Unknown,Transcriptomics,Genomics,Proteomics

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Acid Stress Increases Gene Expression of Proinflammatory Cytokines in Madin Darby Canine Kidney Cells

ABSTRACT: Metabolic acidosis exacerbates chronic kidney disease, in part, by stimulating renal endothelin production. To determine if acidosis alters expression of other genes, we examined the effect of exposure of MDCK cells to pH 7.4 and pH 7.0 for 24 hours on gene expression using a canine derived microarray. Exposure to this pH stress for 24 hours led to increased expression of 278 genes (2.2% of the transcriptome) by at least two fold and 60 of these (21%) were upregulated by more than 3 fold. On the other hand, 186 genes (1.5% of the transcriptome) were down regulated by at least two fold and 16 of these (9%) were down regulated by 3 fold or more. Ten percent of the genes upregulated by at least three fold encode proteins that are pro-inflammatory cytokines, including colony stimulating factor 2, chemokine ligand 7, chemokine ligand 2 0, chemokine ligand 8, and interleukin 1M-NM-1. Two others encode metallopeptidase . The most highly upregulated gene encodes a protein, lubricin, shown to be important in preventing cartilage damage and in tissue injury or repair.Upregulation of four genes were confirmed by qPCR. Housekeeping genes were not increased. There were a total of three biological replicates, each containing one control (incubated at pH 7.4) and one acidic (pH 7.0) sample, whose RNA were extracted on different dates. Microarray studies performed on these six samples were done in duplicate.

ORGANISM(S): Canis lupus familiaris

SUBMITTER: Thomas Nguyen

PROVIDER: E-GEOD-36848 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Penicillins inhibit cell wall synthesis; therefore, H. pylori must be dividing for these antibiotics to be effective. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens. Aim: To determine the effect of acidity on bacterial growth and the bactericidal efficacy of ampicillin. Methods: H. pylori were incubated in dialysis chambers suspended in 1.5L of media at various pHs with 5mM urea, with or without ampicillin, for 4, 8 or 16 hours, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined. The bacterial load of H. pylori infected gerbils was determined with and without profound acid inhibition. Results: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division genes and penicillin-binding proteins. In the presence of ampicillin, viability and survival declined at pH 4.5 and 7.4 but not at pH 3.0. Profound acid inhibition of H. pylori infected gerbils increased the antral bacterial load >3 fold, showing that elevation of intragastric pH stimulated growth. Conclusions: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes at pH 3.0. Therefore, at pH 3.0, the pH at the gastric surface, the bacteria are non-dividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 hours/day should enhance the efficacy of triple therapy and of amoxicillin, even allowing dual therapy for H. pylori eradication. There were a total of three biological replicates, each containing one control (incubated at pH 7.4) and one acidic (pH 3.0) sample, whose RNA were extracted on different dates. Microarray studies performed on these six samples were done in triplicate. One of the triplicates for two of the biological control triplicates were removed from the data set due to poor hybridization

Project description:Lactic acidosis and hypoxia are two prominent tumor microenvironmental stresses that are both known to exert important influences on gene expression and phenotypes of cancer cells. But very little is known about the cross-talk and interaction between these two stresses. We performed gene expression analysis of MCF7 cells exposed to lactic acidosis, hypoxia and combined lactic acidosis and hypoxia. We found the hypoxia response elicited under hypoxia was mostly abolished upon simultaneous exposure to lactic acidosis. The repression effects are due to loss of HIF-1Î± protein synthesis under lactic acidosis. In addition, we showed lactic acidosis strongly synergizes with hypoxia to activate the unfold protein response (UPR) and inflammation response which are highly similar to amino acid deprivation responses (AAR). The statistical factor analysis of hypoxia and lactic acidosis responses indicated that ATF4 locus, an important activator in the UPR/AAR pathway, is amplified in subsets of breast tumors and cancer cell lines. Varying ATF4 levels dramatically affect the ability to survive the post-stress recovery from hypoxia and lactic acidosis and may suggest its selection of ATF4 amplification in human cancers. These data suggest that lactic acidosis interacts with hypoxia by both inhibiting the canonical hypoxia response and while activating the UPR and inflammation response. Gain of ATF4 locus may offer survival advantages to allow successful adaptation to frequent fluctuations of oxygen and acidity in tumor microenvironment. Collectively, our studies have provided linkage between the short-term transcriptional responses to the long term selection of the DNA copy number alterations (CNAs) under tumor microenvironmental stresses. RNAs from MCF7 cells exposed to control condition (ambient air ~21% O2, no lactate and neutral pH), lactic acidosis (ambient air, 10 mM Lactate and pH 6.7), hypoxia (1% pO2, no lactate and neutral pH) and the combined lactic acidosis and hypoxia (1% pO2, 10 mM Lactate and pH 6.7) condition for 24 hours were extracted by miRVana kits (Ambion) and hybridized to Affymetrix Human genome 133A 2.0 arrays with standard protocol.

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Acid Stress Increases Gene Expression of Proinflammatory Cytokines in Madin Darby Canine Kidney Cells

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