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P38alfa and ATF2 act differentially depending on DUSP1 expression in NCSCL in response to cisplatin

ABSTRACT: DUSP1 is involved in different cellular pathways including cancer cell proliferation, angiogenesis, invasion and resistance to chemotherapy. To gain insight into the cellular signaling pathways involving DUSP1 actions and the response to Cisplatin (CDDP) in non small cell lung cancer (NSCLC), we have used a double strategy that combines microarray and SiRNA technology. This strategy provided a differential expression profile of genes involved in CDDP response in NSCLC cell line regulated by DUSP1 using H460 and H460cri and a time course to CDDP. KEYWORDS: Expression profiling by array in cells with genetic modification in response to CCDP treatment The human non small lung cancer cell line H460 used in this study was from ATCC (American Type Culture Collection). The cell line was maintained in RPMI (Gibco, Invitrogen) supplemented with 10% bovine serum and transfected by Lipofectamine Plus Reagent from Life Technologies as directed by the manufacturer. The DUSP1 pSuperRetro-derived vectors were constructed as described (Chattoppadhyay et al., 2006). Clones expressing siRNA for DUSP1 were selected for their ability to grow in the presence of puromycin and kept for selection. Stable transfection was confirmed by Western blotting. We analyzed the differences in expression of 47000 genes in H460 cells expressing DUSP1SiRNA (H460cri) compared with the parental cell line H460pSuperRetro vector (H460v) after 0, 1, 3 and 6 hours of CDDP exposure, in order to know the different pathways that were activated by CDDP treatment dependent on expression of DUSP1. RNA was extracted and quality control was checked before hybridization on Affymetrix microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Bandres Eva

PROVIDER: S-ECPF-GEOD-26704 | biostudies-other |

REPOSITORIES: biostudies-other

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p38alfa and ATF2 act differentially depending on DUSP1 expression in NCSCL in response to cisplatin

2012-01-01 | GSE26704 | GEO

p38alfa and ATF2 act differentially depending on DUSP1 expression in NCSCL in response to cisplatin

Project description:DUSP1 is involved in different cellular pathways including cancer cell proliferation, angiogenesis, invasion and resistance to chemotherapy. To gain insight into the cellular signaling pathways involving DUSP1 actions and the response to Cisplatin (CDDP) in non small cell lung cancer (NSCLC), we have used a double strategy that combines microarray and SiRNA technology. This strategy provided a differential expression profile of genes involved in CDDP response in NSCLC cell line regulated by DUSP1 using H460 and H460cri and a time course to CDDP. KEYWORDS: Expression profiling by array in cells with genetic modification in response to CCDP treatment The human non small lung cancer cell line H460 used in this study was from ATCC (American Type Culture Collection). The cell line was maintained in RPMI (Gibco, Invitrogen) supplemented with 10% bovine serum and transfected by Lipofectamine Plus Reagent from Life Technologies as directed by the manufacturer. The DUSP1 pSuperRetro-derived vectors were constructed as described (Chattoppadhyay et al., 2006). Clones expressing siRNA for DUSP1 were selected for their ability to grow in the presence of puromycin and kept for selection. Stable transfection was confirmed by Western blotting. We analyzed the differences in expression of 47000 genes in H460 cells expressing DUSP1SiRNA (H460cri) compared with the parental cell line H460pSuperRetro vector (H460v) after 0, 1, 3 and 6 hours of CDDP exposure, in order to know the different pathways that were activated by CDDP treatment dependent on expression of DUSP1. RNA was extracted and quality control was checked before hybridization on Affymetrix microarrays.

2012-01-01 | E-GEOD-26704 | biostudies-arrayexpress

The two-faced role of ATF2 on cisplatin response in gastric cancer depends on p53 context.

