Study On SphK-1 Signal Pathway Actived By P210～(bcr/abl)

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210^bcr/abl fusion protein, which is generated by the reciprocal translocation t(9;22), inhibits apoptosis and increase proliferation. P210^bcr/abl plays a central role in the pathophysiology of CML. Sphingomyelin is not only a structural component of cellular membranes but also serves as the precursor for the potent bioactive lipids, such as ceramide and sphingosine 1-phosphate (SIP). Ceramide and sphingosine usually inhibit proliferation and promote apoptosis, while the further metabolite S1P, which acts as not only as an extra cellular mediator but also as an intracellular second messenger, stimulates proliferation and inhibits cell apoptosis in various types of cells. The balance between the cellular concentrations of ceramide and S1P determines whether a cell proliferates or undergoes apoptosis. S1P is formed by phosphorylation of sphingosine catalyzed by sphingosine kinase-1 (SphK-1). And SphK-1 can be activated by a number of growth factors and intracellular oncoproteins. Although substantial progress has been made in the characterization of P210^bcr/abl dependent signaling pathways, whether SphK-1 is involved in the P210^bcr/abl signal and its role in the pathology of CML remain unclear. The goal of this study was to determine the regulatory roles of SphK-1 inP210^bcr/abl oncoprotein signals.Firstly, we detected the expression of SphK-1 and S1P receptor in bcr/abl positive cells by using RT-PCR. Both bcr/abl positive K562 cell line and primary cells from peripheral blood of CML chronic phase patients expressed SphK-1 and S1P receptor mRNA. To determine whether P210^bcr/abl involves in activating SphK-1, we employed STI571, the specific inhibitor of P210^bcr/abl, to inhibit the P210^bcr/abl tyrosine kinases of K562 cells and primary CML cells, and then assayed the intracellular SphK-1 activity. SphK-1 activity in K562 cells was reduced by STI571 (36.7%⁵5.8% decrease). SphK-1 activity in CML cells also was reduced by STI571 (16.8%⁴1.9% decrease).After we had found that STI571 could inhibit SphK-1 activity in K562 cells and CML cells, we construct bcr/abl expression vector to study if P210bcr/abl can activate SphK-1 directly. Full-length b3a2 bcr/abl cDNA was subcloned into the pLXSN expression vector to generate the pLXSN-P210 plasmid. pLXSN-P210 and pLXSN plasmid was transfected into packing cells to get recombinant retrovirus. Mo7e cells were infected with recombinant retrovirus supernatant. The SphK-1 activity increaseed(23³2) % in the Mo7e cell line transfected with bcr/abl gene. Then we further confirmed the effect of P210bcr/ab! on activatation of SphK-1 in the Tet-off inducible expression system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to generate the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasimid and selected the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. Individual subclones were tested for the inducible expression of bcr/abl after withdrawing doxycycline. The selected gene-transfected 293 cell line clone that can induce/inhibit bcr/abl expression without/with doxycycline was treated to measure SphK-1 activity. In the Tet-off inducible expression system for constitutively bcr/abl expression in 293 cell line, p21obcr/abi can increase the SphK_i activity about (46.5 + 5.3) %.Raf-MEK-ERKl/2 pathway and PI-3K/Akt pathway have already been proved to play important roles in the downstreams of P210bcr/abl signal cascades. In order to investigate whether the Raf-MEK-ERKl/2 pathway and PI-3K/Akt pathway mediate that P210bcr/abl activate SphK-1. We treated K562 cells and CML cellswith STI 571 and confirmed that the phosphoration of ERK1/2 were inhibited. Then we treated Mo7e-P210 cells and 293-P210 cells with PD98059 and Wortmannin, the specific inhibitors for ERK1/2 pathway and PI-3K pathway respectively, and measured the intracellular SphK-1 activity. Neither in the Mo7e-P210 cells nor in the 293-P210 cells, PD98059 and Wortmannin didn't affect the SphK-1 activity. Those results indicated that the Raf-MEK-ERKl/2 and PI-3K/Akt pathway are dispensable for activation of SphK-1...