Transcriptional responses to NNK and selenocystine in mouse lungs.
ABSTRACT: This study evaluated transcriptional effects of the lung carcinogen NNK ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) injection and selenocystine consumption on the murine lung. Female A/J mice at 5 weeks of age were obtained from The Jackson Laboratory. Animals were stabilized on an unsupplemented AIN-76 diet for one week prior to being given the selenium supplemented diet. The basal level of selenium in the diet is 0.35 ppm Se, the selenocytine supplemention was at 15 ppm. NNK was administered i.p. as a single 10 uM injection in 0.2 mL saline. With this protocol, 100% of animals reproducibly develop lung tumors after 3 months. Three days after NNK administration, animals were provided AIN-76A diets supplemented with selenocystine at 15 ppm selenium ad libitum for 10 days. Animals were sacrificed thirteen days after NNK administration. Lung tissue was harvested, immediately homogenized in Trizol and frozen. The organic extracted RNAs were run over Qiagen RNeasy columes before quantifying and qualifying them. All samples had RNA quality index's greater than 9. Four groups of A/J mice were utilized with 4 biological replicates per group. 1) Untreated - controls on the AIN-76 diet (0.35 ppm Se). 2) NNK treated - single injection of NNK, maintained on norma AIN-76 diet, sacraficed after 13 days. 3) SECY - selenocystine supplemented (15 ppm) for 10 days on a AIN-76 diet then sacraficed. 4) NNK plus SECY - single injection of NNK, after 3 days, selenocystine supplemented (15 ppm) diet for 10 days then sacraficed. RNAs from the the four untreated mice were combined to phenotypically anchor the dual color expression profile. mouse lung responses to NNK injection and/or selenocystine dietary supplementation
Project description:Purpose: Chronic pulmonary inflammation in the form of chronic obstructive pulmonary disease (COPD) has been consistently shown to increase the risk of lung cancer. Therefore, identification of chemopreventive agents with anti-inflammatory effects, in addition to antiproliferative and apoptotic activities, is indispensable. Recently, we found that combinations of silibinin (Sil) and indole-3-carbinol (I3C) significantly inhibited lung tumorigenesis induced by 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) and enhanced by chronic treatment with the inflammatory agent lipopolysaccharide (LPS). In this study, we described gene expression profiling of lung tissues using RNA-seq to determine the gene expression signature in inflammation-driven lung tumors and modulation of this signature by the chemopreventive agents Sil and I3C. Methods: Total RNA extracted from lung tissues of control and treated mice were processed for mRNA sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed for transcript abundance at the gene level using CLC Bio Genomics Workbench and differential gene expression analysis was performed using the built-in Empirical Analysis of Differential Gene Expression (DGE) based on 'Exact Test' method. qRT–PCR validation was performed using SYBR Green assays. Results and conclusions: We found that 330, 2,957, and 1,143 genes were differentially regulated in mice treated with NNK, LPS, and NNK + LPS, respectively. The expression of inflammation-and immunity-related genes was significantly more deregulated in lung tissues of mice treated with LPS alone compared to mice treated with NNK + LPS. Among 1,143 genes differentially regulated in the NNK + LPS group, the expression of 162 genes and associated signaling pathways were significantly modulated by I3C and/or Sil + I3C. These genes include cytokines, chemokines, and genes with a well-established role in inflammation and/or tumorigenesis such as c-ros oncogene 1 (Ros1), the EGFR ligands amphiregulin and epiregulin, Cyp1a1, and the circadian rhythm genes Arntl, and Npas2. To our knowledge, this is the first report that provides insight into genes that are differentially expressed during inflammation-driven lung tumorigenesis and the modulation of these genes by chemopreventive agents. Lung tissue mRNA profiles of mice treated with control vehicle, NNK, LPS, NNK+LPS, or NNK+LPS supplemented with chemopreventive agent(s) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.
Project description:In order to assess the alteration in lncRNA expression in rat lung carcinogenesis induced by NNK, we determined the lncRNA expression profiles in 3 rat lung tumor samples and matched normal lung tissues and 2 blood samples from the control and NNK treatment group in the 95th week using Arraystar Rat lncRNA Microarray. We induced lung cancer using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in a rat model and determined the lncRNA expression profiles in lung cancer tissues and rat blood samples.
