PCR Application: Can Breast Cancer be Cured?
Jan 15, 2016
PCR Application: Can Breast Cancer be
Cured?
Normal, Healthy Cells
• Cells can change or differentiate to become specialised according to the tissue that they make up
• A healthy cell performs the correct functions according to its type and specialised state
• This feature of cells is controlled by our DNA
Cell division
Cell specialisation/differentiation
Cell adhesion
Properties of a healthy cell
Normal, Healthy Cells
Very common to get DNA mutations
How does a healthy cell deal with this?
Repair DNA e.g. BRCA2
protein
Cell suicide –removes cells
with badly damaged DNA
When things go wrong
Normal, Healthy Cells
DNA MutationsMutations are a change in the base nucleotide
sequence of our DNA
e.g. Deletion A T G C G A T A T C G T T
e.g. Insertion A T G C G A A T G G C G A
G
Changes in the DNA sequence can change the amino acid sequence of the protein.
DNA Mutations
• If a gene becomes mutated, cells can start to lose control and become cancerous
• This can lead to the formation of a tumour
Cancer Cells
Uncontrollable cell division
Reduced cell death - cancer
cells do not always respond to signals telling
them to dieNo cell specialisation - cancer cells lose their specialised features
Reduced cell adhesion - cancer cells
lose their ability to stick together
Cancer cells arise from genetic mutations
Cancerous Cells
External Factors causing genetic mutations
Physical CarcinogensE.g. UV Radiation from sunlight
Biological Carcinogens E.g. viruses such as HPV can create an opportunistic environment for cancer
Chemical CarcinogensE.g. Tobacco, alcohol
Inheritance of these genetic mutations
External Factors causing genetic mutations
Genotype
Genetic makeup of a cell – the set of alleles that a person has.
Phenotype
The observable (way it looks) physical or biochemical appearance of an organism. Determined by genotype and environmental influences.
TYPICAL FORM MUTANT FORM (“WILD TYPE” - WT)
Example
Genotype Terminology
• Heterozygous – when the cells contains 2 different alleles at a gene locus e.g. Bb
• Homozygous – when identical alleles of the gene are present on both homologous chromosomes e.g. BB or bb
Inherited Mutations
• The BRCA2 gene codes for a protein that repairs DNA. Some people can inherit a BRCA2 mutation (Bb genotype for BRCA2)
• These people are more susceptible to accumulating DNA damage and are consequently at a higher risk of breast cancer
• Tumours arise from cells where Bb has spontaneously mutated to become bb with time (since this means that they have no BRCA2 in these cells)
What Does Higher Risk of Breast Cancer Mean?
• 12% of women without any inherited mutations will develop breast cancer in their lives
• 45% of women with a harmful BRCA2 mutation will develop breast cancer by 70 years of age
Managing Risk
Angelina Jolie has an inherited BRCA2 mutation and elected to have her breasts removed as a preventative
measure
How did she know?
• A DNA sample would have been taken (from her blood or saliva) and this sample would have been sent to a testing laboratory
• Then the BRCA2 gene locus would have been amplified by PCR and her genotype analysed by gel electrophoresis
Managing Risk
Other options;
• Enhanced screening – frequent mammograms
• Chemoprevention – possibly tamoxifen or ralaxifene may help to reduce risk, but this has not yet been proven
How can we kill cancer cells with BRCA2 Mutations?
Cancer cell’s repair mechanisms cannot copeCancer cells commit suicide(Healthy cells also commit suicide)
The Problem with mutated BRCA
• The mutation in the BRCA2 (bb) gene in tumour cells is not enough to induce cell suicide
• So BRCA2 mutated cells do not respond very well to chemotherapy or radiotherapy
• This is because other DNA repair mechanisms exist in the body. The other major one is the PARP pathway
Overcoming this Problem
• Treating cancers caused by BRCA2 mutations by inhibiting the other DNA repair pathway:
Synthetic Lethality
BRCA
PARP
BRCA
PARP
BRCA
PARP
BRCA
PARP
Cellsurvives
Cellsurvives
Cellsurvives
Cell Death
BRCA2 Mutant CellNormal Cell
Normal Cell with PARP inhibitor
drug
BRCA2 mutant cell with PARP inhibitor drug
DNA Damage – chemotherapy/radiotherapy
Advantage of synthetic lethality– the person’s healthy cells are not damaged –
only their cancer cells
BRCA
PARP
BRCA
PARP
Cellsurvives
Cell Death
Normal Cell with PARP inhibitor
drug
BRCA2 mutant cell with PARP inhibitor drug
DNA Damage
Conventional chemotherapy and radiotherapy both cause a lot of damage to healthy cells
Synthetic Lethality
NO BRCA2 + NO other = CELL DEATH mechanisms of cancer cells of DNA repair
+ Chemotherapy or Radiotherapy
Synthetic lethal treatments are currently being researched in Cardiff University and elsewhere as a potential new therapy for breast cancer
INHERITED MUTATION+ PARP INHIBITOR
DRUG
Your Task
Your PCR samples are from 4 individuals with or without BRCA2 mutations
Your tasks are to:
1. GENOTYPE each sample
2. PHENOTYPE each sample