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