title: Why expression of phosphatidylethanolamine N-methyltransferase does not rescue Chinese hamster ovary cells that have an impaired CDP-choline pathway.
abstract: The mutant Chinese hamster ovary cell line (CHO), MT58, has a temperature-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), preventing phosphatidylcholine (PC) synthesis at 40 degrees C which results in apoptosis. Previous studies (Houweling, M., Cui, Z., and Vance, D. E. (1995) J. Biol. Chem. 270, 16277-16282) showed that expression of wild-type CT-alpha rescued the cells at 40 degrees C, whereas expression of phosphatidylethanolamine N-methyltransferase-2 (PEMT2) did not, even though PC levels appeared to be maintained at wild-type levels after 24 h at the restrictive temperature. We report that the failure of PEMT2 to rescue the MT58 cell line is due to inadequate long term PC synthesis. We found that changing the medium every 24 h rescued the PEMT2-expressing MT58 cells grown at 40 degrees C. This was due to the uptake and utilization of lipids in the serum. At 40 degrees C, PC levels in the wild-type CHO cells and CT-expressing MT58 cells increased over time whereas PC levels did not change in both the MT58 and PEMT2-expressing MT58 cell lines. Further investigation found that both the PEMT2-expressing MT58 and MT58 cell lines accumulated triacylglycerol at 40 degrees C. Pulse-chase experiments indicated that lyso-PC accumulated to a higher degree at 40 degrees C in the PEMT2-expressing MT58 cells compared with CT-expressing MT58 cells. Transfection of the PEMT-expressing MT58 cells with additional PEMT2 cDNA partially rescued the growth of these cells at 40 degrees C. Inhibition of PC degradation, by inhibitors of phospholipases, also stimulated PEMT-expressing MT58 cell growth at 40 degrees C. Best results were observed using a calcium-independent phospholipase A(2) inhibitor, methyl arachidonyl fluorophosphonate. This inhibitor also increased PC mass in the PEMT2-expressing MT58 cells. When the cells are shifted to 40 degrees C, PC degradation by enzymes such as phospholipases is greater than PC synthesis in the mutant PEMT2-expressing MT58 cells. Taken together, these results indicate that PEMT2 expression fails to rescue the mutant cell line at 40 degrees C because it does not maintain PC levels required for cellular replication.
authors.affiliation: Department of Biochemistry and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
authors.forename: K A
authors.initials: KA
authors.lastname: Waite
authors.forename: D E
authors.initials: DE
authors.lastname: Vance
mesh_terms: D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics* / Q000378:metabolism; D002999:Clone Cells; D006224:Cricetinae; D003470:Culture Media; D003566:Cytidine Diphosphate Choline / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D050918:Phosphatidylethanolamine N-Methyltransferase; D010740:Phospholipases / Q000037:antagonists & inhibitors; D013696:Temperature; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D010713:Phosphatidylcholines; D014280:Triglycerides; D003566:Cytidine Diphosphate Choline; D008715:Methionine; D008780:Methyltransferases; D050918:Phosphatidylethanolamine N-Methyltransferase; D019916:Choline-Phosphate Cytidylyltransferase; D010740:Phospholipases