apoptosis - exam

Question 1

WHAT
cellular response to…

Answer 1

environmental cues

Question 2

What defines programmed cell death vs accidental cell death?

Answer 2

Programmed is controlled and there are cues and steps

Question 3

what is apoptosis

Answer 3

a major form a programmed cell death

Question 4

other forms of programmed cell death

Answer 4

o Autophagy (self-eating) – degradation of cytoplasmic materials via lysosomes
o Necroptosis – programme necrosis (it can accidental)
o Many more types as well

Question 5

major cell death subroutines

Answer 5

RDC: regulated cell death
ICD: immunogenic cell death
ADCD: autophagy-dependent cell death
LDCD: lysosome-dependent cell death
MPT: mitochondrial permeability transition

Question 6

APOPTOSIS:
morphological features

Answer 6

cell shrinks, chromatin condenses

budding

apoptotic bodies are phagozytosed; no inflammation

Through this process membrane is intact and cellular contents are not released

Question 7

necrosis morphological features

Answer 7

necrosis (cell swells)

cells becomes leaky, blebbing

cellular and nuclear lysis causes inflammation

Question 8

APOPTOSIS:
more to discover
early and late

Answer 8

Early apoptotic events: apical actin accumulation, actin bundles connecting nuclei, apical migration of nuclei

Late apoptotic events: basal cell delamination, chromatin condensation, DNA fragmentation

Question 9

APOPTOSIS:
why is it important

Answer 9

• How many cells die each day due to apoptosis in a human adult?
  o 50-70 billion cells
• How much weight of dead skin cells do you lose in a year
  o 8 pounds

Question 10

APOPTOSIS:
when does it occur
developmentally programmed

Answer 10

o Patterning/morphogenesis
 Sculpturing structures; deleting unwanted tissues; regulating cell numbers

Question 11

APOPTOSIS:
when does it occur
stress-induced

Answer 11

eliminates damaged cells

stress e.g. environmental toxins

Question 12

APOPTOSIS:
when does it occur
degenerative disease

Answer 12

Defective tissue homeostasis: degenerative diseases

Question 13

APOPTOSIS:
what does it inhibit

Answer 13

cancer

• Evasion of apoptosis is a hallmark of cancer

Question 14

APOPTOSIS:
how does cancer evade it

Answer 14

sustaining proliferative signalling

evading growth supressors

activating invasion and metatasis

enabling replicative immortality

inducing angiogenesis

resisting cell death

Question 15

HOW DOES APOPTOSIS OCCUR

Answer 15

• Intrinsic/extrinsic pathways mainly through mitochondria/death receptors and caspases

Question 16

HOW DOES APOPTOSIS OCCUR
conserved?

Answer 16

Evolutionary conserved core apoptotic machinery

• caspases

Question 17

HOW DOES APOPTOSIS OCCUR
apoptotic caspases

Answer 17

• A family of cysteine proteases that are synthesized as inactive precursor enzymes or proenzymes (cut up proteins)
• Pro-capases are activated by cleavage events and formation of a heterotetramer of two large (p20) and two small (p10) subunits
• Two groups of caspases: prodomain and function
• Two classes – separated due to structure and function

Question 18

HOW DOES APOPTOSIS OCCUR
apoptotic caspases
effector caspases

Answer 18

shorter prodomain

Question 19

HOW DOES APOPTOSIS OCCUR
apoptotic caspases
initiator caspase

Answer 19

long prodomain
- DED: death effector domain
- CARD: caspase-recruitment domain
once activated prodomain chopped off

Question 20

HOW DOES APOPTOSIS OCCUR
mechanisms

Answer 20

Release of mitochondrial cytochrome c and other pro-apoptotic proteins

Then (two pathways):
1. Inhibition of IAPs
2. Formation of apoptosome (important structure in initiating caspase)

Intrinsic vs extrinsic pathways

Question 21

HOW DOES APOPTOSIS OCCUR
mechanisms
Release of mitochondrial cytochrome c and other pro-apoptotic proteins

Answer 21

There is caspase independent apoptosis
* Caspase-independent apoptosis, also known as caspase-independent cell death (CICD), is a type of cell death that occurs when caspases are not activated. It’s a programmed cell death that can occur in response to most intrinsic apoptotic cues.
- The major ones goes through caspases

