Lecture 22 - introduction to practical Flashcards
How can DNA repair mechanisms be used to fight disease?
Targeting DNA repair proteins with drugs/inhibitors makes cancer cells either more susceptible to damage (e.g. combination therapies).
Therefore studying DNA repair proteins is essential for development of new treatment options.
What is the DNA damage response?
Dysregulation of the DNA damage response leads to uncontrolled cell growth, an increase in genetic instability and resistance to chemotherapeutics
What is P53?
The guardian of the genome
- first tumour suppressor gene to be discovered
- mutated in over 50% of all cancers
- acts as molecular switches to determine the fate of the cell
Establishing the P53 status of cancer cells is therefore vitally important for understanding cancer progression, prognosis (outlook/predicted course) & treatment outcomes for patients
What does P21 do?
P21 inhibit CDK activation = halts the cell cycle
Why is the DNA damage response important of cancer?
Dysregulation of the damage response (including DNA repair) leads to accumulation of mutations and therefore cancer
Classical therapies for cancer work by inducing high levels of DNA damage and this cell death
inhibitors of the DNA damage response can sensitize to chemo- and radiotherapy (cancer cells more likely to die)
ANY protein involved in the DNA damage response has the potential to be a target for inhibitors.
How can we study DNA damage?
- survival assays/proliferation assays - sensitivity to DNA damaging agents
- immunofluorescence - what/are proteins activated?
- comet assays - what type/level of damage is happening (involves electrophoresis of a damaged cell & damaged DNA - tail - travels further)
- many more functional experiments
All these experiments give an indication of the amount if DNA happening and the mechanisms involved.
What is western blotting?
this technique used in molecular biology and biochemistry to analyse protein levels, expression & interactions
What are the main steps in Western Blotting?
- Separate proteins
- Transfer proteins
- Probe with antibodies
- Detect bound antibodies
How do you separate proteins?
Use gel electrophoresis to separate proteins in a sample based on their size.
How do you transfer proteins?
Transfer the separated proteins from the cell to a membrane.
How do you probe with antibodies?
Use antibodies specific to the target protein to probe the membrane
How do you detect bound antibodies?
use a chemical or radioactive tag to detect the binding of the antibody
What was the aim of the practical?
to determine the expression levels of P53 in 2 breast cancer cell lines to determine which one would respond better to treatment