Project description:BackgroundActivating transcription factor-2 (ATF2) is a member of the basic leucine zipper family of DNA-binding proteins, which exhibits both oncogenic and tumor suppression activity in different tumors. However, the molecular mechanism of its dual function in cancer chemotherapy especially in gastric cancer has still not been elucidated.MethodsThe protein expression and location of ATF2 in gastric cancer tissues was detected with immunohistochemistry assay, and the clinical significance was analyzed using TCGA and GEO database. The activation and impact of ATF2 in cisplatin treated cells were evaluated with western blot, incucyte live cell analysis, clone formation and tumor xenografts assays. Interaction between ATF2 and p53 was confirmed with immunoprecipitation and GST-pull down. Potential molecular mechanism of ATF2 in different p53 status cells was analyzed with RNA sequencing and real-time quantitative PCR.ResultsATF2 mainly located in the nucleus of cancer cells, higher ATF2 level was associated with poor five-year survival of gastric patients, especially in those undergone chemotherapy treatment. Cisplatin treatment significantly activated ATF2 in p53 mutant cells. ATF2 could interact with the trans-activation domain of p53 and enhance cisplatin sensitivity in p53 wild type cell lines, while promoted cell survival in mutant p53 cancer cells by affecting ERK1/2 pathway.ConclusionsThis study confirmed the effect of ATF2 on cisplatin sensitivity was associated with the functional status of p53 in gastric cancer cells. Integrated analysis of ATF2 expression and P53 status could be used to evaluate the chemotherapy sensitivity and prognosis of gastric cancer patients.

| S-EPMC9153165 | biostudies-literature

Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES.

Project description:Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed AMD1. To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. In the original study, we tested this hypothesis, by placing the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: Here we employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors. Results: With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter's ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g., AMD1 tail inhibitory effect. We further show by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of RNA levels. Conclusions: While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of readthrough permissive sequences and of IRES elements, though these require a separate investigation.

| S-EPMC8831848 | biostudies-literature

Cisplatin induces a mitochondrial-ROS response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions.

Project description:Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy.

| S-EPMC3834214 | biostudies-literature

<i>Marinobacter atlanticus</i> electrode biofilms differentially regulate gene expression depending on electrode potential and lifestyle.

Project description:Marinobacter spp. are opportunitrophs with a broad metabolic range including interactions with metals and electrodes. Marinobacter atlanticus strain CP1 was previously isolated from a cathode biofilm microbial community enriched from a sediment microbial fuel cell. Like other Marinobacter spp., M. atlanticus generates small amounts of electrical current when grown as a biofilm on an electrode, which is enhanced by the addition of redox mediators. However, the molecular mechanism resulting in extracellular electron transfer is unknown. Here, RNA-sequencing was used to determine changes in gene expression in electrode-attached and planktonic cells of M. atlanticus when grown at electrode potentials that enable current production (310 and 510 mV vs. SHE) compared to a potential that enables electron uptake (160 mV). Cells grown at current-producing potentials had increased expression of genes for molybdate transport, regardless of planktonic or attached lifestyle. Electrode-attached cells at current-producing potentials showed increased expression of the major export protein for the type VI secretion system. Growth at 160 mV resulted in an increase in expression of genes related to stress response and DNA repair including both RecBCD and the LexA/RecA regulatory network, as well as genes for copper homeostasis. Changes in expression of proteins with PEP C-terminal extracellular export motifs suggests that M. atlanticus is remodeling the biofilm matrix in response to electrode potential. These results improve our understanding of the physiological adaptations required for M. atlanticus growth on electrodes, and suggest a role for metal acquisition, either as a requirement for metal cofactors of redox proteins or as a possible electron shuttling mechanism.

| S-EPMC8233155 | biostudies-literature

Adrenergic Stimulation of DUSP1 Impairs Chemotherapy Response in Ovarian Cancer.