Project description:The hepatocyte growth factor (HGF)/c-Met signaling pathway is known to mediate vascularization. We have previously demonstrated that expression of a human HGF transgene in the small airways produced mice (HGF TG) that were more susceptible to the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). We also have observed that HGF TG mice display significantly enhanced vascularization in the lungs that increases over time compared to wild-type (WT) littermates. To analyze which genes might contribute to increased vascularization from HGF overexpression in the airways, RNA and protein were isolated from whole lungs of individual HGF TG and WT adult mice. We profiled the mRNA expression of several hundred genes representative of six biological pathways involved in transformation, angiogenesis, and tumorigenesis using two commercial microarrays. Significant changes in expression over a 1.5-fold boundary were also observed in lung tumors derived from NNK-treated HGF TG mice. Lung tumors were induced by exposing mice to four weekly i.p. injections of 3mg NNK (15μg/μl) over 2 weeks. Whole lungs from control untreated animals were dissected after sacrifice at 10, 20, or 40 wks of age, and NNK induced lung tumors were dissected from the animals at 20 or 40 weeks of age. Total RNA was extracted from whole lung or isolated tumors from HGF TG or WT mice using TRIzol reagent and the Array Grade Total RNA Isolation Kit. The cDNA was generated and labeled using the TrueLabeling-AMP Linear RNA Amplication Kit. RNA was analyzed from 28 mice in total. The angiogenesis array was used to analyze 10 samples taken from 40 week old mice (HGF TG untreated [n=4], WT untreated [n=4], HGF TG NNK treated [n=1], WT NNK treated [n=1]) and 6 samples from 20 week old mice (HGF TG untreated [n=2], WT untreated [n=2], HGF TG NNK treated [n=1], WT NNK treated [n=1]). The cancer gene array was used to analyze 8 samples taken from 40 week old mice (HGF TG untreated [n=2], WT untreated [n=2], HGF TG NNK treated [n=2], WT NNK treated [n=2]) and 4 samples from 20 week old mice (HGF TG untreated [n=2], WT untreated [n=2]).
Project description:Identification of genes associated with exposure to the carcinogen Nitrosamine (NNK) in mouse lungs of susceptible and resistant strains. Microarrays were used to capture all of the up and down regulated genes in two strains of mice. Lungs were excised and analyzed between 3-12 weeks after exposure to NNK before mature tumors had formed
Project description:Specific pathogen free wild-type C57Bl/6 male mice fed ketogenic diet (Bio-Serv AIN-76-A) for 4 weeks Keywords: RNA Expression Array Overall design: Hearts from 12 week-old mice that were maintained on a standard polysacchardide-rich chow until the age of 8 weeks, at which time they were switched to a ketogenic diet (ad libitum) and maintained for 4 additional weeks prior to collection of tissues
Project description:Specific pathogen free wild-type C57Bl/6 male mice fed ketogenic diet (Bio-Serv AIN-76-A) for 4 weeks Keywords: RNA Expression Array Hearts from 12 week-old mice that were maintained on a standard polysacchardide-rich chow until the age of 8 weeks, at which time they were switched to a ketogenic diet (ad libitum) and maintained for 4 additional weeks prior to collection of tissues
Project description:The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early-stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. In these studies, we systematically profiled gene expression in normal (NHBE), immortalized (BEAS-2B) and fully transformed (NNK-BEAS-2B) human bronchial epithelial cells, as well as a non-small cell lung cancer (NSCLC) cell line (H157) from a smoker. Expression profiles that accompany the immortalization and/or transformation of bronchial epithelial cells were generated, and expression of 28 genes was validated by immunoblotting. 4 of them were further evaluated in immunohistochemical analyses of tissue microarrays that contain NSCLC specimens, surrounding non-diseased tissues and non-pulmonary normal tissues. Although all 4 genes were predominantly expressed in tumor tissues, loss of expression of cytoplasmic CDK1 was clinically important because it was associated with a poor prognosis for NSCLC patients. This poor prognostic value may be associated with therapeutic resistance because decreasing levels of cytoplasmic CDK1 in vitro increased resistance to standard chemotherapies used in the treatment of NSCLC, especially microtubule agents where resistance was almost complete. These studies illustrate how a combined microarray approach can facilitate the identification of new, relevant targets in cancer. Fluorescently labeled cDNA were synthesized from 100 microgram RNA by oligo(dT)-primed reverse transcription in the presence of Cy3- or Cy5-dUTP (Amersham Bisciences, Piscataway, NJ). Purified Cy3/Cy5-labelled probes were combined and hybridized in the presence of 2x Denhart's solution, 3.2x saline sodium citrate (SSC), and 0.5% sodium dodecyl sulfate (SDS) in a humidified chamber at 65°C overnight. Prior to scanning (Agilent Technologies, Foster City, CA), slides were successively washed at 22°C in 0.5x SSC/0.1% SDS for 2 min, 0.5x SSC/0.01% SDS for 2 min, and 0.06x SSC for 2 min. Image analyses were performed with the IPLab software (Fairfax, VA). The reference cell (NHBE) was included in every individual hybridization to allow for normalization of each clone’s expression relative to the reference for each cell line (BEAS-2B, NNK-BEAS-2B or H157). A self-to-self hybridization with dye reversal was performed to exclude preferential differences in probe labeling (not included here). Every sample was labeled with Cy5 and Cy3 and hybridized twice. The two fluorescent images (red and green channels) obtained from the scanner constituted the intensity raw data from which differential gene expression ratios and quality control values were calculated. All data were entered into a relational database, using the FileMaker Pro 5 software (Santa Clara, CA). Genes were identified as differentially regulated only if corrected red/green hybridization signals differed by at least two-fold. The genes in each group were alphabetically listed and the ratios of BEAS-2B/NHBE, NNK-BEAS-2B/NHBE or H157/NHBE were graphically visualized by Cluster and TreeView programs (http://rana.lbl.gov/EisenSoftware.htm). These methods fulfilled the MIAME criteria (http://www.mged.org/miame).
