FTY720 mediates activation suppression and G0/G1 cell cycle arrest in a concanavalin A-induced mouse lymphocyte pan-activation model

Abstract

FTY720 is a potent drug for multiple sclerosis treatment. To biologically address its possible applications to more generalized diseases with aberrant inflammation, we are testing whether FTY720 can function as a lymphocyte cell cycle blocker and activation suppressor via a concanavalin A (ConA)-mediated mouse lymphocyte pan-activation model. Mouse lymphocytes were obtained from lymph nodes and subjected to ConA and/or FTY720 treatment. Cell viability was assayed by MTT and mitochondrial assays. Early and late activation, cell cycle, proliferation and intracellular Ca(2+) concentration ([Ca(2+)](i)) were analyzed by flow cytometry. At concentrations of less than 500 nM, FTY720 significantly down-regulated both CD69 and CD25 expressions of T cells, as well as inhibiting proliferation of activated lymphocytes. In addition, FTY720 blocked the ConA-induced mitogenesis, exhibiting lymphocyte G(0)/G(1) phase cell cycle arrest with significant reduction of cells in S and G(2)/M phases. Meanwhile, a significant decline in [Ca(2+)](i) was observed. The correlation of [Ca(2+)](i) and cell cycle arrest were validated by employing a [Ca(2+)](i) inhibitor SK&F 96365 and testing with and without FTY720 treatment. We demonstrated that FTY720 induces G(0)/G(1) phase cell cycle arrest, resulting in proliferation inhibition upon lymphocyte pan-activation, which may be related to reduction of overall intracellular Ca(2+) load.