In vitro models of investigating cancer Flashcards
1
Q
What is necrosis?
A
cell death is unprogrammed, unprepared and premature opposed to apoptosis (programmed cell death)
2
Q
What are the 3 forms of cell death?
A
- anoikis - loss of cell-cell contact and with ECM, caspase dependent
- autophagy - homeostatic mechanism, stress-induced, use of organelles for energy gain, vacuolated phenotype
- necrosis - caused by hypoxia, nutrient deprivation and chemotherapy
3
Q
How does cell death deregulation help cancer?
A
- evasion of apoptosis and anoikis = tumourigenesis = metastasis reoccurrence
- employment of autophagy = treatment resistance = metastasis recurrence
- anti-cancer treatment = hypoxia = autophagic cell death = cell membrane disruption and release of signals = exacerbated inflammation
4
Q
What are the different ways of deregulating pathways?
A
- prevention caspase activation
- neutralisation of active caspases
- suppression of caspase gene expression
5
Q
What in vitro methods can be used in investigation?
A
- appropriate cell culture model 2D or 3D
- ‘right’ assay tool
- selection of inhibitors, agonists, mimetics and cytoxicity assays
- appropriate modality (time-lapse, microscopy, flow cytometry)
- determination of end point
6
Q
How do we visualise effects of cell death in vitro?
A
- membrane integrity assays
- DNA fragmentation
- mitochondrial changes
- use of fluorescent/non-fluorescent dyes (DAPI, PI)
- use of fluorescent assays (TUNNEL staining, annexin-V)
- phenotypic observation (time-lapse microscopy)
7
Q
What is fluorescence microscopy?
A
eg DAPI - blue fluorescent dye, stains dsDNA by strongly binding to adenine-thymine rich regions = assesses DNA fragmentation
8
Q
Describe calcein-AM
A
- non fluorescent, lipophilic = esterase conversion to green fluorescent in live cells
9
Q
Describe PI
A
- propidium iodine - impermeable to live cells, enters necrotic cells through their damaged membranes
- 40-fold enhancement in fluorescence intensity when bound to DNA
10
Q
Describe TUNEL
A
- terminal deoxynucleotidy transferase dUTP nick end labelling
- detects apoptotic cells with extensive DNA degradation = late stage apoptosis
- TdT = enzymes that catalyses attachment of deoxynucleotides in blunt ends of double stranded DNA breaks
- dUTP - BrdU and detection by antibody with biodin-streptavidin + fluorophores activity
11
Q
How is membrane disruption assessed?
A
flow cytometry - sorting live from dead cells
- fluorescence activated cell sorter (FACS)
- viable cells = no labelling
- apoptotic cells = annexin V - FITC
- necrotic cells = PI
- dot plot shows cells stained with annexin V:FITC vs PI
12
Q
What is time lapse microscopy?
A
- apoptosis detected in real time with live cells
- qualitative and quantitative validation of apoptotic cell death based on morphology (eg, shrinkage, membrane blebbing, nuclear condensation)
