Barbara Wolff

LEPTOMYCIN B IS AN INHIBITOR OF NUCLEAR EXPORT : INHIBITION OF NUCLEO-CYTOPLASMIC TRANSLOCATION OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) REV PROTEIN AND REV-DEPENDENT MRNA

BACKGROUND The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev is required for unspliced and incompletely spliced viral mRNAs to appear in the cytoplasm and thus for viral replication. Translocation of Rev from the nucleus to the cytoplasm is essential if Rev is to function. We wanted to identify inhibitors of this transport process because they would be potential antiviral agents. RESULTS The Streptomyces metabolite, leptomycin B, and other antibiotics of the leptomycin/kazusamycin family were identified as inhibitors of the nucleo-cytoplasmic translocation of Rev at nanomolar concentrations. Rev-dependent export of mRNA into the cytoplasm is also blocked by leptomycin B, which inhibits Rev-dependent, but not Rev-independent gene expression in a short-term transfection assay. In primary human monocytes, leptomycin B suppresses HIV-1 replication. CONCLUSIONS Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.

Some aspects of IL-8 pathophysiology III: chemokine interaction with endothelial cells

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.

1α,25-Dihydroxyvitamin D3 decreases DNA binding of nuclear factor-κB in human fibroblasts

1α,25‐Dihydroxyvitamin D3 (1,25‐(OH)2‐D3), the active metabolite of vitamin D, can inhibit NF‐κB activity in human MRC‐5 fibroblasts, targeting DNA binding of NF‐κB but not translocation of its subunits p50 and p65. The partial inhibition of NF‐κB DNA binding by 1,25‐(OH)2‐D3 is dependent on de novo protein synthesis, suggesting that 1,25‐(OH)2‐D3 may regulate expression of cellular factors which contribute to reduced DNA binding of NF‐κB. Although NF‐κB binding is decreased by 1,25‐(OH)2‐D3 in MRC‐5 cells, IL‐8 and IL‐6 mRNA levels are only moderately downregulated, demonstrating that inhibition of NF‐κB DNA binding alone is not sufficient for optimal downregulation of these genes.

Inhibition of Vascular Endothelial Growth Factor Cotranslational Translocation by the Cyclopeptolide CAM741

The cyclopeptolide CAM741 inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide. We now describe the identification of the signal peptide of vascular endothelial growth factor (VEGF) as the second target of CAM741. The mechanism by which the compound inhibits translocation of VEGF is very similar or identical to that of VCAM1, although the signal peptides share no obvious sequence similarities. By mutagenesis of the VEGF signal peptide, two important regions, located in the N-terminal and hydrophobic segments, were identified as critical for compound sensitivity. CAM741 alters positioning of the VEGF signal peptide at the translocon, and increasing hydrophobicity in the h-region reduces compound sensitivity and causes a different, possibly more efficient, interaction with the translocon. Although CAM741 is effective against translocation of both VEGF and VCAM1, the derivative NFI028 is able to inhibit only VCAM1, suggesting that chemical derivatization can alter not only potency, but also the specificity of the compounds.

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.