Question 22

HOW DOES APOPTOSIS OCCUR
mechanisms
two pathways

Answer 22

1. Inhibition of IAPs
   - IAP antagonists (e.g. Smac, HtrA2) stops action of inhibitor of apoptosis proteins (e.g. XIAP)
2. Formation of apoptosome (important structure in initiating caspase)

Question 23

HOW DOES APOPTOSIS OCCUR
mechanisms
extrinsic pathway

Answer 23

• Extrinsic (death receptor)
  o Surface cell death signal (e.g. TNFa/TNFR, FasL/FasR)
  o –> death-inducing signalling complex (DISC)
  o –> caspase 8 activtation
  o –> caspase 3/7 activation
  o –> apoptosis

Question 24

HOW DOES APOPTOSIS OCCUR
mechanisms
intrinsic pathway

Answer 24

• Intrinsic (mitochondrial)
  o DNA damage signals (or cell stress)
  o –> p53
  o –> Bcl-2 family proteins (e.g. Bax, Bak, Bcl-2) [Bax and Bak activate, Bcl-2 inhibits]
  o –> mitochondrial Cyt C release
  o –> caspase 3/7 activation
  o –> apoptosis

Question 25

HOW DO WE DETECT APOPTOSIS effector capases act on: 1. Inactivation of DNA repair enzymes

Answer 25

* poly (ADP-ribose) polymerase (PARP) * When damage is too much to be repaired cell needed to be killed (checkpoints + cell cycle)

Question 26

HOW DO WE DETECT APOPTOSIS effector capases act on: 2. Inactivation of anti-apoptotic proteins

Answer 26

* Bcl-2 and Bcl-XL * In response to stimuli, allows balance to shift towards apoptosis

Question 27

HOW DO WE DETECT APOPTOSIS effector capases act on: 3. Breakdown of structural nuclear proteins

Answer 27

* Degradation of Lamins by Caspase 6 * DNA needs to be fragmentated at the end of apoptosis, allows DNA to be exposed and cleaved

Question 28

HOW DO WE DETECT APOPTOSIS effector capases act on: 4. Endonuclease activation and chromosomal DNA fragmentation

Answer 28

* ICAD (inhibitor of caspase activated DNase) and CAD * Need to be inactivated most the time otherwise DNA would be continually fragmented

Question 29

HOW DO WE DETECT APOPTOSIS effector capases act on: 5. Phosphatidylserine (PtdSer or PS) exposure: “eat-me” signal for macrophages

Answer 29

* Caspase(3/6/7)-mediated cleavage of phospholipid flippase * Exposure of PS on membranes (stops it from going back inside)

Question 30

HOW DO WE DETECT APOPTOSIS

Answer 30

morphological features cellular markers apoptosis pathway components

Question 31

HOW DO WE DETECT APOPTOSIS - Morphological features:

Answer 31

o Apoptotic bodies o Chromatin condensation: Hoechst 33258 staining

Question 32

HOW DO WE DETECT APOPTOSIS - Cellular markers

Answer 32

Vital dyes e.g. acridine orange DNA fragmentation - Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) or DNA laddering Nuclear breakdown - Loss of lamin B from nuclear envelope Caspase activity - Cleaved caspase 3 antibody or cleavage reporters Annexin V labelling of phosphatidylserine

Question 33

HOW DO WE DETECT APOPTOSIS - Apoptosis pathways components:

Answer 33

p53, Bax, Cyt c and IAPs

Question 34

WHY DO WE STUDY APOPTOSIS

Answer 34

- Context-dependent roles of apoptosis and caspases

Question 35

WHY DO WE STUDY APOPTOSIS cancer

Answer 35

Apoptosis inhibits cancer - Evasion of apoptosis is a hallmark of cancer - Therefore apoptosis inducing therapies can be anti-cancer therapies Apoptosis promotes cancer - Apoptosis-induced proliferation Apoptosis promotes carcinogenesis: context dependent

Question 36

WHY DO WE STUDY APOPTOSIS non-apoptotic function of caspases

Answer 36

- Caspase activation o Cell death o Dendritic pruning o Learning/memory o Sperm individualisation o Compensatory proliferation o Immunity o Differentiation o Enucleation