Proc. Natd Acad. Sci. USA Vol. 79, pp.1.698-1702, March 1982 Biochemistry Animal cells dependent on exogenous phosphatidylcholine for membrane biogenesis (Chinese hamster ovary cells/CDP-choline synthetase mutant/liposomes/jysophosphatidylcholine/lipid bilayer assembly) JEFFREY D. ESKO*, MASAHIRO NISHIJIMAt, AND CHRISTIAN R. H. RAETZt Department of Biochemistry, College ofAgricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706 Communicated by M.J. Osborn, November 5, 1981 ABSTRACT A Chinese hamster ovary cell (CHO) mutant sophosphatidylcholine added as dispersions to the medium, in (strain 58), defective in CDP-choline synthetase (cholinephosphate contrast to the situation with serum phospholipids. Phospho- cytidylyltransferase; CTP:cholinephosphate cytidylyltransferase, lipid uptake- under these conditions results in the suppression EC 2.7.7.15), is temperature sensitive for growth and contains less ofthe temperature-sensitive phenotype ofmutant 58 (indicating than halfofthe normal amount ofphosphatidylcholine under non- that the phosphodiester bond is left intact) and supplies as much permissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc. as 50% of the phosphatidylcholine required for membrane as- NatL Acad. Sci USA 77, 5192-5196]. We now report that the ad- sembly in this setting. Mutant 58 incorporates much more of dition of 40 ,uM egg phosphatidylcholine or-lysophosphatidylcho- the added choline phosphoglycerides than the parental cells do, line to the medium suppresses the temperature sensitivity of mu- suggesting that phospholipid incorporation by CHO cells is tant 58 and permits the growth of colonies at the restrictive temperature. Phospholipids with different polar headgroups, li- regulated. poprotein-bound phospholipids, sphingomyelin, and glycerophos- phocholine do not support prolonged growth at 40C, whereas EXPERIMENTAL PROCEDURES phosphatidylcholine analogs, such as phosphatidyldimethyleth- anolamine, D-phosphatidylcholine, and f-phosphatidylcholine Materials. 32Pi (carrier-free) was obtained from New England are quite effective. A broad range of saturated phosphatidylcho- Nuclear. Ham's F-12 culture medium, trypsin, and fetal bovine lines, especially those with fatty acids 12-18 carbons in length, serum were obtained from GIBCO. Dilauroyl, dimyristoyl, and suppresses the phenotype. Phospholipids containing ether-linked distearoyl phosphatidylcholines and lauroyl, myristoyl, palmi- hydrocarbons are.ineffective, whereas polyunsaturated phospha- toyl, stearoyl, and oleoyl lysophosphatidylcholines were ob- tidykholines are toxic. Residual endogenous synthesis of phos- tained from Sigma. Bovine brain sphingomyelin was purchased phatidylcholine by.the mutant is not stimulated under conditions from Applied Science Laboratories, and dipalmitoyl phospha- of phenotypic bypass, but the uptake of exogenous lipid is en- tidylmonomethylethanolamine was supplied by Calbiochem- hanced considerablycompared to thewildtype. Our findings dem- Behring. All other phospholipids were obtained from Serdary onstrate that exogenous phospholipid can provide at least 50% of Research Laboratories, Ontario, Canada. Hexanoyl, octanoyl, the phosphatidylcholine required for membrane biogenesis in an- and decanoyl lysophosphatidylcholines were prepared from imal cells and that uptake of exogenous phospholipids may be their respective phosphatidylcholines by phospholipase A2 regulated. treatment as described below. All phospholipids were judged >95% pure by thin-layer chromatography (6),§ except.diarachi- Previous reports from this laboratory (1, 2) have described the donoyl phosphatidylcholine, which was first purified by thin- isolation ofstrain 58, atemperature-sensitive mutant ofChinese layer chromatography. Organic solvents, including methyletha- hamster ovary (CHO) cells defective.in CDP-choline synthetase nolamine and dimethylethanolamine, were redistilled before (cholinephosphate cytidylyltransferase; CTP:choline phosphate use. cytidylyltransferase, EC 2.7.7.15). Under nonpermissive con- Egg phosphatidylcholine was purified by a modification of ditions (40°C), de novo synthesis ofphosphatidylcholine is dra- the procedures of Singleton et al. (8). Egg lysophosphatidyl- matically reduced, resulting is a reduction to one-half to one- choline was generated from, this material by digestion with quarter in the content ofphosphatidylcholine'compared to pa- phospholipase A2 (9). Final purification was achieved by chro- rental cells (1, 2). The observation that mutant 58 is temperature matography on silicic acid. sensitive for growth in the presence of fetal bovine -serum is Cell Lines and Media. CHO-K1 were obtained from the especially intriguing. Serum, which is commonly