Analysis of ataxia-telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)-regulated gene expression patterns

Abstract

Purpose: Ataxia-telangiectasia (A-T) is a progressive, degenerative, complex autosomal recessive disease characterized by cerebellar degeneration, immunodeficiency, premature aging, radiosensitivity, and a predisposition to cancer. Mutations in the ataxia-telangiectasia mutated (atm) gene, which phosphorylates downstream effector proteins, are linked to A-T. One of the proteins phosphorylated by the ATM protein is Nijmegen Breakage Syndrome protein (NBS, p95/nibrin), which was recently shown to be encoded by a gene mutated in the Nijmegen breakage syndrome (nbs), an autosomal recessive disease with a phenotype virtually similar to that of A-T. The similarities in the clinical and cellular features of NBS and A-T have led us to hypothesize that the two corresponding gene products may function in similar ways in the cellular signaling pathway. Thus, we sought to identify genes whose expression is mediated by the atm and nbs gene products.

Material and methods: To identify genes, we performed an analysis of oligonucleotide microarrays using the appropriate cell lines, isogenic A-T (ATM-) and control cells (ATM+), and isogenic NBS (NBS-) and control cells (NBS+).

Results: We examined genes regulated by ATM and NBS, respectively. To determine the effect of ATM and NBS on gene expression in detail, we classified these genes into different functional categories, including those involved in apoptosis, cell cycle/DNA replication, growth/differentiation, signal transduction, cell-cell adhesion, and metabolism. In addition, we compared the genes regulated by the ATM and NBS to determine the relationship of their signaling pathways and to better understand their functional relationship.

Conclusions: We found that, while ATM and NBS regulate several genes in common, both of these proteins also have distinct patterns of gene regulation, findings consistent with the functional overlap and distinctiveness of these two conditions. Due to the role of ATM and NBS in tumor suppression and the response to chemotherapy and radiotherapy, these findings may assist in the development of a more rational approach to cancer treatment, as well as a better understanding of tumorigenesis.

Fig. 1a,b

Expression profiles of 22232 genes from the oligonucleotide microarrays. a Signal intensities from ATM⁺ cells (y axis) relative to those from ATM^- cells (x axis) were analyzed using Microarray Suite 5.0 software. Log-transformed data are shown. Guidelines show two- and threefold signal ratios; b Signal intensities from NBS⁺ cells (y axis) relative to those from NBS^- cells (x axis); cells were analyzed as in (a)

Fig. 2a,b

Expression of selected genes based on the microarray results. a RT-PCR amplification of the ATM and AKR1C3 mRNAs was shown as a positive control or representative ATM-regulated gene, respectively. The gene-specific primers are shown in Materials and Methods. GAPDH mRNA was amplified as an internal control; b Expression of ESDN and FOLR3 genes were evaluated by RT-PCR, as representative NBS-regulated genes

Fig. 3a–d

Differential functional groups of ATM- or NBS-regulated genes. a Expression profiles of genes up-regulated > twofold in the presence of ATM. These genes were in the functional categories of those related to apoptosis, cell cycle/DNA replication, growth/differentiation, signal transduction and cell-cell adhesion (see Table 1and Table 2); b Expression profiles of genes down-regulated > twofold in the presence of ATM. These genes were in the functional categories of those related to apoptosis, cell cycle/DNA replication, growth/differentiation, signal transduction, metabolism, and cell-cell adhesion (see Table 1 and Table 2); c Expression profiles of genes upregulated by NBS. These genes were in the functional categories of those related to apoptosis, cell cycle/DNA replication, growth/differentiation, signal transduction and cell-cell adhesion (see Table 3 and Table 4); d Expression profiles of genes downregulated by NBS. These genes were in the functional categories of those related to cell cycle/DNA replication, growth/differentiation, signal transduction, cell-cell adhesion, metabolism, and chromatin remodeling (see Table 3 and Table 4)

Fig. 4a,b

Comparison of ATM and NBS-regulated genes. Venn diagrams were created of genes regulated by ATM and/or NBS. a Genes upregulated in ATM⁺ and NBS⁺ cells; b Genes downregulated in ATM⁺ and NBS⁺ cells

Fig. 5a,b

Cluster analysis of representative ATM- and NBS-regulated genes. a The NBS-regulated genes clustered on the basis of their expression in ATM⁺ and ATM^- cells; b ATM-regulated genes clustered on the basis of their expression in NBS⁺ and NBS^- cells