Hanada K, Nishijima M, Fujita T, Kobayashi S
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan.
Biochem Pharmacol. 2000 May 15;59(10):1211-6. doi: 10.1016/s0006-2952(00)00251-3.
In the present study, we demonstrate a model cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) cell mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the mutant. Although LY-B cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 cells under sphingosine-supplied conditions, indicating that, in CHO cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. Cultivation of LY-B/cLCB1 cells in sphingolipid-deficient medium in the presence of 10 microM sphingofungin B and ISP-1 (myriocin, thermozymocidin), potent inhibitors of SPT activity, caused severe growth inhibition with approximately 95% inhibition of de novo sphingolipid synthesis. The growth inhibition by sphingofungin B and ISP-1 was rescued substantially by exogenous sphingosine, whereas the cytotoxicity of two other types of SPT inhibitor, L-cycloserine and beta-chloro-L-alanine, was hardly rescued. Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [(3)H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not L-cycloserine or beta-chloro-L-alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity.
在本研究中,我们展示了一种用于评估丝氨酸棕榈酰转移酶(SPT)抑制剂特异性的模型细胞系统,SPT是催化鞘脂生物合成第一步的酶。LY - B菌株是一种在SPT方面存在缺陷的中国仓鼠卵巢(CHO)细胞突变体,而LY - B/cLCB1菌株是该突变体的基因校正回复株。尽管LY - B细胞在鞘脂缺陷培养基中生长缓慢,但在提供鞘氨醇的条件下,其生长恢复到了LY - B/cLCB1细胞的水平,这表明在CHO细胞中,SPT失活引起的生长抑制几乎完全通过鞘脂的代谢互补得以挽救。在10微摩尔的鞘氨醇菌素B和ISP - 1(嗜热栖热放线菌素、杀稻瘟菌素)(SPT活性的强效抑制剂)存在的情况下,将LY - B/cLCB1细胞培养于鞘脂缺陷培养基中,会导致严重的生长抑制,从头合成鞘脂受到约95%的抑制。外源性鞘氨醇可显著挽救鞘氨醇菌素B和ISP - 1引起的生长抑制,而另外两种SPT抑制剂L - 环丝氨酸和β - 氯 - L - 丙氨酸的细胞毒性几乎无法通过外源性鞘氨醇挽救。在提供鞘氨醇条件下培养的SPT缺陷型LY - B细胞的生长中,也观察到了这些抑制剂类似的细胞毒性模式。这些SPT抑制剂不影响外源性[³H]鞘氨醇向复合鞘脂的代谢转化。因此,鞘氨醇菌素B和ISP - 1的细胞毒性,而非L - 环丝氨酸或β - 氯 - L - 丙氨酸的细胞毒性,很大程度上是由于通过抑制SPT活性来抑制鞘脂合成所致。