lipids of cultured hepatoma cells: i. effect of serum lipid levels on cell and media lipids

Lipids of Cultured Hepatoma Cells" I. Effect of Serum Lipid Levels on Cell and Media Lipidsl RANDALL WOOD, Division of Gastroenterology, Departments of Medicine and Biochemistry, University of Missouri SchooJ of Medicine, Columbia, Missouri 65201

A B S T R A C T

Minimal deviation hepatoma cells were cultured as monolayers to confluency in roller flasks containing modified Swim's medium, supplemented with decreasing amounts of serum, lipid-free serum, and lipid-flee serum containing added fatty acids. Good cell growth was observed until serum levels fell below 5% of the medium. Media containing lipid-flee serum or lipid-flee serum plus linoleic or palmific acids did not support good growth. Lipids were extracted from cells; media, obtained during the first and last half of the incubation period, resolved into neutral and phospholipid fractions; fatty acid composition of each fraction analyzed by gas liquid chromatography; and lipid class distributions compared by thin layer chromatography. The data showed that the media contained more neutral lipids and phospholipids after incubation than initially, indicating that minimal deviation hepatoma cells excreted lipids into the media. The class composition of the excreted lipids re- sembled that of the serum. A comparison of media, cells, and serum fatty acid compositions indicated that the lipids secreted into the media were of cellular origin. Although some differences were noted, in general, cells grown on the nine different media had the same ca. neutral lipid and phospholipid class and fatty acid compositions. In contrast, dramatic differences were observed in the class and fatty acid compositions of the serums from that of the cells and media. These results indicate that exogenous serum lipids had little influence on cellular class and fatty acid compositions of the minimal deviation hepatoma cells. This neoplasm did not contain detectable levels of glyceryl ether diesters, indicating that this compound is not characteristic of all tumors. Lipid class profiles and fatty acid compositions of cells grown on various media suggest that the minimal deviation hepatoma cells can synthesize most, if

1presented at the AOCS Meeting, New Orleans, April 1973.

not all, neutral lipid and phosphoglyc- eride classes found in liver.

I N T R O D U C T I ON

The last decade of biochemical research has brought lipids from a point at which they were considered a good source of energy to the present recognition of their importance in many biological processes. Lipids are major components of all membranes (1-3), essential components of many enzyme systems (4), and precursors of a potent class of hormones (5,6). Lipids are involved in biological mineralization (7), ion transport (8), sugar transport (9), and numerous other important cellular functions. Some investigators in cancer research have recognized the possible direct or indirect in- volvement of lipids in neoplasia and have obtained data to indicate numerous differences between the lipid metabolism of normal and neoplastic tissue. Much data on the biochemistry and metabolism of tumor lipids, compiled from many laboratories, has appeared recently (10).

Although differences in lipid metabolism occur between normal and neoplastic tissue, few consistently appear in all tumors because of their differences in origin, stage of neoplasia, nutritional state of host, and numerous other factors. The development by Morris and col- leagues (11) of a series of "minimal" deviated hepatomas eliminated many of the difficulties in deciding which normal tissue should be compared with neoplasm data. The host effect on metabolism of a minimal deviation hepatoma also can be eliminated by growing hepatoma cells in tissue culture.

Structural and metabolism studies on lipids, derived from minimal deviation hepatoma cells grown in culture, have been limited. The incorporation of activity from various radioactive substrates into the nonsaponifable lipids (12) and the composition and biosynthesis of gangliosides in cultured minimal deviation hepatoma cells have been reported (13). Recently Ontko (14) and Goodridge (15) described fatty acid biosynthesis and metabolism studies obtained with normal liver hepatocytes cultured for relatively short periods of time. The lack of lipid data from minimal deviation hepatoma cells, grown in culture which have numerous advantages, and the potential availability of

690

CULTURED HEPATOMA CELL LIPIDS

TABLE I

Quantity of Neutral Lipids and Phospholipids Derived from Minimal Deviation Hepatoma Cells Grown on Media Containing Varying Amounts of Serum and Lipid a

691

#g of lipid/ #g of lipid/ mg of lipid-

Cell Doubling 106 cells free dry wt Percentage Basal medium numbers time supplement b x 106c (days) PL d NL d PL NL PL NL

