Prolonged Survival of Chronic Lymphocytic Leukemia Cells Induced by Sphingosine 1-Phosphate Is Mediated by the G Protein-Coupled Receptor S1P1 and Involves Erk/MAP Kinase, but Not Akt Signaling.

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator which is generated by the hydrolysis of sphingomyelin and released predominantely by activated platelets but also by other cells upon activation. In normal blood plasma it is found in nanomolar to micromolar concentrations. By qualitative and quantitative (TaqMan) RT-PCR, mRNA expression of the S1P receptor S1P1 was detected in CLL cell lines (EHEB, MEC-1) and in all samples of primary chronic lymphocytic leukemia (B-CLL) cells, analyzed after isolation with a CD19 antibody and magnetic cell sorting. mRNA expression of other S1P receptors was less consistently found. When primary CLL cells were cultured in serum-free medium (RPMI 1640 supplemented with fatty acid-free BSA), viability decreased starting from 3 days of culture and was completely lost after 1–2 weeks depending on the individual sample. However, when the cultures were supplemented with S1P (1 nM - 5 μM) the number of viable cells was increased up to 8-fold in a time- and dose-dependent manner as measured using the WST-1 reagent. Moreover, S1P induced a rapid (maximum at 1 min) phosphorylation of the p44/42 Erk/MAP kinase in CLL cell lines and even more prominent in primary CLL cells. The time- and dose dependent phosphorylation was blocked by pretreatment (24h) of the cells with FTY720 (10–100 nM), a compound that binds predominantly to S1P1 and induces loss of cell surface expression by receptor internalization. FTY720 also abrogated the prolonged survival of CLL cells in cultures supplemented with S1P, demonstrating that both the Erk/MAP kinase phosphorylation and the consecutive increased survival were mediated by the S1P1 receptor. Activation of MAP kinase was also inhibited by pertussis toxin, which selectively blocks Gi proteins, the only class of G proteins known to be coupled to S1P1. Phosphorylation of Akt was not observed in CLL cell lines and primary CLL cells after stimulation with S1P. B-CLL cell lines showed a pronounced chemotactic response to S1P at low concentrations. In primary CLL cells however, S1P did neither induce phosphorylation of the focal adhesion kinase (FAK) nor chemotaxis. We conclude that S1P, constitutively present in the blood, prolongs survival of CLL cells involving the Erk/MAP kinase pathway. Since compounds that induce S1P1 receptor internalization like FTY720 are already used as immunosuppressants in humans, our results suggest inclusion of these drugs also in the treatment of B-CLL, where they may potentiate the effect of conventional antiproliferative therapy.