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Dose- and Time-Dependent Transcriptional Response of Ishikawa Cells Exposed to Genistein.

ABSTRACT: To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10?pM, 1?nM, 100?nM, and 10??M) and time points (8, 24, and 48?h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P???0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest dose of GES evaluated (10??M). The GES' estrogenic activity was identified by comparing the Ishikawa cells' response to GES versus 17 ?-ethynyl estradiol (EE, at equipotent doses, ie, 10??M vs 1??M, respectively) and was defined by changes in the expression of 284 unique genes elicited by GES and EE in the same direction, although the magnitude of the change for some genes was different. Further, comparing the response of the Ishikawa cells exposed to high doses of GES and EE versus the response of the juvenile rat uterus exposed to EE, we identified 66 unique genes which were up- or down regulated in a similar manner in vivo as well as in vitro Genistein elicits changes in multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response and offer an in vitro model to assess this mode of action.

SUBMITTER: Naciff JM

PROVIDER: S-EPMC4914796 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Dose- and Time-Dependent Transcriptional Response of Ishikawa Cells Exposed to Genistein.

Naciff Jorge M JM Khambatta Zubin S ZS Carr Gregory J GJ Tiesman Jay P JP Singleton David W DW Khan Sohaib A SA Daston George P GP

Toxicological sciences : an official journal of the Society of Toxicology 20160210 1

To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10 pM, 1 nM, 100 nM, and 10 μM) and time points (8, 24, and 48 h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P ≤ 0.0001 ...[more]

PMID: 26865667

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Project description:Gene expression in endometrial cancer cells (Ishikawa) after short time exposure to progestins Sample and RNA preparation: The series is composed of sixteen hybridizations for analysis of differentially expressed genes in endometrial cancer cells (Ishikawa). In eight independent experiments, Ishikawa cells in monolayer received with 30 ug/ml progestin and were grown in RPMI-1640 medium (Sigma, Sty. Louis) with 10% dialyzed foetal calf serum (PAA-laboratories, Austria) without phenol red and antibiotics. Cells were seeded at a density of 1 x 105 cells/ml in 25 cm2 culture flasks in a humidified atmosphere (5% CO2) at 37°C for 4 hours. Cells not treated with progestin served as control groups. Total RNA extraction was performed using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: Total RNA was reverse transcribed and labeled with Cy3- and Cy5- attached dendrimer, respectively, using the Genisphere 3DNA 350HS kit (Genisphere, Montvale, NJ) as described in the manufacturer's protocol. Hybridizations of transcribed probes were carried out in a TECAN HS4800 instrument using the formamide-based hybridization buffer from Genisphere containing 5% dextrane sulfate and 5.5 ng/ul COT1 DNA (GIBCO BRL Life technologies) at 37º for 23 hours. 3DNA dendrimer hybridizations were carried out in formamide-based hybridization buffer alone. Post-hybridization washes were carried out at room temperature with 2xSSC for 1 min, 0.2% SDS /2xSSC for 1 min and finally with 0.2xSSC for 30 sec. Sixteen hybridizations including dye-swap have been performed for eight independent experiments. Scanning, image analysis and data processing: The arrays were scanned with the GenePix 4000B scanner (Axon Instruments Inc). The features were extracted from the arrays using GenePix Pro 6.0 (Axon instruments Inc., Union City, CA). All subsequent statistical analyses were performed using the statistical package R (R Development Core Team). Spots that displayed a signal-to-noise ratio of less than 2, or that were significantly saturated (more than 20 % saturation among foreground pixels) were filtered out. The median was used as the averaging measure of the foreground pixels. After quality control, genes that were present in less than 50 % of the arrays were filtered out. The arrays were normalized using dye-swap normalization. One array, whose dye swap twin failed was normalized using the lowess method. Statistical significance was assigned to the genes using the SAM methodology. The bioconductor SAMR package was used in the actual analysis. Missing expression levels were imputed using 10 nearest neighbour's imputation. The local FDR threshold was set to 25 %. Keywords: Progestin treatment, Control-treatment

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Dose- and Time-Dependent Transcriptional Response of Ishikawa Cells Exposed to Genistein.

Publications

Dose- and Time-Dependent Transcriptional Response of Ishikawa Cells Exposed to Genistein.

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