Universiteit Gent Vakgroep Medische basis-wetenschappen, radioprotectie
Breast cancer is a disease that affects many women in the world. A small number of patients have a hereditary mutation in BRCA1 or BRCA2 increasing the risk to develop breast cancer to approximately 60-80%. Both genes are very important in the homologous recombination (HR) pathway. HR is a pathway activated in the S- and G2-phase of the cell cycle to repair DNA double strand breaks, caused for example by ionizing radiation (IR). A mutation in either BRCA1 or BRCA2 leads to an impaired DNA double strand breaks repair which can result in genomic instability and increases the risk to develop breast cancer. Women with a mutation who repeatedly undergo exposure to IR for example because of repeated mammography screenings could be at an increased risk of developing a radiation-induced tumor.
The purpose of this study is to optimize protocols to analyse:
- the expression of the BRCA1 and BRCA2 protein using a western blot
- the impact of a reduction of BRCA1/2 on the efficiency of HR using an RAD51 immunostaining
We observed DNA bands on the western blot at the height of 100 kDa (actinin) and 250 kDa (BRCA1) using protein extracted from MCF10A cells, however, no knockdown in a BRCA1 knockdown cell line could be observed.
Using the RAD51 immunostaining, we observed repeatable results. Furthermore, an increase in foci was observed in the dose-response curve from 0 Gy to 7,5 Gy, followed by a decrease in foci when the dose reached 10Gy. The initial increase was expected. The higher the given dose radiation, the more double strand breaks are induced and the more DNA double strand breaks can be restored with HR. However, the decrease at higher doses was not expected and is probably caused by apoptosis of the radiated cells.
Both protocols need further optimization before they can be used as diagnostic tests on patient’s samples. These protocols are very interesting because when a defect HR pathway is detected, the mutation carrier can be referred to alternative screening methods without exposure to IR, like MRI or ultrasound.
Breast cancer is a disease that affects many women in the world. A small number of breast cancer patients have a hereditary mutation in BRCA1 or BRCA2. Both genes are known as caretaker genes and are involved in the homologous recombination (HR). HR is a pathway activated in the S- and G2/M-phase of the cell cycle for the repair of DNA double strand breaks (DSB), caused by for example exposure to ionizing radiation (IR). A mutation in either BRCA1 or BRCA2 leads to a high risk in developing breast cancer and might also lead to an impaired DSB repair which can result in genomic instability. Women with a mutation who repeatedly undergo exposure to IR for diagnostic (for example mammography screening) or therapeutic purposes could be at higher risk of developing a radiation-induced tumor.
We aim to analyze HR capacity in lymphocytes of BRCA1 and BRCA2 mutation carriers with a RAD51 foci assay. This protocol will be optimized using both continuous cell lines (control cell line and BRCA2 knockdown cell line) and lymphocytes from non-mutation carriers. Flow cytometry is performed to confirm that an acceptable fraction of the analyzed cells is in the S- or G2/M-phase of the cell cycle. Additionally, a PARP-inhibitor is added to maximize the RAD51 foci formation to allow a clear distinction in HR capacity from lymphocytes from mutation carriers and from a control population.
To achieve an optimal RAD51 foci staining with minimal background staining, the incubation period of the first antibody is preferred to be 24 hours in 4° C. Furthermore, for optimal S-/G2-/M-phase nucleus selection, a classifier with the contour radius of the nuclei set to 60-140 μm2 is used. To obtain an optimal foci formation and maintain a good cell quality, the lymphocytes are preferably irradiated with a 5 gray dose to induce DSB’s. Lastly, a concentration of 2 μM PARP-inhibitor seems to generate the best results to analyze the HR-capacity.
The protocol needs further optimization before analyzing HR capacity in lymphocytes of mutation carriers. When a defect HR pathway is detected, the patient can be referred to alternative screening methods without exposure to IR, like MRI or echography. Furthermore, when a tumor develops in patients with a defect HR pathway, treatment with PARP-inhibitor could be implemented.
Prof. Anne Vral