Etude in vitro des mécanismes d'action impliqués dans la réponse cellulaire à une radioimmunothérapie à l'iode 125

La radioimmunothérapie (RIT) consiste à associer la spécificité de ciblage d'un anticorps monoclonal à la forte toxicité d'un atome radioactif. Afin d'évaluer l'efficacité d'une RIT à l' 125 I, émetteur d'électrons Auger, les lignées cellulaires de carcinome colique HCT 116 p53 (+/+) et p53 (-/-) exprimant les récepteurs HER1 et CEA ont été exposées pendant 48h à des activités croissantes d'anticorps radiomarqué permettant d'obtenir une localisation finale de l'émetteur cytoplasmique (anticorps internalisant anti-HER1) ou membranaire (anticorps non-internalisant anti-CEA). L'évaluation rigoureuse de l'efficacité de cette RIT a nécessité de déterminer la dose d'irradiation moyenne délivrée au noyau par le formalisme du MIRD cellulaire. Nos résultats montrent qu'un taux de survie cellulaire similaire (40%) est observé pour les 2 localisations alors qu'une dose moyenne d'irradiation au noyau beaucoup plus faible (1,2 versus 42,9 Gy) est délivrée dans le cas de l'anticorps non-internalisant. Cette toxicité plus importante associée à une localisation membranaire de l'125I est observée quel que soit le statut p53. Ces résultats suggèrent que la membrane est une cible sensible aux forts dépôts d'énergie associés aux électrons Auger. Afin d'étudier l'origine de cette toxicité accrue, la formation des lésions au niveau de l'ADN en fonction de la dose délivrée au noyau a d'abord été évaluée. Les résultats montrent, qu'en dépit de doses d'irradiation au noyau très différentes, un niveau similaire de cassures de brins de l'ADN et de micronoyaux est observé quel que soit la localisation finale de l'125I, dans les deux lignées cellulaires. Rapportée au Gy, la formation des lésions est plus importante lorsque le débit de dose est plus faible. Nous avons aussi observé une accumulation de la protéïne p53 et une activation de la protéïne p21 dans la lignée HCT 116 p53 (+/+), accompagnée d'un arrêt du cycle cellulaire en G2/M et d'une induction d'apoptose. Ces données suggèrent que les lésions de l'ADN produites sont bien détectées par la cellule et que la mort par apoptose ne joue pas un rôle majeur dans la toxicité de l'125I au niveau membranaire. L'autophagie semble toutefois être impliquée dans la réponse à la RIT à l'125I et reste à déterminer dans de futurs travauxRadioimmunotherapy (RIT) results from the combinaison between a monoclonal antibody (mAb) directed against tumor associated antigen and the strong toxicity of a radioactive isotope. In order to assess the efficiency of RIT based on 125I, an Auger electron emitter, carcinoma colic cell lines HCT 116 p53 (+/+) and p53 (-/-) expressing HER1 and CEA antigens were exposed to increasing activities of either internalizing 125I-mAbs (anti-HER1, cytoplasmic localization) or of non-internalizing 125I-mAbs (anti CEA, cell membrane localization). In order to truly compare efficiency of both 125I final localizations, mean nucleus irradiation dose was determined using MIRD cellular approach. Similar cell survival (40%) was observed while much lower irradiation doses were delivered to the nucleus (1,2 versus 42,9 Gy) using the non-internalizing mAb. The latter toxicity due to cell membrane 125I decays was observed whatever p53 status. These results suggest thet cell membrane is a sensitive target to 125I decays. In order to investigate the mechanisms involved, the level of DNA damage was measured. Although strongly different irradiation doses were delivered, we observed that levels in DNA breaks, together with micronuclei formation was similar for the two final localizations of 125I and for the two cell lines. Expressed as a function of delivered Gy, DNA damge yield was much higher for low dose-rate irradiation (membrane localization). We also observed p53 accumulation and subsequent p21 activation in HCT 116 p53 (+/+) cell line. Cell cycle arrest in G2/M and radio-induced apoptosis were also measured in the latter cell line. These data suggest that low level of radiation-induced DNA damage are properly detected by the cells and that apoptotic death is not involved in the toxicity of 125I at the cell membrane level. However, autophagy might be involved but further studies are required to assess its involvement in the observed biological effectsMONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy.: Signaling pathways induced by 125I-RIT

