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Gene expression changes in human lung cells exposed to arsenic, chromium, nickel or vanadium indicate the first steps in cancer.

ABSTRACT: The complex process of carcinogenesis begins with transformation of a single cell to favor aberrant traits such as loss of contact inhibition and unregulated proliferation - features found in every cancer. Despite cancer's widespread prevalence, the early events that initiate cancer remain elusive, and without knowledge of these events cancer prevention is difficult. Here we show that exposure to As, Cr, Ni, or vanadium (V) promotes changes in gene expression that occur in conjunction with aberrant growth. We exposed immortalized human bronchial epithelial cells to one of four metals/metalloid for four to eight weeks and selected transformed clonal populations based upon anchorage independent growth of single cells in soft agar. We detected a metal-specific footprint of cancer-related gene expression that was consistent across multiple transformed clones. These gene expression changes persisted in the absence of the progenitor metal for numerous cell divisions. Our results show that even a brief exposure to a carcinogenic metal may cause many changes in gene expression in the exposed cells, and that from these many changes, the specific change(s) that each metal causes that initiate cancer likely arise.

SUBMITTER: Clancy HA

PROVIDER: S-EPMC3563094 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Gene expression changes in human lung cells exposed to arsenic, chromium, nickel or vanadium indicate the first steps in cancer.

Clancy Hailey A HA Sun Hong H Passantino Lisa L Kluz Thomas T Muñoz Alexandra A Zavadil Jiri J Costa Max M

Metallomics : integrated biometal science 20120619 8

The complex process of carcinogenesis begins with transformation of a single cell to favor aberrant traits such as loss of contact inhibition and unregulated proliferation - features found in every cancer. Despite cancer's widespread prevalence, the early events that initiate cancer remain elusive, and without knowledge of these events cancer prevention is difficult. Here we show that exposure to As, Cr, Ni, or vanadium (V) promotes changes in gene expression that occur in conjunction with aberr ...[more]

PMID: 22714537

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Project description:To determine early changes leading to human cell transformation (cancer) we exposed an immortalized human bronchial epithelial cell line, BEAS-2B, to one of four different metals that may cause cancer via inhalation in humans or rodents: 2.0 micro-Molar soluble sodium arsenite (NaAsO2), 0.50 micro-Molar potassium chromate (K2CrO4), 250 micro-Molar nickel (II) sulfate (NiSO4), 10 micro-Molar sodium meta-vanadate (NaVO3), or were left untreated (control). After a 30-60 day exposure, cells were rinsed of metals and seeded in soft agar. A small number of the cells formed colonies in the soft agar, demonstrating the potential for anchorage independent growth, a characteristic of cancer. These colonies that originated from a single cell were extracted from the agar and grown out in monolayer for 3-4 weeks. The RNA data provided here is taken from these cells. The significance it that the metal exposure was stopped many generations before the analysis, yet each sample demonstrates changes in gene expression based on the original metal exposure. A total of 39 samples were analyzed, consisting of 3 parental cell lines (BEAS-2B cells that were never seeded in agar or exposed to any metal), 11 controls cells (grouped into three sets, based on those seeded at the same time as the V-treated clones, the Cr-treated clones, or the As-treated and Ni-treated clones), and 25 metal-treated clones (7 each for Ni and As, processed in two separate batches of 3 and 4 clones each, 6 Cr clones and 5 Vanadium clones). Because the samples were processed in different batches, the data was batch-normalized using the COMBAT package in R prior to importing into GeneSpring. Once in GeneSpring the data was base-lined with each sample compared to its control samples (Batch 1: Cr 1-6, P-2, Ctrls 6-8; Batch 2: As1-3, Ni 1-3, P-1 and Ctrl 1-3; Batch 3: As4-7, Ni 4-7, and Ctrl 4-5; Batch 4: V 1-5, P-3 and Ctrl 9-11). When the same analysis was performed but with baseline of samples to all controls combined, a similar result was obtained, albeit with less tight clustering. Arsenic clones 2 and 5 appear to be outliers as their removal nicely cleans up the PCA, but this appears to be genuine biological variation as we did not identify any significant problems with the data.

