Apoptosis and Cell Necrosis

Group 5 Mallare, Melvin Manabat, Armand Jeric Manalang, Kerwin Manalili, Maria Minerva Manarang, Grace Haziel Mancia, Chester

APOPTOSIS

programmed cell death

is a normal component of the development and health of multicellular organisms. is a process in which cells play an active role in their own death.

Biochemical events lead to characteristic cell changes (morphology) and death. blebbing loss of cell membrane asymmetry and attachment cell shrinkage nuclear fragmentation chromatin condensation chromosomal DNA fragmentation.

(A). Typically, the cell begins to shrink following the cleavage of lamins and actin filaments in the cytoskeleton

(B). The breakdown of chromatin in the nucleus often leads to nuclear condensation and in many cases the nuclei of apoptotic cells take on a "horseshoe" like appearance

(C) Cells continue to shrink

The end stages of apoptosis are often characterized by the appearance of membrane blebs (D) or blisters process. Small vesicles called apoptotic bodies are also sometimes observed

There are a number of mechanisms through which apoptosis can be induced in cells. The sensitivity of cells to any of these stimuli can vary depending on a number of factors such as the expression of pro- and anti-apoptotic proteins .

NECROSIS

is the premature death of cells and living tissue.

Cells which die due to necrosis do not usually send the same chemical signals to the immune system that cells undergoing apoptosis do.

This prevents nearby phagocytes from locating and engulfing the dead cells
caused by external factors, such as infection, toxins or trauma.

APOPTOSIS NATURAL YES

NECROSIS NO

EFFECTS

BENEFICIAL
Physiological or pathological Single cells Energy dependent Cell shrinkage Membrane integrity maintained

DETRIMENTAL
Always pathological

Sheets of cells Energy independent Cell swelling Membrane integrity lost

APOPTOSIS Role for mitochondria and cytochrome C

NECROSIS No role for mitochondria

No leak of lysosomal enzymes

Characteristic nuclear changes Apoptotic bodies form DNA cleavage Activation of specific proteases Regulatable process

Leak of lysosomal enzymes

Nuclei lost Do not form No DNA cleavage No activation Not regulated

Evolutionarily conserved
Dead cells ingested by neighbouring cells

Not conserved
Dead cells ingested by neutrophils and macrophages

Internal Mechanism

Caspases

Caspases are cysteine proteases that cleave peptide bonds next to an aspartate residue.

Types:
1. Initiator Caspases - cleave other procaspases 2. Execution Caspases - cleave other cellular proteins involved in maintaining cellular integrity

Intracellular signals
Oxidative damage from free radicals, Radiation, Virus infection, Nutrient deprivation, Pro-apoptotic Factors Damage to the mitochondrial membrane increasing permeability Entry of Cytochrome C into the cytoplasm Cytochrome C binds to Apaf-1 forming an apoptosome Apoptosome activates procaspase-9 to caspase-9 Caspase-9 cleaves and activates caspase-3 and caspase-7.

This executioner caspases activate a cascade of proteolytic activity that leads to: Chromatin condensation, DNA fragmentation, Protein cleavage, Membrane permeability

Controlling Apoptosis The Bcl-2 family members are decision-makers that integrate

prodeath and antideath signals to determine whether the cell should commit suicide. Members:

Anti-apoptotic BCL-2 and BCL-xL

can block the release of cytochrome C from the mitochondria Bcl-2 can also bind to Apaf-1 (mentioned above) and inhibit its activation facilitate apoptosis by stimulating cytochrome C release.

Pro-apoptotic BAX, BAD, BAK, BID

In addition, other proteins called IAP's (inhibitors of apoptosis) can inhibit caspase or other apoptotic proteins. SMAC and DIABLO binds to IAPs and inactivates them therefore allowing apoptosis to proceed

External Mechanism

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 initiates a cascade of caspase activation leading to
phagocytosis

of the cell or apoptosis.

 Example

: When cytotoxic T cells recognize or 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.

Role of Apoptosis
Why should a cell commit suicide?

There are two different reasons.

1. Programmed cell death is as needed for proper development as mitosis is.

Example:

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

Role of Apoptosis

In Embryogenesis
The

development and maintenance of multicellular biological systems depends on a sophisticated interplay between the cells forming the organism, it sometimes even seems to involve an altruistic behavior of individual cells in favour of the organisms as a whole. During development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues [Meier, 2000]

Role of Apoptosis

In Development and Differentiation of Tissues

an

integral part of both plant and metazoan (multicellular animals) tissue development does not resemble the sort of reaction that comes as a result of tissue damage due to accident or pathogenic infection (cell death by necrosis).

swelling and bursting - hence spilling their possibly damaging internal contents into extracellular space

apoptotic

cells and their nuclei shrink, and often fragment. In this way, they can be efficiently phagocytosed (and, as a consequence of this, their components reused) by macrophages or by neighboring cells.

Role of Apoptosis

2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism.

Role of Apoptosis

Apoptosis and AIDS

hallmark-

the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (l) of blood) responsible, directly or indirectly (as helper cells), for all immune responses

In

Cancer

Virus associated cancer Several human papilloma 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).

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. 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). 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

In 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.

Determination of cells undergoing apoptosis

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Cytomorphological alterations II. DNA fragmentation and micronuclei formation III. Externalization of membrane Phosphatidylserine IV.Activation and presence of caspases, cleaved substrates regulators and inhibitors
I.

Externalization of Phosphatidylserine of the inner cell membrane

Cells that undergo PCD display an eat me signals to elicit phagocytosis The signal is Phosphatidylserine (PS) of the inner leaflet of the cell membrane Apoptotic cell flips the PS to the outside leaflet to let the phagocytic scavengers recognize the signal

This signaling of dying cells let the scavenger cells phagocytose without eliciting any inflammatory response
The lack of this PS receptors can lead to the accumulation of undigested cells that have undergone apoptosis

DNA fragmentation & micronuclei formation

Fragmentation of the chromatin result in degradation of the nucleus and the formation of nuclear blebbing Nucleic acid staining by the use of fluorescent dyes can reveal micronuclei bodies that are inherent to apoptotic cells

Nuclear fragmentation and micronuclei formation

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DNA fragmentation
Chromatin materials condensed and show a ladder pattern of low molecular weight DNA fragments when electrophoresis technique is used.

Cytomorphological alterations

Cytoskeleton degradation and asymmetry of the cell membrane Change in the permeability of the cell membrane

Degradation of cytoskeleton
Caspases digest the cytoskeletal proteins of the apoptotic cells This leads to the asymmetry of the cell membrane

Change in membrane permeability

Apoptotic cells have different membrane permeability than normal cells Determination of possible apoptotic cells is through the use of stains (can either be fluorescent or not) that take advantage of this change in permeability Stains able to label the apoptosome with the dye

Detection and presence of Caspase

Caspase are cysteine-aspartic acid specific proteases that mediates the events that are associated with programmed cell death
Caspase-3

key effector in the apoptosis pathway, amplifying the signal from initiator caspases and signifying full commitment to cellular disassembly

Caspase-8

acts as an initiator of the caspase activation cascade