Palmieri, Dario (2009) Critical role of HMGA proteins in regulation of ATM expression and in cellular response to DNA damage. [Tesi di dottorato] (Unpublished)

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MGA proteins are a family of chromatin remodelers involved in many cellular processes including regulation of gene expression, apoptosis and cell proliferation. They have a crucial role in cell transformation and their overexpression is a constant feature of human malignant neoplasias, representing a poor prognostic index. Sequence analysis of HMGA proteins revealed the presence of a consensus site for ATM phosphorylation, which is highly conserved among different species and the different HMGA family members. Since ATM is the main cellular sensor to DNA damage, we decided to investigate the role of HMGA proteins in the ATM pathway and in the cellular response to DNA-damaging agents. In this study we demonstrate that both members of HMGA family, HMGA1 and HMGA2, interact and are phosphorylated by ATM in response to DNA damage. Interestingly, RT-PCR and Western Blot analysis on Mouse Emrbyonic Fibroblasts (MEFs) lacking Hmga1, Hmga2 or both genes, revealed that, in the absence of HMGA proteins, mRNA and protein levels of ATM are strongly reduced. Moreover, p53 phosphorylation and the consequent activation of p21 transcription, induced by ATM kinase activity, were significantly affected by the absence of HMGA proteins. Consistent with these data, ChIP and luciferase assays demonstrated that HMGA proteins bind and positively regulate the promoter of the ATM gene. Moreover, induction of ATM kinase activity increased HMGA-mediated activation of ATM promoter, suggesting that, following DNA damage, ATM is able to trigger an HMGA-dependent positive feedback loop on its own promoter. Accordingly with the reduced expression of ATM, MEFs lacking both Hmga genes showed a significant impairment of the DNAdamage response. In fact, Hmga1/Hmga2 double knock-out MEFs displayed a reduction in the DNA repair activity and in the stress-induced senescence following the induction of DNA double strand breaks (DSBs), compared to wild-type MEFs. It has been also demonstrated that inhibition of ATM determines cell sensitization to DNA damaging therapies. Thus, we investigated the role of HMGA proteins in cellular sensitivity to genotoxic drugs. On this purpose, we treated transformed thyroid cell lines with antisense oligonucleotides to abrogate the expression of HMGA proteins, and evaluated their response to DNA-damaging agents such as ionizing radiation and UV-light. As expected, down regulation of HMGA proteins reduced cellular levels of ATM. Moreover, following the exposure to DNA-damaging agents, absence of HMGA proteins determined alteration of cell-cycle checkpoints, reduced cell proliferation and promoted apoptosis. Taken together our data indicate that HMGA proteins play a crucial role in the cellular response to DNA damage, and may represent a new target to improve tumour sensitivity to conventional anti-cancer therapies.

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