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lsm 4243 - tumour biology > Lecture 1 - Apoptosis (Pathways & Detection techniques) > Flashcards

Lecture 1 - Apoptosis (Pathways & Detection techniques) Flashcards

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1
Q

Difference between programmed cell death (PCD) and apoptosis?

A
  1. Programmed cell death (PCD) originally used to describe cells that die at predictable times (not random, genetically programmed) & places during development
  2. Apoptosis is morphological description of cells dying (Different from Necrosis death)
  3. PCD = Apoptosis (major) + Necroptosis / Pyroptosis
    Hence PCD and apoptosis now used interchangeably
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2
Q

Describe apoptosis in depth.

A

Basic phenomenon:
a. Controlled cell deletion occurs under physiological conditions
b. Morphologically distinct from accidental death or necrosis
c. Active, controlled process, the deregulation of which could lead to many diseases including cancer.
Release of apoptotic bodies which are membrane bound
d. Hyper-proliferation of cells (underregulation of apoptosis) was thought to be the underlying problem –> Cancer!

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3
Q

What is senescence?

A

Form of permanent arrest, seen with ageing as divisions gets reduced due to shortening of telomeres till it is impossible to replicate further, thus, cell stops dividing.

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4
Q

What is DNA laddering?

A

Features that can be observed when DNA fragments, resulting from apoptotic DNA fragmentation, are visualised after separation by gel electrophoresis.

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5
Q

In what situations would you want your cells to die? (6)

A

2 systems depend heavily on apoptosis → Immune system & Nervous system:

  1. Virally infected
  2. Surplus of cells
  3. Non-functional / damaged cells
  4. Auto-immune cells
  5. Mutated DNA
  6. Photo-reactive cells
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6
Q

What is the significance of apoptosis?

A
  1. Development and morphogenesis
    a. Limb formation
    b. Ablation of cells during metamorphosis
    c. Formation of reproductive organs
    d. Development of the nervous system**
    * *Super dependent on the apoptosis of neutrons (cells in digits die to form limbs)
  2. Tissue Homeostasis
    a. Homeostasis in the immune system:
    several million T&B cells are generated and a majority (>95%) die during maturation (death by neglect, negative selection)
    AICD of peripheral immune cells**
    **AICD (Activation-induced cell death) is a negative regulator of activated T lymphocytes that results from repeated stimulation of their T-cell receptors (TCR) and helps to maintain peripheral immune tolerance. … The AICD effector cell is one that expresses FasL, and apoptosis is induced in the cell expressing the Fas receptor.
  3. Deletion of damaged & dangerous cells
    a. Cells with severely damaged DNA
    b. Autoreactive cells of the immune system
    c. Elimination of infected cells
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7
Q

List the consequences of the balance between number of new cells (cell growth) and cell death.

A 
# 1 Cell formation = Cell death
Normal: Homeostasis
# 2 Cell formation < Cell death
Neurodegeneration, immunodeficiency, infertility
# 3 Cell formation > Cell death
Cancer, Autoimmunity
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8
Q

List the 3 categories of apoptosis inducers. Provide examples for each categories.

Random side note:
SPS containing growth factors are used as lab medium to grow cultures.

A 
1. Physiological activators
TNF family
TGFβ
Growth factor withdrawal
Glucocorticoids
2. Damage related inducers
Heat shock
Viral infection
Oncogenes
Tumour suppressors p53
Free radicals
  1. Therapy associated agents
    Chemotherapeutic drugs (i.e. Cisplatin, Doxorubicin)
    Gamma irradiation
    UV irradiation
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9
Q

What is the link between apoptotic research & cancer?

A

1988: BCL-2 found to inhibit apoptosis
BCL is an oncogene which was initially thought to increase proliferation of cell, found that NOOOO it actually inhibits apoptosis. → Realise cancer is not purely a proliferation of cells but rather also the inhibition of cell death.

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10
Q

Why the explosion in studies related to apoptosis?

A

+ Identification of the role of apoptosis in oncogenesis
Bcl2 (B cell lymphoma gene) oncogene shown to be a regulator of apoptosis

+ Genetic studies in C. elegans (roundworms)
Led to the identification of the molecular regulators of apoptosis

  • Evolutionary conservation between nematodes, mammals & fruit flies
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11
Q

Why is apoptosis an essential process?

A

+ Plays an important role in normal development & homeostasis
+ Activated through 2 principal signaling pathways: Intrinsic & Extrinsic
+ Normal cells undergo apoptosis in response to stress-inducing events in the cell (i.e. DNA damage)
+ Cancer is often initiated by DNA damage
+ Dysregulation of apoptosis is critical for cancer development and tumour cell survival.

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12
Q

What are the 2 apoptotic pathways? What are their triggers?

A
  1. Extrinsic: Death receptor mediated (TNF, Fas, etc.)
    Not conserved: More modern, not an ancient pathway
  2. Intrinsic: Mitochondrial (in response to UV, y-irradiation, cytotoxic drugs serum deprivation, oxidative stress
    Passed on for many generations hence considered conserved.
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13
Q

Describe the extrinsic pathway in greater details.

