Role of oxidative stress in radio-induced DNA damage and in RET/PTC rearrangement in papillary thyroid cancer

Objective: The thyroid gland is one of the most susceptible organs to the carcinogenic effects of ionizing radiation and about 90% of these cancers are papillary, presenting a RET/PTC chromosomal rearrangement in 70% of cases. The objective of this study was to demonstrate the role of the oxidative stress caused by up-regulation of NADPH oxidase DUOX1 in DNA damage and in RET/PTC rearrangement in thyroid cells at post-irradiation. Methods: We analyzed, in vitro, the effect of irradiation in NTHY cells in terms of oxidative and replicative stress. Moreover, we analyzed DUOX1 expression in 28 papillary thyroid cancers (PTC) (RET/PTC positive and negative, irradiated and not irradiated) and we correlated this data with the expression of DUOX1 in 28 normal thyroid tissues (NTT). Results: Preliminary data from human thyroid cells show that chromatin loading and activation from day 3 post-irradiation of the kinase ATR, which is crucial for genome integrity, is impaired respectively, by catalase, a scavenger of H2O2, and diphenileniodonium (DPI), an inhibitor of NADPH oxidases. Analysis of replication speed by DNA combing shows a decrease of the speed from day 3 post-IR, which is also reversed by DPI. Chromatin Immunoprecipitaton-quantitative PCR (ChIP-QPCR) analysis shows that, while GAPDH, CCDN2 genes, in addiction to RET and CCD6 genes, break 30 min after irradiation due to stochastic damage, there is only an enrichment of γH2AX (a marker of double-strand breaks) in genomic regions mapping between RET and CCDC6 gene at day 4 post-irradiation. Immunohistochemistry performed on thyroid tissues showed that 15/28 (54%) PTC tissues had a DUOX1 moderate-strong staining pattern. At variance, only 3/28 (11%) NTT showed a DUOX1 moderate-strong staining pattern. Conclusions: These data suggest that a radio-induced oxidative stress may promote a replicative stress involved in DNA breakage in a region between RET and CCDC6. The impact of DUOX1 as a major source of radioinduced H2O2 on the endogenous replicative stress underlying the formation of RET/PTC1 translocation is under investigation. Moreover, the expression of DUOX1 is greater in PTC tissues than in normal thyroid tissue.