Cancer Res Treat.  2007 Sep;39(3):125-130.

Activation of ATM-dependent DNA Damage Signal Pathway by a Histone Deacetylase Inhibitor, Trichostatin A

• Lee JS

Abstract

PURPOSE: Ataxia-telangiectasia mutated (ATM) kinase regulates diverse cellular DNA damage responses, including genome surveillance, cell growth, and gene expression. While the role of histone acetylation/deacetylation in gene expression is well established, little is known as to whether this modification can activate an ATM-dependent signal pathway, and whether this modification can thereby be implicated in an ATM-mediated DNA damage response.
MATERIALS AND METHODS
Formation of H2AXgamma foci was examined in HeLa and U2OS cells following treatment with a histone deacetylase inhibitor, Trichostatin A (TSA). We determine an ATM-dependency of the TSA-induced DNA damage signal pathway using isogenic A-T (ATM square) and control (ATM+) cells. We monitored the phosphorylation of ATM, an ATM-downstream effector kinase, Chk2, and H2AXgamma to detect the activation of the ATM-dependent DNA damage signal pathway.
RESULTS
Exposure of cells to TSA results in the formation of H2AXgamma foci in HeLa and U2OS cells. The TSA-induced formation of H2AXgamma foci occurs in an ATM-dependent manner. TSA induces phosphorylation of serine 1981 of ATM, accumulation of phosphorylated H2AX and Chk2, and formation of H2AX foci, in a manner analogous to genotoxic DNA damage.
CONCLUSION
In this work, we show that TSA induces a DNA damage signaling pathway in an ATM-dependent manner. These results suggest that ATM can respond to altered histone acetylation induced by the histone deacetylase inhibitor, TSA.

Keyword

ATM; HDAC inhibition; DNA damage signal pathway

MeSH Terms

Acetylation
Ataxia Telangiectasia
DNA Damage*
DNA*
Gene Expression
Genome
Histone Deacetylase Inhibitors*
Histone Deacetylases*
Histones*
Phosphorylation
Phosphotransferases
Serine
Signal Transduction*
DNA
Histone Deacetylase Inhibitors
Histone Deacetylases
Histones
Phosphotransferases
Serine

Figure

• 

  Fig. 1 TSA induces the formation of H2AX foci. (A) Formation of H2AX foci following γ-irradiation. The formation of H2AX foci in HeLa cells was examined at 1 h and 4 h after exposure of 1 Gy or 12 Gy irradiation. Formation of H2AX foci following TSA treatment in HeLa cells (B) and U2OS cells (C). These cells were incubated for 12 h, 24 h, and 36 h with 0.33, 1, or 10 µM TSA. H2AX foci were observed in the TSA-treated cells, as seen in the irradiated cells. Additionally, cells were stained with anti-H2AXγ (green) and DAPI (blue).

• 

  Fig. 2 ATM is required for the formation of H2AX foci following treatment with TSA. ATM+ (A) and ATM- (B) cells were treated with 0.33 and 1 µM TSA for 24 h. Following treatment with 0.33 µM TSA for 24 h, formation of H2AX foci was induced in ATM+ cells (A), but not in ATM- cells (B). These cells were stained with anti- H2AXγ (green) and DAPI (blue).

• 

  Fig. 3 TSA induces the phosphorylation of H2AX. (A) Immunoblotting of γ-H2AX following γ-irradiation. Phosphorylation of H2AX (γ-H2AX) was monitored in 293T cells irradiated at 1 Gy or 12 Gy. Cells were harvested at the indicated time points (1 h, 4 h, 8 h, 12 h, 24 h, and 36 h) following irradiation. Induction of phosphorylation of H2AX was seen in the irradiated cells within 12 h after irradiation. (B) Immunoblotting of γ-H2AX following TSA treatment. The cells were incubated for 1 h, 8 h, 12 h, 24 h, and 36 h with 0.33, 1, or 10 µM TSA. Phosphorylation of H2AX occurred 24 h after TSA treatment. Etoposide (eto) was used as a DNA damage control. (C) ATM is required for the phosphorylation of H2AX foci following treatment with TSA. ATM- and ATM+ cells were treated with 0.33 and 10 µM TSA for 24 h. Phosphorylation of H2AX was detected in 0.33 µM TSA-treated ATM+ cells. β-actin was used as a loading control.

• 

  Fig. 4 TSA induces the phosphorylation of ATM. ATM- and ATM+ cells were treated with 0.33, 1, and 10 µM TSA for 24 h. (A) Confocal analysis of the levels of endogenous phospho-ATM in ATM+ cells. TSA induced the phosphorylation of ATM in ATM+ cells. (B) Western blot analysis of the levels of ATM phosphorylation in 293T cells transfected with ATM-WT or ATM-KD prior to treatment with 1 µM TSA. In the presence of the ATM-WT, phosphorylation of ATM occurred following treatment with TSA or etoposide. β-actin was used as a loading control. (C) Phosphorylation of Chk2 at Thr68 in U2OS cells treated with 1 µM TSA. These cells were stained with anti-phospho-ATM (green), anti-phospho-Chk2 (green), and DAPI (blue).

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