Search form

Universiteit Gent Vakgroep Medische basis-wetenschappen, radioprotectie

Contact details
Traineeship proposition
Abstract bachelorproef 2016-2017The RAD51-foci test for the detection of radiosensitivity in BRCA1 and BRCA2 mutation carriers

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:

  1. the expression of the BRCA1 and BRCA2 protein using a western blot
  2. 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.

Abstract bachelorproef 2015-2016Optimization of the RAD51 foci assay for analysis of radiation sensitivity of BRCA1 and BRCA2 mutation carriers 

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. 

Samenvatting eindwerk 2013-2014: Inductie en herstel van DNA dubbelstrengbreuken na bestraling met mammograaf en 60Co-bron in borstweefsel
Breast cancer is the most common cancer and cause of death in women. Screening programs for the early detection of breast cancer have been set up and supervised by the Centre for Breast Cancer Detection, targeting Flemish women between age 50 and 70. These programs result not only in a better prognosis, but are also a beneficial factor for the ultimate goal: a decrease in mortality. For mammography screening low energy X-rays (30 kV) are used. As X-rays are potent inducers of DNA damage, especially double strand breaks they are carcinogenic. By this, their use, besides the beneficial effect also implies a risk for the development of radiation-induced breast cancer even if the dose is very low. Especially in individuals having a high breast cancer risk due to a genetic predisposition  (BRCA1 and 2 mutation carriers) a safe use is very important. As BRCA1 and 2 proteins are both involved in the repair of DNA double strand breaks women carrying a mutation are radiosensitive and at high breast cancer risk. To allow proper risk estimation of the effects of low-dose 30 kV X-rays in normal and sensitive individuals research is needed to investigate the ‘relative biological effectiveness’ (RBE) of mammography X-rays. Up to now most RBE research is performed in peripheral blood lymphocytes, primary fibroblasts or cell lines, but never on the actual exposed  breast tissue. In this study we wanted to investigate if exposure of healthy breast tissue to low doses of 30 kV X-rays, as given in mammography, results in detectable radiation induced DNA double strand breaks. To quantify the number of radiation-induced DNA double strand breaks the Y-H2AX foci test was used. RBE values as well as repair kinetics for radiation induced DNA double strand breaks (30 kV X-rays versus 60Co gamma-rays) were analysed.
Healthy breast tissue, obtained from women undergoing breast reduction surgery or mastectomy was used. Thin 2 mm sections of the fibroglandular tissue were made, placed in cell culture medium and irradiated in vitro. After irradiation, the fibroglandular tissue was further incubated at 37°C for 30 minutes or 24 hours. The 30 minutes incubation time was used to determine the RBE, the 24 hours incubation time was performed to investigate the repair capacity of the breast epithelial cells.
The results obtained in this study are preliminary and must be treated with caution.  They indicate that a biological effect already can be detected at very low doses of 2+2 mGy and 4 mGy 30 kV X-rays, such as used for mammography. The results also show that mammography X-rays are more harmful than 60Co gamma-rays in the low-dose region. The RBE derived from the fitted dose response curves in the low-dose area (0-10 mGy) is 5,19. The repair-kinetic analysis shows that after exposure to 60Co gamma-rays the amount of radiation-induced foci is reduced to background levels after a post irradiation time of 24 hours. However, in case of 30 kV X-rays the number of residual foci remains above background levels after 24 hours. This may indicate that 30 kV X-rays cause more complex damage in breast tissue, making it more difficult to repair.
Preliminary results show that low doses of mammography X-rays produce more DNA double strand breaks then 60Co gamma-rays. Data about the genotoxic effects of mammography X-rays are important for correct radiation risk assessment and for assisting those who design screening guidelines, in particular for young high-risk women.
Samenvatting eindwerk 2012-2013: Optimalisatie van de ‘cytokinesis-block’ micronucleustest en ‘cell survival’ testen in fibroblasten voor het bepalen van de stralings-gevoeligheid bij patiënten met een primaire immuundeficiëntie
Exogene factoren zoals ioniserende straling en bepaalde chemische agentia (o.a. chemotherapeutica) zijn effectief in het induceren van DNA-dubbelstrengbreuken (DSB). Daarnaast kunnen DNA DSB ook ontstaan tijdens het proces van V(D)J (variabele, ‘diversity’, ‘joining’) recombinatie bij de ontwikkeling van lymfocyten. Bijgevolg geven mutaties in DNA DSB herstelgenen aanleiding tot stralingsgevoeligheid, immunodeficiëntie en een verhoogd risico op het ontstaan van kanker. Een bepaling van de in vitro chromosomale radiosensitiviteit is daarom aangewezen als bijkomende test bij de verdere diagnose en behandeling van patiënten met een immunodeficiëntie syndroom waarvan het gemuteerde gen nog onbekend is.
Verschillende stralingsgevoeligheidstesten zoals o.a. de ‘cytokinesis-block’ micro-nucleustest en celoverlevingstesten kunnen gebruikt worden om de stralingsgevoeligheid van een patiënt met een immuundeficiëntie na te gaan. De radiosensitiviteit wordt doorgaans onderzocht via in vitro bestraling van lymfocyten uit een bloedstaal. Vanwege de kans op het ontstaan van infecties, lymfoom of leukemie, is een beenmergtransplantatie vaak noodzakelijk bij patiënten met een immunodeficiëntie syndroom. Bijgevolg kan de stralingsgevoeligheid na transplantatie niet meer worden getest op lymfocyten van de patiënt. Daarom worden enkele radiosensitiviteitstesten geoptimaliseerd voor fibroblasten en toegepast op enkele controlepersonen en een patiënt met een primaire immuundeficiëntie.
Uit de micronucleustest kan men voorzichtig afleiden dat de patiënt meer stralings-gevoelig is in vergelijking met een controlepersoon. Bij de ‘crystal violet’ cel-proliferatietest blijkt er echter geen grotere daling in celoverleving op te treden bij toename van de stralingsdosis, vergeleken met twee controlepersonen. Aangezien de micronucleustest specifieker is dan de celproliferatietest voor de detectie van stralingsgevoeligheid ten gevolge van een defect in DNA DSB herstel, is het resultaat van deze eerste test vermoedelijk het meest betrouwbaar. Verder onderzoek met meer controlecellijnen in een lager passage zou deze hypothese moeten bevestigen.




Traineeship supervisor
Prof. Anne Vral
Traineeship supervisor
Julie Depuydt
Traineeship supervisor
Annelot Baert
Via Map