Cell Death

Question 0

Intrinsic mechanism of apoptosis initiated by

Answer 0

Irreparable DNA damage
Hypoxia
Increase Ca levels
Severe oxidative stress

Question 1

Steps in apoptosis

Answer 1

Cell shrinkage
Chromatin condensation/ margination
Membrane blebbing
Nuclear collapse
Apoptotic body formation
Lysis of apoptosis bodies

Question 2

Intrinsic pathway - apoptosis

Answer 2

Increase p53, increase BAX.
Permeable mitochondria membrane.
Release of SMAC/ DIABLO -> inhibits inhibitors of apoptosis IAPs
Release of cytochrome C, binds apoptosis protease activity factor Apaf 1. Forms apoptosome which activates caspase 9.
Activation of effector caspase a 3, 6, 7.

Question 3

Extrinsic pathway - apoptosis

Answer 3

Proapoptotic ligand a bind receptor (TNF/Fas)
Recruitment of death domains (TRADD/FADD)
Procaspase 8 (initiator caspase) binds and forms DISC - death inducing signaling complex.
Procaspases brought into close proximity, auto catalytic processing.
Activates effector caspase a 3,6,7.

Question 4

What do caspases do?

Answer 4

Activate endonucleases and proteases
3,8 regulate phosphatidyl serine expression
3,6,7 cleave substrates.

Question 5

Biochemical features of apoptosis

Answer 5

Protein cleavage
Protein cross linking
DNA breakdown
Phosphatidyl serine expression
Caspases

Question 6

Assays for apoptosis

Answer 6

DNA fragmentation - TUNEL assay
- caspase activated DNase cut internucleosomal sites
Caspases detection - ELISA
- Western BLOT
Alteration in membrane - annexing V assay
Mitochondrial assay - laser scanning confocal microscopy LSCM

Question 7

Types of necrosis

Answer 7

Liquifactive
Coagulative 
Haemorrhagic
Toxin induced
Viral
Caseous

Question 8

Necrosis - changes in cell morphology

Answer 8

Increase size, organelles swell, plasma membrane rupture, loss of cell contents.

Question 9

Nuclear changes in necrosis

Answer 9

Chromatin clumping, karyohexis, pyknosis, karyolysis.

Question 10

Triggers of autophagy

Answer 10

Protein aggregation
Organelle damage
Nutrient depletion
Growth factor deprivation
Microbial infection

Question 11

Three types of autophagy

Answer 11

Microautophagy
Chaperone mediated autophagy
Macroautophagy

Question 12

Process of microautophagy

Answer 12

Cytoplasmic contents enter directly into lysosomes. Degraded by hydrolases

Question 13

Process of chaperone mediated autophagy

Answer 13

Hsc70 recognizes KFERQ.
Co chaperones unfold protein.
Lamp2 translocates protein into lysosomes.
Degraded by hydrolases.

Question 14

Process of macroautophagy

Answer 14

Engulfment of components by phagophore.
Phagophore extends creating double membrane vehicle = autophagosome.
Fuses with lysosome. Inner membrane and protein content hydrolysed = autolysosome.

Question 15

Pathways regulating autophagy

Answer 15

mTOR dependent

mTOR independent

Question 16

Uses of autophagy

Answer 16

Adaption for longer periods of starvation
Elimination of cytoplasmic contents
Simple pathway from cytosol to lysosomes
Sequestration with degradation eg ER stress

Question 17

Hayflick limit

Answer 17

Finite replicative potential of normal somatic cells in culture

Question 18

Use of senescence

Answer 18

Prevent genomic instability

Prevent cancer

Question 19

Two types of senescence

Answer 19

Replicative senescence

Accelerated senescence

Question 20

Bypassing senescence in culture

Answer 20

Grow on fibroblast feeding layer
Ectopic expression of hTERT
Suppression of p53
Simian virus 40 infection

Question 21

Markers of senescence

Answer 21

Fail to proliferate in response to growth factors
Not sensitive to apoptotic stimuli eg serum withdrawal
P53, p16, RB levels
Morphology - large, thin, stress fibers

Question 22

Assay for senescence

Answer 22

B-gal positive at pH 6

Question 23

Quiescence

Answer 23

Reversibly growth arrested cells

Question 24

Markers of quiescence

Answer 24

Sensitive to apoptotic stimuli
GFs cause proliferation
B-gal neg
Morphology - cobblestone.