Dynamic analyses of lymphoblast membranes exposed to alpha interferon using flow cytometry and fluorescence recovery after photobleaching.
Interferons represent a major group of the biologic response modifiers which exert multipotent effects upon cell growth, cyto-differentiation and immune functions. Previous experimental studies with alpha interferon (IFN-alpha) have suggested that modulation of transmembrane signaling could be a critical determinant in the bioregulatory diversity. To determine whether any initial changes at the plasma membrane would directly correlate with one or more actions of IFN-alpha, we investigated cultures of Daudi lymphoblasts which are uniquely susceptible to growth inhibition. Complementary biophysical techniques were applied. In one approach, changes in plasma membrane ion flux were measured by flow cytometry, using a fluorescent dye indicator of membrane potential: Cells briefly exposed (5-10 min) to a DNA-recombinant IFN-alpha 2 (100 to 800 U/ml) manifested a consistent plasma membrane hyperpolarization (-60 to -90 mV) which could be blocked by ouabain. In a second approach, changes in diffusion coefficients of plasma membrane-associated macromolecules were determined by measuring the fluorescence redistribution after pulse photobleaching (FRAP): Individual plasma membrane proteins (sIgM, Leu 12 or Leu 16) were labelled with FITC conjugated goat antibodies [F(ab')2 or Fab'] or with phycoerythrin-B conjugated monoclonal mouse antibodies. Statistical comparisons of cells exposed to IFN-alpha 2 for 10 to 30 min showed immediate 27 to 88% increases in mean lateral diffusion rates. Mutant Daudi cells, cloned for resistance to growth inhibition showed no plasma membrane hyperpolarization with IFN-alpha 2 (up to 1000 U/ml), and baseline lateral diffusion coefficients matched those of IFN-alpha 2-treated, non-resistant cells. We conclude that biophysical status and responses of the plasma membrane must be closely linked to the molecular mechanisms of anti-proliferative signal transduction.
Balint
E
E
F. Edward Hebert School of Medicine, USUHS, Bethesda, MD 20814.
Aszalos
A
A
Pine
P S
PS
Grimley
P M
PM
D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology*; D008214:Lymphocytes / Q000378:metabolism / Q000648:ultrastructure*; D008223:Lymphoma / Q000378:metabolism / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects; D010042:Ouabain / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D007370:Interferon Type I; D010042:Ouabain