The dose of curative radiotherapy (RT) for cancer is commonly limited by adverse effects presenting years later. Late reacting normal tissues are, on average, more sensitive to the size of daily doses (fractions) than early reacting normal tissues and cancers. Clinical trials have shown breast cancers to be one exception to this rule, in that they are as sensitive to fraction size as the late reacting normal tissues. This has led to the adoption of hypofractionation (use of fractions >2.0 Gy) in the UK for the adjuvant therapy of women with early breast cancer. An understanding of the molecular basis of fraction size sensitivity is necessary to improve radiotherapy outcome. In this respect, it is relevant that late reacting normal tissues have lower proliferative indices than early reacting normal tissues and most cancers. Here, we test the hypothesis that tissue sensitivity to fraction size is determined by the DNA repair systems activated in response to DNA double strand breaks (DSB), and that these systems vary according to the proliferative status of the tissue. Clinical data suggest that sensitivity of epidermis to fraction size varies over a 5-week course of RT. It resembles a late reacting normal tissue in its sensitivity to fraction size in the first week of RT and loses fractionation sensitivity by weeks 4 & 5. We used this feature of human epidermis to test how fractionation sensitivity and DNA repair changed over 5 weeks of RT. Breast skin biopsies were collected 2 h after the 1st, 5th and last fractions from 30 breast cancer patients prescribed 50 Gy/25fractions/5weeks. Sections of epidermis were co-stained for Ki67, cyclin A, p21, RAD51, 53BP1 and β1-Integrin. After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per mm of basement membrane (p=0.002), of which the majority were in S/G2 phase as judged by cyclin A staining (p<0.0003). The p21 index rose from 2.8% to 87.4% (p<0.0001) after 25 fractions, indicating cell cycle arrest in the basal epidermis. By week 5, there was a 4-fold increase (p=0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, confirming an association between activation of homologous recombination (HR) and loss of tissue fractionation sensitivity. Subsequently, CHO cell lines deficient in specific DNA repair genes were used to test molecular pathways involved in sensitivity to fraction size. We irradiated AA8 (WT), irs-1SF (XRCC3-), V3-3 (DNA-PK-) and EM9 (XRCC1-) with 16 Gy gamma-rays in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA double-strand break (DSB) repair kinetics (RAD51 & 53BP1 staining) and cell cycle analysis using flow cytometry. We found that wild-type and DNA repair defective cells acquire resistance to fractionated radiotherapy by accumulation in the late S/G2 phase of the cell cycle and increased use of HR. In contrast, the irs1SF cells, defective in HR, failed to acquire radioresistance and remained equally sensitive to ionizing radiation throughout the 3-week treatment. We also demonstrated that sensitivity to fraction size is associated with functional NHEJ. It was undetectable in V3-3 cells lacking NHEJ and thereby likely relying on HR. The high fidelity of HR, which is independent of induced DNA damage levels and hence, of fraction size, may explain the low fractionation sensitivity of cells using HR to repair radiation induced DSBs. We then wanted to investigate the modifying effects of small molecule inhibitors of DNA repair on fractionation responses. To this end we tested the effects of adding selected ATM, PARP, and DNAPK inhibitors to fractionated radiotherapy in WT CHO cells. Our results showed that the ATM inhibitor had a significant radiosensitising effect when combined with fractionated RT and resulted in loss of sparing effect of fractionation in wild type CHO cells, an observation that may be clinically relevant. We also examined DNA DSB repair kinetics (RAD51 & 53BP1 foci) with these drugs in the context of fractionated IR.

