9 - Control of Cell Death

Question 1

Types of cell death

Answer 1

• Programmed cell death
• Necrosis

Question 2

Programmed cell death

Answer 2

• A physiological process where cells are eliminated during development and other normal biological processes
• E.g. Apoptosis, Autophagy

Question 3

Apoptosis

Answer 3

Type 1 cell death

Question 4

Autuphagy

Answer 4

Type 2 cell death

Question 5

Necrosis

Answer 5

Pathological process after exposure to serious physical or chemical insult

Question 6

Anoikis

Answer 6

Delayed cell death associated with build up of autophagy vesicles

Question 7

Cornification

Answer 7

Epithelial cell specific process to produce outer (dead) layer of the skin

Question 9

Pyroptosis, pyronecrosis

Answer 9

Infection induced death
of macrophages

Question 10

Necroptosis

Answer 10

‘regulated’ necrosis

Question 11

Morphological features of apoptosis

Answer 11

• No loss of membrane integrity
• Aggregation of chromatin at the nuclear membrane
• Shrinking of the cytoplasm and condensation of nucleus
• Fragmentation of cell into apoptotic bodies
• Leaky mitochondria due to pore formation

Question 12

Biological features of apoptosis

Answer 12

• Strictly regulated process
• Energy (ATP) dependent
• Ladder pattern of DNA fragmentation (non random)
• Prelytic DNA fragmentation
• Alteration in membrane asymmetry

Question 13

Physiological significance of apoptosis

Answer 13

• Evoked by physiological stimuli (growth factors etc)
• Affects individual cells
• Phagocytosis by macrophages or adjacent cells
• No inflammatory response

Question 14

Morphological features of necrosis

Answer 14

• Loss of membrane integrity
• Swelling of cytoplasm and mitochondria
• Total cell lysis
• No vesicle formation
• Disintegration (swelling) of organelles

Question 15

Biological features of necrosis

Answer 15

• Loss of regulation of ion homeostasis
• No energy requirement
• Smear pattern of DNA (random digestion)
• Postlytic DNA fragmentation
• Possibility for recovery after reversible injury (unlike apoptosis)

Question 16

Physiological significance of necrosis

Answer 16

• Evoked by non-physiological disturbance
• Affects groups of cells
• Phagocytosis by macrophages
• Significant inflammatory response

Question 17

What mediates the events associated with apoptosis

Answer 17

Caspases

Question 18

Pathways to apoptosis

Answer 18

• Can be extrinsic or intrinsic
• Apoptosis induction –> Initiator caspase activation –> Effector caspase activation –> Death substrate cleavage –> Apoptosis

Question 19

Regulation of apoptosis

Answer 19

• Positive modulators (pro-apoptosis)
• Negative modulators (anti-apoptosis)

Question 20

BCL-2 Family

Answer 20

• Inhibitors of apoptosis (anti-apoptosis): Bcl-2, Bcl-xL
• BH3 only (pro-apoptosis): Bid, Bim Bad
• Effectors (pro-apoptosis): Bax, Bak, Bok

Question 21

Anti-apoptotic Bcl-2 family structure

Answer 21

Four Bcl-2 homology domains (BH) as well as a putative trans
-membrane domain (TM) responsible for their preferred localization at inner membranes.

Question 22

Effector Bcl-2 family structure

Answer 22

Bax subfamily resemble Bcl-2
closely in structure possessing three out of four (multiple) BH domains

Question 23

BH3 only Bcl-2 family structure

Answer 23

Only share one BH3 domain with all other Bcl-2 family members.

Question 24

Balance between anti-apoptotic and pro-apoptotic Bcl-2 family members

Answer 24

Determines if effector members are free to initiate apoptosis

Question 25

Positive modulators of apoptosis (inducers)

Answer 25

- Cytochrome c - Apoptosis protease activating factor 1 (APAF1) - Caspases - Apoptosis inducing factor (AIF) - Endonuclease G (Endo G) - Granzyme A (GrA)

Question 26

Cytochrome-c

Answer 26

Activates APAF1

Question 27

APAF1

Answer 27

Critical component of apoptosome, cleaves caspase 9

Question 28

AIF

Answer 28

Induces chromatin condensation and DNA degradation

Question 29

EndoG

Answer 29

Facilitates chromatin condensation with AIF

Question 30

GrA

Answer 30

Serine protease released by cytotoxic T cells

Question 31

E2F

Answer 31

- Transcriptionally activates many pro-apoptotic genes - E.g. Bax, Bad, APAF1

Question 32

Negative modulators of apoptosis (inhibitors)

Answer 32

- Bcl-2 family genes - Inhibitor of apoptosis proteins (IAPs) that block caspase activation - Pro-thymosin-α (ProTα) that blocks apoptosome formation - E1B: acts like Bcl2 to bind Bcl2 family effectors

Question 33

NF-kB

Answer 33

- Activation leads to enhanced survival - Inhibition of NF-kB promotes apoptosis

Question 34

Reasons for studying apoptosis

Answer 34

Utilised in studies of: - Immunology - Embryology - Aging - AIDS - Neurology - Cancer

Question 35

Methods for studying apoptosis

Answer 35

- Protease activity (caspase 3) - Membrane alterations - DNA fragmentation assay - DNA strand breaks

Question 36

Membrane alterations

Answer 36

- Phosphatidylserine translocation to outside of cell when cell undergoes apoptosis - Annexin V (a phospholipid-binding protein with a high affinity for phosphatidylserine)

Question 37

DNA fragmentation assay

Answer 37

- Apoptotic DNA ladder - Gel electrophoresis

Question 38

DNA strand breaks

Answer 38

TUNEL assay (terminal deoxynucleotidyl transferasemediated dUTP nick end labelling)

Question 39

Internal pathways of how cancer cells avoid apoptosis

Answer 39

- p53 loss of function - Rb inactivating mutations - Myc gene amplification - Bcl2 activation - Bax inactivating mutations - Caspase inactivating mutations

Question 40

External pathways of how cancer cells avoid apoptosis

Answer 40

- Loss of pro-apoptotic signaling molecules - Viral infection can prevent apoptosis (e.g. HPV E6 protein blocking p53 function) - Interaction with other cells (hide from CTLs) - Interaction with chemicals

Question 41

Apoptosis and cancer therapy

Answer 41

- Inducing apoptosis of cancer cells is an ideal therapeutic approach as it prevents inflammation and damage due to necrotic cell death and harnesses the cells own apoptotic machinery - But cancerous cells are resistant to apoptosis - Need to know affected pathways to produce targeted therapies