TY - THES ID - 138790324 TI - De generatie en karakterisatie van isogene HNSCC cellijnen AU - Janssen, Reile AU - Nuyts, Sandra AU - Dok, Rüveyda AU - KU Leuven. Faculteit Geneeskunde. Opleiding Master in de biomedische wetenschappen (Leuven) PY - 2019 PB - Leuven KU Leuven. Faculteit Geneeskunde DB - UniCat UR - https://www.unicat.be/uniCat?func=search&query=sysid:138790324 AB - Introduction: Currently, up to 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) develop loco-regional recurrences after radiotherapy (RT) treatment. Resistance of cancer cells to RT plays a substantial role in the development of these loco-regional recurrences. However, the understanding of radioresistance mechanisms is limited due to the lack of appropriate study models. In this thesis, we aim to generate radiotherapy resistant (RTR) cell lines from established HNSCC cells and to characterize these isogenic radioresistant cell lines, to study RT resistance in HNSCC. Material and methods: Four established HNSCC cell lines were treated with fractionated RT, 2 Gy per fraction to a total dose of 60 Gy, to generate RTR cell lines. These RTR cell lines were subjected to a limiting dilution assay (LDA) to generate individual cell clones. Radioresistant clones were selected using a sulforhodamine B (SRB) assay and a clonogenic assay. Possible differences in morphology, clonogenic growth and doubling time between the parental cell line and the radioresistant clones were assessed. In addition, expression levels of important DNA damage response (DDR) proteins were assessed by immunoblotting. Finally, the effect of a PARP-inhibitor and a DNApk-inhibitor, both DDR-inhibitors, in combination with RT was assessed by a SRB assay. Results: Fractionated treatment resulted in the generation of three radioresistant HNSCC cell lines. The RTR cell line with the easiest growth condition and a significant increase in RT resistance was selected for further experiments, which was RTR-SCC61. LDA resulted in 123 clones from RTR-SCC61 of which 23 clones were radioresistant. Two RTR cell clones, R61-1 and R61-4, were selected based on their radioresistant profiles. These cell clones have a similar morphology and clonogenic growth pattern compared to their parental cell line SCC61. However, the cell clones proliferate faster than the parental cell line. Differences in expression levels of DDR proteins were observed between the RTR clones and the parental cell line. Results of the combination of DDR-inhibiting drugs with RT suggest that both clones rely more on non-homologous end joining (NHEJ) for DSB repair than their parental cells. Discussion: We successfully generated two RTR cell clones, R61-1 and R61-4, with a faster proliferation rate and different DDR protein expressions compared to the parental cell line SCC61. The RTR cell clones may serve as a new model for studying radioresistance in HNSCC. In addition, the results provide important insights into the biology of acquired radioresistance in HNSCC. ER -