Abstract
125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN.
Highlights
K662 transferrin receptor antibodies that inhibit transferrin bindand Daudicells markedly increasedafter exposure of ing has been shownto block cellularproliferation (19,ZO). the cells to culture conditions that stimulated prolif- Interferons (IFNs') are a protein family with a wide range eration.Treatmentofthese cells withinterferon-a of biologicaleffects on cells,including inhibition of viral (IFN-a) resulted in concurrent inhibiotfiocnell growth replication, inhibition of cell growth,and modulation of the and of the rise in transferrin binding
Binding sites occurs in lymphocytes undergoing mitogen-or Ce2b"uman lymphoblastoid K562 [35] and Daudi [36] cells were antigen-inducedproliferation [13,14,15,16],as well as in stationary routinely propagated in suspension in RPMI 1640 medium supplecells exposedto conditions that stimulate proliferation (&,lo, mented with 10%fetal calf serum (Eurobio Laboratories, France)
Aliquots of the final preparation of radioiodinated transferrin were precipitated by 10%trichloroacetic acid, which demonstrated that 95% of the counts tion (Fig. 1B). These results are in good agreement with all previous reports showing that transferrin receptor expression on the cell surface is strongly correlated with the cell proliferation rate
Summary
IFN-a(Le) or IFN-aA reduces the number of their transferrin receptors. Transferrin, a serum glycoprotein,is the major ironcarrier in vertebrates [1].The first step in iron transport across the EXPERIMENTALPROCEDURES plasma membrane is the binding of diferric transferrin to M ~ ~ e r ~ s - P u r h~uemdan transferrin and p h ~ ohemagglut ~ n specific,high affinity receptors (reviewed in Ref. 2). Aliquots of the final preparation of radioiodinated transferrin were precipitated by 10%trichloroacetic acid, which demonstrated that 95% of the counts tion (Fig. 1B) These results are in good agreement with all previous reports showing that transferrin receptor expression on the cell surface is strongly correlated with the cell proliferation rate. Transferrin, 1 X lo K562 cells, Daudi cells, or lymphocytes were washed once with RPMI medium at 20 "C and resuspended in 200 pl of cold RPMI 1640 medium containing 1 mg/ml BSA The number cells, both types of inhibition were significant with 250 IU of IFN. Nonspecific binding was determined by allowing the binding to proceed in the presence of a 1,000-fold excess of unlabeled iron-loaded dependent on the concentration of IFN and compared the dose-response curve for its antiproliferative effect with the curve for its inhibition of 1251-transferrinbinding (Fig. 2). Cells were permeabilized a t 4 "Cin 50 mM Tris.HC1 (pH7.2), 150mM NaC1, 1.5 mM MgClZ, mM phenylmethylsulfonyl fluoride, 0.5 mg/ml BSA, and 0.1% Triton nated by the fact that theaddition of 250 IU of IFN-a(Le)or IFN-aA to the binding assay at 4 "C did not modify lZ5Itransferrin binding (data not shown)
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