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Rôle de la sénescence dans l'initiation des sarcomes secondaires survenant en territoire irradié

Abstract : Radiotherapy is a major technique used in cancer treatment. It aims to cause the death of cancer cells,mainly through the induction of DNA Double-Strand Breaks (DSBs) and Single-Strand Breaks (SSBs).Paradoxically, radiotherapy is associated in rare cases with the occurrence of second cancers. Datafrom the literature show that these second cancers are mostly sarcoma which develop preferentiallyin the margin of the Planning Target Volume (PTV). This margin is composed of non-tumoral cells andis characterized by a low dose irradiation with a photon energy spectrum shifted towards lowerenergies. The first objective of my thesis project was to characterize the DNA damages encounteredby Normal Human Dermal Fibroblasts (NHDFs) irradiated in the margin of the PTV. For that purpose,the team developed an experimental setting allowing the irradiation of NHDFs positioned in themargin of the PTV. By using this system, we observed that a fractionated irradiation protocolmimicking those commonly applied to patients induces an accumulation of SSBs in the margin,without inducing DSBs. This accumulation of SSBs was associated with a repair defect correlated witha decrease of PARP activity, an essential enzyme for SSB repair.The second objective was to study the effect of this SSB accumulation on NHDF outcome. For thispurpose, we followed the growth of NHDFs irradiated in the margin. Our results show that cellspositioned in the margin of an irradiated PTV underwent growth arrest. We measured their cell deathlevel and showed that NHDF irradiated in the margin of the PTV did not die. Then, we made thehypothesis of a senescence induction. Cell senescence is a cell cycle arrest state associated with along-term survival. By using different markers, we have shown that the NHDFs irradiated in the marginenter in premature senescence.The population of cells irradiated in the margin being heterogenous regarding the occurrence ofsenescence, we sorted fully senescent cells harbouring marked senescent markers and monitoredthem up for long time. We observed that a few of them were able to escape from the cell cycle arrestto give rise to proliferating daughter cells, harbouring mutations and invasive capacities. However, thexenografting of these cells in SCID mice did not induce tumoral development, suggesting that theseemerging cells are only pre-cancerous.By combining a hydrogen peroxide treatment with a PARP inhibitor (Veliparib), we have demonstratedthat an SSB induction is sufficient to induce NHDF senescence and escape from cell cycle arrestassociated to senescence.In vivo, we have confirmed part of these results. We have evidenced that SSBs are generated in thedermal cells of mice positioned in close contact with an irradiated phantom. Moreover, by performingpreliminary experiments with the p16-LUC mice model, we have some data, that have to beconfirmed, that senescence would be induced in mice positioned in close contact with a phantomirradiated with a protocol mimicking those commonly applied to patients.To conclude, these different results show that SSBs are induced in normal fibroblasts positioned atthe margin of a PTV and, consequently, that the cells enter in premature senescence. We have alsoshowed that a few of senescent cells can escape from the cell cycle arrest to give a rise to precancerousdaughter cells. We thus suggest that SSBs and senescence could play a role in thedevelopment of second sarcoma occurring in irradiated field.
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Submitted on : Tuesday, February 1, 2022 - 8:59:08 AM
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Erwan Goy. Rôle de la sénescence dans l'initiation des sarcomes secondaires survenant en territoire irradié. Médecine humaine et pathologie. Université de Lille, 2021. Français. ⟨NNT : 2021LILUS034⟩. ⟨tel-03550375⟩

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