Characterization of the Ah receptor and aryl hydrocarbon hydroxylase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benz(a)anthracene in the human A431 squamous cell carcinoma line.
Certain human cell lines previously have been shown to exhibit substantial induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P450IA1) when treated in culture with aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz(a)anthracene. Yet the Ah receptor, which is known to mediate the AHH induction process in rodent cells and tissues, has not previously appeared to be present at a significant level in any human cell line. In the human A431 squamous cell carcinoma line we found that cytosolic Ah receptor was present in high concentration (approximately 200 fmol/mg cytosol protein at maximal saturation); this corresponds to approximately 10,000 Ah receptor sites per cell in the human A431 line compared with about 35,000 receptor sites per cell in the mouse Hepa-1 hepatoma cell line in which Ah receptor previously has been extensively characterized. Detection of Ah receptor in A431 cytosol required modification of assay techniques, especially reduction in the amount of charcoal used to adsorb nonspecifically bound radioligand. The specific binding peak from A431 cytosol sedimented approximately 9S on sucrose gradients, the same as the cytosolic receptor from the well-characterized mouse Hepa-1 hepatoma cell line. In addition to [3H]TCDD, specific binding to Ah receptor in A431 cytosol also was detected with [3H]3-methylcholanthrene and with [3H]benzo(a)pyrene as radioligands. A specific [3H]TCDD-Ah receptor complex was extracted from nuclei of A431 cells incubated in culture at 37 degrees C with [3H]TCDD. The nuclear form of Ah receptor sedimented approximately 5S, the same as the nuclear receptor from mouse Hepa-1 cells. AHH activity was induced in A431 cells treated in culture with TCDD or benz(a)anthracene. The maximum level of induced AHH activity that could be achieved in A431 cells was about 20% of the maximally induced level in the mouse Hepa-1 cell line. However, the dose-response curves for AHH induction by TCDD or benz(alpha)anthracene in A431 cells were shifted about one log unit to the right of the curves for Hepa-1 cells. The lower sensitivity of A431 cells to AHH inducers was in proportion to the lower affinity with which cytosolic Ah receptor in A431 cells bound [3H]TCDD. The saturation curve for binding of [3H]TCDD to cytosolic Ah receptor in A431 cells also was shifted about one log unit to the right of the curve for saturation of the cytosolic receptor from mouse Hepa-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Harper
P A
PA
Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
Golas
C L
CL
Okey
A B
AB
D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis*; D001551:Benz(a)Anthracenes / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000032:analysis*; D002467:Cell Nucleus / Q000032:analysis; D003600:Cytosol / Q000032:analysis; D004147:Dioxins / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D008748:Methylcholanthrene / Q000378:metabolism; D008982:Molybdenum / Q000494:pharmacology; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism / Q000494:pharmacology*; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000032:analysis* / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000187:drug effects
D001551:Benz(a)Anthracenes; D004147:Dioxins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D011965:Receptors, Glucocorticoid; C044659:molybdate; D008748:Methylcholanthrene; D008982:Molybdenum; C030935:benz(a)anthracene; D001189:Aryl Hydrocarbon Hydroxylases