Elias, BenjaminDecottignies, AnabelleBoulle, RoxaneRoxaneBoulle2025-05-142025-05-142025-05-142024https://hdl.handle.net/2078.2/37722Most cancer cells are immortal because they manage to maintain their telomere length, either by reactivating telomerase (TEL+) or through an alternative telomere lengthening mechanism (ALT+). Targeting these mechanisms to induce cell death, offers interesting perspectives for new anticancer therapies. More specifically, there is growing interest in targeting DNA G-quadruplex (G4) structures, formed by the stacking of four guanines and with crucial roles in telomere regulation and protection. In addition to their impact on telomere homeostasis, G4s are also present in the promotor sequences of proto-oncogenes, making them important targets for drug development. The development of new powerful anti-G4 antibodies may help understanding G4 physiology and assessing the efficacy of potential G4-targeting drugs. Today, there are only few antibodies targeting G4s, but the research team of Eric Defrancq at the University of Grenoble has successfully developed two new antibodies against anti-parallel G4s, namely T13 and T20. In this master’s thesis, these antibodies were first tested in cultured cells using an optimized immunofluorescence protocol and results were compared to those obtained with the commercially available 1H6 anti-G4 antibody, that is not specific to any G4 conformation. Further experiments were carried out to test the specificity of these antibodies, including the siRNA-mediated depletion of ATRX chromatin remodeler to increase telomeric G4s. A comparative analysis was also carried out between TEL+ and ALT+ cells as telomeric G4s are expected to be more abundant in ALT+ cells. Our experiments revealed that the new T13 antibody performed well in immunofluorescence, with staining results comparable to those obtained with the 1H6 antibody. Furthermore, immunofluorescence signals of the T13 antibody disappeared upon DNAse treatment, supporting the specificity of T13 against DNA structures. Additionally, as expected from the loss of ATRX in ALT+ cells, staining with T13 and 1H6 antibodies showed an increased frequency of G4-positive telomeres in ALT+ compared to TEL+ osteosarcoma cell lines. In the past years, G4 ligands were developed to stabilize these structures, leading to replication stress and inhibition of DNA replication. Subsequently, metal complexes targeting G4s have been developed that bind G4s and induce DNA damage or replication stress. In this context, Justin Weynand in the laboratory of Professor Benjamin Elias at Louvain-la-Neuve developed and characterized photoactivable dinuclear ruthenium(II) complexes. These complexes showed strong affinity for telomeric G4s in vitro, but evidences for the targeting of telomeric G4s in cellulo were missing. Using the new T13 antibody from Grenoble, we were able to show a decrease in the abundance of telomeric G4 signals after illumination of cells, thus supporting the activity of the tested dinuclear ruthenium(II) complex in cellulo.G-quadruplexAntibodiesTelomeresCancerMetal complextext::thesis::master thesisthesis:46470