Project description:Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in airway remodeling associated with V2O5-induced bronchitis. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Expression data were preprocessed using RMA with a log2 transformation. Statistical analysis was performed in R using the affylmGUI package using a linear model with contrasts between untreated control and V2O5-exposed fibroblasts. Genes identified as statistically significant were filtered by selecting only those genes that exhibited a > 2-fold change. Quantitative real-time RT-PCR was utilized to confirm expression of selected genes. More than 2000 genes were significantly changed in response to V2O5 over the time course of our experiment. Genes altered by V2O5 were involved in biologic processes related to cell growth and differentiation, oxidative stress responses, immune regulation, and interferon signaling and apoptosis. In particular, V2O5 induced genes that encode growth factors involved in epithelial repair (HB-EGF) or angiogenesis (VEGF), peroxide generating enzymes (SOD2), pro-inflammatory enzymes (PGHS2), while suppressing genes involved in growth arrest (GAS1, STAT-1) and cell cycle inhibition (CDKN1B). Our study also identified a variety of novel genes that could be used as biomarkers of V2O5-induced bronchitis or could serve as candidate genes for disease progression. Experiment Overall Design: Experimental Design Experiment Overall Design: Normal adult human lung fibroblasts (ATCC 16 Lu) were purchased from American Type Culture Collection (Rockville, MD). Fibroblasts were seeded into 175 cm2 plastic culture flasks and grown to confluence in 10% fetal bovine serum (FBS)/Dulbeccoâs modified Eagleâs medium (DMEM), then trypsin-liberated, and seeded into 150 mm dishes. Confluent monolayers were rendered quiescent for 24 hrs in serum-free defined medium (SFDM) that consisted of Hamâs F-12 medium with 0.25% BSA with an insulin/transferrin/selenium supplement. Cells were treated with 10 Âµg/cm2 vanadium pentoxide, V2O5 (Aldrich Chemical, Milwaukee, WI) or SFDM and RNA was harvested from the fibroblast cultures at 1, 4, 8, 12 and 24 hrs post-treatment. RNA from an SFDM control was harvested at each of these time points to normalize the V2O5 treatment at the same corresponding time point. Three replicate chips were analyzed for SFDM and V2O5 treatment groups at each of the five time points tested. Experiment Overall Design: Microarray Hybridizations and Data Analysis Experiment Overall Design: Human lung fibroblast RNA was isolated using RNeasy columns (Qiagen, Valencia, CA). RNA quality was verified by spectrophotometry and gel electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Probe preparation and hybridization to the microarray was performed in the CIIT Gene Expression Core Facility using standard Affymetrix procedures. Double-stranded cDNA was synthesized from RNA using an oligo-dT24-T7. Biotinylated cRNA was synthesized from an aliquot of the cDNA template using the T7 RNA Transcript Labeling Kit (ENZO Diagnostics, Farmingdale NY). The labeled cRNA was then fragmented, hybridized to Affymetrix Human Genome U133A 2.0 arrays (Affymetrix, Santa Clara, CA), and stained using phycoerythrein-conjugated streptavidin (Molecular Probes, Eugene, OR). Experiment Overall Design: Statistical Analysis and Data Processing Experiment Overall Design: The microarray data were preprocessed using RMA with a log base 2 (log2) transformation. Statistical analysis of the data was performed in R using the affyImGUI package. To identify genes with significant changes in expression following V2O5 exposure, all treatment groups were analyzed using a linear model with contrasts between untreated fibroblasts and V2O5-exposed fibroblasts at each time point. Probability values were adjusted for multiple comparisons using a false discovery rate of 5% (FDR = 0.05). Genes identified as statistically significant were subject to an additional filter by selecting only those genes that exhibited a > 2-fold change from the untreated fibroblasts.

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Gene expression changes in human lung cells exposed to arsenic, chromium, nickel or vanadium indicate the first steps in cancer.

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Gene expression changes in human lung cells exposed to arsenic, chromium, nickel or vanadium indicate the first steps in cancer.

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