requires
searching around in the nucleus for the homolog — a task
sufficiently uncertain that G1 cells usually prefer to mend their
DSBs by NHEJ. Two of the proteins used in homologous
recombination are encoded by the genes BRCA-1 and BRCA-2.
Inherited mutations in these genes predispose women to breast
and ovarian cancers.
APOPTOSIS
NECROSIS
NECROSIS
 A pathological response to cellular injury
 Chromatin clumping
 Mitochondria swelling and rupture
 Plasma membrane lyses
 Cell contents spill out
 General inflammatory response is triggered
APOPTOSIS
 A normal physiological response to specific suicide signals or lack of
survival signals
 Chromatin condenses and migrates to nuclear membrane.
Internucleosomal cleavage leads to laddering of DNA at the
nucleosomal repeat length (200bp)
 Cytoplasm shrinks without membrane rupture
 Blebbing of plasma and nuclear membranes
 Cell contents are packaged in membrane bounded bodies, to be
engulfed by phagocytes
 No spillage, no inflammation
The phosphoipid phosphatidylserine, which is normally hidden within
the plasma membrane, is exposed on the surface.This is bound by
receptors on phagocytic cells which then engulf the cell fragments.
The phagocytic cells secrete cytokines that inhibit inflammation (e.g.,
IL-10 and TGF-B)
WHY SHOULD A CELL COMMIT SUICIDE?
1. PROGRAMMED CELL DEATH IS AS NEEDED FOR
PROPER DEVELOPMENT AS MITOSIS IS.
Examples:
The resorption of the tadpole tail at the time of its
metamorphosis into a frog occurs by apoptosis.
The formation of the fingers and toes of the fetus requires the
removal, by apoptosis, of the tissue between them.
The sloughing off of the inner lining of the uterus (the
endometrium) at the start of menstruation occurs by apoptosis.
The formation of the proper connections (synapses) between
neurons in the brain requires that surplus cells be eliminated by
apoptosis
APOPTOSIS IN EMBRYONIC AND FETAL
DEVELOPMENT
Normal
Apoptosis
Dysfunctional
Apoptosis
WHY SHOULD A CELL COMMIT SUICIDE?
2. PROGRAMMED CELL DEATH IS NEEDED TO DESTROY
CELLS THAT REPRESENT A THREAT TO THE INTEGRITY
OF THE ORGANISM.
Examples:
CELLS INFECTED WITH VIRUSES
One of the methods by which cytotoxic T lymphocites (CTLs) kill virusinfected cells is by inducing apoptosis
CELLS OF THE IMMUNE SYSTEM
As cell-mediated immune responses wane, the effector cells must be
removed to prevent them from attacking body constituents. CTLs
induce apoptosis in each other and even in themselves. Defects in the
apoptotic machinery is associated with autoimmune diseases such as
lupus erythematosus and rheumatoid arthritis.
WHY SHOULD A CELL COMMIT SUICIDE?
2. PROGRAMMED CELL DEATH IS NEEDED TO DESTROY
CELLS THAT REPRESENT A THREAT TO THE INTEGRITY
OF THE ORGANISM.
Examples:
CELLS WITH DNA DAMAGE
Damage to its genome can cause a cell to disrupt proper embryonic
development leading to birth defects or to become cancerous.
Cells respond to DNA damage by increasing their production of p53.
p53 is a potent inducer of apoptosis. Is it any wonder that mutations in
the p53 gene, producing a defective protein, are so often found in
cancer cells (that represent a lethal threat to the organism if
permitted to live)?
CANCER CELLS
Radiation and chemicals used in cancer therapy induce apoptosis in
some types of cancer cells.
WHAT MAKES A CELL DECIDE TO
COMMIT SUICIDE?
WITHDRAWAL OF POSITIVE/SURVIVAL SIGNALS
The continued survival of many cells requires that they
receive continuous stimulation from other cells and, for
many, continued adhesion to the surface on which they
are growing.
WHAT MAKES A CELL DECIDE TO
COMMIT SUICIDE?
RECEIPT OF NEGATIVE/SUICIDE SIGNALS
increased levels of oxidants within the cell
damage to DNA by these oxidants or other agents like
•Ultraviolet light
•X-rays
•Chemotherapeutic drugs
accumulation of proteins that failed to fold properly
molecules that bind to specific receptors on the cell surface and signal
the cell to begin the apoptosis program. These death activators include:
•Tumor necrosis factor-alpha (TNF-α ) that binds to the TNFreceptor
•Lymphotoxin (also known as TNF-β ) that also binds to the TNF
receptor;
•Fas ligand (FasL), a molecule that binds to a cell-surface receptor
named Fas (also called CD95).
