Cell Death – Mechanisms

K Siva Prasad

• The balance between cell viability and cell death is critical for all organisms, and involves a network of proteins and communication amongst several signalling pathways.

Why ??

• Interesting Fact! Without apoptosis, 2 tons of bone

marrow and lymph nodes and a 16-km intestine would probably accumulate in a human by the age of 80

• Impaired cell death is associated with various human pathologies, including

1. Immunodeficiency2. Autoimmunity3. neurodegenerative diseases 4. and cancer• Similar to proliferation, cell death is also essential

for embryonic and postnatal development and for tissue homeostasis.

What is Death ?

In the absence of a clearly defined biochemical event that can be considered as the point-of no-return,

the NCCD proposes that a cell should be considered dead when any one of the following molecular or morphological criteria is met:

(1) the cell has lost the integrity of its plasma membrane, as defined by the incorporation of vital dyes (e.g., PI) in vitro;

(2) the cell, including its nucleus, has undergone complete fragmentation into discrete bodies (which are frequently referred to as ‘apoptotic bodies’); and/or

(3) its corpse (or its fragments) has been engulfed by an adjacent cell in vivo.

Cell death can be classified according to its: 1. Morphological Appearance (which may be

apoptotic, necrotic, autophagic or associated with mitosis),

2. Enzymological Criteria (with and without the involvement of nucleases or of distinct classes of proteases, such as caspases, calpains, cathepsins and transglutaminases),

3. Functional Aspects (programmed or accidental, physiological or pathological)

4. Immunological Characteristics (immunogenic or nonimmunogenic).

Three Pathways of Cell Death

APOPTOSIS

NECROSIS

AUTOPHAGY

Life and Death Matter • Autophagy, which has been proposed as a third mode of cell

death, is a process in which cells generate energy and metabolites by digesting their own organelles and macromolecules

• Autophagy allows a starving cell, or a cell that is deprived of growth factors, to survive.

• However, cells that do not receive nutrients for extended periods ultimately digest all available substrates and die (autophagy-associated cell death)

• While initially most of the focus has been placed on apoptosis, which is a highly regulated mechanism involving interplay among a large number of pro and antiapoptotic proteins,

• mounting evidence has emerged to support the genetic regulation of necrosis

Apoptosis

• Apoptosis (or type I programmed cell death) is a genetic pathway for rapid and efficient killing of unnecessary or damaged cells that was initially described by Vogt (1842)

• Apoptosis (commonly pronounced ap-a-tow' -sis), coined from the Greek apo or from, and ptosis or falling, to make the analogy of leaves falling off a tree.

Caenorhabditis elegans

Triggers ....

• Apoptosis is triggered in mammalian cells in response to

• endogenous stimuli (eg, growth factor deprivation) or exogenous stimuli (eg, irradiation or genotoxic chemotherapeutic drugs).

• Apoptosis is also induced in response to inadequate cell–matrix interactions and this specific type of apoptosis is known as anoikis.

• Physiological during development

BCL-2 Family Members and Their Roles inApoptosis

• The mammalian Bcl-2 gene, a homolog of the C. elegans Ced-9, is a prototypical member of the BCL-2 family that includes

• antiapoptotic proteins such as BCL-2, BCL-XL,• BCL-W, A1, and MCL-1, and• proapoptotic proteins, such as BAX, BCL-XS,

BAK, BAD, BIK, BIM, BID, and PUMA

BAXBCL-XS

BAKBADBIK

BIM BID, and PUMA

• BCL-2• BCL-XL• BCL-W• A1• MCL-1

• “BCL-2 homology” (BH) regions including BH1, BH2, BH3, and BH4

• These domains allow the formation of homo- and heterodimers between BCL-2 family members and are essential for the function of these proteins.

• Overexpression of BCL-2 also prolongs the survival of cells and increases their resistance to a variety of apoptotic stimuli including

1. glucocorticoids, 2. phorbol esters,3. ionomycin, 4. anti-CD3 monoclonal antibodies,5. irradiation, 6. and chemotherapeutic agents.

Caspases

• “cysteine-dependent aspartate specific proteases,” are the orthologs of the C. elegans protein Ced-3.

• The first caspase (ICE or CASP1) was identified in 1993 on the basis of its similarity to Ced-3.

• More than 14 mammalian caspases have since been cloned

• Although caspases are primarily known for their apoptotic function,

• caspases such as CASP1 and CASP11 play important roles in inflammation.

