Requirements for the simultaneous presence of phorbol esters and calcium ionophores in the expression of human T lymphocyte proliferation-related genes

We have investigated the synergistic effects of phorbol ester and calcium ionophore on human T lymphocyte proliferation and the expression of the proliferation-related genes, c-myc, c-fos, interleukin 2 receptors (IL-2R) and interleukin 2 (IL-2). Incubation of T lymphocytes with both the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin, leads to the expression of a series of proliferation-related genes, followed by T cell proliferation. In contrast, stimulation of T cells sequentially with PDB and then ionomycin did not induce mitogenesis, demonstrating that simultaneous exposure to both agents is necessary for proliferation. Exposure of T cells to both agents together for different time periods resulted in a proliferative response in proportion to the duration of the exposure, with more than 6 hr required for maximum proliferation. In contrast, a 1-hr exposure to both drugs was sufficient for maximum expression of c-fos or c-myc proto-oncogene mRNA. The expression of IL-2R and the production of IL-2 were also dependent on the duration of simultaneous exposure to both phorbol ester and calcium ionophore. Levels of IL-2 mRNA became detectable at 1 hr and peaked at 3 hr after stimulation. The induction of IL-2 mRNA occurred only in the presence of both agents and became undetectable within 2 hr after the drugs were removed. In contrast, the expression of IL-2R mRNA became detectable at 1 hr, but was maintained even after the drugs were removed and reached a peak at 24 hr. Both IL-2 and IL-2R mRNA accumulated in proportion to the duration of the exposure. Augmentation of cell proliferation by exogenous IL-2 was observed in T cells exposed to the drugs for less than 3 hr. These data demonstrated that the induction of maximum expression of the nuclear proto-oncogenes c-myc and c-fos was not sufficient for PDB-ionomycin-induced T cell proliferation. The level of IL-2 mRNA accumulation and resultant IL-2 secretion is one of the limiting factors for proliferation of T cells exposed to the drugs for less than 3 hr, but not for longer exposures. Additional events such as accumulation of IL-2R mRNA and protein triggered by a long exposure to the drugs were obligatory for obtaining maximum proliferation.