Project description:Selenium (Se) is an essential cofactor of the antioxidant enzyme glutathione peroxidase beside other functions. The evaluation of optimal selenium supplementation in chicken feed and the subsequent effects on animal health and performance requires comprehensive knowledge of the overall metabolic effects of selenium. Therefore the gene expression was measured in the control group with a standard diet and in the group with a Se supplemented diet (0.5mg Se/kg diet) to determine significantly altered gene expression. The selenium was supplemented in the form of selenized yeast (Se-yeast), which mainly consists of organic Se in the form of L-selenomethionine and L-selenocysteine. The control group received a diet, which contained 70μg of Se / kg diet and the Se-yeast group 620μg of Se / kg diet (analyzed). Overall design: The one-day old broiler chicks were separated into two groups and received the control or the Se-supplemented diet ad libitum for 35 days. After slaughter the gene expression was determined in the liver of four control and five samples from the Se-yeast group. One sample from the control group did not correspond to the quality requirements and was excluded from the analysis.
Project description:Selenium (Se) is an essential cofactor of the antioxidant enzyme glutathione peroxidase beside other functions. The evaluation of optimal selenium supplementation in chicken feed and the subsequent effects on animal health and performance requires comprehensive knowledge of the overall metabolic effects of selenium. Therefore the gene expression was measured in the control group with a standard diet and in the group with a Se supplemented diet (0.5mg Se/kg diet) to determine significantly altered gene expression. The selenium was supplemented in the form of selenized yeast (Se-yeast), which mainly consists of organic Se in the form of L-selenomethionine and L-selenocysteine. The control group received a diet, which contained 70μg of Se / kg diet and the Se-yeast group 620μg of Se / kg diet (analyzed). The one-day old broiler chicks were separated into two groups and received the control or the Se-supplemented diet ad libitum for 35 days. After slaughter the gene expression was determined in the liver of four control and five samples from the Se-yeast group. One sample from the control group did not correspond to the quality requirements and was excluded from the analysis.
Project description:Increasing the understanding of the impact of changes in oncogenes and tumor suppressor genes is essential for improving the management of lung cancer. Recently, we identified a new mouse lung-specific tumor suppressor - the G-protein coupled receptor 5A (Gprc5a). We sought to understand the molecular consequences of Gprc5a loss and towards this we performed microarray analysis of the transcriptomes of lung epithelial cells cultured from normal tracheas of Gprc5a knockout and wild-type mice to define a loss-of-Gprc5a gene signature. Moreover, we analyzed differential gene expression patterns between Gprc5a knockout normal lung epithelial cells as well as lung adenocarcinoma cells isolated and cultured from tumors of NNK-exposed Gprc5a knockout mice. Overall design: Gprc5a wild type cells (WT-NLE) and Gprc5a knockout cells (NULL-NLE) were isolated and cultured from trachea of three week old Gprc5a wild type and knockout mice, respectively. MDA-F471 mouse lung adenocarcinoma cells were derived de novo from an adenocarcinoma that was found in the lung of a female Gprc5a-knockout mouse sacrificed 16 months after NNK tobacco carcinogen i.p. injection based on a protocol approved by the M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. Following RNA extraction and purification, the transcriptome of the Gprc5a wild type and knockout cells were analyzed by microarray analysis using the Affymetrix MG-430 2.0 murine array platform.