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1.
J Toxicol Sci ; 44(3): 213-224, 2019.
Article in English | MEDLINE | ID: mdl-30842373

ABSTRACT

The human cell line activation test (h-CLAT) is a skin sensitization test that measures the expression of cell surface proteins CD86 and CD54 to evaluate the skin sensitization potential of test chemicals. However, some skin irritants have been reported to induce dramatically high CD54 expression leading to false-positive h-CLAT results. Furthermore, CD54 expression is strongly induced by cytokines, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, or danger signals that activate its signaling pathways. In this study, we focused on the relationship between CD54 expression and the Nucleotide binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome, a protein complex that plays a pivotal role in intra-cellular inflammation. We observed the activation of caspase-1 and production of IL-1ß after exposure of THP-1 cells to 2,4-dinitrochlorobenzene (DNCB, sensitizer), octanoic acid (OA, non-sensitizer), and salicylic acid (SA, non-sensitizer), implying NLRP3 activation. These observations confirmed the activation of the inflammasome by CD54-only positive chemicals. CD54 expression, induced by OA and SA, was suppressed by potassium chloride, a typical inhibitor of NLRP3 inflammasome activation. These results suggested that the NLRP3 inflammasome may be activated in THP-1 cells resulting in the expression of CD54, and subsequently leading to false-positive results.


Subject(s)
Haptens/toxicity , Inflammasomes/immunology , Intercellular Adhesion Molecule-1/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , B7-2 Antigen/immunology , Caspase 1/immunology , False Positive Reactions , Humans , Interleukin-1beta/immunology , Reactive Oxygen Species/metabolism , THP-1 Cells , Toxicity Tests
2.
J Toxicol Sci ; 43(5): 299-309, 2018.
Article in English | MEDLINE | ID: mdl-29743441

ABSTRACT

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na+/H+ exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.


Subject(s)
B7-2 Antigen/genetics , B7-2 Antigen/metabolism , Gene Expression , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Skin Tests , Allergens/immunology , Biomarkers/metabolism , Dendritic Cells/immunology , Dinitrochlorobenzene/immunology , Humans , Hydrogen-Ion Concentration , Skin/cytology , Sodium-Hydrogen Exchangers/physiology , THP-1 Cells , Urea/analogs & derivatives , Urea/immunology
3.
Cancer Discov ; 6(4): 430-45, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26603525

ABSTRACT

UNLABELLED: Loss-of-function mutations in the CBP/CREBBP gene, which encodes a histone acetyltransferase (HAT), are present in a variety of human tumors, including lung, bladder, gastric, and hematopoietic cancers. Consequently, development of a molecular targeting method capable of specifically killing CBP-deficient cancer cells would greatly improve cancer therapy. Functional screening of synthetic-lethal genes in CBP-deficient cancers identified the CBP paralog p300/EP300 Ablation of p300 in CBP-knockout and CBP-deficient cancer cells induced G1-S cell-cycle arrest, followed by apoptosis. Genome-wide gene expression analysis revealed that MYC is a major factor responsible for the synthetic lethality. Indeed, p300 ablation in CBP-deficient cells caused downregulation of MYC expression via reduction of histone acetylation in its promoter, and this lethality was rescued by exogenous MYC expression. The p300-HAT inhibitor C646 specifically suppressed the growth of CBP-deficient lung and hematopoietic cancer cells in vitro and in vivo; thus p300 is a promising therapeutic target for treatment of CBP-deficient cancers. SIGNIFICANCE: Targeting synthetic-lethal partners of genes mutated in cancer holds great promise for treating patients without activating driver gene alterations. Here, we propose a "synthetic lethal-based therapeutic strategy" for CBP-deficient cancers by inhibition of the p300 HAT activity. Patients with CBP-deficient cancers could benefit from therapy using p300-HAT inhibitors.


Subject(s)
Apoptosis/genetics , CREB-Binding Protein/deficiency , E1A-Associated p300 Protein/genetics , Neoplasms/genetics , Proto-Oncogene Proteins c-myc/genetics , Synthetic Lethal Mutations , Animals , Cell Line, Tumor , Chromatin Assembly and Disassembly , Disease Models, Animal , E1A-Associated p300 Protein/antagonists & inhibitors , E1A-Associated p300 Protein/metabolism , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation, Neoplastic , Heterografts , Histone Acetyltransferases/antagonists & inhibitors , Humans , Mice , Mutation , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , RNA Interference , Transcription, Genetic
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