NCOA4 depletion leads to replication stress and premature senescence
Bellelli, Roberto (2011) NCOA4 depletion leads to replication stress and premature senescence. [Tesi di dottorato] (Inedito)
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The Nuclear Receptor Coactivator 4 (NCOA4) gene (also known as RFG/ELE1/ARA70) is frequently targeted by chromosomal rearrangements in papillary thyroid carcinoma (PTC). These events join the N-ter (exons 1-5, encoding amino acids 1-238) of the NCOA4 gene to the DNA sequence encoding the tyrosine kinase (TK) domain of the receptor tyrosine kinase RET, generating the RET/PTC3 chimeric gene, whose protein product displays oncogenic activity. The NCOA4 N-ter mediates homodimerization of the RET TK domain, followed by RET kinase activation and, in turn, gain of transforming activity. Disruption of NCOA4 gene might contribute to the neoplastic phenotype as well, however to date no cellular phenotype has been associated with NCOA4 deficiency. To gain insight into the physiological function of NCOA4, we disrupted NCOA4 in mice. NCOA4-deficient mice did not exhibit any apparent phenotypic alteration. However, when grown at 20% O2, NCOA4-/- mouse embryonic fibroblasts (MEFs) undergo premature senescence, characterized by block of cell proliferation and BrdU incorporation, as well as SA(senescence-associated)-β-galactosidase positive staining. This phenotype is associated with accumulation of DNA damage and activation of a DNA damage response (DDR). Using a fiber stretching assay, that monitors DNA replication fork progression, we show that NCOA4-depleted cells accumulate replication fork stalling lesions and feature increased DNA replication origin activation. In addition, NCOA4-/- MEFs, grown at 3% O2 concentration, feature increased sensitivity to genotoxic stress, particularly that induced by S-phase specific DNA damaging agents. In conclusion, these findings highlight a novel role for NCOA4 in the prevention of replication stress, DNA damage, and premature senescence and, thus, in the maintenance of genome stability. Loss of such NCOA4 function may contribute to thyroid cancer formation.
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