APOPTOSIS OVERVIEW
DNA damage
Apoptosis is a regulated physiological process leading
to cell death characterized by cell shrinkage,
membrane blebbing and DNA fragmentation.
Caspases, a family of cysteine proteases, are central
regulators of apoptosis. Initiator caspases (including
caspase-2, -8, -9, -10, -11 and -12) are closely coupled
to pro-apoptotic signals. Once activated, these
caspases cleave and activate downstream effector
caspases (including caspase-3, -6 and -7), which in
turn execute apoptosis by cleaving cellular proteins
following specific Asp residues. Activation of Fas and
TNFR by FasL and TNF, respectively, leads to the
activation of caspase-8 and -10. DNA damage leads to
the activation of caspase-2. Cytochrome c released
from damaged mitochondria is coupled to the
activation of caspase-9, a caspase critical for
intracellular amplification of apoptotic signals.
Caspase-11 is induced and activated by pathological
proinflammatory and pro-apoptotic stimuli and leads
to the activation of caspase-1 to promote inflammatory
response and apoptosis by directly processing caspase3. Caspase-12 is specifically activated under ER stress
conditions. Anti-apoptotic ligands including growth
factors and cytokines activate Akt and p90RSK. Akt
inhibits Bad by direct phosphorylation and prevents
the expression of Bim by phosphorylating and
inhibiting the Forkhead family of transcriptional
factors.
THE MECHANISMS OF APOPTOSIS
There are two different mechanisms by which a cell commits suicide
by apoptosis.
 One generated by signals
arising within the cell;
 another triggered by death
activators binding to
receptors at the cell
surface:
• TNF-α
• Lymphotoxin
• Fas ligand (FasL)
APOPTOSIS TRIGGERED BY INTERNAL SIGNALS:
THE INTRINSIC OR MITOCHONDRIAL PATHWAY
APOPTOSIS TRIGGERED BY INTERNAL SIGNALS:
THE INTRINSIC OR MITOCHONDRIAL PATHWAY
In a healthy cell, the outer membranes of its mitochondria display the
protein Bcl-2 on their surface. Internal damage to the cell (e.g., from
reactive oxygen species) causes Bcl-2 to activate a related protein, Bax,
which punches holes in the outer mitochondrial membrane, causing
cytochrome c to leak out.
The released cytochrome c binds to the protein Apaf-1 ("apoptotic
protease activating factor-1"). Using the energy provided by ATP, these
complexes aggregate to form apoptosomes. The apoptosomes bind to
and activate caspase-9. Caspase-9 is one of a family of over a dozen
caspases. They are all proteases. They get their name because they
cleave proteins — mostly each other — at aspartic acid (Asp) residues).
Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3
and -7). The activation of these "executioner" caspases creates an
expanding cascade of proteolytic activity which leads to digestion of
structural proteins in the cytoplasm, degradation of chromosomal DNA,
and phagocytosis of the cell.
APOPTOSIS TRIGGERED BY INTERNAL SIGNALS:
THE INTRINSIC OR MITOCHONDRIAL PATHWAY
The Bcl-2 family of proteins regulate
apoptosis by controlling mitochondrial
permeability and the release of cytochrome
c. The anti-apoptotic proteins Bcl-2 and
Bcl-xL reside in the outer mitochondrial
wall and inhibit cytochrome c release. The
proapoptotic Bcl-2 proteins Bad, Bid, Bax
and Bim may reside in the cytosol but
translocate to mitochondria following death
signaling, where they promote the release
of cytochrome c. Bad translocates to
mitochondria and forms a pro-apoptotic
complex with Bcl-xL. This translocation is
inhibited by survival factors that induce the
phosphorylation of Bad, leading to its
cytosolic sequestration. Cytosolic Bid is
cleaved by caspase-8 following signaling
through Fas: its active fragment (tBid)
translocates to mitochondria. Bax and Bim
translocate to mitochondria in response to
death stimuli, including survival factor
withdrawal. p53, activated following DNA
damage, induces the transcription of Bax,
Noxa and PUMA. Upon release from
mitochondria, cytochrome c binds Apaf1
and forms an activation complex with
caspase-9. Although the mechanism(s)
regulating mitochondrial permeability and
the release of cytochrome c during
apoptosis are not fully understood, Bcl-xL,
Bcl-2 and Bax may influence the voltagedependent anion channel (VDAC), which
may play a role in regulating cytochrome c
release.
