title: Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout.
abstract: Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.
authors.affiliation: Novartis Forschungsinstitut, Brunner Strasse 59, A-1235 Vienna, Austria. barbara.wolff-winiski@pharma.novartis.com
authors.forename: Barbara
authors.initials: B
authors.lastname: Wolff
authors.forename: Andreas
authors.initials: A
authors.lastname: Billich
authors.forename: Waltraud
authors.initials: W
authors.lastname: Brunowsky
authors.forename: Gerda
authors.initials: G
authors.lastname: Herzig
authors.forename: Ivan
authors.initials: I
authors.lastname: Lindley
authors.forename: Peter
authors.initials: P
authors.lastname: Nussbaumer
authors.forename: Edith
authors.initials: E
authors.lastname: Pursch
authors.forename: Christa
authors.initials: C
authors.lastname: Rabeck
authors.forename: Gottfried
authors.initials: G
authors.lastname: Winkler
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D045744:Cell Line, Tumor; D018914:Cells, Immobilized; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008832:Microchemistry / Q000379:methods; D015394:Molecular Structure; D051381:Rats; D012680:Sensitivity and Specificity; D043266:Steryl-Sulfatase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D043266:Steryl-Sulfatase