Intracellular Localization of Migration Inhibitory Factor-Related Protein (MRP) and Detection of Cell Surface MRP Binding Sites on Human Leukemia Cell Lines

Abstract

The migration inhibitory factor-related proteins (MRPs) MRP-8 and MRP-14 were detected in differentiated human leukemia cell lines (THP-1 and HL-60) by immunocytochemical analysis. They were induced and colocalized in the cytoplasm and in lesser amounts in the nucleus when THP-1 and HL-60 cells were induced to differentiate by 1alpha,25-dihydroxyvitamin D3 or retinoic acid. In a search for a protein capable of binding MRPs, both MRPs were individually produced in insect cells (Sf21) infected with recombinant baculovirus. The purified recombinant MRPs were electrophoretically and antigenically indistinguishable from the native proteins, and their ability to form the MRP8/14 complex was retained. The presence of MRP binding sites was investigated by a binding assay using recombinant MRPs and specific monoclonal antibodies. MRP binding sites were detected on the cell membrane of the human leukemia cell lines THP-1, Raji, and MOLT-4. HL-60 cells treated with 1alpha, 25-dihydroxyvitamin D3 did not express MRP binding sites on the cell membrane, but a high level of MRPs accumulated in the cells. The occurrence of MRP binding sites on the cell surface of leukemia cell lines of monocyte and lymphocyte origin suggests that MRPs, released from neutrophils under certain conditions, may contribute to the activation and recruitment of effector cells to inflammatory lesions.

Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions

“…Several binding sites for S100A8/A9 have been reported on various human leukemia [42] and endothelial cells [43][44][45][46][47]; however, the cell-surface receptor of S100A8/A9 is still in debate. The binding site(s) on the colon carcinoma cell lines by which S100A8/A9 induces its apoptotic effect are the issue of our current research.…”

Two Proteins Modulating Transendothelial Migration of Leukocytes Recognize Novel Carboxylated Glycans on Endothelial Cells

“…Recent studies show that S100A8/A9 binds to unsaturated fatty acids with high affinity and may be involved in arachidonic acid metabolism (30 -32). Cell surface S100A8/A9 binding sites have also been detected on human leukemia cell lines (6). S100A9 also is shown to modulate adhesion of neutrophils to fibrinogen via ␤ 2 integrin Mac1, by recognizing a distinct but uncharacterized pertussis toxin-sensitive G protein-coupled receptor on neutrophils (33).…”

“…The protein in the supernatant was adjusted to 2 mg/ml with the above buffer, and the solution stirred on ice. Solid ammonium sulfate was slowly added to a final concentration of 75% (w/v) and stirred for 1 h. The solution the was centrifuged at 10,000 ϫ g for 30 min and the supernatant dialysed against 3ϫ 4 liters of the above buffer for 24 h. The preparation was concentrated on YM10 membranes and applied to a 10-ml column of Phenyl-650C resin equilibrated in 0.1% trifluoroacetic acid (6). The unbound proteins were washed off with 3 column volumes of starting buffer and the bound proteins eluted with a gradient of 0 -50% acetonitrile in the starting buffer.…”

“…17). We exploited the high solubility of S100 proteins in ammonium sulfate (5) to obtain a preliminary enrichment of ϳ80% before further purification of the proteins (to ϳ95% purity) on a hydrophobic column (6). The proteins stayed as heteromeric complexes through purification, were even retained on YM10 and YM30 ultrafiltration membranes, and could not be separated into individual proteins without denaturation.…”

Retinoic Acid Increases Expression of the Calcium-Binding Protein S100P in Human Gastric Cancer Cells

“…Expression of S100A6 was induced during an early phase of cell differentiation in neuroblastoma cells induced to differentiation by RA [36]. Expression of S100A8 and S100A9 proteins were upregulated during RA-mediated differentiation of HL-60 and THP-1 leukemia cells [21]. Also, the increase in expression of S100 family proteins, such as MDR8, MDR14, is associated with differentiated phenotypes [28].…”