International audienceINTRODUCTION: Low dose-rate radioimmunotherapy (RIT) using (125)I-labelled monoclonal antibodies ((125)I-mAbs) is associated with unexpected high cytotoxicity per Gy. METHODS: We investigated whether this hypersensitivity was due to lack of detection of DNA damage by the targeted cells. DNA damage was measured with the alkaline comet assay, gamma-H2AX foci and the micronucleus test in p53(-/-) and p53(+/+) HCT116 cells exposed to increasing activities of internalizing anti-HER1 (125)I-mAbs or non-internalizing anti-CEA (125)I-mAbs. The expression of proteins involved in radiation response and progression of cells through the cycle were determined. RESULTS: Cell hypersensitivity to low absorbed doses of anti-CEA (125)I-mAbs was not due to defect in DNA damage detection, since ATM (ataxia telangiectasia mutated gene), gamma-H2AX, p53 and p21 were activated in RIT-treated HCT116 cells and G2/M cell cycle arrest was observed. Moreover, the alkaline comet assay showed that DNA breaks accumulated when cells were placed at 4°C during exposure but were repaired under standard RIT conditions (37°C), suggesting that lesions detected under alkaline conditions (mostly DNA single strand breaks and alkali-labile sites) are efficiently repaired in treated cells. The level of gamma-H2AX protein corroborated by the level of foci measured in nuclei of treated cells was shown to accumulate with time thereby suggesting the continuous presence of DNA double strand breaks. This was accompanied by the formation of micronuclei. CONCLUSION: Hypersensitivity to non-internalizing (125)I-mAbs is not due to lack of detection of DNA damage after low absorbed dose-rates. However, DNA double strand breaks accumulate in cells exposed both to internalizing and non-internalizing (125)I-mAbs and lead to micronuclei formation. These results suggest impairment in DNA double strand breaks repair after low absorbed doses of (125)I-mAbs

Apoptosis and p53 are not involved in the anti-tumor efficacy of (125)I-labeled monoclonal antibodies targeting the cell membrane.: Running title: 125I-RIT-induced cell death mechanisms

International audienceINTRODUCTION: (125)I-labeled monoclonal antibodies ((125)I-mAbs) can efficiently treat small solid tumors. Here, we investigated the role of apoptosis, autophagy and mitotic catastrophe in (125)I-mAb toxicity in p53(-/-) and p53(+/+) cancer cells. METHODS: We exposed p53(-/-) and p53(+/+) HCT116 cells to increasing activities of internalizing (cytoplasmic location) anti-HER1 (125)I-mAbs, or non-internalizing (cell surface location) anti-CEA (125)I-mAbs. For each targeting model we established the relationship between survival and mean nucleus absorbed dose using the MIRD formalism. RESULTS: In both p53(-/-) and p53(+/+) HCT116 cells, anti-CEA (125)I-mAbs were more cytotoxic per Gy than anti-HER1 (125)I-mAbs. Sensitivity to anti-CEA (125)I-mAbs was p53-independent, while sensitivity to anti-HER1 (125)I-mAbs was higher in p53(-/-) HCT 116 cells, suggesting that they act through different signaling pathways. Apoptosis was only induced in p53(+/+) HCT116 cells and could not explain cell membrane radiation sensitivity. Inhibition of autophagy did not modify the cell response to (125)I-mAbs. By contrast, mitotic death was similarly induced in both p53(-/-) and p53(+/+) HCT116 cells by the two types of (125)I-mAbs. We also showed using medium transfer experiments that γ-H2AX foci were produced in bystander cells. CONCLUSION: Cell membrane sensitivity to (125)I-mAbs is not mediated by apoptosis and is p53-independent. Bystander effects-mediated mitotic death could be involved in the efficacy of (125)I-mAbs binding cell surface receptors