Project description:The two-stage cell transformation assay is an in vitro model cell culture system to identify the ability of chemicals to act as initiators or promoters of cell transforma- tion and also to study the cellular and molecular mechanisms of chemically induced morphological and neoplastic cell transformation. The global gene expression profiles of 3- methylcholanthrene (MCA)+12-O-tetradecanoylphorbol-13- acetate (TPA)-transformed C3H/10T1/2 cells are not known. Therefore, we have investigated the global transcriptional profile of MCA+TPA-transformed C3H10T1/2 cells using an 8×60 k probe microarray. The study revealed a differential regulation of pathways and gene expressions. Multifold dysregulation was seen in pathways of cancer, phagosomal activity, and tumor cell microenvironment information pro- cessing systems, notably the neuroactive ligand–receptor in- teraction, actin cytoskeleton regulation, tight junction, axon guidance, and cell adhesion molecules. The genes FGF1, EIF4E1B, MAGI1,and GRIA3 showed upregulation; these encoded the pluripotent fibroblast growth factor, the transla- tion initiation factor, the tight junction scaffolding protein, and the antiapoptotic as well as the enhancer of proliferation and migration, respectively. The genes CXCL7/CXCL5/CXCL12, H2DMB1,and HSPA1A showed downregulation; these encoded the chemotactic agent protein, the protein involved Total mRNA was isolated from the control as well as the MCA+TPA-transformed C3H/10T1/2 cells and complemen- tary RNA (cRNA) was prepared from mRNA (1 μg). One- color microarray processing was performed. Acceptable qual- ity of the total RNA sample was ascertained by its electropho- resis trace and integrity assay using a Bioanalyzer which profiled RNA by RIN interpretation. The T7 promoter-based linear amplification was used to generate labeled complemen- tary RNA to amplify target material and incorporate cyanine 3-labeled CTP using Agilent’s low-input RNA linear amplifi- cation kit one color (cat no. 5188-5339). The fluorescence- labeled cRNA samples were hybridized onto a Genotypic- designed Custom Whole Genome Mouse 8x60k slide (AMADID no. 26986) in duplicate using Agilent’sinsitu hybridization kit (no. 5184-3568). Hybridization was carried out in Agilent’s Surehyb Chambers at 65°C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers (part no. 5188-5327). Fluorescence data were collected using an Agilent Microarray Scanner (G2567AA) and analyzed with program Gene Spring GX, version 11.5 (Agilent Technologies, Bangalore, India). Significantly dysregulated genes were identified. Statistical t test p value was calculated based on volcano plot using Gene spring GX Software.
Project description:Murine or human cancer cells have high glutathione levels. Depletion of the elevated GSH inhibits proliferation of cancer cells. Molecular basis for this observation is little understood. In an attempt to find out the underlying mechanism, we reproduced these effects in transformed C3H10T1/2 and BALB/c 3T3 cells using diethyl maleate and studied cytogenomic changes in the whole mouse genome using spotted 8 × 60K arrays. Transformed cells revealed an increase in GSH levels. GSH depletion by DEM inhibited the growth of transformed cells. The non-cytotoxic dose of DEM (0.25 mM) resulted in GSH depletion, ROS generation, cell cycle arrest, apoptosis, decrease in anchorage independent growth, gene expression changes and activation of all three members of the MAPK family. Increase in intracellular GSH levels by GSHe countered the effect of DEM. These results support the physiological importance of GSH in regulation of gene expression for transformed cell growth restraint. This study is of interest in not only understanding the molecular biology of the transformed cells, but also in identifying new targets for development of gene therapy together with the chemotherapy. Agilent one-color experiment; Organism: Mus musculus; Agilent Custom Mouse Whole Genome Mouse 8x60k gene expression designed by Genotypic Technology Private Limited; Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442).
