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The ataxia-telangiectasia mutated (ATM) protein kinase is best known for its role in the DNA damage response, but recent findings suggest that it also functions as a redox sensor that controls the levels of reactive oxygen species in human cells. Here, we review the evidence supporting the conclusion that ATM can be directly activated by oxidation, as well as various observations from ATM-deficient patients and mouse models that point toward the importance of ATM in oxidative stress responses. We also discuss the roles of this kinase in regulating mitochondrial function and metabolic control through its action on tumor suppressor p53, AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 (HIF-1), and how the regulation of these enzymes may be affected in ATM-deficient patients and in cancer cells.

Splicing and nuclear export are vital components of eukaryotic gene expression. Defects in splicing due to cis mutations are known to cause a number of human diseases. Here we present a dual reporter system that can be used to look at splicing or export deficiencies resulting from an insufficiency in components of the cotranscriptional machinery. The constructs use a bidirectional promoter to coexpress a test reporter and a control reporter. In the splicing construct, maximal expression of the test reporter is dependent on efficient splicing and splicing-related nuclear export, whereas the control reporter is an intronless complementary DNA expression cassette. The dual reporters allow a robust ratiometric output that is independent of cell number or transfection efficiency. Therefore, our construct is internally controlled and amenable to high-throughput analysis. As a counterscreen, we have a nonsplicing control construct in which neither reporter bears an intron. We demonstrate the sensitivity of our construct to defects in nuclear export by depleting UAP56 and NXF1, essential components of the cotranscriptional machinery.

Ferritin plays an important role in the storage and release of iron, an element utilized in cellular processes such as respiration, gene regulation, and DNA replication and repair. Ferritin in animals is composed of 24 ferritin L (FTL) and ferritin H (FTH) subunits in ratios that vary in different cell types. Because the subunits are not functionally interchangeable, both L and H units are critical for maintaining iron homeostasis and protecting against iron overload. FTL and FTH are regulated primarily at a post-transcriptional level in response to cellular iron concentrations. Individual regulation of FTL and FTH is of much interest, and although transcriptional differences between FTL and FTH have been shown, differences in their post-transcriptional regulation have not been evaluated. We report here that FTL and FTH are differentially regulated in 1% oxygen on a post-transcriptional level. We have designed a quantitative assay system sensitive enough to detect differences between FTL and FTH iron regulatory elements (IREs) that a standard electrophoretic mobility shift assay does not. The FTL IRE is the primary responder in the presence of an iron donor in hypoxic conditions, and this response is reflected in endogenous FTL protein levels. These results provide evidence that FTL and FTH subunits respond independently to cellular iron concentrations and underscore the importance of evaluating FTL and FTH IREs separately.

The Ataxia-Telangiectasia Mutated (ATM) kinase initiates a rapid cascade of protein phosphorylation in response to double-strand breaks in chromosomal DNA. The critical targets of ATM during this response include many tumor suppressors involved in cell cycle progression and apoptosis. The DNA damage response is thus defective in cells lacking ATM; however, there are also many indications that cells lacking ATM are defective in responses to other forms of stress, particularly oxidative stress. Mammalian cells lacking ATM exhibit high levels of reactive oxygen species and indications of chronic oxidative stress. In addition, the shortened lifespan, haematopoietic stem cell loss, and increased lymphomagenesis seen in A-T mouse models can be suppressed by antioxidants, suggesting that oxidative stress constitutes an important part of the A-T phenotype and is important for cancer progression. We have observed robust ATM activation in human cell lines in response to oxidative stress, in the absence of apparent DNA damage. In addition, oxidation of purified recombinant ATM in vitro induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide-crosslinked dimer containing several disulfide bonds. Mutation of a critical cysteine residue involved in disulfide bond formation specifically blocks activation via the oxidation pathway, both in vitro with purified components and in human cells. Identification of this pathway explains many observations of ATM activation under conditions of oxidative stress, and shows that ATM is an important sensor of reactive oxygen species (ROS) in vivo. Here we investigate how the oxidation pathway of ATM activation is affected by small molecules, specifically by resveratrol and related compounds, and how this affects cell cycle checkpoint responses in human cells. We also show in vitro that ATM is a direct target of these compounds and investigate the mechanism underlying the effects on ATM function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY11-03. doi:1538-7445.AM2012-SY11-03

While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future.

Ferritin plays an important role in the storage and release of iron, an element utilized in cellular processes such as respiration, gene regulation, and DNA replication and repair. Ferritin in animals is composed of 24 ferritin L (FTL) and ferritin H (FTH) subunits in ratios that vary in different cell types. Because the subunits are not functionally interchangeable, both L and H units are critical for maintaining iron homeostasis and protecting against iron overload. FTL and FTH are regulated primarily at a post-transcriptional level in response to cellular iron concentrations. Individual regulation of FTL and FTH is of much interest, and although transcriptional differences between FTL and FTH have been shown, differences in their post-transcriptional regulation have not been evaluated. We report here that FTL and FTH are differentially regulated in 1% oxygen on a post-transcriptional level. We have designed a quantitative assay system sensitive enough to detect differences between FTL and FTH iron regulatory elements (IREs) that a standard electrophoretic mobility shift assay does not. The FTL IRE is the primary responder in the presence of an iron donor in hypoxic conditions, and this response is reflected in endogenous FTL protein levels. These results provide evidence that FTL and FTH subunits respond independently to cellular iron concentrations and underscore the importance of evaluating FTL and FTH IREs separately.

Background: Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in the signaling response to DNA double strand breaks (DSBs) by phosphorylating multiple targets involved in DNA repair and cell cycle control. In response to DSBs, ATM is activated through interactions with the Mre11-Rad50-Nbs1 (MRN) complex. Recent work from our group identified a second mechanism of ATM activation through direct oxidation that is independent of both MRN and DNA DSBs. ATM signaling through the p53 tumor suppressor in response to elevated ROS is known to prevent aneuploidy driven tumorigenesis and ATM deficiency is linked to oxidative stress. While ATM is best known for its role within the nuclear DNA damage response, recent studies implicate ATM in cytoplasmic signaling pathways. ATM activation through direct oxidation may function as a primary tumor suppressor mechanism and may serve to regulate the intracellular redox balance, yet the biological role of oxidized ATM remains to be elucidated. Methods: To investigate the role of ATM kinase activity in redox regulation, the levels of reactive oxygen species (ROS) were analyzed and compared in normal fibroblasts and various cancer cell lines with different genetic backgrounds following incubation with a specific inhibitor of the ATM kinase and mock treatment. To analyze the role of ATM activation through direct oxidation in redox regulation, ROS levels were measured and compared in human cell lines overexpressing wild-type ATM and a mutant form of ATM (C2991L) that is unresponsive to activation through direct oxidation. Cellular fractionation experiments were used to analyze the intracellular distribution of ATM following treatment with an oxidizing agent. Findings: Inhibition of ATM kinase activity for 24 hours resulted in increased ROS levels in normal human fibroblasts, WT and p53 -/- HCT116 colon cancer cell lines and hela cells, which are deficient in LKB1. Stable expression of the C2991L mutant form of ATM in 293 cells resulted in elevated levels of ROS when compared to 293 cells overexpressing wild-type ATM. Induced expression of the C2991L ATM mutant in cells lacking native ATM resulted in elevated ROS levels 24 hours post-induction when compared to the induced expression of wild-type ATM. Using cellular fractionation studies, we show that ATM levels in the cytoplasmic fraction increased at 30 minutes post-treatment with hydrogen peroxide and remained elevated up to 2 hours post-treatment without an accompanying increase in γ-H2AX, a widely utilized marker of DSBs. Conclusions: These findings indicate that oxidized ATM functions in regulating intracellular ROS levels and that this function is independent of ATM signaling through the p53 and LKB1 signaling pathways. These findings further show that ATM signaling in response to oxidative stress may not be restricted to known nuclear signaling pathways activated by DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2063. doi:1538-7445.AM2012-2063