Development and characterization of a tissue culture cell line ... - PNAS

Proc. Nati Acad. Sci. USA Vol. 79, pp. 7654-7658, December 1982 Biochemistry

Development and characterization of a tissue culture cell line with essential fatty acid deficiency (arachidonic acid/prostaglandins/phospholipids/fatty acid desaturase)

MICHAEL LAPOSATA, STEPHEN M. PRESCOTT*, TERESA E. BROSS, AND PHILIP W. MAJERUSt Division of Hematology-Oncology, Departments of Internal Medicine and of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

Communicated by Roy Vagelos, September 10, 1982

with arachidonate and it produces the diagnostic fatty acid of EFA deficiency A3Ach.

ABSTRACT We have developed an essential fatty acid-deficient cell line from a parental cell line, HSDMICI, which metabolizes arachidonic acid to prostaglandin E2 (PGE2). This cell line, designated EFD-1, is depleted of arachidonate, is unable to synthesize PGE2 in response to bradykinin, and has changes in fatty acid composition characteristic of tissues from animals with essential fatty acid deficiency. Within 15 min of repletion by arachidonate, the ability to synthesize PGE2 is restored. Linoleate also is able to restore PGE2 synthesis, indicating that deficient cells contain both the rate-limiting 46 desaturase enzyme and the A5 desaturase enzyme, which are required to form arachidonate. When parental cells are incubated in lipid-free medium, there is rapid induction of the ability to convert linoleate to arachidonate. Arachidonate prevents this induction, suggesting that icosanoid precursor availability controls the rate of arachidonate formation.

MATERIALS AND METHODS Development and Maintenance of the EFA-Deficient Cell Line (EFD-1). Cells from the HSDM1Cj cell line (ATCC CCL 148) were obtained from the American Type Culture Collection (Rockville, MD). These cells were maintained and passed in control medium: Ham's F10 (KC Biological, Lenexa, KS) containing 12.5% dialyzed horse serum (GIBCO), 2.5% dialyzed fetal calf serum (KC Biological), penicillin at 100 units/ml (GIBCO), streptomycin at 100 ,ug/ml (GIBCO), and 1 mM Larginine HCI (Sigma). Delipidated horse serum was prepared from the dialyzed horse serum noted above according to the method of Albutt (10). More than 98% of total lipid phosphate was removed by the delipidation procedure. The serum was stored at 40C and was used within 3 months. To establish the EFD-1, one million HSDM1Cj cells were plated into a plastic 100-mm Petri dish (no. 3003, Falcon Plastics, Cockeysville, MD) and were maintained in delipidated medium with daily medium change. This medium was Ham's F10 that contained 4.2% delipidated horse serum with penicillin, streptomycin, and arginine at the same concentrations noted above. Initially, the cells grew slowly and in the first passage, only about 50% of the cells were removed from the surface of the dish. However, within two passages the doubling time had shortened significantly and all of the cells were transferred in each subsequent passage. The HSDM1Cj cells grown in delipidated serum, hereafter known as EFD-1 cells, were fed daily with a complete change of medium. The HSDM1Cj cells grown in regular serum, hereafter known as control cells, also were fed daily, although medium change was not essential to their survival as it was for the EFD-1 cells. Lipid Extraction, HPLC, and Gas/Liquid Chromatography. Cells adherent to the surface of a 100-mm or 60-mm plastic Petri dish were washed three times with Tris-buffered saline (140 mM NaCl/35 mM Tris, pH 7.4) and then were precipitated with 3 ml of cold 10% trichloroacetic acid. While on ice the precipitate was harvested with a rubber policeman and was combined with two subsequent 10% trichloroacetic acid rinses of the Petri dish in an acid-washed glass centrifuge tube. The precipitate was collected by centrifugation at 4°C, washed in 6 ml of 0.15 M Tris (pH 8.35), and centrifuged again to remove