APOPTOSIS TRIGGERED BY EXTERNAL SIGNALS:
THE EXTRINSIC OR DEATH RECEPTOR PATHWAY
When cytotoxic T cells recognize (bind to) their target, they produce more FasL at
their surface. This binds with the Fas on the surface of the target cell leading to
its death by apoptosis.
APOPTOSIS TRIGGERED BY EXTERNAL SIGNALS:
THE EXTRINSIC OR DEATH RECEPTOR PATHWAY
Fas and the TNF receptor are integral membrane
proteins with their receptor domains exposed at the
surface of the cell. Binding of the complementary death
activator (FasL and TNF respectively) transmits a signal
to the cytoplasm that leads to activation of caspase 8.
Caspase 8 (like caspase 9) initiates a cascade of caspase
activation leading to phagocytosis of the cell.
APOPTOSIS TRIGGERED BY EXTERNAL SIGNALS:
THE EXTRINSIC OR DEATH RECEPTOR PATHWAY
Apoptosis can be induced through the
activation of death receptors including
Fas, TNFR, DR3, DR4 and DR5 by their
respective ligands. Death receptor
ligands characteristically initiate
signaling via receptor oligomerization,
which in turn results in the recruitment of
specialized adaptor proteins and
activation of caspase cascades. FasL
binding induces Fas trimerization, which
recruits initiator caspase-8 via the
adaptor protein FADD. Caspase-8 then
oligomerizes and is activated via
autocatalysis. Activated caspase-8
stimulates apoptosis via two parallel
cascades: it directly cleaves and activates
caspase-3, and it cleaves Bid (a Bcl-2
family protein). Truncated Bid (tBid)
translocates to mitochondria, inducing
cytochrome c release, which sequentially
activates caspases 9 and 3. TNF and DR3L can deliver pro- or anti-apoptotic
signals. TNFR and DR3 promote
apoptosis via the adaptor proteins
TRADD/FADD and the activation of
caspase-8. Alternatively or
simultaneously, interaction of TNF with
TNFR may activate the NF-κB pathway
via an adaptor protein complex including
RIP and induce survival genes including
IAP. Induction of apoptosis via Apo2L
requires caspase activity.
APOPTOSIS INHIBITION
Cell survival requires the
active inhibition of apoptosis,
which is accomplished by
inhibiting the expression of
pro-apoptotic factors as well
as promoting the expression
of anti-apoptotic factors
The PI3K pathway, activated by many survival factors,
leads to the activation of Akt, an important player in
survival signaling. Activated Akt inhibits the pro-apoptotic
Bcl-2 family member Bad, caspase-9, GSK-3 and FKHR
by phosphorylation. Many growth factors and cytokines
induce anti-apoptotic Bcl-2 family members. The Jaks
and Src phosphorylate and activate Stat3, which in turn
induces the expression of Bcl-xL and Bcl-2. Erk1/2 and
PKC activate p90RSK, which activates CREB and
induces the expression of Bcl-xL and Bcl-2. These Bcl-2
family members protect the integrity of mitochondria,
preventing cytochrome c release and the subsequent
activation of caspase-9. TNF may activate both proapoptotic and anti-apoptotic pathways: TNF can induce
apoptosis by activating caspase-8 and -10, but can also
inhibit apoptosis signaling via NF-κB, which induces the
expression of IAP, an inhibitor of caspases 3, 7 and 9.
APOPTOSIS AND CANCER
Some viruses associated with cancers use tricks to prevent apoptosis of
the cells they have transformed. Several human papiloma viruses (HPV)
have been implicated in causing cervical cancer. One of them produces a
protein (E6) that binds and inactivates the apoptosis promoter p53.
Epstein-Barr Virus (EBV), the cause of mononucleosis and associated
with some lymphomas produces a protein similar to Bcl-2, produces
another protein that causes the cell to increase its own production of
Bcl-2. Both these actions make the cell more resistant to apoptosis (thus
enabling a cancer cell to continue to proliferate).
APOPTOSIS AND CANCER
Even cancer cells produced without the participation of viruses may have tricks
to avoid apoptosis. Some B-cell leukemias and lymphomas express high levels of
Bcl-2, thus blocking apoptotic signals they may receive. The high levels result
from a translocation of the BCL-2 gene into an enhancer region for antibody
production.
Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by
inhibiting the expression of the gene encoding Apaf-1.
Some cancer cells, especially lung and colon cancer cells, secrete elevated levels
of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind
Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells.
Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells
(CTL) that try to kill them because CTL also express Fas (but are protected
from their own FasL).
