Unknown,Transcriptomics,Genomics,Proteomics

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ERK activation, gene dysregulations and cell transformation at submicromolar doses of Cr6+ in C3H10T1/2 and BALB/c 3T3 cells: interaction with Lipoic acid

ABSTRACT: The molecular mechanism of Cr6+ carcinogenicity is not well understood. Studies advocate a key involvement of the altered cytogenomics and dysregulated pathways in Cr6+ toxicity. However, the dissimilarity in expression of genes and pathways in cells transformed by Cr6+ has led us to supplement more data on the subject. We report here genomics of cells transformed after acute exposure to submicromolar (0.01or1ÂµM) test doses of Cr6+; the non-cytotoxic submicromolar test concentrations transformed the mammalian cells; the characteristic gain of anchorage independent growth potential was demonstrable in soft agar assay. C3H10T1/2 and BALB/c 3T3 cell transformation assay has been used which is established in literature as the in vitro carcinogenesis test system. Gene expression profile of the transformed cells was found to be dysregulated at both the toxicant doses. PARP1 gene was significantly up regulated and SFN, MTIF2, IRGQ, RAVER2, SLC2A6, MLLT3, EPB4.1L3, JAK1, RALGPS1, EXTL3, PRMT6, PREB, SERPINE1, MORC4 genes were significantly down regulated commonly at two test concentrations. These genes were found to be involved in tumour suppression, DNA repair, cell cycle control, energy metabolism and biosynthesis.Cr6+ also induced MAPK signalling via ERK phosphorylation. Activation of ERK signalling, dysregulation of gene expressions, and cell transformation was prevented by alpha lipoic acid (LA) in equimolar concentrations. The acute exposure to submicromolar concentrations of Cr6+ can induce cell transformation and gene dysregulations in mammalian cell. The influenced genes are crucial for progression of Cr6+ toxicity and their mitigation by LA shows critical role of redox reactions in Cr6+ toxicity. Agilent one-color experiment,Organism: Mus musculus ,Agilent-Whole Genome Mouse 4x44k (AMADID: 14868) , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442) C3H10T1/2 cells treated with 1uM or 0.01uM Cr(V1)

ORGANISM(S): Mus musculus

SUBMITTER: Dr Sushil Kumar

PROVIDER: E-GEOD-43156 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 8M-CM-^W60 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 ligandM-bM-^@M-^Sreceptor 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 M-NM-<g). 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 AgilentM-bM-^@M-^Ys 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 AgilentM-bM-^@M-^Ysinsitu hybridization kit (no. 5184-3568). Hybridization was carried out in AgilentM-bM-^@M-^Ys Surehyb Chambers at 65M-BM-0C 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.

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ERK activation, gene dysregulations and cell transformation at submicromolar doses of Cr6+ in C3H10T1/2 and BALB/c 3T3 cells: interaction with Lipoic acid

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