Essential fatty acid (EFA) deficiency (1) is a nutritionally induced disorder characterized by impaired growth, infertility, and skin and renal lesions, which is thought to result from inadequate levels of prostaglandin precursors (2). Prostaglandins are derived from essential polyunsaturated fatty acids, the most predominant being 5,8,11,14-icosatetraenoic acid or arachidonic acid. This fatty acid is the precursor of icosanoids (3), including the 2-series of prostaglandins, thromboxanes, and prostacycins and the 4-series leukotrienes. Arachidonate is esterified at the 2-position of phospholipids and its release in response to appropriate agonists initiates the production of arachidonate metabolites. Animals that have been fed a diet deficient in EFA become depleted in arachidonate and lose a measure of their capacity for prostaglandin synthesis (4, 5). However, the study of EFA deficiency in animals is difficult because: (i) long periods of dietary restrictions are required to induce the deficient state; (ii) animals do not become totally depleted in arachidonate or in the ability to form prostaglandins and other icosanoids; and (iii) in whole animal studies it is difficult to determine whether individual cells synthesize or take up and store fatty acids. A variety of cultured cell lines will grow in lipid-free medium (6-8), but in most cases these cells do not metabolize arachidonate nor do they synthesize the 5,8,11-icosatrienoic acid (A3Ach) that is characteristic of EFA deficiency. Therefore, we set out to induce EFA deficiency in a cell line known to metabolize arachidonate. We chose HSDM1Cj cells (ATCC CCL 148), originally obtained from a mouse fibrosarcoma, because they produce large amounts of prostaglandin E2 (PGE2) in response to bradykinin (9). We have adapted these cells to grow in lipid-free medium and the cell line has 3.5 times that in control cells. Third, A3Ach was present only in EFD-1 cells, with a A3Ach/arachidonate ratio of 5.4, well above the value of 0.4 suggested by Holman as an appropriate criterion for EFA deficiency (15). It is possible that 16:1 A9 also was increased in the EFD-1 cells, but the butylated hydroxytoluene added to the system to prevent fatty acid oxidation had a retention time that overlapped that of 16:1 A9 and made its estimation impossible. The total fatty acid in EFD-1 cells was 75% of that in control cells and the difference in fatty acid content approximated the difference in total cell protein. In two experiments, control cells contained (±SEM) 88.8 ± 8.4 and 85.0 ± 7.0 /.kg of protein per 106 cells, as compared to values (± SEM) of 75.5 ± 2.8 and 74.9 ± 4.0 ,ug of protein per 106 cells for EFD-1 cells. The time course for development of EFA deficiency is shown in Fig. 1. The cells were plated in 60-mm dishes so that approximately two cell doublings would result in confluence (i.e., 0.75 to 3 X 106 cells per dish). Cells initially grew very slowly and within 4 days showed decreases in arachidonate and linoleate with increased levels of oleate and the appearance of A3Ach. The cells became confluent after 10 days in culture. After each passage the cells grew somewhat faster, reaching confluence in 8 and 5 days after the second and third passages, respectively. The level of A3Ach was variable and appeared to be greatest when cells were near confluence as shown in Fig. 1 (e.g., on day 10 and on day 29 in the cultures not passed on day 18). In another experiment, cells were maintained at confluence for 5 days, by which time A3Ach was 6.6% of total fatty Table 1. Fatty acid composition of cells Fatty acid content, nmol per 106 cells* Control EFD-1 Fatty acid 10.7 ± 0.2 8.7 ± 0.2 16:0 17.3 ± 0.5 9.8 ± 0.3 18:0 8.3 ± 0.2 29.8 ± 1.2 18:1A9 24.4 ± 0.7 2.5 ± 0.1 18:2 A9"12 Trace 0.1 ± 0.1 el9,12 18:3 0.5 ± 0.0 18:3 e9l12,15 0.2 ± 0.1 0.1 ± 0.0 1.8 ± 0.0 20:1 All 2.0 ± 0.1 20:2 &11,14 ND 0.9 ± 0.1 20:3 A8,11,14 ND ND 1.3 ± 0.1 20:3 A5,8,11 8.1 ± 0.3 20:4 AS6l1,14 0.2 ± 0.1 0.4 ± 0.0 20:5 &5,.811.14,17 ND ND 22:1 A13 0.6 ± 0.2 2.4 ± 0.1 22:4 A7,10,13,1"6 ND 0.4 ± 0.0 22:5 A4,7,10,13,16 ND 2.2 ± 0.0 22:5 &7,l0,l3,16,19 ND 3.0 ± 0.1 22:6 A4,7,10,13,1619 3.1 ± 1.4 24:1 A15 ND 0.7 ± 0.3 0.2 1.9 Other 80.9 60.7 Total ND, none detected. EFA are shown in boldface. Cell cultures in 100mm Petri dishes were allowed to grow to confluence, at which time the cells were harvested, the lipids were extracted, and fatty acid methyl esters were prepared and measured by gas chromatography. Recovery of fatty acids was based upon recovery of [1-'4C]linoleate label in PtdEtn. * Values are the mean ± SEM of three separate cultures.