APOPTOSIS IN THE IMMUNE SYSTEM
The immune response to a foreign invader involves the proliferation of
lymphocytes — T and/or B cells. When their job is done, they must be removed
leaving only a small population of memory cells. This is done by apoptosis.
Very rarely humans are encountered with genetic defects in apoptosis. The most
common one is a mutation in the gene for Fas, but mutations in the gene for FasL
or even one of the caspases are occasionally seen. In all cases, the genetic
problem produces autoimmune lymphoproliferative syndrome or ALPS.
Features:
an accumulation of lymphocytes in the lymph nodes and spleen greatly enlarging
them.
the appearance of clones that are autoreactive; that is, attack "self" components
producing such autoimmune disorders as
hemolytic anemia
thrombocytopenia
the appearance of lymphoma — a cancerous clone of lymphocytes.
In most patients with ALPS, the mutation is present in the germline; that is,
every cell in their body carries it. In a few cases, however, the mutation is
somatic; that is, has occurred in a precursor cell in the bone marrow. These
latter patients are genetic mosaics — with some lymphocytes that undergo
apoptosis normally and others that do not. The latter tend to out-compete the
former and grow to become the major population in the lymph nodes and blood.
APOPTOSIS AND AIDS
The hallmark of AIDS (acquired immunodeficiency syndrome) is the decline
in the number of the patient's CD4+ T cells (normally about 1000 per
microliter (µl) of blood). CD4+ T cells are responsible, directly or indirectly
(as helper cells), for all immune responses. When their number declines below
about 200 per µl, the patient is no longer able to mount effective immune
responses and begins to suffer a series of dangerous infections.
What causes the disappearance of CD4+ T cells?
HIV (human immunodeficiency virus) invades CD4+ T cells, and one might
assume that it this infection by HIV that causes the great dying-off of
these cells. However, that appears not to the main culprit. Fewer than 1 in
100,000 CD4+ T cells in the blood of AIDS patients are actually infected with
the virus. So what kills so many uninfected CD4+ cells?
The answer is clear: apoptosis.
The mechanism is not clear. There are several possibilities. One of them:
All T cells, both infected and uninfected, express Fas.
Expression of a HIV gene (called Nef) in a HIV-infected cell causes
the cell to express high levels of FasL at its surface
while preventing an interaction with its own Fas from causing it to selfdestruct.
However, when the infected T cell encounters an uninfected one (e.g. in a
lymph node), the interaction of FasL with Fas on the uninfected cell kills it by
apoptosis.
APOPTOSIS AND ORGAN TRANSPLANTS
APOPTOSIS AND ORGAN TRANSPLANTS
For many years it has been known that certain parts of the body such as
the anterior chamber of the eye, the testes are "immunologically
privileged sites". Antigens within these sites fail to elicit an immune
response. It turns out that cells in these sites differ from the other
cells of the body in that they express high levels of FasL at all times.
Thus antigen-reactive T cells, which express Fas, would be killed when
they enter these sites. This finding raises the possibility of a new way
of preventing graft rejection. If at least some of the cells on a
transplanted kidney, liver, heart, etc. could be made to express high
levels of FasL, that might protect the graft from attack by the T cells
of the host'scell-mediated immune system. If so, then the present need
for treatment with immunosuppressive drugs for the rest of the
transplant recipient's life would be reduced or eliminated.
So far, the results in animal experiments have been mixed. Allografts
engineered to express FasL have shown increased survival for kidneys
but not for hearts or islets of Langerhans.
THINGS TO KNOW
EXIT FROM THE CELL CYCLE-G 0
EVIDENCES OF SPF AND MPF
CDKs/CYCLINS OF HIGHER EUKARYOTES
CDK REGULATION BY PHOSPHORILATION-DEPHOSPHORILATION
THE G1/S CELL CYCLE CHECKPOINT, p53, ATM, Rb
THE G2/M CELL CYCLE CHECKPOINT, CHK1, CDC25
THE META/ANAPHASE TRANSITION, COHESIN, SEPARIN, SECURIN, APC
SPINDLE ASSEMBLY CHECKPOINT, THE ROLE OF MAD2
MUTATIONS
REPAIR MECHANISMS
THE SIGNS OF NECROSIS AND APOPTOSIS
APOPTOSIS IN DEVELOPMENT
SUICIDE/SURVIVAL SIGNALS
APOPTOSIS: THE MITOCHONDRIAL PATHWAY
APOPTOSIS: THE DEATH RECEPTOR PATHWAY