Aberrant expression of sphingosine kinase and its pathological significance of malignant tumors and neuronal degenerative diseases

1.采用定量RT-PCR方法检测骨髓增生异常综合征（MDS）和急性白血病患者鞘脂代谢酶基因表达。结果表明，鞘磷脂激酶1 （SPHK1）在高危MDS和急性白血病中升高，中性鞘磷脂酶2 （NSMase2）在高危MDS和急性白血病骨髓样品中降低。结果表明，SPHK1信息、SPHK1蛋白和SPHK酶活性与抗癌药物柔红霉素（dada）的IC50有良好的相关性。采用LC/MS-MS（液相色谱-串联质谱法）测定鞘脂代谢产物。DA处理可调节各鞘脂代谢物水平。DA的IC50和SPHK的表达水平存在显著的异质性。在低剂量DA处理下，与DA抗性细胞株相比，DA敏感细胞株神经酰胺含量显著增加，鞘氨醇1-磷酸（S1P）含量显著降低。神经酰胺/S1P的比值随DA的IC50剂量变化较大。我们的研究结果首次表明，Spiegel等人提出的鞘脂变阻器模型可以应用于血液恶性肿瘤。利用神经胶质细胞系衍生神经营养因子（glial cell line derived neurotrophic factor， GDNF）反应性神经母细胞瘤细胞系TGW，我们发现SPHK1随着GDNF的增加而增加，并且这种反应是通过GDNF受体RET介导的，因此认为REF基因突变是MEN2型肿瘤的发病机制。我们也证实了SPHK1基因在MEN2型肿瘤中表达升高，SPHK1基因表达升高参与了其肿瘤的发生。

1.Sphingolipid metabolic enzymes gene expression of myelodysplastic syndrome (MDS) and acute leukemia was measured by quantitative RT-PCR method. It was shown that sphingosine kinase 1 (SPHK1) was increased in high risk MDS and acute leukemia and that neutral sphingomyelinase 2 (NSMase2) was decreased in bone marrow samples from high risk MDS and acute leukemia Using established leukemia cell lines, further analysis was performed. It was revealed that SPHK1 message, SPHK1 protein and SPHK enzyme activity were well correlated with IC50 of anti-cancer drug, daunorubicin (DA) . Sphingolipid metabolites were measured by LC/MS-MS (liquid column chromatography followed by tandem mass spectrometry) . DA treatment modulated each sphingolipid metabolite level. There was a remarkable heterogeneity of IC50 of DA as well as SPHK expression level. With low dose of DA, DA-sensitive cell lines showed remarkable increase of ceramides as well as decrease of sphingosine 1-phosphate (S1P) compared with DA-resistant cell lines. The ratio of ceramide/S1P was changed greatly with IC50 dose of DA. Our results showed, for the first time, that sphingolipid rheostat model proposed by Spiegel, et. al. can be applied in hematological malignancies.2.Using a GDNF (glial cell line derived neurotrophic factor) -responsive neuroblastoma cell line, TGW, we showed that SPHK1 was increased with GDNF and that the response was mediated through the GDNF receptor, RET. Mutated REF gene was recognized as the pathogenesis of MEN2 type tumors. We also proved that SPHK1 gene expression was increased in MEN2 type tumors and that increased SPHK1 gene expression was involved in its oncogenesis.