Figure 1
FTY720-P is produced in the nucleus of breast cancer cells by SphK2. Breast cancer cell lines, murine 4T1 (a, b), human MDA-MB-231 (c, d) and human MCF7 (f) were treated with 5 μM FTY720. (a, c) Nuclear levels of FTY720-P and S1P were determined by liquid chromatography, electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) at the indicated times. Equal amounts of protein from nuclear and cytosolic fractions were analyzed by immunoblotting with SphK2 antibody. Antibodies against histone H3 or laminA/C and tubulin were used as nuclear and cytosol markers. (b, d) Total intracellular and secreted FTY720-P were determined in 4T1 cells after 8 h and MDA-MB-231 cells after 6 h of FTY720 treatment, respectively. MDA-MB-231 cells (e) and MCF7 cells (g) transfected with vector, SphK2 or catalytically inactive SphK2G212E (ciSphK2) were treated with vehicle or 5 μM FTY720 for 6 and 24 h, respectively. Nuclear levels of FTY720-P and S1P were determined by LC-ESI-MS/MS. Data are mean±s.d. *P<0.005 compared with vector; #P<0.005 compared with vehicle. Equal expression of nuclear SphK2 was confirmed by immunoblotting.