A
  1. Death factor (Fas ligand / TNF / TRAIL) binds to Death receptor (Fas, TNR-R, DR3)
  2. Results inactivation of adaptor FADD / TRADD which pulls in caspase 8
  3. DD (Death domain) & DED (Death effector domain) engage in homophilic interactions
  4. Receptor, adaptor & caspase 8 forms a complex (DISC = Death inducing signaling complex)
  5. Caspase 3 gets activated and cleaves many proteins / caspases vital to cells (i.e. proteolysis of PARP, lamins, actin, etc.)
  6. Caspase 3 will also activate Caspase activated DNAse (CAD) byl breaking down the complex of CAD & ICAD.
  7. CAD will then migrate to the nucleus and then degrades DNA
  8. ICAD will be degraded.
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14
Q

Describe the intrinsic pathway in greater details.

AKA Mitochondrial poison cabinet

A
  1. Targets (Any kind of stress): y-ray, anti-cancer drugs, factor deprivation, etc.
  2. Triggers activation of the tumour suppressor gene p53.
  3. P53 then activates Puma, Noxa (BH3 only proteins)
  4. Activated Puma & Noxa & Bad (another protein) binds to BCL-2 receptors on mitochondria, inhibiting it.
    Bax moves into the mitochondria and then neutralises it.
  5. Bax then initiates release of cytochrome C by making holes in the mitochondria (Mitochondrial potential decreases)
  6. Cytochrome C forms complex with adaptor (Apaf-1) and pro-caspase 9 to form apoptosome.
  7. Caspase 9 then activates caspase 3.
  8. Caspase 3 gets activated and cleaves many proteins / caspases vital to cells (i.e. proteolysis of PARP, lamins, actin, etc.)
  9. Caspase 3 will also activate Caspase activated DNAse (CAD) byl breaking down the complex of CAD & ICAD.
  10. CAD will then migrate to the nucleus and then degrades DNA
  11. ICAD will be degraded.
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15
Q

What happens to the cells after caspases CAD degrade their DNA?

A

+ DNA fragmentation leads to the sending of signals
+ This activates membrane flipping
+ Which then exposes Phosphatidylserine (PS) on the membrane.
+ This will signal for phagocytosis.
+ The engulfment of apoptotic cells by phagocytes
+ They are then digested by lysosomal enzymes
+ This process does not lead to inflammation since the contents are not exposed.

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16
Q

Why is apoptosis an essential process?

A

a. Plays an important role in normal development & homeostasis
b. Activated through 2 principal signaling pathways: Intrinsic & Extrinsic
c. Normal cells undergo apoptosis in response to stress-inducing events in the cell (i.e. DNA damage)
d. Cancer is often initiated by DNA damage
e. Dysregulation of apoptosis is critical for cancer development and tumour cell survival.

17
Q

What are the ways to detect apoptotic cells?

A
  1. Morphology
  2. Plasma membrane alterations
  3. Caspase activity
  4. DNA fragmentation
18
Q

Briefly describe what is DNA fragmentation analysis.

A

+ Intact DNA will stay at the top of the agarose gel electrophoresis
+ Those that undergo DNA fragmentation via endonuclease will lead to many bands
+ One nucleosome (one core histone and DNA) = 180bp
+ So many fragments which can travel further away leading to more bands formation.

19
Q

Degradation of DNA during apoptosis is mediated by 2 systems. What are they?

A
  1. Cell autonomous which occurs in the dying cell and is mediated mainly by Caspase activated Dnase (CAD) → DNA is first cleaved into fragments of ~50kb with intact nucleosomes, followed by degradation into nucleosomal units.
  2. Dying cell is engulfed by macrophages which recognise phosphatidylserine that is exposed on the cell surface in a caspase-dependent manner and the DNA is further degraded by lysosomal enzymes (DNAsell) into nucleotides (sub-units).
20
Q

What are the properties of CAD/ICAD?

A

`. Since CAD is an extremely potent DNAse, it always exists as a heterodimer with ICAD.
2. ICAD has 2 caspase recognition sites -ASP117 & 224
Caspases 3 & 7 cleave ICAD and cleavage at both sites are required to lose CAD binding activity
3. CAD carries an NLS (nuclear localisation site) in its C-terminus.
4. CAD/ICAD complex likely exists in cytoplasm and CAD moves to the nucleus
5. CAD is specific for double stranded DNA with a preference for AT-rich regions, and does not cleave single stranded DNA or RNA.
6. Generates 5’ phosphate and 3’ hydroxyl group which can be substrates for terminal transferase used for TUNEL

21
Q

Briefly describe the process of TUNEL.

A

a. TUNEL stands for TdT-mediated dUTP Nick End Labeling
b. Digoxigenin-dNTP will then binds to the fragmented DNA (5’ phosphate)
c. Then peroxidase-conjugated anti-digoxigenin antibody then binds.
d. Free 3’ hydroxyl end can then be tagged with colouring (DAB, H2O2)
e. DNA shown as blue, fragmentation shown as red.
f. Able to detect spatial distribution of apoptosis.

