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Genome-wide gene expression analysis of HL60 cells and its alpha particle-resistant derivative RA11, gamma resistant-derivative RG8, and vincristine resistant-derivative RV+ at baseline and following 8Gy gamma IR and ~2Gy alpha IR

ABSTRACT: The purpose of this study was to identify genes that were differentially expressed in radiation-sensitive, naïve HL60 cells, and the derivatives created in our laboratory as indicated in the 'sample title'. We wanted to identify genes that were differentially expressed both before and after ionizing radiation (IR) exposure, from both a single dose of gamma rays and a single dose of alpha particles, at 4h following IR exposure. The data were used to identify genes that could be driving radioresistance in each respective cell line.

ORGANISM(S): Homo sapiens

PROVIDER: GSE35372 | GEO | 2012/01/28

SECONDARY ACCESSION(S): PRJNA152603

REPOSITORIES: GEO

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Project description:Folate deficiency, arsenic exposure, and gamma-IR are known developmental toxicants and have carcinogenic effects. The mechanism by which IR is known, but the effect of arsenic or folate deficiency remains unclear. Their effect may be mediated by epigenetic alterations, leading to miRNA expression changes, and this experiment examined this hypothesis We used microarrays to detail the miRNA expression profiles of TK6 cells treated with 2 uM sodium arsenite for 6 days, folate-deficient media for 6 days, or 2.5 Gy IR exposure either acutely at 4 hours post exposure or long-term at 6 days post exposure, as well as requiste controls, all in biological triplicate. Keywords: exposure differences TK6, were cultured in standard RPMI 1640 (Invitrogen Inc., Gaithersburg, MD) or folate-deficient RPMI 1640 (Invitrogen). Growth media was supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin; dialyzed fetal bovine serum (Invitrogen) was added to the folate-deficient medium in order to eliminate folic acid in the serum. For controls, folate-deficiency, arsenic exposure, and 6-day ?-IR exposure groups, 106 cells were diluted into 50ml of appropriate growth media. For the 4-hour post-?-IR exposure group, 107 cells were diluted into 50ml of growth media. For the arsenic exposed group, sodium arsenite was added to the media to a concentration of 2 ?M. For the ?-IR exposure groups, cells were diluted and allowed to incubate for 4 hours prior to irradiation treatment, and were exposed at a dose rate of 86.76 rad/min to a final dose of 2.5 Gy using a Philips MGC-40 X-ray source. Following exposure, cells were returned to the incubator. After fours hours, the short-term post-??-IR exposure group was collected for RNA isolation, as well as a mock (control) group. All experimental and control conditions were performed in triplicate. For all other groups, cells were cultured for 6 days, with the media changed and renewed, with the appropriate treatment, on day 3.

Project description: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.

Dataset Information

Genome-wide gene expression analysis of HL60 cells and its alpha particle-resistant derivative RA11, gamma resistant-derivative RG8, and vincristine resistant-derivative RV+ at baseline and following 8Gy gamma IR and ~2Gy alpha IR

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