Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

被引：19

作者：

Wolff, B ^{[1
]}

Billich, A ^{[1
]}

Brunowsky, W ^{[1
]}

Herzig, G ^{[1
]}

Lindley, I ^{[1
]}

Nussbaumer, P ^{[1
]}

Pursch, E ^{[1
]}

Rabeck, C ^{[1
]}

Winkler, G ^{[1
]}

机构：

[1] Novartis Forschungsinst, A-1235 Vienna, Austria

来源：

ANALYTICAL BIOCHEMISTRY | 2003年 / 318卷 / 02期

关键词：

steroid sulfatase; enzyme assay; 4-methylumbelliferyl sulfate; estrone-3-O-sulfamate;

D O I：

10.1016/S0003-2697(03)00256-2

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2003 Elsevier Science (USA). All rights reserved.

引用

页码：276 / 284

页数：9

共 31 条

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