Unknown,Transcriptomics,Genomics,Proteomics

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ATM-dependent Transcriptional Responses to 1.5 Gy Gamma Radiation in Human Lymphocytes

ABSTRACT: Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration, telangiectasias, acute cancer predisposition, and hypersensitivity to ionizing radiation (IR). The gene defective in AT, ATM (for AT-mutated), encodes a protein, pATM that has been found to have IR-inducible kinase activity. Cells from individuals with AT exhibit severely attenuated cell cycle checkpoints in response to gamma radiation exposure. pATM has been hypothesized to act as part of a complex that senses DNA damage, in particular, DNA double strand breaks. We are studying the pATM-dependent gene expression responses to a dose of 1.5 Gy radiation in lymphoblastoid cell lines from multiple individuals with either wild type or mutated ATM. The gene expression analyses were performed on Agilent Human 1A Oligo chips containing approximately 16,000 60mer probes. We identified a set of genes whose gene expression changes are ATM-dependent following exposure to 1.5 Gy IR. This set of genes was tested by real time quantitative PCR analysis. The study consists of one dose group - 1.5 Gray gamma radiation. Samples were collected 6 hr following irradiation (treated) or mock irradiation (controls) for each culture. Each culture has a treated sample and a control sample. There are cultures from 6 wild-type ATM individuals and cultures from 6 individuals that are ATM-deficient, for a total of 12 cultures. Hybridizations compared RNA extracted from each individual culture's treated sample against its corresponding control sample, and were performed as dye-flip duplicates (1 replicate in each direction). Hybridizations were performed on the Human 1A Oligo Chips using low input, direct labeling at Paradigm Genetics Laboratory. Microarray scanning was performed with the Agilent G2565AA Scanner. Paradigm Standard Operating Procedures were followed for quality analysis, sample labeling, microarray hybridization and washing, scanning, image analysis and initial data analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: NIEHS Microarray Core

PROVIDER: E-GEOD-19287 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Radiotherapy has a critical role in the treatment of small cell lung cancer (SCLC). Effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using a Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 induce apoptosis in SCLC cells by inducing hallmarks of apoptosis (cleaved caspase-3, sub-G1 fraction induction and induction of the mitochondrial caspase activation pathway). S44563 markedly enhanced sensitivity of H146 cells and H69 cells to radiation in clonogenic assay. In addition, the combination S44563 and cisplatin-based chemo-radiation showed a dramatic tumor growth delay and increased overall survival in mouse xenograft models. In vitro experiments demonstrated a greater induction of apoptosis (sub-G1 fraction and cleaved caspase-3) with the combination as well as in vivo caspase-3 immunostaining. This positive interaction between S44563 and radiation was greater when S44563 was given after the completion of the radiation, which may be explained by the radiation-induced over expression of anti-apoptotic proteins (Bcl-2 and Bcl-XL) through the NF-?B pathway activation. Taken together, these data underline the critical role of sequence administration of targeted therapies with conventional therapies. SCLC cells which survive to IR highly rely on the induction of anti apoptotic proteins in part through NF- ?B activation for their survival yielding to the concept of 'contextual oncogene addiction' to the Bcl-2/Bcl-XL pathway providing rational for targeting survival pathways to potentiate IR. For transcriptome analysis, 106 cells were seeded in T25 flasks, allowed to growth for 24 h, and then left untreated or treated with 2 Gy radiation. After 24 hours, cells were harvested, lysed for the extraction of RNA, and processed to analyze gene expression.The design of this study consists to 6 hybridizations in dual color with Agilent Human Genome 4x44K (design 014850). The reference is always the untreated condition.

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ATM-dependent Transcriptional Responses to 1.5 Gy Gamma Radiation in Human Lymphocytes

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