Human Lymphoblastoid Proteome Analysis Reveals a Role for the Inhibitor of Acetyltransferases Complex in DNA Double-Strand Break Response

Abstract

A DNA double-strand break (DSB) is highly cytotoxic; it emerges as the type of DNA damage that most severely affects the genomic integrity of the cell. It is essential that DNA DSBs are recognized and repaired efficiently, in particular, prior to mitosis, to prevent genomic instability and eventually, the development of cancer. To assess the pathways that are induced on DNA DSBs, 14 human lymphoblastoid cell lines were challenged with bleomycin for 30 and 240 minutes to establish the fast and more prolonged response, respectively. The proteomes of 14 lymphoblastoid cell lines were investigated to account for the variation among individuals. The primary DNA DSB response was expected to occur within the nucleus; therefore, the nuclear extracts were considered. Differential analysis was done using two-dimensional difference in gel electrophoresis; paired ANOVA statistics were used to recognize significant changes in time. Many proteins whose nuclear levels changed statistically significantly showed a fast response, i.e., within 30 minutes after bleomycin challenge. A significant number of these proteins could be assigned to known DNA DSB response processes, such as sensing DSBs (Ku70), DNA repair through effectors (high-mobility group protein 1), or cell cycle arrest at the G 2 -M phase checkpoint (14-3-3 Z). Interestingly, the nuclear levels of all three proteins in the INHAT complex were reduced after 30 minutes of bleomycin challenge, suggesting that this complex may have a role in changing the chromatin structure, allowing the DNA repair enzymes to gain access to the DNA lesions.

Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF‐7 and MCF‐10A

“…The first step for DSB repair is the recognition of the damage by sensor proteins like the ATP-dependent helicase II (Ku70). This enzyme is found in increased levels 30 minutes after DSB induction [ 36 ]. Both of these proteins were found to be induced in samples treated with SEO, after 1 hour of exposure.…”

“…Dirksen et al (2006) studied protein expression in 14 human lymphoblast cell lines after induction of DSBs using bleomycin. 14-3-3 ζ , a protein involved in cell cycle regulation, was found among the proteins that were expressed in the samples [ 36 ]. Upon induction of DNA damage, 14-3-3 ζ binds to Cdc25 and removes it from the nucleus, halting the cell cycle [ 43 ].…”

Differential Proteomics Identifies Protein Biomarkers That Predict Local Relapse of Head and Neck Squamous Cell Carcinomas

“…To our knowledge, our study is the first to use detailed genetic characterizations to ascertain the selection of strictly normal and precancerous tissues. We selected the 2D-DIGE technology as a comparative proteomics approach as it has been proven to be a valuable platform for the large-scale proteome analysis of multiple paired clinical samples (22,23,40), due to the use of a common internal standard and the 4-log dynamic range of protein detection. With only 10 μg of protein per sample, high-quality 2D gel images were acquired and 24 different patient tissues could be compared, which would be unfeasible with any other current quantitative proteomics platform.…”

“…With only 10 μg of protein per sample, high-quality 2D gel images were acquired and 24 different patient tissues could be compared, which would be unfeasible with any other current quantitative proteomics platform. Notwithstanding, the use of 2D gel electrophoresis restricts the proteome analysis to the approximately 1,000 most abundant proteins, unless cellular subfractions are investigated (40).…”

Utility of lymphoblastoid cell lines

“…There have been initiatives to produce a proteome map and database of lymphoblastoid cells by characterizing protein spots on a two‐dimensional electrophoresis (2‐DE) map (Joubert‐Caron et al, 2000; Caron et al, 2002). Proteomes from LCLs have been reportedly used in the elucidation of protein expression profile analysis of cellular response to DNA double‐strand break, an approach now recognized as differential proteome analysis (Dirksen et al, 2006). Again, LCLs are suitable for this role because they provide continually sufficient biomaterials for these analyses, which is not possible from direct specimen sampling of patients.…”