***Background story:

Antigen-specific lymphocytes are activated through their antigen receptors. They undergo blast transformation and clonal expansion so lymphocytes specific for the infecting antigen increase in number and dominate the cell population. The activated T cells differentiate into effector cells and remove the pathogen, ending the infection. The activated effector T cells that are now no longer needed undergo apoptosis.

I. There are two ways in which cells die:

• they are killed by injurious agents
• they are induced to commit suicide

1. Death by injury = "necrosis"

Cells that are damaged by injury, such as by

• mechanical damage
• exposure to toxic chemicals

undergo a characteristic series of changes:

• they swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted)
• the cell contents leak out, leading to
• inflammation of surrounding tissues

B. Death by suicide = "apoptosis"

Cells that are induced to commit suicide:

• shrink
• have their mitochondria break down with the release of cytochrome c
• develop bubble-like blebs on their surface
• have the chromatin (DNA and protein) in their nucleus degraded
• break into small, membrane-wrapped, fragments which are usually
• engulfed by nearby phagocytes cells like macrophages
• do not induce inflammation

*** Overall idea: The pattern of events in death by suicide is so orderly that the process is often called programmed cell death (PCD), which is also called apoptosis. It plays an important role in the sculpting of a developing organism and in the elimination of undesirable cells, such as cancerous cells.

 

II. Why should a cell commit suicide?

There are two different reasons.

A. Programmed cell death is needed for proper development.

Examples:

• 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 (synapes) between neurons in the brain requires that surplus cells be eliminated by apoptosis.

B. Programmed cell death is needed to destroy cells that represent a threat to the organism.

Examples:

• Cells infected with viruses

A way cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis.

• Cells of the immune system

As cell-mediated immune responses decline, the effector cells must be removed to prevent them from attacking body constituents. CTLs induce apoptosis in each other and in themselves. Defects in the apoptotic machinery is associated with autoimmune diseases.

• Cells with DNA damage

Damage to its genome can cause a cell

• to disrupt proper embryonic development leading to birth defects

• to become cancerous

Cells respond to DNA damage by increasing their production of p53, which is a potent inducer of apoptosis. Mutations in the p53 gene produce a defective protein and are often found in cancer cells.

• Cancer cells

Many agents used in cancer chemotherapy achieve their effect by inducing apoptosis in the cancer cells.

 

III. What makes a cell decide to commit suicide?

The balance between:

• the withdrawal of positive signals (signals needed for continued survival)
• the receipt of negative signals

A. Withdrawal of positive signals

The continued survival of most cells requires that they receive continuous stimulation from other cells and, continued adhesion to the surface on which they are growing. Examples of positive signals are:

• growth factors for neurons
• Interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes

B. Receipt of negative signals

• increased levels of oxidants within the cell
• damage to DNA by these oxidants or other agents like

• UV lights

• x-rays

• chemotherapeutic drugs

• molecules that bind to specific receptors on the cell surface and signal the cell to begin apoptosis. These death activators include:

• Tumor necrosis factor (TNF) that binds to the TNF receptor

• Lymphotoxin that also binds to the TNF receptor

• Fas ligand (FasL), a molecule that binds to a cell-surface receptor named Fas (also called CD95)

IV. The Mechanisms of Apoptosis

A cell commits suicide via apoptosis by two different mechanisms.

• by signals arising within the cell

• by death activators binding to receptors at the cell surface.

• TNF

• Lymphotoxin

• Fas ligand (FasL)

A. Apoptosis triggered by internal signals

• In a healthy cell, the outer membranes of the mitochondria, the endoplasmic reticulum (ER), and the nuclear envelope express the protein bcl-2 (isolated from the second B cell lymphoma) on their surface.
• bcl-2 is a member of a small family of closely related genes that can either inhibit or promote apoptosis depending upon which protein is more abundant (this determines if a cell lives or dies)

• proteins that promote death are Bax and Bad
• proteins that inhibit death are bcl-2 and bcl-X_L

1. How apoptosis is promoted:

• Bax binds a molecule of the protein Apaf-1, which is itself bound to a molecule of caspase 9

• Caspase 9 is one of a family of over a dozen caspases. They are all proteases and they cleave proteins - mostly each other - at aspartic acid (Asp) residues.
• The trimer of Bax/Apaf-1/caspase 9 is called an apoptosome.
• Internal damage in the cell causes Bax to release the heterodimer of Apaf-1 and caspase 9.
• These aggregate in the cytosol.
• Caspase 9 cleaves and, in so doing, activates other caspases.
• The sequential activation of one caspase by another creates an expanding cascade of proteolytic activity, which leads to

• digestion of structural proteins in the cytoplasm

• degradation of chromosomal DNA and

• Death of the cell occurs.

1. How apoptosis is inhibited:

• Cytochrome c is found in the organelles of normal cells.

• With apoptosis, the organelle (mitochondria) swells allowing cytochrome c to leak out into the cytosol.

• The cytochrome c interacts and binds to the protein Apaf-1 creating a complex that is now able to activate caspase.

• An activated capase leads to the fragmentation of DNA, and later the death of the cell.

• HOWEVER… bcl-2 binds to the membranes of the organelle to block the swelling and prevent the release or leakage of cytochrome c, thus inhibiting apoptosis.

B. Apoptosis triggered by external signals

(refer to p. 187, fig. 5.22)

• Fas and the TNF receptor are integral membrane proteins (monomers) with their receptor domains exposed at the surface of the cell
• FasL and TNF are death activators
• binding of Fas to FasL causes a trimerization of Fas, which binds death domains (on the cytoplasmic tails of Fas) containing adaptor proteins
• this transmits a signal to the cytoplasm that leads to the activation of caspase 8 by the adaptor proteins
• caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to the death of the cell.

(refer to p. 187, fig. 5.23)

TUNEL staining!!!

It shows apoptotic-stained cells that can be detected by light microscopy.

• TUNEL Staining Steps:

1. DNA in apoptotic cells are fragmented
2. TdT (terminal deoxynucleotidyl transferase -- enzyme) adds nucleotides to the 3' ends of the fragmented DNA
3. The now biotinylated DNA can be detected by using streptavidin, which binds to biotin
4. This reaction generates a colored precipitate (only in apoptotic cells)

VI. What is the importance of understanding apoptosis?

• It plays a critical role in important biological processes:

• Morphogenesis

• Tissue Homeostasis

• Elimination of damaged or infected cells

• Elimination of self-reactive clones from the immune system

• Its activation contributes to many diseases that need to be studied:

• AIDS
• Ischemic injuries
• Neurogenerative disorders

• It’s an important factor in etiology (the study of the cause or origin of diseases):

Janeway, C. A., et al. Immunobiology: The Immune System in Health and Disease, 4^th ed. New