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.
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 M-CM-^W 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:In this study, we demonstrate the consequences of Fe overdosing (achieved by iron(III) ammonium citrate (FAC)) or/and GSH depletion (achieved by the GSH binding molecule diethyl maleate (DEM)) in Caenorhabditis elegans (C. elegans) on Fe homeostasis.
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
Project description:Synergists can counteract metabolic insecticide resistance by inhibiting detoxification enzymes or transporters. In this study we used Illumina RNA-sequencing to investigate genome-wide transcriptional responses in an acaricide resistant strain (JP-R) of the spider mite Tetranychus urticae upon exposure to synergists such as S,S,S-tributyl phosphorotrithioate (DEF), diethyl maleate (DEM), piperonyl butoxide (PBO) and cyclosporin A (CsA).
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.
Project description:Dysregulation of pathways involved in the processing of cancer and microenvironment information in MCA+TPA transformed C3H/10T1/2 cells
Project description:In this study, we have investigated the changes in transformed cells that occur after GSH depletion and may be critical to accentuate the attenuation of carcinogenesis. The in vitro carcinogenesis method and GSH depletor Phorone (PHO) were used. We found that exposure of transformed C3H10T1/2 cells to PHO (2mM) caused attenuation of transformed cell growth as seen in soft agar assay. Loss of cellular GSH content, increase in intracellular ROS generation, and DNA strand break formation also occurred. A microarray based study using 8x60k oligonucleotide array in duplicate revealed changes in global gene expression profile. Our study has identified changes in expression of many genes that may contribute in abatement of carcinogenesis.