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acid. The ratio of 20:3/20:4 fatty acids reached values diagnostic for EFA deficiency by day 18. The EFD-1 line was established in an experiment similar to that in Fig. 1 (as described in Materials and Methods) and has been grown continuously for 1 yr. Growth curves for control and EFD-1 cells are shown in Fig. 2. The control cells doubled every 25 hr, compared with 40 hr for EFD-1 cells. The maximal cell density for EFD-1 cells was one-third that ofcontrol cells. The control cells were fibroblastic in appearance and had a greater propensity for overlapping one another than did EFD1 cells, which had an epithelioid morphology. Newly plated EFD-1 cells assumed the morphology ofthe control line within 3 days when transferred to control medium and control cells similarly assumed the morphology of the EFD-1 line when transferred to delipidated medium (not shown). Control cells synthesized and secreted PGE2 in response to bradykinin as shown in Fig. 2. As cells reached confluence there was a diminution in PGE2 synthesis in response to bradykinin. EFD-1 cells, which had virtually no arachidonate, did not produce PGE2 when challenged with bradykinin. Neither cell line produced PGE2 when treated with buffer alone (not shown). Phospholipids of Control and EFD-1 Cells. The fatty acid content of the major phospholipids from control and EFD-1 cells is shown in Table 2. Although A3Ach levels are much lower in these cells than those used for the experiment in Table 1, the fatty acid composition of each phospholipid from EFD-1 cells reflects EFA deficiency-i.e., decreased arachidonate and linoleate and increased oleate and A3Ach. In addition, the total content of PtdCho and PtdEtn was decreased in EFD-1 cells. The decreased levels of these two major phospholipids can account for the diminished total fatty acid content per cell de-

scribed in Table 1. In contrast, the level of PtdIns was increased in EFD-1 cells. This increase was documented in other experiments in which PtdIns was isolated from control and EFD-1 cells by HPLC and the PtdIns content was measured by lipid phosphorus determination. Control cells contained (±SEM) 2.4 ± 0.25 nmol of Ptdlns per 106 cell vs. 3.1 ± 0.4 nmol of PtdIns per 106 EFD-1 cells. This difference was significant by a paired t test (t = 2.4, P < 0.05, n = 6). Repletion of EFD-1 Cells. When EFD-1 cells were incubated with [1-14C]arachidonate, they incorporated it rapidly into phospholipids as shown in Fig. 3. At early time points arachidonate was incorporated exclusively into PtdCho and Ptdlns; later, PtdEtn and lyso-PtdEtn (l-PtdEtn) (derived from plasmalogen PtdEtn) also were labeled. No incorporation into PtdSer was detected. The newly incorporated arachidonate rapidly restored the ability of EFD-1 cells to synthesize PGE2 in response to bradyldnin as shown in Fig. 4. At 2 ,uM supplemental arachidonate, PGE2 synthesis was restored to 25% of maximal by 15 min. At 25 t&M arachidonate, PGE2 synthesis was 75% of maximal by 30 min. Linoleate also was able to restore the ability of EFD1 cells to synthesize PGE2, indicating that these cells contain the A6 and A5 desaturases and the chain elongation enzymes required to convert linoleate to arachidonate. The parent cell line HSDMj has been reported previously to lack the A6 desaturase (16). Our results suggested that arachidonate deficiency may induce the enzymes required for its formation. We explored this possibility by incubating control and EFD1 cells with 5 ILM [1-'4C]linoleate as shown in Fig. 5. Control cells converted