title: CYP1B1 detection.
abstract: This unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real-time PCR (qRT-PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7-ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3',5'-Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC₅₀ of 3 nM for EROD and ~90 nM for E2 4-hydroxylation. Binding studies with purified CYP1B1 suggests that TMS interferes in the proximity of the heme region of CYP1B1 with high affinity. Compared to other potent inhibitors such as α-naphthoflavone, which is a known CYP1 family inhibitor with no selectivity between CYP1B1 and CYP1A2, TMS is ~50- and 520-fold selective for inhibition of CYP1B1 when compared to CYP1A1 and CYP1A2, respectively. Thus, TMS can serve as a helpful chemical scalpel for dissecting CYP1B1 activity from the overall activity of CYP1 family members against ethoxyresorufin.
authors.affiliation: Methods and Technologies Branch, Division of Cancer Control and Population Sciences, National Cancer Institute, NIH, Bethesda, Maryland, USA.
authors.forename: Rao L
authors.initials: RL
authors.lastname: Divi
authors.forename: Andreas
authors.initials: A
authors.lastname: Luch
authors.forename: Mukesh
authors.initials: M
authors.lastname: Verma
authors.forename: Brinda
authors.initials: B
authors.lastname: Mahadevan
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D065633:Cytochrome P-450 CYP1B1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010078:Oxazines / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D013267:Stilbenes / Q000494:pharmacology; D014116:Toxicology / Q000379:methods*
chemical_list: C440850:2,4,3',5'-tetramethoxystilbene; D004791:Enzyme Inhibitors; D010078:Oxazines; D013267:Stilbenes; D004958:Estradiol; C007649:ethoxyresorufin; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1