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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.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev, which is required for the cytoplasmic expression of unspliced and incompletely spliced viral mRNAs, is located predominantly in the nucleolus. In this study, we show that Rev translocates from the nucleolus to the cytoplasm in HeLa and COS cells transfected with Rev under conditions where rRNA synthesis is inhibited (e.g., with actinomycin D). Dominant-negative mutants with mutations in the activation domain of Rev, which are known to inhibit wild-type Rev function in trans, are unable to leave the nucleus upon actinomycin D treatment. More importantly, when present in excess, these mutants inhibit the translocation of wild-type Rev. This correlation of inhibitory activities suggests that Rev function depends on its transport to and presence (at least transient) in the cytoplasm. In this context, we discuss the possibility that Rev is actively involved in the transport of HIV-1-specific mRNAs containing the Rev response element (a highly structured RNA sequence, which is specifically recognized by the Rev trans-activator). We also discuss the potential of nucleocytoplasmic export of Rev as a target for anti-HIV chemotherapy.

In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular protein (colorimetric assay). The quantitative and qualitative determination of intracellular IL-8 was achieved by immunofluorescence using the ELF-detection system (Molecular Probes, Eugene, OR), which is approximately 10 times more sensitive than conventional immunofluorescence detection systems. Thus, a 32× objective magnification (without immersion oil) is sufficient to precisely assess the subcellular localization of IL-8. Experiments were carried out with human umbilical vein endothelial cells. Drugs interfering with transcription or translation and inhibitors of protein kinase C inhibited both production and secretion of IL-8. Brefeldin A (BFA), colchicine, and the HMGCoA-reductase inhibitor fluvastatin disrupted the characteristic Golgi localization of IL-8, but only BFA inhibited its secretion. This assay can therefore be used to distinguish drugs that inhibit both IL-8 synthesis and secretion from those that inhibit IL-8 secretion only. It can easily be adapted to other cellular proteins for which a sensitive detection method is required.

Chemolrines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not yet clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilised on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1α on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.