20% Bovine serum + 5% fetal calf serum 446 1.18 37 21 106 62 63 37 10% Bovine serum + 5% fetal calf serum 665 1.18 23 15 -- -- 57 43 5% Bovine serum + 5% fetal calf serum 635 1.32 31 24 110 8"7 60 40 5% Fetal calf serum 565 1.44 17 12 119 80 60 40 5% Bovine serum 384 1.53 18 8 87 39 69 31 2.5% Fetal calf serum 342 2.07 22 7 -- -- 77 23 5% Lipid-free fetal calf serum 194 2.5 18 7 71 30 71 29 5% Lipid-free fetal calf serum+ palmitic acid d 94 5.5 45 47 208 222 49 51 5% Lipid-free fetal calf serum + linoleic acid e 92 3.5 53 55 107 111 49 51

aAll data obtained from cells grown in roller cultures for five-six days. bBasal medium was a modified Swim's 77. CTotal number of cells harvested from two roller culture flasks and analyzed as a single sample. dpL = phospholipids, NL = neutral lipids. e37.5 pg per ml of medium.

comparab le data f rom norma l h e p a t o c y t e s p r o m p t e d us to init iate a s tudy to examine the metabol i sm and s t ruc ture of lipids at the molecular species level f rom the minimal devia- t ion h e p a t o m a 7288C(HTC) grown in tissue culture (16).

This article represents the first in a series on the data ob ta ined wi th cul tured HTC cells and a solid minimal deviat ion h e p a t o m a 7288CTC ob ta ined by the r e in t roduc t ion of HTC cells in to a hos t (17). These data show the effects of exogenous serum lipid levels on g rowth rate of HTC cells and class and fa t ty acid compos i t ions of cellular and media neutra l l ipid and phospho - lipids.

MATERIALS AND METHODS

Cells

Starter cultures of HTC cells were ob ta ined f rom W.D. N o t e b o o m , Depa r tmen t of Biochem- istry, Universi ty of Missouri, who had ob ta ined the cells f rom G.M. Tomkins , the developer of this line of cul tured minimal deviat ion hepatoma cells (16,18). Cells were ob ta ined at the 17th passage. Stock cul tures were main ta ined on a modi f ied Swim's 77 med ium supple- m e n t e d wi th 20% bovine serum and 5% fetal

calf serum. The med i u m modi f i ca t ion included the addi t ion of g lutamine (292 mg/1) , sodium bicarbonate (500 mg/1) , calcium chlor ide dihy- drate (265 mg/1), Sod ium Penicillin G (60 rag / l ) , L-cystine (12 rag / l ) , and phenol red (5 rag / l ) . All media were steril ized by f i l t rat ion th rough 0.2/~ pore size filters.

Gram quant i t ies of cells grown on various amounts of serum and lipids (Table I and II) were ob ta ined f rom roller cultures. Cells (30-50 million) were p lan ted in two 10 x 20 cm glass roller cul ture bo t t l es con ta in ing ca. 100 ml of med ium each and incuba ted at 37 C. The roller bot t les were ro ta t ed at the rate of 0.8 RPM for the first four-six hr to ensure an even distribu- t ion of cells, a f ter which t ime the rate was reduced to 0.25 RPM. The media were changed on the second or thi rd day of incubat ion . Af te r five-six days, when the ceils had reached confluency or failed to grow fur ther , the med ium from the second half o f incuba t ion was collected. The cells were released enzymat ica l ly f rom the roller flasks, coun ted , washed wi th serum-free Swim's 77 med ium, and t ransferred to tared lyophi l iza t ion flasks. Medium from the first half or last half o f incuba t ion was centri- fuged for 6 x 106 g-rain to sed imen t any par t iculate mat ter .

LIPIDS, VOL. 8, NO. 12

692 R. WOOD

TABLE 1I

Accumulation of Phospholipids and Neutral Lipids in the Media of Cultured Minimal Deviation Hepatoma Ceils

Cells a Media (rag)

Initial lipid Basal medium Numbers Lipid (mg) content/200 ml supplement x 106 PL NL PL NL

