WS1: Data Analysis I

Question 1

Cancer research pathway:

How are new drugs created?

Answer 1

1. Determine which genes and proteins are responsible for aberrant cell growth/cell-cycle/apoptosis resistance in cancer cells
2. Identify possibly cancer-specific ‘targets’
3. Develop drugs (small molecules, antibodies..) to inhibit ‘target’
4. Examine the growth inhibitory/cytotoxic effects of anti-cancer drugs

Question 1

How do cell viability assays work? Give 3 examples:

Answer 1

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)

Question 2

MTT assay in detail:

Answer 2

• 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

Question 3

Disadvantages of colorimetric assays:

Answer 3

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

Question 4

Basis of clonogenic assays:

Answer 4

• 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

Question 5

One advantage and 3 disadvantages of clonogenic assays:

Answer 5

• 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

Question 6

Types of cell cycle analysis:

Answer 6

• Flow cytometry
• Specific marker
• Single cell analysis

Question 7

Flow cytometry basis:

Answer 7

• 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

Question 8

Advantage and 3 disadvantages of flow cytometry population analysis:

Answer 8

• 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

Question 9

Alternate method of population analysis:

Answer 9

• 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

Question 10

Single cell analysis:

Answer 10

• 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

Question 11

Bcl2 family:

Answer 11

• 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

Question 12

How can bcl2 become oncognic?

Answer 12

• Overexpression -> inhibiting Bax-like
• Disregarding BH3-only signal even in times of cellular stresses

Question 13

Cellular stresses which upregulate Bh3-only

Answer 13

• Oncogene signalling
• Genome instability
• Ionising radiation

Question 14

How can apoptosis be measured?

Answer 14

• 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