Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout.
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.
Wolff
Barbara
B
Novartis Forschungsinstitut, Brunner Strasse 59, A-1235 Vienna, Austria. barbara.wolff-winiski@pharma.novartis.com
Billich
Andreas
A
Brunowsky
Waltraud
W
Herzig
Gerda
G
Lindley
Ivan
I
Nussbaumer
Peter
P
Pursch
Edith
E
Rabeck
Christa
C
Winkler
Gottfried
G
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
D004791:Enzyme Inhibitors; D043266:Steryl-Sulfatase