The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane-water-lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D3 and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-gamma (IFN-gamma) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-gamma treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-gamma resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.

The effect of various tyrosine protein kinase inhibitors on processes involved in the antiproliferative effect of interferon-gamma on WISH cells was studied. Following 24 hr treatment interferon-gamma inhibited thymidine incorporation into DNA and thymidine kinase activity, but no significant effect on cell number was observed. The isoflavonoid, genistein, which is a specific inhibitor of tyrosine protein kinase, reversed the inhibition in thymidine incorporation caused by the cytokine in a dose dependent manner. Prunetin, a member of the same group, did not significantly antagonize this effect. N alpha-tosyl-L-lysyl-chloromethane, a serine protease inhibitor which also serves as a tyrosine protein kinase inhibitor, partially reversed the effect of interferon-gamma at a concentration of 100 microM. The bioflavonoid, quercetin, a non-specific tyrosine protein kinase inhibitor, at a concentration of 30 microM completely abolished the action of interferon-gamma on thymidine incorporation. Genistein completely reversed the inhibition of thymidine kinase exerted by interferon, while quercetin had only a slight effect. However, the drugs could not antagonize the antiproliferative effect of interferon following 48 hr incubation, as measured by reduction of cell number. The results indicate that tyrosine protein kinase may play a role in the effects of interferon on thymidine metabolism and thymidine kinase activity. The differential effects of the inhibitors on thymidine metabolism and cell proliferation could support dissociation between the effect of interferon-gamma on these processes. Alternatively, this dissociation of effects could point to the limited use of inhibitors in clarifying modes of action as described.