used for grow- American Type Culture Collection (CCL-61), Rockville, MD, ing cells in tissue culture (3, 4) provides a considerable amount and were grown in Ham's F-12 medium (GIBCO), supple- of choline-linked phospholipid bound to various serum pro- mented with 10% fetal calf serum, penicillin-G (100 units/ml), teins, especially lipoproteins (5). If CHO cells were able to streptomycin sulfate (100 ,ug/ml), and NaHCO3 at 1.176 g/ utilize the intact phospholipid molecules present in 10% serum, the temperature-sensitive phenotype of the mutant would be Abbreviations: CHO, Chinesehamster ovary; PINaCl, phosphate-buf- suppressed. Because phenotypic suppression does not occur fered saline; PtdCho, phosphatidylcholine; l-PtdCho, lysophosphati- (1, 2) under typical growth conditions, CHO cells may not pos- dylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidyl- sess adequate mechanisms for intact utilization of serum inositol; PtdSer, phosphatidylserine; SPH, sphingomyelin; PDME, phospholipids. dipalmitoyl phosphatidyldimethylethanolamine. We now that mutant 58 in tissue cul- * Present address: Molecular Biology Institute, University ofCalifornia, demonstrate growing Los Angeles, CA 90024. ture can utilize large quantities of phosphatidylcholine and ly- t Present address: The National Institute of Health, 10-35, 2-Chome, Kamiosaki, Shinagawa-Ku, Tokyo, 141 Japan. The publication costs ofthis article were defrayed in part by page charge t To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertise- § Silica gel 60 (E. Merck) plates were employed. These were incorrectly ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. designated silica gel "G" in previous publications (6, 7). 1698 Downloaded by guest on October 3, 2021 Biochemistry: Esko et aL Proc. Natl. Acad. Sci. USA 79 (1982) 1699 liter. Bovine pancreatic insulin (Sigma) was also included at 20 ,ug/ml. The isolation and characterization of mutant 58 were described in previous publications (1, 2). Cultures were main- nio~o10~M0| Q4 ILM tained at 330C or 40.0 ± 0.20C in a 5% CO2 atmosphere at 100% 40OAM / 8 p relative humidity. In all of the studies reported here, only di- alyzed fetal calf serum (10) was employed. When phospholipids were added to the growth medium, concentrated lipid stocks were prepared in the following ways. 1.0 120 IAM Pure lipids in chloroform or 2:1 (vol/vol) chloroform/methanol 0.5 were dried in acid-washed glass centrifuge tubes under a stream of nitrogen. Phospholipid samples were resuspended at 2-6 20 40 60 80 100 120 20 40 60 80 100 120 mM in phosphate-buffered saline (PjNaCl) at pH 7.2 (11) and sonically irradiated twice for 5 min at power setting 5 with a Time, hr W185F ultrasonic disrupter (Heat System Ultrasonics, Plain- FIG. 1. Concentration dependence of the phosphatidylcholine by- view, NY) equipped with a no. 419 micro-tip. Unsaturated lipids pass phenomenon. Mutant and wild-type (parental) cells cultured at were prepared at 0C, whereas saturated lipids were dispersed 330C were harvested with trypsin (14) and added to multiple 60-mm- without cooling. Lysophosphatidylcholines were dissolved at diameter plastic tissue culture dishes containing 5 ml of growth me- liver diumtoyield6 x 104 and4 x 104cells, respectively. After 24 hr at 330C, 2mM in PjNaCl without sonication. Pig phosphatidyleth- an appropriate amount of 2 mM phosphatidylcholine liposomes pre- anolamine and phosphatidylmonomethylethanolamine (and pared in P/NaCl (10) was added to the cultures to give the indicated also in some instances phosphatidylcholine) were dissolved at concentration of added lipid. The dishes also received an aliquot of Pi 4 mM in distilled ethanol. All samples were sterilized with NaCl so that the final dilution of growth medium was the same in all Swinnex 13-mm-diameter, 0.22-,um-pore filters (Millipore, cultures (6%, vol/vol). The cells were then shifted to 40'C. At the in- Bedford, MA). The recovery ofphospholipids after filtration was dicated times the cells from duplicate cultures were harvested with dispersions or trypsin and counted with a model B Coulter Counter. (A) Mutant 58 typically 90% or more, and lysophospholipid at 400C. (B) Wild type at 400C. All cultures also contained 100 XM cho- ethanol solutions gave quantitative recovery. line, a component of F-12 medium. Other Procedures. Quantitation ofcellular phospholipid was achieved by perchloric acid digestion (7, 12) ofsamples obtained from two-dimensional thin-layer chromatography (6). Protein divided every 24 hr, whereas the parental cells doubled every was measured by the method of Lowry et al. (13). 14 hr independently of added phospholipid. The final cell den- sities attainable by the mutant were still one-half to one-third