Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant.
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.
Hanada
K
K
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan. hanak@nih.go.jp
Nishijima
M
M
Fujita
T
T
Kobayashi
S
S
D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D009154:Mutation; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis*; D013379:Substrate Specificity
D000596:Amino Acids; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D013107:Sphingolipids; C076078:sphingofungin B; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase