cell cycle dna repair cell death

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 ground-breaking 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.

DNA replicationDNA replication

mitosismitosis

cytokinesiscytokinesis

THE PHASES OF THE CELL CYCLE

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

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.

EXIT FROM THE CELL CYCLE-G0

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.

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.

Accumulation and degradation of cyclin B

THE M PHASE PROMOTING FACTOR (MPF) -IDENTIFICATION OF CYCLIN

Sea urchin adult/embryo.

THE M PHASE PROMOTING FACTOR (MPF) -IDENTIFICATION OF CYCLIN

Cdk 1

Cyclin B

THE M PHASE PROMOTING FACTOR (MPF): CIKLIN+CDK

MPF IS ACTIVE ONLY AT HIGH CYCLIN CONCENTRATIONS

THE M PHASE PROMOTING FACTOR (MPF): CIKLIN+CDK

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

M G1 G2S M G1

Start

Cell cycle phases

Cyclin-dependent kinases

D E A B(A)cyclins

Cdk4 Cdk2 Cdk1

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.

Control of 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.

Steps in the cell cycle

MECHANISMS OF CDK REGULATION


PHOSPHORILATION-DEPHOSPHORILATION

INHIBITORY PROTEIN (P27/CKI) BINDING INACTIVATES THE CYCLIN-CDK COMPLEX



UBIQUITIN-DEPENDENT PROTEIN DEGRADATION

CELL CYCLE CHECKPOINTS

DNA

ProteinProtein kinase kinase

activationactivation

p53 foszforilációp53 foszforiláció

p53 degradation

Stable,Active p53

p53 binds to the promoter of the p21 gene

transcription

translationP21 mRNS

P21 (CKI)

ACTIVEACTIVE INACTIVEINACTIVE

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.

THE G1/S CELL CYCLE CHECKPOINT

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.

p53

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.

ATM (ataxia telangiectasia mutated)

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 retinoblastomaFamilial 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 retinoblastomaIn 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) removed.)

RB - the retinoblastoma gene

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

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 DNA-PK/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 cyclin-dependent kinases including cdk1.

THE G2/M CELL CYCLE CHECKPOINT

cdk1

THE META/ANAPHASE TRANSITION

What if There Were No Checkpoint?

THE META/ANAPHASE TRANSITION-SPINDLE ASSEMBLY CHECKPOINT

Unattached Kinetochores Cause a Checkpoint Delay


APC is the target of the spindle assembly checkpoint


Mad2 cycles through kinetochore and inhibits cdc20


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.

MAD AND CANCER

G1G1--CdkCdkG1G1--CdkCdk G1G1/S-/S-CdkCdkG1G1/S-/S-CdkCdk S-S-CdkCdkS-S-CdkCdk M-M-CdkCdkM-M-CdkCdk APCAPCAPCAPCHct1CKI Cdc25

p53

CH

EC

KP

OIN

TS

KE

Y P

LA

YE

RS

AN

D

EV

EN

TS

G1 S G2 M G1

Inappropiate environment

DNAdamage

mitogenstimulation

non replicated

DNA

DNA damage

Unattached kinetochores

Synthesis of G1/S cyclin

Synthesis of S cyclin DNA replication

CELL CYCLE CONTROL MECHANISMS

MUTATION

DNA REPAIR

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 lead to unusual base pair formation (so-called mismatches)

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.

In their rare forms A* pairs with C, T* with G, G* with T and C* with A


If tautomeric shift happens at the time of replication, BASE PAIR SUBSTITUTION happens in the DNA



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.

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.

SPONTANEOUS MUTATIONS–BP. ADDITION & DELETION

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

INDUCED MUTATIONS

The formation of mutagenic substance from the nonreactive benzopyrene.

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.

INDUCED MUTATIONS

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.

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.


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.


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

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.

STRUCTURAL CHANGES DUE TO CHROMOSOME BREAKS


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

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.


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.

MUTATIONS IN THE GERM LINE AND SOMA

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)

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.

XERODERMA PIGMENTOSUM (XP)

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 Base-Excision 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 End-Joining (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.


http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.html

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.html

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.html

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

A pathological response to cellular injury

Chromatin clumping

Mitochondria swelling and rupture

Plasma membrane lyses

Cell contents spill out

General inflammatory response is

triggered

NECROSIS

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 inflammationThe 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-)

APOPTOSIS

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

NormalApoptosis

DysfunctionalApoptosis


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 virus-infected 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.


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 TNF-receptor•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

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 caspase-3. 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.

DNA damage

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)

THE MECHANISMS OF APOPTOSIS

There are two different mechanisms by which a cell commits suicide by apoptosis.

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.


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 voltage-dependent 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.

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 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 DR-3L 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

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 pro-apoptotic 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.

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

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 AND CANCER

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 IN THE IMMUNE SYSTEM

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 self-destruct.

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 AIDS

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.

APOPTOSIS AND ORGAN TRANSPLANTS

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

THINGS TO KNOW

cell cycle dna repair cell death

Documents

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cell cycle regulation

cell morphology

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g2arrested cell

g0 cells

factor mpfidentification

g2 phase primary oocyte