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Human AhR Reporter Assay Kit
Product Description and Product Data
This is an all-inclusive cell-based luciferase reporter assay kit targeting the Aryl Hydrocarbon Receptor. INDIGO’s AhR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the AhR. In addition to AhR Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human AhR. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.
Features
Ready to Use Upon Receipt
- Includes All Needed Components
- Contains Transfected Reporter Cells
- Eliminates Cell Licensing Fees
- Clear, Reproducible Results
- Consistent Results Lot to Lot
Product Specifications
| Target Type | Nuclear Hormone Receptor | ||
| Species | Human | ||
| Receptor Form | Native | ||
| Assay Mode | Agonist, Antagonist | ||
| Kit Components |
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| Shelf Life | 6 months | ||
| Orthologs Available | Yes | ||
| Shipping Requirements | Dry Ice | ||
| Storage temperature | -80C |
Data
Target Background
Although technically not a member of the Nuclear Receptor (NR) superfamily, the AhR shares many of the same attributes. The AhR is a member of the basic helix-loop-helix, Per-Arnt-Sim (bHLH-PAS) family of transcription factors and is responsible for the toxicologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, often referred to as simply “dioxin”) and several other related polycyclic aromatic hydrocarbons. The basic mechanism of action of dioxin and related compounds has been extensively studied, in particular as it relates to regulation of cytochrome P450 1A1 (CYP1A1).
AhR is present in most cell types and in the unactivated state is cytosolic and exists in a complex with chaperone proteins such as heat shock protein 90 (Hsp90). Binding of TCDD and related molecules to AhR leads to nuclear translocation and heterodimerization with its partner protein ARNT, another bHLH-PAS family member. The AhR-ARNT heterodimer binds to specific cognate DNA sequence elements known as dioxin/xenobiotic response elements (DRE/XRE) present in the regulatory region of specific genes such as CYP1A1. Binding of the AhR:ARNT heterodimer to these elements, and subsequent recruitment of transcription coactivator complexes, leads to increased transcription of the specific gene, known as “target genes.” There is a battery of genes affected in this manner and targets include certain xenobiotic-metabolizing enzymes, such as CYP1A1,CYP1A2, CYP2B1, and UGT1A6. In addition, genes affected directly and indirectly by the TCDD/AhR-complex code for both inhibitory and stimulatory growth factors and their gene products affect cellular growth and differentiation leading to tumor promotion and carcinogenicity as well as other forms of toxicity.
INDIGO’s Aryl Hydrocarbon Receptor (AhR) Assay is a cell-based genetic reporter assay that utilizes the luciferase reporter gene Aryl Hydrocarbon Receptor Agonist and Antagonist Assays functionally linked to an AhR-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in AhR activity.
The principle application of this reporter assay system is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human AhR.
Citations
As the most classic and extensively studied transcription factor in response to environmental toxic chemicals, the human aryl hydrocarbon receptor (AHR) has been implicated in mediating some oncogenic responses also. Limited information is available, however, on whether arsenic, a widely presented environmental carcinogen, can regulate AHR to exert its carcinogenic activity. Through chromatin immunoprecipitation and sequencing (ChIP-seq), CRISPR-Cas9 gene editing, RNA-seq, and immunohistochemistry (IHC), in this report we provided evidence showing that arsenic enforces TGFβ and other oncogenic signaling pathways in bronchial epithelial cells through disrupting the tumor suppressor-like activity of AHR. AHR is normally enriched on a number of oncogenic genes in addition to the known phase I/II enzymes, such as genes in TGFβ and Nrf2 signaling pathways and several known oncogenes. Arsenic treatment substantially reduced the binding of AHR on these genes followed by an increased expression of these genes. CRISPR-Cas9-based knockout of AHR followed by RNA-seq further demonstrated increased expression of the TGFβ signaling and some oncogenic signaling pathway genes in the AHR knockout cells. IHC studies on human tissue samples revealed that normal human lung tissues expressed high level of AHR. In contrast, the AHR expression was diminished in the lung cancer tissues. Accordingly, the data from this study suggest that AHR has tumor suppressor-like activity for human lung cancer, and one of the carcinogenic mechanisms of arsenic is likely mediated by the inhibition of arsenic on the tumor suppressor-like activity of AHR.
There has been considerable scientific effort dedicated to understanding the biologic consequence and therapeutic implications of aberrant tryptophan metabolism in brain tumors and neurodegenerative diseases. A majority of this work has focused on the upstream metabolism of tryptophan; however, this has resulted in limited clinical application. Using global metabolomic profiling of patient-derived brain tumors, we identify the downstream metabolism of tryptophan and accumulation of quinolinate (QA) as a metabolic node in glioblastoma and demonstrate its critical role in promoting immune tolerance. QA acts as a metabolic checkpoint in glioblastoma by inducing NMDA receptor activation and Foxo1/PPARγ signaling in macrophages, resulting in a tumor supportive phenotype. Using a genetically-engineered mouse model designed to inhibit production of QA, we identify kynureninase as a promising therapeutic target to revert the potent immune suppressive microenvironment in glioblastoma. These findings offer an opportunity to revisit the biologic consequence of this pathway as it relates to oncogenesis and neurodegenerative disease and a framework for developing immune modulatory agents to further clinical gains in these otherwise incurable diseases.
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