22
Q

Briefly describe Annexin V staining for the detection of apoptotic cells.

A

+ This is a popular test
+ Quantitative (amount of apoptosis that happened)
+ Normal cells will have inner leaflets of membrane containing PS.
+ Apoptotic cells will undergo externalisation of PS where the membrane is flipped inside out.
+ The PS are then conjugated to form Annexin V-PE conjugate.
+ This reaction occurs very strongly in the presence of Ca2+ ions.

23
Q

What are the principles behind the advantages & disadvantages of using Annexin V and Propidium iodide staining to determine apoptosis?

A

TESTS | Normal cells | Apoptotic cells | Necrotic cells

  1. Annexin V Staining | (-) | (+) | (+)
  2. Propidium Iodide staining | (-) | (-) | (+)

Why is Annexin V positive for both?
→ Apoptotic (Flipping of membrane to expose PS).
→ Necrotic (Cells burst open, Annexin V enter cell and bind to PS on the inner side)

Why is PI staining - for apoptosis and + for necrosis?
→ PI stains DNA.
→ Apoptosis is where the cell is intact and PI unable to enter the nucleus
→ Necrosis (cell bursting)

Can we do both Annexin V and PI concurrently? NO POINT!

How can we use Annexin V + PI staining using Flow Cytometry to detect necrotic and apoptotic cells?

24
Q

What about tracking caspase activity to detect apoptosis?

A

Tracking caspase activity is one of the best methods to show that caspase has been activated
→ Caspase activity as hallmark for apoptosis
→ Use caspase antibodies like M30 to detect cleaved and uncleaved forms of caspase
Cleaved forms: Apoptosis (brown) ; normal: (purple/blue)

25
Q

What about using the electron microscope to analyse apoptosis?

A

Normal cell morphology compared to apoptotic cell morphology
→ Apoptotic cell morphology: cell in a late stage of apoptotic cell death will experience, cell shrinkage, nuclear condensation and final fragmentation of the whole cell

Downside (X): Apopototic bodies can be seen but normally quite hard to identify unless one is very experienced and trained. Em is also very expensive. Most people don’t use EM for regular analysis as it is costly and time consuming.

26
Q

What are the methods for detection of apoptosis & provide a brief description?

A

Method –> Description

  1. DNA fragmentation assays –> DNA ladders (200-5000 bp fragments)
  2. TUNEL staining –> In situ end-labelling of free 3’ ends of DNA fragments by using chromogenic agents
  3. Annexin V binding–> In vitro detection of membrane PS is achieved with annexin V
  4. Changes in membrane potential (cytochrome C, hallmark of intrinsic pathway) –> Assessed with voltage sensitive dyes, western blots (release of cyto c from mito & caspase activation)
  5. Caspase cleavage –> Western blots, immunohistochemistry, detection of proteolytic cleavage products
  6. Flow cytometry–> Detection of number of cells in sub G1/G0
  7. Electron microscopy–> Morphologically can look for apoptotic bodies
27
Q

Difference between apoptosis and necrotic death?

A

Apoptotic VS Necrotic
1. Description
Naturally occurring programmed & targeted cause of cellular death.
VS
+ originally thought to be the only form of death
+ Caused by external factors to cell/tissue
+ Infection, toxins, trauma (unregulated digestion of cell components)

  1. Nucleus
    Chromatin condensation
    VS
    Nuclear swelling
  2. Cell size
    Cell shrinkage
    VS
    Cell swelling
  3. Membrane
    Budding
    VS
    Leaky, Blebbing
  4. Organelles fate
    Preservation of organelles & cell membranes
    VS
    Disruption of organelles; Rupture of cells & release of cellular contents
  5. Trigger inflammation?
    Rapid engulfment by phagocytes, No inflammation
    VS
    Release of cellular contents, Inflammatory response
  6. Biochemical hallmark?
    Yes! DNA fragmentation
    VS
    No! No DNA fragmentation
28
Q

Characteristic features of different types of cell death

A

Type–> Morphological changes–> Biochemical features–> Nucleus–> Membrane–> Cytoplasm

1. Apoptosis
\+ Chromatin condensation; DNA laddering
\+ Blebbing (protrusions of cell membrane/formation of apoptotic bodies) 
\+ Fragmentation
\+ Caspase dependent**

**Unique to apoptosis

  1. Necrosis
    Clumping & random degradation of nuclear DNA
    Swelling, rupture
    Organelle degradation
  2. Mitotic catastrophe
    Multiple micronuclei nuclear fragmentation (errors in mitosis)
    High CDK1/cyclin B
    (Drives mitosis → not cyclase dependent)
  3. Senescence
    (Condition/process of deterioration with age)
    Distinct heterochromatic structure

Flattening, increased granularity
SA-b-gal activity
(Enzymatic activity that occurs during senescence)

5. Autophagy
(self-eating) 
Partial chromatin condensation; no laddering
Blebbing
Increased number of autophagic vesicles
Caspase-independent

lsm 4243 - tumour biology (1 decks)

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