Oddmund Bakke

A flow cytometric and immunofluorescence microscopic study of tumor necrosis factor production and localization in human monocytes

The production and localization of tumor necrosis factor (TNF) in human monocytes were investigated by using monoclonal and polyclonal antibodies against recombinant human TNF together with flow cytometry and immunofluorescence microscopy. Lipopolysaccharide (LPS) induced a rapid and transient accumulation of TNF in perinuclear vesicles which was detected 20 min after the addition of LPS. The fluorescence intensity of the vesicles peaked at 40 min of LPS exposure, concomitantly with the release of TNF into the medium. Thus, our results indicate that the secretion of TNF is typical for secretory proteins as it involves passage through the secretory apparatus. Additional studies demonstrated that plasma membrane-associated TNF could not be detected in live monocytes not exposed to LPS. However, after 90 min with LPS, a small population of monocytes expressed membrane-associated TNF, and by 24 hr approximately 50% of the monocytes displayed TNF on the plasma membrane. Furthermore, our results indicate that plasma membrane-associated TNF does not represent released TNF bound back to its own receptor. Thus, our findings support the view that TNF exists as a surface trans-membrane protein in LPS-stimulated monocytes.

Glucocorticoid mechanism of action: Monoclonal antibodies as experimental tools

In odder to provide a further insight into glucocorticoid receptor (GR)-mediated action of glucocorticoid hormones, we produced ten monoclonal antibodies against rat GR. In studies combining physicochemical separation methods with antibody methodology, we established that the molybdate-stabilised GR contains one steroid-binding monomer. Using a monoclonal anti-GR antibody-based immunoaffinity chromatographic procedure, we purified two non-ligand-binding proteins, with molecular weights of 80,000 and 90,000, present in the molybdate-stabilised GR complex. These proteins are not recognised by monoclonal antibodies directed against GR. The possible relation of these two proteins to heat shock proteins remains to be established.Immunohistochemical studies of GR in the central nervous system of the rat provided new information on the distribution of GR, particularly in the hypothalamus. Studies of intracellular receptor localisation in rat brain after endocrine manipulations gave results in support of the classical concept of translocation of GR from cytoplasm to cell nucleus. Studies with a cell culture system also supported the existence of GR in the cytoplasm as well as in the cell nucleus.

Effects of potassium dichromate on the cell cycle of an established human cell line (NHIK 3025).

The effect of K2Cr2O7 exposure on the cell cycle phases of the human cell line NHIK 3025 has been studied. Inhibition of cell proliferation was found to depend on the concentration and length of exposure. An effect on cell proliferation was observed 6-9 h after addition of K2Cr2O7, whereas cell death was not observed until 3-4 days later. The cells were most sensitive in G2 phase. After exposure to 8 mumol/l K2Cr2O7 the greatest prolongation of the cell cycle was in G2 + M phase, but an increase of S phase was also observed. No prolongation of G1 phase could be measured. Our results thus indicate that K2Cr2O7 delays progression through the cell cycle.

Retinoic acid induces a specific membrane glycoprotein in human epithelial cell lines

Retinoic acid (RA) inhibits growth, increases the cytokeratin content, and alters the cytoskeleton of the human cervical cell line NHIK 3025. Using RA-treated NHIK 3025 cells as immunogen we prepared murine monoclonal antibodies (IgG1) which recognized an RA-induced cell-surface antigen which could not be detected in untreated NHIK 3025 cells. Analysis of the Triton soluble proteins by SDS-gel electrophoresis and immunoblotting revealed that the cell-surface antigen is a 140-kDa glycoprotein (gp140). gp140 was also shown to be induced by RA in HeLa S3 cells and constitutively expressed in the human trophoblast cell line BeWo. gp140 was also detected in other human epithelial cell lines, but not in human hematopoietic cells. Expression of gp140 was induced in HeLa S3 cells by nanomolar concentrations of RA, and in NHIK 3025 cells by micromolar amounts (1-10 microM). The glycoprotein was detectable 3-6 h following exposure to RA and its expression was reversible upon removal of RA from the medium. Our results indicate that gp140 is a newly identified RA-inducible epithelial membrane glycoprotein which may represent a phenotypic differentiation marker for epithelial cells.

Structure requirements for glucocorticoid growth inhibition of a human cell line (NHIK 3025)

Abstract The human cell line NHIK 3025 has a cytoplasmic dexamethasone receptor. When these cells are exposed to glucocorticoids, the cell cycle time is prolonged. The structural requirements for this effect were investigated by measuring cell number after 4 days exposure to different glucocorticoid analogues at concentrations of 10 −6 M and 10 −7 M. Growth inhibition at 10 −7 M required the 4–5 double bond, the 3,20 ketone—and the 11β, 17α and 21 hydroxygroups of the glucocorticoid structure, e.g. cortisol, dexamethasone and prednisolone. Other synthetic glucocorticoids like bimetrazol and triamcinolone acetonide were also active at this dose whereas corticosterone, lacking the 17α hydroxygroup, was only active at 10 −6 M. The effect was steroid specific and could be inhibited by anti-cortisol antiserum or by glucocorticoid antagonists.

The Association of the Glucocorticoid Receptor with Mr 90,000 Heat Shock Protein and Tubulin

The rat liver glucocorticoid receptor (GR) is associated with an Mr ~ 90 000 heat shock protein (hsp90) which in vitro has been shown to affect the functional status of GR. When GR is associated with a diner of hsp90 it is unable to bind to DNA. By a ligand- and temperature-dependent process GR dissociates from hsp90 and becomes functionally active, in terms of capacity to bind to specific and non-specific DNA-sequences. Hsp90 interaction with GR has been shown to require at least the presence of an intact steroid binding domain of GR. After in vitro expression of rat GR in a reticulocyte lysate system GR interacts with rabbit hsp90 preexisting in the lysate.