Lipid gain during incubation

First half b Last half b

PL NL PL NL

20% Bovine serum + 5% fetal calf serum 446 16.5 9.6 47.7 56.5 -- -- +24.8 c +17.8 c 10% Bovine serum + 5% fetal calf serum 665 15.0 10.3 21.6 26.7 +2.9 +16.2 +19.9 +19.8 5% Bovine serum + 5% fetal calf serum 635 19.4 15.3 15.1 16.6 -- -- +15.9 +8.2 5% Fetal calf serum 565 9.8 6.6 4.3 3.3 -- -- +3.2 +0.8 5% Bovine serum 384 7.0 3.2 10.8 13.3 +10.6 +8.9 +17.7 +11.5 2.5% Fetal calf serum 342 %5 2.3 2.2 1.7 +3.2 -0 .4 +6.6 +0.9 5% Lipid-free fetal calf serum 194 3.5 1.5 1.2 0 +2.8 +0.5 +2.2 +1.4 5% Lipid-free fetal calf serum + palmitic acid d 94 4.2 4.4 1.2 7.5 d +3.7 +0.6 +6.6 +1.1 5% Lipid-free fetal calf serum + linoleic acid d 92 4.9 5.1 1.2 7.5 d +4.4 +2.0 +3.7 +3.4

aCell numbers represent the total number of cells harvested from two roller flasks after five-six days of incubation and analyzed as a single sample. Each flask contained between 15 and 25 million cells at the start of incubation.

bNeutral lipid (NL) and pbospholipid (PL) weights represent the difference between lipids known to be in the media at the start of incubation (given in columns 4 and 5) and that found in the first or last half incubation media.

CRepresents the total media lipids for both first and last half (media not changed). d37.5 /zg per ml of medium.

Lipid Extraction and Fractionation

Lipid-free fetal calf s e r u m was p repa red by son ica t ing lyophi l i zed s e r u m wi th 20-30 vol- u m e s of c h l o r o f o r m - m e t h a n o l , 2:1 (v/v), un t i l a h o m o g e n o u s suspens ion , free o f c l umps , was obta ined . Solvent was r e m o v e d by f i l t ra t ion t h r o u g h a f ine poros i ty f i t t ed disc glass f unne l and washed wi th a n o t h e r 50 v o l u m e s of chloro- f o r m - m e t h a n o l . The c h l o r o f o r m - m e t h a n o l ex- t r a c t s w e r e c o m b i n e d , evapora ted , and weighted to d e t e rm in e e x t e n t of lipid ext rac- t ion. The lipid-free p ro te in was t rans fe r red to a 100 ml pea r - shaped flask wi th c h l o r o f o r m and m e t h a n o l and the so lven t f lash evapora ted , leaving a t h in f i lm on the wall of the flask wh ich faci l i ta ted the r emova l o f the last t races of so lven t u n d e r h igh v a c u u m . When linoleic and pa lm/ t ic acid were added to the m e d i u m , they were i n t r o d u c e d at this po in t before the solvent was f lash evapora ted . The l ipid-free fetal calf s e r u m or l ipid-free s e r u m s c o n t a i n i n g added f a t t y acids were r e c o n s t i t u t e d in e i ther water or Swim ' s 77 m e d i u m .

Lyoph i l i zed cells, media , and s e r u m s for lipid d e t e r m i n a t i o n s were ex t r ac t ed by the Bligh and Dyer p ro cedu re (19) to ob ta in total

lipids. A second e x t r a c t i on was p e r f o r m e d by read jus t ing c h l o r o f o r m - m e t h a n o l c o n c e n t r a t i o n to give a single phase again. Each t ime the s u s p e n s i o n was sonica l ly d i s rup t ed for ca. five rain to faci l i tate lipid ex t r ac t ion . Neut ra l lipids were sepa ra ted f r o m the phospho l i p id s by silicic acid c h r o m a t o g r a p h y (20). Percen tages of neu t r a l lipid and p h o s p h o l i p i d f rac t ions were d e t e r m i n e d gravimetr ical ly . A qual i ta t ive e xa m- ina t ion of the neu t r a l lipid f rac t ion was m a d e by th in layer c h r o m a t o g r a p h y (TLC) on adsorb- en t layers of Silica Gel G, deve loped in a so lvent s y s t e m of h e x a n e d i e t h y l e ther -ace t ic acid 80 :20 : 1, v/v. The qual i ta t ive and quan t i t a - tive e x a m i n a t i o n of indiv idual p h o s p h o l i p i d classes was carr ied ou t by TLC on a d s o r b e n t layers o f Silica Gel HR, deve loped in a so lvent s y s t e m o f c h l o r o f o r m - m e t h a n o l - a c e t i c acid- 0.9% saline, 5 0 : 2 5 : 8 : 4 , v/v.

