Apoptosis

Question 1

What causes cell death?

Answer 1

• All cells in multi-cellular organisms undergo growth and death
• cellular death is essential for an organism to grow and survive.
• there are two main ways by which a cell undergoes death,
  
  • either through exposure to harmful environment and/or injury (Necrosis) or through a pre-planned and regulated process of disintegration (apoptosis)

Question 2

What are the characteristics of necrosis?

Answer 2

• Nuclear swelling
• Cell swelling
• Disruption of organelles
• Rupture of cell and release of cellular contents
• Inflammatory response

Question 3

What are the characteristics of apoptosis ?

Answer 3

• Chromatin condensation
• Cell shrinkage
• Preservation of organelles and cell membranes
• Rapid engulfment by neighboring cells/phagocytosis

Biochemical hallmark:
-DNA fragmentation

-Caspases

Question 4

What is apoptosis?

Answer 4

Enables an organism to eliminate unwanted and defective cells through an orderly process of cellular disintegration with the advantage of not inducing an undesirable inflammatory response

Question 5

What are the molecular markers of apoptosis ?

Answer 5

A. Apoptosis

B. Engulfed apoptotic cell

C. Necrosis

Phosphatidylserine on the surface of an apoptotic cell is the is the “eat me” signal

Question 6

How does phosphatidylserine (PS): work as a marker for apoptotic cells?

Answer 6

• The negatively charged PS is normally found on the inner leaf of the lipid bilayer
• In apoptotic cells, PS flips to the outer leaf
• PS helps signal macrophages to phagocytosis the dying cell
• PS dependent engulfment of apoptotic cells inhibits production of inflammation inducing signal proteins (cytokines) by the phagocytes cell

Question 7

What markers determine if a cell is apoptotic or a cell treatment gives rise to apoptosis ?

Answer 7

Apoptotic DNA shows a characteristic periodicity of fragmentation

Endonucleases cleave chromosomes in the linker region between the Nucleosomes

• Caspase-activated DNAse
• With necrosis, all DNA would be decayed, small remnants at the bottom of the gel

Question 8

Explain the value of apoptosis

Answer 8

Apoptosis is needed for proper development
-proper formation (sculpting) of fingers and toes in the fetus

 - Formation of proper connections between neurons in the brain
 - Resorption of tadpole tail

Apoptosis is needed to destroy unwanted cells
-Cells with DNA damage

  - Infected cells (e.g. cells infected by viruses)
  - Camcer cells
  - Elimination of self-reactive components of the imm7ne system

In adult tissues, cell death exactly balances cell division

Question 9

Describe apoptosis in a developing mouse paw

Answer 9

Developing mouse paw, dye for labeling cells that have undergone apoptosis

-One day later, tissue is eliminated between digits and very few apoptotic cells

Question 10

Describe apoptosis in developing chick leg bud

Answer 10

Labeled dUTP is added to the 3’ ends of the DNA fragments to visualize cells that are undergoing apoptosis in a developing chick leg bud

Question 11

What stimulates apoptosis during metamorphosis of a tadpole to a frog?

Answer 11

Increase in thyroid horm9ne stimulates this change

Question 12

How can C. Elegans in understanding the genes involved in apoptosis?

Answer 12

The life cycle of C. Elegans from egg to sexual maturity (and new eggs) is about 3 days

• The adult hermaphrodite consists of exactly 959 somatic cells of precisely determined lineage and function
• Individual cells are named and their relationships to their neighbors are known
• Cell fate of cells within the C. Elegans embryo have been determined by laser ablation

Question 13

How much of C. Elegans cells undergo apoptosis

Answer 13

Overall the 959 cells of adult C. Elegans arise from 1090 original cells

• Exactly 131 cells undergo programmed cell death in the wild type worm
• Of the 1090, 302 are neurons, and many of the programmed deaths also lie in the neuronal lineage

Question 14

Who found out what controls the fate of the 131 cells in C. elegans ?

Answer 14

Horvitz and colleagues mutated the genome of C. elegans and screened for worms that contained ‘un- dead’ cells (that is, cells that should have died, but survived instead)

Tracing the mutations in these worms led them to three intersting genes, ced-3 and ced-4 and ced-9 (called ced for cell death abnormal)

Question 15

What are the results of the Horvitz experiment?

