Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids.
We previously isolated a temperature-sensitive Chinese hamster ovary cell mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 cells near to the parental levels through metabolic utilization and allowed the mutant cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 cell growth was due to the lack of cellular sphingolipids, possibly that of sphingomyelin.
Hanada
K
K
Department of Chemistry, National Institute of Health, Tokyo, Japan.
Nishijima
M
M
Kiso
M
M
Hasegawa
A
A
Fujita
S
S
Ogawa
T
T
Akamatsu
Y
Y
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival; D006224:Cricetinae; D014644:Genetic Variation; D007700:Kinetics; D009154:Mutation; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology*; D013109:Sphingomyelins / Q000494:pharmacology; D013696:Temperature
D013107:Sphingolipids; D013109:Sphingomyelins; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase