The Ataxia telangiectasia Gene Product Is Required for Oxidative Stress-induced G1 and G2Checkpoint Function in Human Fibroblasts

Abstract

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G 1 and G 2 phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.

Cellular response to oxidative stress: Signaling for suicide and survival*

“…This, however, is not the case, as patients with AT exhibit hypersensitivity to IR and a reduced ability to respond appropriately to oxidative challenge (Lavin and Shiloh, 1997;Gatei et al, 2001). In response to oxidative challenges or DNA damage, AT cells show reduced induction of p53 protein and p53 phosphorylation, and they fail to show G1/S and G2/M cell cycle checkpoints as compared to cells with wild type ATM (Shackelford et al, 2001). It is the loss of these critical p53-dependent checkpoint functions that is believed to render AT cells hypersensitive to IR.…”

DNA Protein Kinase–Dependent G2 Checkpoint Revealed following Knockdown of Ataxia-Telangiectasia Mutated in Human Mammary Epithelial Cells

“…The antibody to phospho-ATM (Ser 1981 ) was from Rockland Immunochemicals. The antiserum to total ATM was generated in our laboratory (21); alternatively, we also used anti-ATM purchased from Bethyl. The antibody to phospho-DNA-PKcs (Thr 2609 ) was from Abcam.…”

Chromium (VI) Activates Ataxia Telangiectasia Mutated (ATM) Protein

“…It has been shown by numerous investigators that ATM-deficient cells are hypersensitive to IR-induced clonogenic lethality. Shackelford et al (15) showed that GM03395 ATMϪ/Ϫ and GM02052 ATM mutant cells exhibit increased sensitivity to IR-induced clonogenic lethality compared with normal human fibroblasts. Furthermore, using neocarzinostatin (generous gift of Dr. Irving Goldberg, Harvard Medical School), a radiomimetic agent, we found that 10 and 25 ng/ml neocarzinostatin caused 82 and 35% clonogenic survival in GM03440 ATMϩ/ϩ cells, but only 40 and 7% in GM03395 ATMϪ/Ϫ cells (data not shown).…”