La plupart des traitements anticancéreux, comme la chimiothérapie ou la radiothérapie, sont cytotoxiques et causent des dommages à l'ADN dans le but d’induire la mort des cellules tumorales. Cependant, l’efficacité d’activité de réparation de l'ADN des tumeurs entraine des résistances intrinsèques et acquises aux traitements. L'une des étapes précoces de la réparation de l’ADN est le recrutement de protéines au niveau du site de dommage. Ce recrutement est coordonné par une cascade de modifications et est contrôlé par des protéines senseurs telles que la protéine kinase ADN dépendante (DNA-PK) et / ou la poly (ADP- ribose) polymérase (PARP). Dans ce manuscrit, nous avons identifié et caractérisé le mécanisme d'action de petites molécules d'ADN (les siDNA), mimant des cassures double brin (appelé Dbait) ou simple brin (appelé Pbait), dans l’inhibition des voies de réparation des cassures simple brin (SSBR/BER). Nous démontrons que les molécules Dbait recrutent et activent à la fois PARP et DNA-PK, contrairement aux molécules Pbait qui ne recrutent que la PARP. L'étude comparative de ces deux molécules permet d'analyser les rôles respectifs des deux voies de signalisation: les deux molécules recrutent les protéines impliquées dans la voie de réparation des cassures simple brin (comme PARP, PCNA et XRCC1) et empêchent leurs recrutements aux niveaux des lésions chromosomiques. Les molécules Dbait inhibent par ailleurs le recrutement des protéines impliquées dans la voie de réparation des cassures double brin (NHEJ et HR). Pbait et Dbait désorganisent la réparation de l’ADN et sensibilisent les cellules tumorales aux traitements. L’inhibition de la réparation des cassures simple brin semble dépendre d’un piégeage des protéines directement sur les siDNA ou indirectement sur les polymères PAR. L’inhibition des voies de réparation des cassures double brin (DSB) semble par contre se faire de façon indirecte ; cette inhibition résulterait plutôt de la phosphorylation des protéines de réparation des DSB de part l’activation de DNA-PK. Les molécules Dbait et Pbait induisent un effet de létalité synthétique des cellules tumorales BRCA mutées. Cependant, la mutation BRCA semble être suffisante mais non nécessaire pour induire la sensibilité des cellules tumorales aux traitements Dbait. En effet, nous avons démontré que les molécules Dbait peuvent aussi sensibiliser les cellules ne présentant pas de mutation BRCA mais ayant toutefois une forte instabilité génétique. Nous avons trouvé une corrélation entre le niveau basal de protéines de réparation de l'ADN (ɣH2AX, PARP et PAR), le taux basal de cassures à l’ADN, la présence de micronoyaux (MN) et la sensibilité des cellules tumorales au traitement Dbait. Nous avons émis l’hypothèse que cette instabilité génétique, déterminé par la quantification de MN dans des biopsies tumorales, pourrait être un biomarqueur prédictif de l’effet du Dbait, non seulement dans les cancers du sein, mais aussi dans les glioblastomes, les mélanomes, les mélanomes uvéaux et les cancers du côlon. / Most conventional cancer treatments, such as chemotherapy or radiotherapy, are cytotoxic and cause DNA damages in the tumoral treated cells, which ultimately lead to their death. However, several intrinsic and acquired resistances of tumors to these treatments are due to the tumor efficient DNA repair activities. One of the major early steps of DNA repair is the recruitment of repair proteins at the damage site and this is coordinated by a cascade of modifications controlled by sensor proteins such as DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In this manuscript, we identify and characterize the mechanism of action of short interfering DNA molecules (siDNA), mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) in Single Strand Break Repair pathway (SSBR/BER) inhibition. We demonstrate that Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. The comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both molecules recruit proteins involved in single-strand break repair (such as PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break (DSB) repair. By these ways, Pbait and Dbait disorganized DNA repair, thereby sensitizing cells to treatments. SSB repair inhibition depends upon a direct trapping of the main proteins on both molecules and an indirect trapping in PAR polymers. DSB repair inhibition may be indirect, resulting from the phosphorylation of DSB repair proteins by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations tumoral cell lines. However, BRCA mutation could be sufficient but not necessary to induce breast cancer cell lines and tumors sensitivity to Dbait treatment. In fact, we demonstrate that Dbait molecules could also have a stand-alone effect in BRCA wild type cells with a high genetic instability. We found a correlation between DNA repair proteins basal level (ɣH2AX, PARP and PAR), DNA break basal level, presence of micronucleus (MN) and tumoral cell lines sensitivity to Dbait treatment. We hypothesis that this genetic instability, determined by MN in tumor biopsies, could be a predictive biomarker of Dbait stand-alone effect, not only in breast cancer treatment, but also in glioblastoma, melanoma, uveal melanoma and colon cancer treatment.