DECREASED PHOSPHATIDYLCHOLINE BIOSYNTHESIS AND ABNORMAL DISTRIBUTION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE IN CHOLESTEROL AUXOTROPHIC CHINESE-HAMSTER OVARY CELLS
Mk. Storey et al., DECREASED PHOSPHATIDYLCHOLINE BIOSYNTHESIS AND ABNORMAL DISTRIBUTION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE IN CHOLESTEROL AUXOTROPHIC CHINESE-HAMSTER OVARY CELLS, Journal of lipid research, 38(4), 1997, pp. 711-722
Phosphatidylcholine (PtdCho) biosynthesis was examined in the sterol r
egulatory defective (SRD) Chinese hamster ovary (CHO) cell line SRD 6.
SRD 6 cells do not display transcriptional activation of sterol-regul
ated genes and are cholesterol auxotrophs. Compared to CHO 7 cells (pa
rental line from which the SRD cells were derived), incorporation of [
H-3]choline during a 2-h pulse into PtdCho and sphingomyelin was reduc
ed 3- and 4.5-fold, respectively, in SRD 6 cells grown with or without
cholesterol. SRD 6 cells grown in cholesterol-free medium for 24 h ha
d 8% less phosphatidylcholine (PtdCho) mass compared to CHO 7 cells. C
onsistent with impaired CTP:phosphocholine cytidylyltransferase activi
ty, [H-3] choline-labeled SRD 6 cells bad elevated [H-3] phosphocholin
e and delayed conversion to [H-3]PtdCho during a 2-h chase period. Com
pared to CHO 7 cells, cytosolic cytidylyltransferase activity was elev
ated 2- to 3-fold in SRD 6 cells grown in the absence of cholesterol,
but activity in the total membrane fraction was normal. Immunoblot ana
lysis confirmed that cytidylyltransferase mass was increased P-fold in
SRD 6 total cell extracts and cytosol, but not membranes. The amount
of [P-32] phosphate-labeled cytidylyltransferase in cytosol and membra
nes of SRD 6 cells were similar to controls. Likewise, cytidylyltransf
erase mRNA levels were not significantly different between SRD 6 and C
HO 7. The defect in PtdCho sythesis in SRD 6 cells could be overcome b
y treatment with 150 mu M oleate, such that after 5 h [H-3]choline inc
orporation into PtdCho and phosphocholine in SRD 6 and CHO 7 cells was
similar. Cholesterol auxotrophic SRD 6 cells display reduced PtdCho m
ass and synthesis and elevated levels of cytosolic cytidylyltransferas
e, defects that were only partially corrected by growth in exogenous c
holesterol. These results indicate a requirement for normal cholestero
l regulation and synthesis in the maintenance PtdCho levels and activi
ty of cytidylyltransferase.