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biology of cancer > In-vitro methods of studying cancer cell death > Flashcards

In-vitro methods of studying cancer cell death Flashcards

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1
Q

Why do cells die?

A
  • unrepairable DNA damage
  • cellular death has a morphogenetic role during development s
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2
Q

when does cellular necrosis happen?

A

when a cell is destructed by an unintended/intended external cirucmstance.
it’s unprogrammed and therefore unprepared and premature

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2
Q

apoptosis

A

process of programmed cellular death
- evasion of apoptosis > cancer

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3
Q

what is the cause of cell necrosis?

A

infections, toxins, trauma, hypoxia, acidosis

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4
Q

what is the effect of cell necrosis?

A
  • large cells
  • swelling
  • liquefaction
  • random metabolic collapse
  • vacuolated cytoplasm
  • membrane integrity loss
  • nuclear dissolution
  • non function organelles
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5
Q

what is the tissue reaction during cell necrosis?

A

surrounding tissue damage, inflammation

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6
Q

what is the cause of apoptosis?

A

natural cell death program mediated by caspases (intrinsic and extrinisic pathways)

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7
Q

what is the effect of apoptosis?

A

membrane blebbing, cell shrinkage, apoptotic bodies, chromatin condensation, DNA fragmentation, functional organelles

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8
Q

what is tissue reaction during apoptosis?

A

no inflammation, no secondary tissue damage

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9
Q

types of regulated cell death (RCD)

A
  • apoptosis
  • autophagic cell death
  • mitotic catastrophe
  • necroptosis
  • parthanatos
  • ferroptosis
  • pyroptosis
  • pyronecrosis
  • anoikis
  • cornification
  • entosis
  • NETosis
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10
Q

cell death deregulation aids cancer; cause

A
  • evasion of apoptosis and anoikis and employment of autophagy >
  • tumorigenesis and metastasis and treatment resistance >
  • metastasis recurrence >
    poor outcome
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11
Q

cell death degregulation aid cancers; effect and perpetutation

A
  • anticancertreatment AND/OR aggressive malignancy >
  • hypoxia, metabolic depletion >
  • autophagic cell death, necrosis >
  • cell membrane disruption and release of signals >
  • exacerbated inflammation >
  • poor outcome
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12
Q

caspase induced pathway:

A

initiation of caspases > activation of effector caspaces > execution of apoptosis > phagocytosis and clearance

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13
Q

way of deregulating caspases

A
  • prevention of caspase activation
  • neutralisation of active caspases
  • suppression of caspase gene expression
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13
Q

studying caspases in vitro

A
  • 11 cell death pathways / 11 caspases
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14
Q

state of cell during in vitro study

A

biochemical study vs phenotype of cellular integrity

14
Q

other cell death pathway studies

A
  • inhibitor of apoptosis protein (IAPs)
  • TNF receptor superfamily
  • Bcl-2 family
15
Q

factors to take into account during in vitro study

A
  • appropriate cell culture model (2D/3D)
  • correct assay tool
  • selection of inhibitors, agonists, mimetics and cytotoxicity assays
  • appropriate modality
  • determination of end point
16
Q

types of studies in vitro

A
  • membrane integrity assays
  • DNA fragmentation
  • mitochondrial changes
  • fluorescence microscopy
  • time lapse microscopy
  • flow cytometry
17
Q

how are in vitro study effects visualised?

A
  • use of flurorescent / non-flurorescent / exlusion dyes
  • use of fluorescent assays
  • phenotypic observation
18
Q

what is DAPI?

A

blue fluorescent dye
- stains dsDNA by strongly binding to adenin-thymine-rich regions

19
Q

TUNEL

A

terminal deoxynucleotidyl transferase dUTP nick end labelling
- detects apoptotic cells with extensive DNA degradaiton > late stage apoptosis

20
Q

terminal deoxynucleotidyl transferase (TdT)

A

enzyme that catalyses attachment of deoxynucleorides in blunt end of ds DNA breaks

21
Q

dUTP becomes BrdU (bromodeoxyuridine)

A

and detected by antibody with biodin-streptavidin + fluorophore activity

22
Q

analysis with flow cytometry

A
  • viable cells; no labelling
  • apoptotic cells ; annexin-V-FITC
  • necrotic cells; PI
23
Q

time lapse microscopy

A

qualitative and quantitative (by number of cells dying per frame) validaiton of apoptotic cell death based on morphology (e.g. cell shrinkage, membrane blebbing, nuclear condensation)

24
Q

time lapse microscopy with fluorescence detection

A
  • apoptosis detected in real time using mix and read Casapse 3/7 reagent
  • non fluorescent substrate crosses cell membrane
  • cleaved by activated caspase 3/7 in apoptotic cells > DNA binding green fluorescent label
25
Q

caspase 1/8

A
  • pyroptosis, apoptosis, necroptosis
  • proinflammatory cytokines
  • process IL-1b, IL-18
  • deleterious hyperinflammation
  • loss of airway or alveolar epithelial cells
26
Q

what do effects of Caspase 1/8 lead to in post mortem phenotype

A
  • massive inflitration of inflam. cells
  • necrotic cell debris
  • pulm. interstitial fibrosis

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