Project description:PurposeChronic adrenergic activation has been shown to associate with adverse clinical outcomes in cancer patients, but the underlying mechanisms are not well understood. The focus of the current study was to determine the functional and biologic effects of adrenergic pathways on response to chemotherapy in the context of ovarian cancer.Experimental designIncreased DUSP1 production by sympathetic nervous system mediators (e.g., norepinephrine) was analyzed by real-time quantitative RT-PCR and by Western blotting. In vitro chemotherapy-induced cell apoptosis was examined by flow cytometry. For in vivo therapy, a well-characterized model of chronic stress was used.ResultsCatecholamines significantly inhibited paclitaxel- and cisplatin-induced apoptosis in ovarian cancer cells. Genomic analyses of cells treated with norepinephrine identified DUSP1 as a potential mediator. DUSP1 overexpression resulted in reduced paclitaxel-induced apoptosis in ovarian cancer cells compared with control; conversely, DUSP1 gene silencing resulted in increased apoptosis compared with control cells. DUSP1 gene silencing in vivo significantly enhanced response to paclitaxel and increased apoptosis. In vitro analyses indicated that norepinephrine-induced DUSP1 gene expression was mediated through ADRB2 activation of cAMP-PLC-PKC-CREB signaling, which inhibits JNK-mediated phosphorylation of c-Jun and protects ovarian cancer cells from apoptosis. Moreover, analysis of The Cancer Genome Atlas data showed that increased DUSP1 expression was associated with decreased overall (P= 0.049) and progression-free (P= 0.0005) survival.ConclusionsThese findings provide a new understanding of the mechanisms by which adrenergic pathways can impair response to chemotherapy and have implications for cancer management.

| S-EPMC4818718 | biostudies-literature

Tamoxifen differentially regulates miR-29b-1 and miR-29a expression depending on endocrine-sensitivity in breast cancer cells.

Project description:Endocrine-resistance develops in ?40% of breast cancer patients after tamoxifen (TAM) therapy. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ER? is responsible for TAM regulation of miR-29b-1/a transcription. We also demonstrated that transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest miR-29b-1/a has tumor suppressor activity in TAM-resistant cells and does not appear to play a role in mediating TAM resistance.

| S-EPMC5318263 | biostudies-literature

Control of LPS-response by DUSP1

Project description:Activation of the Mitogen activated protein kinase (MAPK) cascade following Toll-like receptor (TLR) stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase Dual specificity phosphatase-1 (DUSP1) as an essential endogenous regulator of the inflammatory response to LPS. DUSP1-deficient (DUSP1-/-) bone marrow derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1-/- mice with LPS caused increased lethality and overshooting production of IL-6 and TNF?. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, that includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4 and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, IFN? and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock. Keywords: in vivo analysis of splenic mRNA expression

2005-12-15 | GSE3565 | GEO

Inhibition of JNK/c-Jun-ATF2 Overcomes Cisplatin Resistance in Liver Cancer through down-Regulating Galectin-1.

Project description:Due to drug resistance, the clinical response to cisplatin (CDDP) from patients with liver cancer is unsatisfactory. The alleviation or overcoming of CDDP resistance is an urgent problem to be solved in clinics. Tumor cells rapidly change signal pathways to mediate drug resistance under drug exposure. Here, multiple phosphor-kinase assays were performed and c-Jun N-terminal kinase (JNK) was activated in liver cancer cells treated with CDDP. The high activity of the JNK promotes poor progression and mediates cisplatin resistance in liver cancer, leading to a poor prognosis of liver cancer. Mechanistically, the highly activated JNK phosphorylated c-Jun and ATF2 formed a heterodimer to upregulate the expression of Galectin-1, leading to promoting cisplatin resistance in liver cancer. Importantly, we simulated the clinical evolution of drug resistance in liver cancer by continuous CDDP administration in vivo. In vivo bioluminescence imaging showed the activity of JNK gradually increased during this process. Moreover, the inhibition of JNK activity by small molecular or genetic inhibitors enhanced DNA damage and overcame CDDP resistance in vitro and in vivo. Collectively, our results underline that the high activity of JNK/c-Jun-ATF2/Galectin-1 mediates cisplatin resistance in liver cancer and provides an optional scheme for dynamic monitoring of molecular activity in vivo.

| S-EPMC10197891 | biostudies-literature

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