Induction of human interferon-gamma mRNA analyzed by cytoplasmic dot hybridization: effect of Con A, TPA, and A23187.

The cytoplasmic dot hybridization method was improved by eliminating non-specific false signals. The non-specific binding to a probe disappeared almost completely when the hybridized nitrocellulose sheet was treated with pronase. Using this improved method we demonstrated that reagents such as 12-O-tetradecanoylphorbol-13-acetate (TPA), concanavalin A (Con A) and a calcium ionophore (A23187) could induce the expression of IFN-gamma gene of a human T-lymphoblastoid cell line, TCL-Fuj 2M, where TPA functioned synergistically with Con A and A23187. However, when the cells were cultured with the ionophore for more than one day the amount of IFN-gamma mRNA was markedly reduced to a low level.