DECREASED PHOSPHATIDYLCHOLINE BIOSYNTHESIS AND ABNORMAL DISTRIBUTION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE IN CHOLESTEROL AUXOTROPHIC CHINESE-HAMSTER OVARY CELLS

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.