Young Normal Thyrocytes Show A Delayed DNA Repair Kinetics After Irradiation, Not Associated With A Transcriptional Deregulation Of DNA Repair Genes

Sheila LIMA, Instituto Nacional de Câncer - INCA, Brazil
PENHA R. 1 , PINTO L. 1 , FUSCO A. 1,2

1 Programa de Carcinogênese Molecular, Instituto Nacional de Câncer - INCA, Rio de Janeiro, Brazil
2 Istituto di Endocrinologia ed Oncologia Sperimentale - CNR c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli “Federico II”, Naples, Italy

Purpose: Ionizing radiation (IR) is the most well-known risk factor for papillary thyroid cancer (PTC), especially during childhood, mainly due to the gradual loss of DNA repair genes and the accumulation of DNA damage. Recent molecular characterization of PTC revealed alterations of DNA methylation in the promoter of several DNA repair genes. Thus, aberrant methylation of DNA might be a plausible mechanism for the inactivation of suppressor genes in radiation-induced thyroid tumors. Herein, we investigated the association among promoter methylation and expression of DNA repair genes and DNA damage in normal thyrocytes after irradiation. Methods: Thyroid cell lines derived from normal 3-week and 3-months old mice, FRTL5 and PCCL3, were exposed to single 1-10 Gy X-rays doses for 1-48 h or five cycles of 5 Gy with 72 h intervals. The following parameters were evaluated: cell viability (MTT), cell cycle (propidium iodide/citometer), DNA double-strand breaks (western blot and immunofluorescence of γH2AX and 53BP-1), promoter methylation (pyrosequencing), gene expression (real-time PCR) and senescence (morphology and β-galactosidase activity). Results: IR promoted: 1) G2/M arrest, more pronounced in young thyrocytes; 2) a rapid γH2AX accumulation (1h) and an earlier DNA damage resolution in adult thyrocytes, with γH2AX and 53BP1 colocalization in DNA damage sites, 3) biphasic promoter methylation profile of DNA repair genes (Atm, Brca1, Rad50, Lig4, Xrcc4, Xrcc6, Xrcc1) in 5-15% of the cells, with no impact on gene expression. This phenomenon was totally abolished after G0/G1 synchronization with TSH deprivation; 4) senescence when cells were chronically exposed to doses ≥ 15 Gy. Conclusions: Young thyrocytes seem to be more sensitive to deleterious effects of IR. Aberrant methylation of DNA repair genes might not be the mechanism involved in the delay of DNA repair kinetics observed in normal young thyrocytes after IR exposure. Funding source: Ministério da Saúde, CAPES, FAPERJ and CNPQ.