Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM
Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. in maintaining cell survival in the absence of ATM function. INTRODUCTION Ataxia telangiectasia (A-T) is usually a rare multisystemic autosomal recessive disorder. The clinical features of the syndrome include progressive neurological impairment, predisposition to malignancy and hypersensitivity to ionising WAY-100635 maleate salt supplier radiation (1). A-T is usually generally linked with WAY-100635 maleate salt supplier mutations in the A-T mutated WAY-100635 maleate salt supplier (ATM) gene, Rabbit polyclonal to MAP1LC3A which ultimately lead to the synthesis of a dysfunctional ATM protein (2,3). ATM is usually a large serine/threonine kinase belonging to the PI3K-like protein kinase family (4). The protein has been extensively linked with the DNA damage response to DNA strand breaks (5,6) and to reactive oxygen species (ROS) (7). In fact, the presence of common oxidative stress constitutes a major feature in A-T and elevated ROS levels have been detected in ATM knock-out mice (8), as well as in lymphocytes from A-T patients (9). ROS are potentially harmful to a number of cellular macromolecules, including DNA and proteins. Oxidative DNA damage is usually generally dealt with by the DNA base excision repair pathway (BER), which is usually responsible for the clearance of base lesions and DNA single-strand breaks (SSBs) (10). Importantly, endogenous DNA lesions arise spontaneously at an incredible rate, mainly as a result of cellular oxidative metabolism (11), therefore detection and repair of these lesions is usually completely essential to maintain genomic stability. Recent evidence strongly suggests that ATM is usually a vital sensor for endogenous DNA strand breaks, as its activation has been shown to enforce a cell-cycle delay necessary for DNA repair to occur prior to DNA replication (6,12). Accordingly, impairment of ATM functions affects the G1/S checkpoint transition producing in unrestricted replication of damaged DNA and WAY-100635 maleate salt supplier genomic instability (6,12). While the role of ATM in the context of DNA damage has been thoroughly characterised, much less investigated is usually the cellular response to ROS-induced protein damage in ATM-deficient cells. Despite the accumulation of ROS and genomic instability, it is usually obvious that a lack of functional ATM is usually compatible with cell survival, suggesting that adaptation mechanisms must be in place to prevent cell death in the presence of prolonged oxidative stress. Nonetheless, the cellular adjustments that promote survival of ATM-deficient cells have been poorly investigated to date. In this study, we exploit a stable isotope labelling with amino acids in cell culture (SILAC)-based proteomics approach to gain insight into the early adaptation of human fibroblasts to the lack of ATM. Our data confirm that loss of ATM prospects to progressive accumulation of ROS and mitochondrial damage, which start WAY-100635 maleate salt supplier very early on upon depletion of ATM. Furthermore, we show that a serious rearrangement of cellular proteostasis takes place in response to ATM depletion and that this is usually necessary for cells to counter-top protein damage originating from prolonged oxidative stress. Surprisingly, while modulation of proteostasis promotes survival of ATM-depleted cells, this has a considerably unfavorable impact on the BER pathway, whose capacity shows indicators of strong impairment. As a result, spontaneously generated DNA damage cannot be completely repaired in ATM-depleted fibroblasts, leading to accumulation of genomic instability. Our study provides insight into cellular adaptation to the loss of ATM, reinforcing the notion that oxidative stress and impaired DNA repair capacity play a major role in the pathology. Moreover, our data spotlight a previously overlooked role for proteostasis in maintaining cellular viability in the absence of functional ATM. MATERIALS AND METHODS Cell culture, chemicals and siRNA transfections TIG1 and GM03349 normal human fibroblasts, as well as AG03058 A-T fibroblasts were obtained from the Coriell Institute Cell Repository. Cells were produced in DMEM (Life Technologies) supplemented with 15% FBS at 37C in a humidified atmosphere with 5% CO2. H2O2 and MMS were from Sigma, MG132 was from Enzo Life Sciences, bortezomib was from Cayman.