• Mounting evidence supports that CASP8 also possesses nonapoptotic functions.

• “initiators” (eg, CASP8, CASP9 and CASP10) or “executioners” of apoptosis (eg, CASP3, CASP6, and CASP7).

• Initiator procaspases contain large prodomains,

• whereas executioner procaspases contain small prodomains.

• Hundreds of caspase substrates have been identified

1. cytoskeleton proteins (eg, Actin and Gelsolin),2. nuclear proteins (eg, Lamin A and B), 3. proteins involved in DNA damage repair (eg,

PARP, RAD51, and DNA-PKcs),4. cell-cycle proteins (eg, p21, p27, CDC27, and

RB), 5. cytokines (eg, IL-1β and IL-18)6. apoptotic proteins (eg, Caspases, BCL-2, BCL-

XL, BID, BAX, and ICAD)

• The caspase-activated DNase (CAD) is inactive when associated with its inhibitor ICAD (also known as DNA fragmentation factor [DFF]).

• In response to apoptotic stimuli, ICAD is cleaved by caspases allowing the release of the active endonuclease CAD to cleave DNA of apoptotic cells to produce internucleosomal DNA cleavage, a characteristic of apoptosis.

The Death ReceptorApoptotic Pathway

• The death receptor or extrinsic apoptotic pathway is initiated by the interaction of the death receptors, members of the tumor necrosis factor (TNF) receptor superfamily, with their ligands

• The death receptors share the presence of an approximately 80-amino-acid motif known as the death domain (DD) in their cytoplasmic tails.

• Six human death receptors have been identified and include FAS (also known as CD95 or APO-1), TNFR-1, death receptor (DR) 3, DR4 (also known as TRAILR1), DR5 (also known as TRAILR2), and DR6.

• Cellular FLICE-like inhibitory protein (cFLIP), a pseudo-CASP8 protein with a nonfunctional catalytic domain, inhibits the death receptor apoptotic pathway by precluding the recruitment of CASP8 to the DISC.

The MitochondrialApoptotic Pathway

• Intrinsic apoptotic pathway, activate BAX and BAK and lead to mitochondrial outer membrane permeabilization and the release of a number of proteins important for apoptosis (eg, Cytochrome C, SMAC, and OMI)

• 8 human IAPs have been identified, including the X-linked IAP (XIAP), Survivin, and cellular IAP1 and -2 (c-IAP1 and c-IAP2).

• Several IAPs bind directly to caspases such as CASP3, -7, and -9 and inhibit their functions.

• This IAPs-mediated inhibition of caspases can be antagonized by the proteins SMAC and OMI

• When SMAC and OMI are released from the mitochondrial intermembrane space into the cytosol, they bind directly to XIAP and suppress its inhibition of caspases.

Cross Talk

• Communication between the death receptor and mitochondrial apoptotic pathways is best demonstrated by CASP8 cleavage of BID.

• This cleavage generates a truncated form of BID (tBID) that cooperates with BAX to form openings in the outer mitochondrial membrane, leading to release of proteins including Cytochrome C from the mitochondrial intermembrane space

Animal Models

• Bcl-2−/− mice die by 6 weeks of age.• Inactivation of Bcl-XL in mice results in

embryonic lethality by day 14 of gestation• Bax−/− mice display male sterility and mild

lymphoid Hyperplasia• perinatal lethality was observed in

Bax−/−Bak−/− mice.

• The surviving Bax−/−Bak−/− mice also accumulate cells of the central nervous

• system and fail to remove their interdigital webs.

• Casp3−/− and casp9−/− mice exhibit a similar brain defect characterized by ectopic masses of supernumerary cells that escape apoptosis during brain development

• Autoimmune lymphoproliferative syndrome (ALPS), mutations of FAS or FASL

Necrosis

• Necrosis is derived from the Greek word nekros for corpse, and it involves rapid swelling of the cell, loss of plasma membrane integrity, and release of the cellular contents into the extracellular environment, resulting in an acute inflammatory response.

• a type of programmed necrotic cell death, called necroptosis has been identified as induced by interaction of death domain receptors with their respective ligands under conditions of defective o inhibited downstream apoptotic machinery.

• Although necrosis was thought initially to be a nonregulated process, recent studies demonstrate that necroptosis, a programmed type of necrosis, is highly regulated by a network of proteins

Role in Cancer