In-vitro methods of studying cancer cell death Flashcards
Why do cells die?
- unrepairable DNA damage
- cellular death has a morphogenetic role during development s
when does cellular necrosis happen?
when a cell is destructed by an unintended/intended external cirucmstance.
it’s unprogrammed and therefore unprepared and premature
apoptosis
process of programmed cellular death
- evasion of apoptosis > cancer
what is the cause of cell necrosis?
infections, toxins, trauma, hypoxia, acidosis
what is the effect of cell necrosis?
- large cells
- swelling
- liquefaction
- random metabolic collapse
- vacuolated cytoplasm
- membrane integrity loss
- nuclear dissolution
- non function organelles
what is the tissue reaction during cell necrosis?
surrounding tissue damage, inflammation
what is the cause of apoptosis?
natural cell death program mediated by caspases (intrinsic and extrinisic pathways)
what is the effect of apoptosis?
membrane blebbing, cell shrinkage, apoptotic bodies, chromatin condensation, DNA fragmentation, functional organelles
what is tissue reaction during apoptosis?
no inflammation, no secondary tissue damage
types of regulated cell death (RCD)
- apoptosis
- autophagic cell death
- mitotic catastrophe
- necroptosis
- parthanatos
- ferroptosis
- pyroptosis
- pyronecrosis
- anoikis
- cornification
- entosis
- NETosis
cell death deregulation aids cancer; cause
- evasion of apoptosis and anoikis and employment of autophagy >
- tumorigenesis and metastasis and treatment resistance >
- metastasis recurrence >
poor outcome
cell death degregulation aid cancers; effect and perpetutation
- anticancertreatment AND/OR aggressive malignancy >
- hypoxia, metabolic depletion >
- autophagic cell death, necrosis >
- cell membrane disruption and release of signals >
- exacerbated inflammation >
- poor outcome
caspase induced pathway:
initiation of caspases > activation of effector caspaces > execution of apoptosis > phagocytosis and clearance
way of deregulating caspases
- prevention of caspase activation
- neutralisation of active caspases
- suppression of caspase gene expression
studying caspases in vitro
- 11 cell death pathways / 11 caspases
state of cell during in vitro study
biochemical study vs phenotype of cellular integrity
other cell death pathway studies
- inhibitor of apoptosis protein (IAPs)
- TNF receptor superfamily
- Bcl-2 family
factors to take into account during in vitro study
- appropriate cell culture model (2D/3D)
- correct assay tool
- selection of inhibitors, agonists, mimetics and cytotoxicity assays
- appropriate modality
- determination of end point
types of studies in vitro
- membrane integrity assays
- DNA fragmentation
- mitochondrial changes
- fluorescence microscopy
- time lapse microscopy
- flow cytometry
how are in vitro study effects visualised?
- use of flurorescent / non-flurorescent / exlusion dyes
- use of fluorescent assays
- phenotypic observation
what is DAPI?
blue fluorescent dye
- stains dsDNA by strongly binding to adenin-thymine-rich regions
TUNEL
terminal deoxynucleotidyl transferase dUTP nick end labelling
- detects apoptotic cells with extensive DNA degradaiton > late stage apoptosis
terminal deoxynucleotidyl transferase (TdT)
enzyme that catalyses attachment of deoxynucleorides in blunt end of ds DNA breaks
dUTP becomes BrdU (bromodeoxyuridine)
and detected by antibody with biodin-streptavidin + fluorophore activity
analysis with flow cytometry
- viable cells; no labelling
- apoptotic cells ; annexin-V-FITC
- necrotic cells; PI
time lapse microscopy
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)
time lapse microscopy with fluorescence detection
- 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
caspase 1/8
- pyroptosis, apoptosis, necroptosis
- proinflammatory cytokines
- process IL-1b, IL-18
- deleterious hyperinflammation
- loss of airway or alveolar epithelial cells
what do effects of Caspase 1/8 lead to in post mortem phenotype
- massive inflitration of inflam. cells
- necrotic cell debris
- pulm. interstitial fibrosis
