CELL DEATH

Cells die by one of TWO

mechanisms:

Necrosis or Apoptosis

OVERVIEW:

• Necrosis is a pathological process induced by accidental cell damage.

• A number of toxic chemical or physical events can cause necrosis: toxins,

radiation, heat, trauma, lack of oxygen due the blockage of

blood flow, etc.

• As necrotic cells begin to die, they swell (increase in size), holes

appear in the plasma membrane and intracellular materials

spill out into the surrounding tissue inducing an inflammatory

response to the neighbor cells.

• It is completed within several days

E.g. Gangrene : a type of necrosis caused by insufficient blood

supply. It may occur after an injury or infection, or in people suffering

from any chronic health problem affecting blood circulation (Diabetes

and long-term smoking increase the risk of suffering from gangrene).

Necrosis/morphological features

Apoptosis/ morphological features

• Apoptosis is a normal physiological process

(non-pathological) that removes “unwanted” cells

without damaging neighboring cells (cellular

contents are not released) and inflammation

does not occur.

• Apoptotic cells shrinks in size but do not lyse, their plasma

membrane remain intact, part of it bud off (bleb), became

asymmetrical, loss attachment with neighbor tissue.

• Its chromatin condenses,

• the cell fragmented into membrane enclosed structures called

apoptotic bodies which contain: cytosol, condensed chromatin

and organelles

• During apoptosis, a Phosphatidylserine,

(phospholipid) in the inner leaflet of lipid

bilayer, actively held facing cytoplasm by the

enzyme flippase, will invert by scramblase

(floppase) and became exposed on cell surface

using ATP.

• When the phosphatidylserines flip to the

extracellular surface, they act as a signal for

phagocytes cells (e.g. macrophages) to engulf

and degrade the cells (reducing the risk of

inflammation)

• Phagocytes cells also release cytokines (IL-10)

and transforming growth factor (TGF-b) that

inhibit inflammation.

• Apoptosis completes within few hours

• Effect is beneficial

• Causes can be physiologic or pathologic

• Cellular condensation

• Membranes remain intact

• Requires ATP

• Cell is phagocytosed, no tissue reaction

• In vivo, individual cells appear affected

• Effect is detrimental

• Causes always pathologic

• Cellular swelling

• Membranes are broken

• ATP is depleted

• Cell lyses, eliciting an inflammatory reaction

• In vivo, whole areas of the tissue are affected

Necrosis Apoptosis

Causes of apoptosis

Physiologic

Pathologic

Physiologic apoptosis - it’s essential for the proper development and to maintain homeosasis for the

organism:

Apoptosis is used during development of the embryo: There is an extensive cell division during that period leading to an excess of cells that must be removed for normal development and function

* (e.g., in developing nervous system approx. half of the generated nerve cells are immediately undergo apoptosis).

Selective apoptosis sculpt the developing tissues (e.g., Tissues between developing digits, must occur to have separated fingers and toes; incomplete apoptosis can result in abnormal structures)

Homeostasis: In normal adults, the number of cells kept constant by balance between cell division and cell death: If equilibrium is disturbed, abnormal growth (tumors) or loss of cells will occur

Apoptosis in Pathologic conditions

• DNA damage due to radiation, chemotherapy. • Accumulation of misfolded proteins leads to ER stress which ends with apoptosis. • Viral infections that induce apoptosis such as HIV and Adenovirus or • by the host immune response such as hepatitis.

Mechanisms of Apoptosis

Activation of caspases

INTRINSIC

(Mitochondrial pathway)

Involves release of cytochrome c (and other proteins) from mitochondria

EXTRINSIC

(Death receptor pathway)

Activated by the engagement of death receptors on cell surface

Death Receptors

• Belong to tumor necrosis factor receptor (TNFR) superfamily

• Can initiate apoptosis from external signals.

• Only the receptors that have cytoplasmic sequence known as

death domain (DD) can initiate apoptosis (NOT ALL THE

MEMEBERS CAN INITIATE CELL DEATH)

• Adaptor molecules such as FADD (Fas-associated death

domain) and TRADD (TNFR-associated protein) contain such

Death Domains:They interact with the death receptor to

transmit the apoptotic signal to the death machinery via

activation of caspases.

Exemple of Death receptor:

Fas death receptors

• The Fas death receptor is a member of TNFR

superfamily, initiate apoptotic cell death when it

interacts with Fas ligand (FasL, CD178)

• FasL is expressed in T-cytotoxic cells, NK cells, and

mature B cells as well as in sites of immune

privilege such as the eye, thyroid, lung, brain,

placenta, and testis

Caspases family

(cysteine-aspartic proteases)

• Cysteine proteases (degrade proteins) that cleave after an Asp residue in their substrate.

