Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180.
The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.
Harper
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Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, Ontario, Canada.
Prokipcak
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Bush
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Golas
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Okey
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D000345:Affinity Labels; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism* / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000345:Affinity Labels; D001551:Benz(a)Anthracenes; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C030935:benz(a)anthracene; D001189:Aryl Hydrocarbon Hydroxylases