Cancer Res Treat.  2007 Sep;39(3):116-124.

Functional Link between DNA Damage Responses and Transcriptional Regulation by ATM in Response to a Histone Deacetylase Inhibitor TSA

• Lee JS

Abstract

PURPOSE: Mutations in the ATM (ataxia-telangiectasia mutated) gene, which encodes a 370 kd protein with a kinase catalytic domain, predisposes people to cancers, and these mutations are also linked to ataxia-telangiectasia (A-T). The histone acetylaion/deacetylation- dependent chromatin remodeling can activate the ATM kinase-mediated DNA damage signal pathway (in an accompanying work, Lee, 2007). This has led us to study whether this modification can impinge on the ATM-mediated DNA damage response via transcriptional modulation in order to understand the function of ATM in the regulation of gene transcription.
MATERIALS AND METHODS
To identify the genes whose expression is regulated by ATM in response to histone deaceylase (HDAC) inhibition, we performed an analysis of oligonucleotide microarrays with using the appropriate cell lines, isogenic A-T (ATM square) and control (ATM+) cells, following treatment with a HDAC inhibitor TSA.
RESULTS
Treatment with TSA reprograms the differential gene expression profile in response to HDAC inhibition in ATM square cells and ATM+ cells. We analyzed the genes that are regulated by TSA in the ATM-dependent manner, and we classified these genes into different functional categories, including those involved in cell cycle/DNA replication, DNA repair, apoptosis, growth/differentiation, cell- cell adhesion, signal transduction, metabolism and transcription.
CONCLUSION
We found that while some genes are regulated by TSA without regard to ATM, the patterns of gene regulation are differentially regulated in an ATM-dependent manner. Taken together, these finding indicate that ATM can regulate the transcription of genes that play critical roles in the molecular response to DNA damage, and this response is modulated through an altered HDAC inhibition-mediated gene expression.

Keyword

ATM; HDAC inhibition; Transcriptional modulation

MeSH Terms

Apoptosis
Ataxia Telangiectasia
Catalytic Domain
Cell Adhesion
Cell Line
Chromatin Assembly and Disassembly
DNA Damage*
DNA Replication
DNA*
Gene Expression
Histone Deacetylase Inhibitors*
Histone Deacetylases*
Histones*
Metabolism
Oligonucleotide Array Sequence Analysis
Phosphotransferases
Signal Transduction
Transcriptome
DNA
Histone Deacetylase Inhibitors
Histone Deacetylases
Histones
Phosphotransferases

Figure

• 

  Fig. 1 Comparison of the gene expression profiles between the ATM- and ATM+ cells in response to TSA, as was derived from the oligonucleotide microarray analysis. (A) The signal intensities from the TSA-treated ATM+ cells (y-axis) relative to those from the untreated ATM+ cells (x-axis) were analyzed using MicroArray Suite 5.0 software. The log-transformed data is shown; the guideline shows the 2-fold signal ratio. (B) The signal intensities in the ATM- cells. The ATM-dependent and ATM-independent TSA-responsive genes were distinguished from the Venn diagrams (C), and the genes that were significantly enhanced or reduced in the ATM+ cells, as compared to that in the ATM- cells, in response to TSA were visualized by Treeview software (D). The expressions of more genes are regulated in the ATM+ cells, as compared to the ATM- cells, in response to HDAC inhibition.

• 

  Fig. 2 The oligonucleotide microarray results were validated by RT-PCR analysis of the expression of selected genes in the ATM- cells, the ATM+ cells and the HCT116 cells. (A) The expressions of the TSA responsive genes were evaluated by RT-PCR as being representative of the ATM-regulated genes. The TSA-induced increase in the level of CDKN1C mRNA was reduced in the absence of ATM. The TSA-induced reduction of the expressions of ERCC3, BAX, CND1 and ERBB2 mRNAs was observed in the ATM+ cells, but not the ATM- cells. (B) The effect of wortmannin on the expression of the ATM-regulated genes in response to TSA. Inhibition of ATM kinase activity by wortmannin attenuated the TSA-induced transcriptional effects on upregulation of the CDKN1C and CCND2 genes. (C) The ATM-expressing constructs were transiently transfected into HCT116 cells, and the expression levels of ATM-WT or ATM-KD were checked by RT-PCR with using the primer sets ATM-p1 (exon 7~10) and ATM-p2 (exon 10~13). (D) Monitoring of the ATM pathway activation after etoposide treatment by measuring the Chk2 phosphorylation in the HCT116 cells expressing ATM-WT or ATM-KD. In response to etoposide, ATM-WT phosphorylated Chk2 and this resulted in the shifted mobility of the phosphorylated Chk2 proteins, compared with that of nonphosphorylated Chk2. (E) Evaluation of the mRNA expression of the genes regulated by TSA in the HCT116 cells expressing ATM-WT or ATM-KD. The expressions of BAX and CCND1 were reduced in response to TSA in the cells expressing ATM-WT, and that of CDKN1C increased in the cells expressing ATM-WT. TSA-induced downregulation of BAX and CCND1 and upregulation of CDKN1C was rarely detected in the cells expressing ATM-KD. GAPDH mRNA was amplified as an internal control.

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  Fig. 3 Effects of TSA on the expression of MCL1 mRNA. Time course of the effect of TSA on the expression of MCL1 mRNA in the 293T cells. The TSA induced-upregulation of MCL1 mRNA fluctuated during the 1~24 h time course. GAPDH mRNA was amplified as an internal control.

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