Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses

Abstract

Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.

NBS1 is regulated by two kind of mechanisms: ATM-dependent complex formation with MRE11 and RAD50, and cell cycle–dependent degradation of protein

“…HeLa, U2OS, hTERT-immortalized human fibroblast (48BR), SV40-trasformed normal fibroblast (MRC5SV), SV40-transformed AT patient–derived fibroblast (AT5BIVA), SV40-transformed AT-LD patient–derived fibroblast (ATLD2) and SV40-transformed NBS patient–derived fibroblast (GM7166SV) cells [ 12 , 13 ] were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics. NBS cells, expressing wild-type (WT) or mutated NBS1 (serine substituted with alanine at a.a. 278 and 343) were prepared as reported previously [ 14 ].…”

Oxidative stress, mitochondrial abnormalities and antioxidant defense in Ataxia-telangiectasia, Bloom syndrome and Nijmegen breakage syndrome

“…p53, Chk2, Brca1, and Nbs1 [4], [10], [15], [19], [20], [22]. A fraction of ATM is also localised in mitochondria [12] and ATM plays a crucial role in the anti-oxidative response to enhanced ROS levels and accumulated oxidative DNA damage [23], [24], [25], [26]. ATM can therefore be regarded as an important part of the cellular redox regulatory system [21], [22], [27].…”

“…The authors believe that mitochondrial DNA (mtDNA) damage may promote their repair and lead to reduced mROS production and mitochondrial dysfunctions [44]. In contrast, Kobayashi et al [26] reported that elevated oxidative stress levels, expressed as excessive concentrations of endogenous ROS from mitochondrial defects, reduced ATM activation and impaired homologous recombination (HR) repair in AOA3 (ataxia with oculomotor apraxia type 3) lymphoblastoid cells. Resseguie et al [45] showed that prolonged exposure to hyperoxia in A549 cells activated ATM kinase, irrespective of mitochondrial alternations and the accumulation of mROS.…”

Polynucleotide kinase-phosphatase (PNKP) mutations and neurologic disease

“…AOA2 has an older age onset (mean 14.6 years) and affected individuals rarely manifest dystonia and have normal albumin and cholesterol values, but consistently have increased alpha-fetoprotein (Anheim et al, 2012; Schieving et al, 2014). In contrast, AOA3 is reported to result from mutations in the phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) gene, and cell lines from these patients have been linked to defective DNA damage signaling (Al Tassan et al, 2012; Gueven et al, 2007; Kobayashi et al, 2015). Like AOA2, AOA3 also occurs in the second decade of life and is characterized by normal serum cholesterol and albumin, but elevated alpha-fetoprotein (Al Tassan et al, 2012; Anheim et al, 2012).…”