Answer 15

• In worms carrying mutations in ced-3 or ced-4 genes, programmed cell death doesn’t occur, and all 1090 cells survive
• In contrast, in ced-9 mutant animals, all 1090 cells die
• These genetic studies indicate that the CED-3 and CED-4 proteins are required for cell death and that CED-9 suppresses apoptosis

Question 16

In vertebrates, ced homologues are called caspases…

Answer 16

Derivation of the name:
- cysteine-dependent Aspartate directed proteases

Caspases are a family of cysteine proteases that play essential roles in apoptosis

Each Caspase is a cysteine Aspartase which embryos cysteine in its active site to cleave aspartic acid peptide bonds within proteins.

Caspases are regulated at the post translational level
-They are first synthesized as inactive pro-caspases, that consist of a pro domain, a small subunit and a large subunit

Question 17

Describe the conversion of process paste to caspase

Answer 17

Each caspase is made as an inactive pro enzyme (pro caspase ) which is usually activated by proteolytic cleavage by another member of the caspase family

An initial activation of a small number of pro caspase molecules can lead to an amplifying chain reaction activating many caspases

Question 18

What are the analogues of Ced genes that control apoptosis in C. Elegans have been found in humans?

Answer 18

Death signals initiate the pathway by:

• directly inhibiting the actions of anti-apoptotic proteins (Ced-9 in C. elegans and Bcl-2 in humans) or
• by activating factors that are capable of suppressing the actions of these proteins (such as Eagle-1 and BAD )
• Inhibition of Ced-9 or Bcl-2 leads to triggering of the next step in the suicide programme
• Subsequent activation of Ced-4 or Apaf-1 factors sets off the final executors of apoptosis, Ced-3 or caspases

Question 19

What are the regulators, adaptor and effector of apoptosis ?

Answer 19

Ced-9 and Bcl-2(c ekegabs and vertebrates respectively) both function to suppress apoptosis in the presence of trophic factors (survival factors)

Adaptors Ced 4 & Apaf-1(c elegans and vertebrates) interact with both regulators and effectors; in the absence of trophic factors (survival factors), they promote activation of effectors and cell death

-C. Elegans Ced-9 and vertebrate Bcl-2are homologues proteins

Question 20

What are the molecular events of extrinsic pathways of apoptosis ?

Answer 20

Induction of apoptosis by extracellular stimuli

• Activated T-lymphocytes can induce apoptosis in bacteria or virus infected cells
• Fas ligand to Fas receptor recruits FADD adaptor protein
• FADD recruits procaspases 8 and/or 10
• Caspases are activated —> —> —> apoptosis

Question 21

Explain the molecular events if intrinsic pathway of apoptosis

Answer 21

Induction of apoptosis by intracellular stimuli

Damaged or stressed cells can induce apoptosis by releasing cytochrome c from the mitochondria

Cytochrome c binds to adaptor protein (Apache-1). The cytochrome c/Apaf-1 complex aggregates procaspase and their activation

Question 22

What 3 classes of Bcl2 proteins regulate the intrinsic pathway?

Answer 22

The Bcl-2 family of proteins controls a critical step in commitment to apoptosis by regulating permeabilization 9f the mitochondrial outer membrane (MOM)

• Antiapoptotic proteins that inhibit apoptosis at several steps. (Bcl2, Bcl-XL)
• Multiregion pro apoptotic proteins(BH123 protein, Bax, Bak) that directly permeabilize the MOM
• BH3 proteins that directly or indirectly activate the pore forming. Class members(e.g., Bad, Bim, Bid, Puma, Noxa)EGL-1

Question 23

What are the effects of mitogens on apoptosis?

Answer 23

Mitogens (an extracellular factor, example growth factors) stimulate cell division by:

• activating Ras and MAP kinase (mitogen activated protein kinase)
  - which ultimately causes transcription of the MHC gene

When Myc is expressed, it drives cells into the cell cycle.

-Conversely, inhibition of myc expression leads to growth arrest

Question 24

How can uncontrolled cell division can lead can lead to apoptosis?

Answer 24

Caused by uncontrolled mitogen production which can cause the activation of p19^ARF

• p19^ARF binds and inhibits Mdm2 preventing the degradation of p53
• The p53 pathway is then activated

Question 25

What are the roles of survival factors on apoptosis?

Answer 25

• extracellular signals can control the fate of cells
• Some signals can stimulate apoptosis and others inhibit
• Survival factors inhibit apoptosis
• Nerve cells are produced in excess during development and compete for limited amounts of survival factors (e.g., neurotrophins) secreted in target cells
• Nerve cells that receive enough of the survival signals live, others die
• the cells that survive= the number of target cells they connect with