Related Experiment Video
Updated: Aug 6, 2026
Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection
Published on: February 2, 2018
DNA binding properties of the Ah receptor in wild-type and variant mouse hepatoma cells
J Piskorska-Pliszczynska1, V Morrison, T Zacharewski
1Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4466.
Abstract:
Mouse hepatoma Hepa 1c1c7 cells and nonresponsive mutants have been extensively used as models for investigating the molecular mechanism of induction of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Incubation of cytosolic [3H]TCDD-aryl hydrocarbon (Ah) receptor from wild-type Hepa 1c1c7 cells for 16 h at 4 degrees C in 0.4 M KCl resulted in the formation of transformed liganded receptor which exhibited increased binding affinity on DNA-Sepharose columns. The elution properties of the peak with the highest DNA binding affinity were similar to the elution profiles of the nuclear receptor complex isolated from wild-type cells. TAOc1BPrcl (class I) nonresponsive mutant cells were characterized by relatively low levels of the cytosolic and nuclear Ah receptor complex. The BPrcl (class II) variant cell line contained levels of cytosolic receptor which were comparable to those observed in the wild-type cells; however, significantly reduced levels of nuclear receptor complex were observed in the class II variant cell line. Incubation of the nuclear or transformed liganded cytosolic Ah receptor from wild-type cells with a consensus 32P-labeled dioxin responsive element (DRE) in a gel shift assay gave a retarded band associated with the receptor-DRE complex. Incubation of the cytosolic receptor complex from the class I and II mutant cells for 16 h at 4 degrees C in 0.4 M KCl or for 2 h at 20 degrees C did not yield complexes with increasing binding affinities on DNA-Sepharose columns. Moreover, incubation of these complexes with 32P-labeled DRE did not give a retarded band in a gel shift assay. However, coincubation of the liganded class II mutant cytosol with cytosol from class I cells resulted in transformation of the liganded receptor and this was confirmed in both the DNA-Sepharose and gel retardation assays. These results suggest that the failure of class II mutant cells to respond to TCDD is due to a defect in the factors responsible for transformation of the cytosolic receptor complex.
Insights
This study investigated how the aryl hydrocarbon (Ah) receptor complex transforms to activate CYP1A1 gene transcription. Class II mutant cells, lacking a factor for receptor transformation, fail to respond to dioxin (TCDD).
Area of Science:
- Molecular Biology
- Toxicology
- Cell Biology
Background:
- Mouse hepatoma Hepa 1c1c7 cells and mutants are models for CYP1A1 gene induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
- The aryl hydrocarbon (Ah) receptor mediates cellular responses to TCDD.
Purpose of the Study:
- To elucidate the molecular mechanism of Ah receptor complex transformation and its role in CYP1A1 gene induction.
- To identify defects in nonresponsive mutant cell lines.
Main Methods:
- Utilized DNA-Sepharose chromatography to assess receptor binding affinity.
- Employed gel shift assays to detect receptor-dioxin responsive element (DRE) complex formation.
- Compared wild-type Hepa 1c1c7 cells with class I (TAOc1BPrcl) and class II (BPrcl) mutant cell lines.
Main Results:
- Wild-type Ah receptor transformed into a DNA-binding form after incubation in high salt.
- Class I and II mutants showed defective receptor transformation and DNA binding.
- Class II mutant cytosol, when combined with class I cytosol, restored receptor transformation and DNA binding.
Conclusions:
- Class II mutant cells' unresponsiveness to TCDD is due to a defect in factors required for cytosolic Ah receptor complex transformation.
- A factor present in class I mutant cytosol is essential for the transformation of the liganded Ah receptor.
