WS1: Data Analysis I Flashcards
1
Q
Cancer research pathway:
How are new drugs created?
A
- Determine which genes and proteins are responsible for aberrant cell growth/cell-cycle/apoptosis resistance in cancer cells
- Identify possibly cancer-specific ‘targets’
- Develop drugs (small molecules, antibodies..) to inhibit ‘target’
- Examine the growth inhibitory/cytotoxic effects of anti-cancer drugs
1
Q
How do cell viability assays work? Give 3 examples:
A
Based on metabolic activity of cells…
- Reduction of tetrazolium salts (e.g. MTT, MTS) to formazan dyes (colorimetric)
- Reduction resaruzin (e.g. AlamarBlue) to resourfin
- [3H]-thymidine incorporation (and similar)
2
Q
MTT assay in detail:
A
- The intensity of the purple colour depends on the concentration of formazan and is directly proportional to the flux of NADPH in the cell i.e. metabolic activity
- Usually measured by determining absorbance at an appropriate wavelength using a microplate reader
- Quick and semi-automatable process
3
Q
Disadvantages of colorimetric assays:
A
Metabolic activity does not always give a true reflection of cell viability:
- Resting cells may have a low activity resulting in underestimation of cell number
- Dying cells may still have some activity resulting in overestimation of cell number
- Redox changes in cell affect results
4
Q
Basis of clonogenic assays:
A
- Ability of single cells to proliferate and form colonies
- Cells are treated with drug/radiation etc and plated at an appropriate concentration to allow detection of single colonies
- Adherent cells can be plated normally whereas suspension cells are plated in soft agar or media containing methylcellulose to allow colony formation
5
Q
One advantage and 3 disadvantages of clonogenic assays:
A
- Considered the gold standard in proliferation assays as they directly measure the ability to proliferate
- However, they are labour intensive, take a long time to obtain data and not all cells will form clones
6
Q
Types of cell cycle analysis:
A
- Flow cytometry
- Specific marker
- Single cell analysis
7
Q
Flow cytometry basis:
A
- Cells stained with propidium iodide, which is a fluorescent dye that binds DNA
- Analysed by flow cytometry
- Amount of DNA in cells indicates cell cycle phase
- Facilitates population analysis
- Represented as either a histogram or scatter plot
8
Q
Advantage and 3 disadvantages of flow cytometry population analysis:
A
- Can rapidly acquire data for 10s of thousands of cells
- Can’t measure cell cycle timings or correlate cell phenotypes with cell-cycle stage
- Can’t assess heterogeneity of cell populations
9
Q
Alternate method of population analysis:
A
- Staining cells for a specific cell-cycle marker e.g. CyclinB1
- Quantify percentage of cells in that stage
- Still can’t measure timings
- Able to costain with other markers and see correlation of cell cycle stage with other phenotypes e.g. apoptosis
10
Q
Single cell analysis:
A
- Engineer cells to express ‘cell cycle biosensors’
- Proteins which naturally fluctuate through cell cycle tagged with fluorescent proteins
- Cells stably express fluorescently tagged proteins
- Cells imaged live over days, allowing tracking of cell cycle
11
Q
Bcl2 family:
A
- Regulating integrity of mitochondrial outer membrane
- Balance of signals from BH3-only can overpower Bcl-2-like protein signal to activate Bax-like and induce apoptosis
- MOMP induced -> cytochrome C and Smac/Diablo Release -> caspase cleavage and apoptosis
12
Q
How can bcl2 become oncognic?
A
- Overexpression -> inhibiting Bax-like
- Disregarding BH3-only signal even in times of cellular stresses
13
Q
Cellular stresses which upregulate Bh3-only
A
- Oncogene signalling
- Genome instability
- Ionising radiation
14
Q
How can apoptosis be measured?
A
- Measuring changes associated with the various steps involved…
- Annexin-V: Detects externalisation of phosphatidyl serine by fluorescent labelling
- DNA fragmentation can be detected by e.g. Sub-G1 population by Flow-cytometry, TUNEL
- Caspase activation can be detected by e.g. Abs
