Thesis presented July 09, 2021
Abstract: Human cells are under constant threat from DNA-damaging agents. Double-strand breaks (DSBs) are highly deleterious DNA lesions that can induce chromosomal translocations, increase mutation rate and participate in malignant transformation. DSBs are also induced by standard anticancer treatments, in order to trigger cell death mechanisms in cancerous cells. In humans, DSBs are processed by complex repair systems that operate with different speed and fidelity. The last two decades saw the emergence of several repair inhibitors that target major repair pathways and reduce repair capacity in tumor cells. A better understanding of DSB repair processes would improve the identification of tumors that could respond to repair inhibitors. In addition, it may also allow the identification of specific repair profiles associated with an increased risk of treatment-induced adverse effects. Thus, functional approaches allowing the characterization of DSB repair profiles could complement current genotyping methods. In this intent, LXRepair developed a multiplexed assay on biochip that allows the analysis of several DSB repair activities. Repair profiles have been characterized in three human cancer cell lines as part of technical and biological assay validations. Repair activities were studied both at the basal level and following exposure to DSB-inducing chemical doxorubicin, combined or not with various repair inhibitors. In collaboration with the CIBEST team, results were compared to a reference assay based on agarose gel electrophoresis. In addition, 53BP1 foci and repair protein levels or activity were investigated, together with the activity of repair pathways that process lesions other than DSBs, allowing further characterizations of the cellular response to the treatments. This study highlighted the interest of the biochip approach compared to the chosen reference method, both from the technical and biological perspective. The assay reduced experimental workload while providing information on several repair pathways simultaneously. However, this work also reflects the complexity of the regulation of DNA repair systems and raises a set of questions that call for further investigation. Overall, the results obtained within the course of this project highlight the potential of the biochip method for various applications in oncology, fundamental research or biomolecular screening.
Keywords: Radiosensitivity, Biochip, DNA, Cell biology
On-line thesis.