Integrated Bioinformatics for Radiation-Induced Pathway Analysis from Proteomics and Microarray Data

Abstract

Functional analysis and interpretation of large-scale proteomics and gene expression data require effective use of bioinformatics tools and public knowledge resources coupled with expert-guided examination. An integrated bioinformatics approach was used to analyze cellular pathways in response to ionizing radiation. ATM, or ataxia-telangiectasia mutated , a serine-threonine protein kinase, plays critical roles in radiation responses, including cell cycle arrest and DNA repair. We analyzed radiation responsive pathways based on 2D-gel/MS proteomics and microarray gene expression data from fibroblasts expressing wild type or mutant ATM gene. The analysis showed that metabolism was significantly affected by radiation in an ATM dependent manner. In particular, purine metabolic pathways were differentially changed in the two cell lines. The expression of ribonucleoside-diphosphate reductase subunit M2 (RRM2) was increased in ATM-wild type cells at both mRNA and protein levels, but no changes were detected in ATM-mutated cells. Increased expression of p53 was observed 30min after irradiation of the ATM-wild type cells. These results suggest that RRM2 is a downstream target of the ATM-p53 pathway that mediates radiation-induced DNA repair. We demonstrated that the integrated bioinformatics approach facilitated pathway analysis, hypothesis generation and target gene/protein identification.

Targeting ribonucleotide reductase M2 subunit by small interfering RNA exerts anti-oncogenic effects in gastric adenocarcinoma

“…In pancreatic adenocarcinoma, synergism between RRM2 siRNA and gemcitabine resulted in increased tumor apoptosis (28). RRM2 is a downstream target of the ATM-p53 pathway that mediates radiation-induced DNA repair (29) and silencing of RRM2 was found to enhance DNA damage as measured by histone γ-H2AX. Under DNA damage and Chk1 activation mediated by ATM and ATR, upregulation of RRM2 expression level coupled with its nuclear recruitment suggests an active role of RRM2 in the cellular process in response to DNA damage (30).…”

Progesterone and DNA Damage Encourage Uterine Cell Proliferation and Decidualization through Up-regulating Ribonucleotide Reductase 2 Expression during Early Pregnancy in Mice

“…Defective RR often leads to cell cycle arrest, growth retardation, and high mutation rate (8). The high level of RRM2 protein and RR activity in human nasopharyngeal cancer cells results in ionizing radiation resistance through enhancing damage repair during G 2 phase of the cell cycle (1). CHK1 is widely known as a DNA damage checkpoint signaling protein (9).…”

“…2 is the only enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides and a rate-limiting enzyme for the production of 2Ј-deoxyribonucleoside 5Ј-diphosphates (dNTP) required for DNA synthesis and repair (1)(2)(3). RR consists of RR1 (encoded by the Rrm1 gene) and RR2 (encoded by the Rrm2 and Rrm2b genes) subunits, and both proteins are required for enzymatic activity.…”

Omics-Based Molecular Target and Biomarker Identification

“…Direct comparison of expression at both mRNA and protein levels can provide stronger evidences for the underlying changes. For example, the 2D-gel/MS proteomics study identified 412 and 771 proteins that potentially changed in response to radiation treatment in ATM (Ataxia Telangiectasia Mutated) mutated (ATM − ) and wild type (ATM + ) cells, respectively, while the gene microarray study identified 103 and 131 significantly changed genes in the two cells, respectively (54). Among those genes/proteins, only 13 were commonly identified, including RRM2, the catalytic subunit of ribonucleoside-diphosphate reductase (RR), a rate-limiting enzyme required for synthesis of dNDP and thus of DNA synthesis in human (55).…”

“…Pathway profiling also led to the identification of “purine metabolism pathway” that showed notable differences between radiation-treated vs. untreated ATM − and ATM + cells (Fig. 5a) (54). …”