Project description:Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). Phorbol esters, butyrates and N-nitroso compounds are known chemical carcinogens in foodstuffs and cigarettes that have been implicated as risk factors contributing to the development of NPC. We have demonstrated previously that low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.1 microg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation (Chem Biol Interact 188: 623-634). Since residents of areas with a high risk of NPC are reported to contact with these carcinogens (TPA, SB or nitrosamines) frequently, we sought to determine the consequence of repeated exposure of EBV-harboring nasopharyngeal cells to these carcinogens in a long-term, low dose, repeated manner. An NPC cell line latently infected with EBV, NA, was periodically treated with TPA/SB combined with MNNG for recurrent EBV reactivation. After 10 times of chemically-induced recurrent reactivation of EBV, the expression profile analysis indicates that many carcinogenesis-related genes were altered in recurrent reactivated NA cells when compared to the parental NA cells. The expression profile was analyzed in the recurrent EBV reactivated NA cells and the parental NA cells. The expression profile of two NA cells, NA-P10/TS-MG and NA-P1/mock, were analyzed in this study. NA-P10/TS-MG cells were subject to 10 times of repeated chemical exposure by incubating these cells periodically with TPA/SB (10 ng/ml and 1.0 mM) in combination with MNNG (0.1 microg/ml). The NA cells at the beginning of this experiment were defined as passage 0 (P0) cells. 24 h after seeding, the cells were subjected to a 24 h period of mock or chemical treatment. After this 24 hr treatment, the cells were recovered by replacing the medium with fresh (treatment-free) medium and incubation for a further 48 h. The resulting cells were defined as passage 1 (P1) cells. Cells from P1 were trypsinized and transferred and the process repeated again to obtain the P2 cells. A total of ten treatments were applied to the cells in this study. “NA-P10/TS-MG” refers to NA cells with 10 times of combined TPA/SB and MNNG (TS-MG) treatments. NA-P1/mock is mock-treated NA cells with one time of passage that was used as reference in this study. Replicates were included for each samples.
Project description:BACKGROUND: Cadmium (Cd) is implicated in prostate carcinogenesis but its oncogenic action remains unclear. OBJECTIVES: This study aims to decipher changes in cell growth and the transcriptome in an immortalized human normal prostate epithelial cell line (NPrEC) following exposure to low-dose Cd. METHODS: Synchronized NPrEC cells were exposed to different doses of Cd and assayed for cell viability and cell-cycle progression. Temporal changes in transcriptome were investigated by global profiling. Ingenuity Pathways Analyses were used to develop propositions about functional connections among differentially expressed genes. A neutralizing antibody was used to negate the effect of Cd-induced upregulation of TNF in NPrEC. RESULTS: Exposure of NPrEC to 2.5 microM of Cd enhanced cell viability and accelerated cell-cycle progression. Global expression profiling identified 48 genes that exhibited ≥1.5-fold changes in expression after 4, 8, 16 and 32 hr of Cd treatment. Pathway analyses inferred a functional connection among 35 of these genes in one major network with TNF as the most prominent node. Fourteen out of the 35 genes in the network are related to TNF with 11 exhibited an average of ≥2-fold changes in gene expression. Real-time RT-PCR confirmed the upregulation of 7 of the 11 genes (ADAM8, EDN1, IL8, IL24, IL13RA2, COX2/PTGS2 and SERPINB2) and uncovered a 28-fold transient increase in TNF expression in Cd-treated NPrEC. A TNF-neutralizing antibody effectively blocked Cd-induced elevations in the expression of these genes. CONCLUSIONS: Non-cytotoxic, low-dose Cd has growth-promoting effects on NPrEC and induces transient overexpression of TNF, leading to upregulation of genes with oncogenic and immunomodulation functions. Experiment Overall Design: A total of 19 samples were analyzed, each experimental condition having two biological replicates, except Cadmium 4hr which has only 1 sample. Cadmium-treated and control samples were taken at 0, 4, 8, 16, and 32 hours.
Project description:Invastigation of whole genome gene expression level changes in human osteosarcoma cell line MNNG/HOS sarcospheres,hypoxia-induced sarcospheres and TGF-beta1 induced sarcospheres. A three chip study using total RNA cover from three cultures of human osteosarcoma cell line MNNG/HOS sarcospheres, hypoxia-induced sarcospheres and TGF-beta1-induced sarcospheres. Each chip measures the expression level of 45033 genes from osteosarcoma cell line MNNG/HOS.
Project description:Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we report here the first large-scale identification of Mediator kinase substrates in human cells (HCT116). Among over 16,000 quantified sites, we identified 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, CA effects on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, which tracked about 7,000 proteins across six time points (0-24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation; contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest roles beyond transcription, including metabolism and DNA repair.
Project description:Transcription profiling by array of Bacillus amyloliquefaciens strain FZB42 after interaction exudates (IE) treatment, at OD600=1.0 and at OD600=1.0 respectively. IE was the root exudates prepared from maize plants growing with FZB42. The reference was treated with the root exudates (RE), prepared from maize plants grown in an axenic system.
Project description:Transcriptional profiling by array of Bacillus amyloliquefaciens FZB42 at log phase(OD600=1.0) and early stationary phase(OD600=3.0) after maize root exudate treatment of three concentrations(0.25mg/ml,0.50mg/ml,1.00mg/ml).