• Synthesized as pro-enzymes (inactive) and

then activated when needed (post-translational

modification)

CARD= caspase-recruitment domain DED= death effector domain L= large catalytic subunit S= small catalytic subunit

• About 11 members in human

• Classified based on function

• Some not involved in apoptosis like:

- Caspases 1& 4 & 5 involved in inflammation

- Caspase 14 important in skin development

The remaining caspases are

involved in apoptosis, grouped

into either initiators or effectors

caspases

Classification of caspases

Initiator caspases

• Trigger onset of apoptosis by activating the caspases cascade

• Include Caspase 2, 8, 9 & 10.

• These caspases have characteristic region or domains that enable them to

interact with their substrates (effector caspases) and activate them

CARD domain (caspase recruitment domain) in caspase 2 & 9

and

DED domain (death effector domain) in caspase 8 & 10

are executioner caspases: they undertake the

actual work of destroying critical components

of cell

are: 3, 6 & 7,

Effector caspases

•FasL interacts with Fas-death receptor

causes trimerization of the Fas receptor

in host cells (infected with virus).

•This result in the clustering of the

receptors DD which then recruit the

cytosolic adaptor protein FADD by

binding FADD death domain.

•FADD contain another domain called

death effector domain that bind to a

domain in pro-caspase 8:

•The complex:

Fas receptor trimer+FADD+caspase 8

is called Death Inducing Signaling

Complex (DISC).

•Upon recruitement procaspase 8

activate itself and caspase 8 then

activates other caspase downstream

such as caspase 3 and 7 that mediate

Extrinsic (Death receptor initiated) pathway

cell killing.

Intrinsic-Mitochondrial pathway

•Initiates by signal within the cell

•Key protagonists:

- Cytochrome C

- Bcl-2 family members

- APAF-1 (apoptotic protease-activating factor 1)

• Cytochrome is released from the mitochondria as the membrane become permeable and associates with APAF-1 constituting the apoptosome (the “wheel of death”). This structure binds to procaspase-9 promoting its activation (see picture in the next slide)

The cyt c activates Apaf-1, adaptor protein, which in turn activates caspase 9, of the caspase

proteolytic cascade, that will destroy cell proteins and DNA to cause cell death by apoptosis.

Bcl-2 family proteins

• Bcl-2 family members directly regulate the release of cytochrome c. • This family contains both pro- and anti-apoptotic proteins. - ANTI-APOPTOTIC: Bcl-2; Bcl-XL; Bcl-W; A1; Mcl-1 - PRO-APOPTOTIC: Bax family (Bax; Bak; Bok); BH3-only family (Bid; Bim, Bik, Bad, Bmf, Hrk, Noxa; Puma) The level between pro- and anti- apoptotic proteins determines if cytochrome c is released from the mitochondrion: IMBALANCE initiates the

death pathway!

Expression of anti-apoptotic proteins (Bcl-2 over-expression in follicular B-cell lymphoma; over-expression of IAPs in different types of cancers including neuroblastoma)

Inactivation of pro-apoptotic genes (BAX mutation; APAF-1 in melanomas)

Alteration of p53 pathway (p53 mutation)

Altered survival signalling

Deregulation of apoptosis/ Cancer

• Accelerated cell death is found in degenerative diseases and immunodeficiency

• Insufficient apoptosis found in cancer or autoimmunity

• Tumour cells can acquire resistance to apoptosis by :

-Normal p53 binds p53-response

elements within gene promoter of

protein Bax (pro-apoptotic protein),

triggering apoptosis.

-Mutant p53 can not produce Bax

protein and the cell continues to survive

and divide.

DNA Damage

Alteration Mechanism of anti-apoptotic

action Types of tumors

CASP3 repression Inactivation of executioner caspase Breast carcinomas

p53 mutation Loss of ability to induce pro-apoptotic genes

Many types

NF-kB constitutive activation Induction of anti-apoptotic genes Many types

Mdm2 over-expression Suppression of p53 levels Sarcomas

APAF-1 methylation Loss of proacaspase-9 activation by Cytochrome c

Melanomas

BAX mutation Loss of pro-apoptotic protein Colon carcinomas

Bcl-2 over-expression Closes mitochondrial channels ~ 50% of human tumours

Akt/PKB activation Phosphorylation and inactivation of pro-apoptotic Bcl2-like proteins

Many types

Caspase cascade

END!