Other Procedures

P h o s p h o r o u s was d e t e r m i n e d by the m e t h o d of R o u s e r et al. (21). C h r o m a t o p l a t e s u se d for qual i ta t ive analyses were sp r a ye d wi th su l fur ic acid, charred, and d o c u m e n t e d by pho tog ra - phy . Class iden t i f i ca t ion was based u p o n the


CULTURED HEPATOMA CELL LIPIDS

use of phosphorous and ninhydrin spray reagents (22), gas liquid chromatography (GLC) of hydrolysis products, and cochromatography with authentic phospholipid and neutral lipid standards in two or more TLC solvent systems. Methyl esters of the total neutral lipid and phospholipid fractions were prepared by acid catalyzed esterification and analyzed by GLC, as described previously (23).

Materials

Single lots of fetal calf serum, bovine serum, and Swim's 77 medium obtained from Grand Island Biological Co., Grand Island, N.Y., were used for these experiments. Fatty acids and neutral lipid standards were purchased from Nu-Check-Prep, Inc., Elysian, Minn. Phospho- lipid standards were purchased from Supelco, Inc., Bellefonte, Pa. All solvents were glass- distilled and obtained from Burdick and Jack- son Laboratories, Muskegon, Mich. Other chem- icals and reagents used were reagent grade, or better, and used without further purification.

RESULTS

Cell Total Lipids

Numbers of cells harvested, doubling time, and quantity of neutral lipids and phospholipids, derived from HTC cells cultured on Swim's 77 medium containing varying amounts of serum and lipids, are given in Table I. The doubling time, which included the lag phase, increased when serum levels decreased below 10%, and growth was poor in media containing less than 5% serum. Removal of lipids from the serum further increased the doubling time, and the addition of either palmitic or linoleic acid to the lipid-free fetal calf serum did not improve growth. Except for the growth conditions where fatty acids were added, the phospholipids represented between 60 and 70% of the total celt lipids. The quantity of phospholipid/given number of cells or/given amount of dry wt showed less variation than the neutral lipi ds.

Media Total kipids

The total lipid of fetal calf and bovine serums represented 2.2 and 4.4%, respectively, of the dry wt. Fetal calf serum and bovine serum contained 3.6 and 5.6% dry wt, respectively, of the wet wt. These values were used to calculate the amount of neutral lipid and phospholipid in the original medium of each experiment. When chloroform-methanol was used to extract the lipids from fetal calf serum for the experiments using lipid-free fetal calf serum, only 85% of the lipids were removed

693

relative to the amount obtained by the Bligh and Dyer extraction procedure. The difference was probably due to nonlipid components being extracted by the Bligh and Dyer procedure. However, to remove bias from the data, the chloroform-methanol extracted serum was assumed to contain 15% unextracted phospholipid, and this was taken into account in the calculation of accumulated lipid. The differences between the amount of lipid in the media at the start of the incubations and that found in the media after the first or last half of the incubations are given in Table II. The media from all HTC cell growth conditiong showed a gain in the neutral lipid (one exception) and phospholipid fractions. Generally, the last half of the incubation showed more lipid accumulation than the first half. Table I1 shows that the quantity of lipid found in the media exceeds that found in the HTC ceils in all cases where media were collected from both incubation periods,

TLC of Cell Neutral Lipids

The distribution of major neutral lipid classes derived from HTC cells cultured in Swim's 77 medium, supplemented with various levels of serum and lipids, is shown in Figure 1, along with the distribution of neutral lipids found in bovine and fetal calf serums. Sterol esters, triglycerides, free fatty acids, and sterols were the major neutral lipid classes present in HTC cells from all experiments. Triglycerides appeared to be the most abundant component in all growth conditions, except one, while the serum triglyceride levels in both serums were low. Cells grown on lipid-free fetal calf serum contained all the major neutral lipid classes. Supplementation of the lipid-free fetal calf serum with free palmitic or linoleic acids did not elevate the free fatty acids in the cells above other growth conditions but did appear to reduce the level of sterol esters. Glyceryl ether diesters were not detected on these heavily loaded plates nor when even heavier loads were examined.

TLC of Cell Phospholipids

Figure 2 shows the distribution of the major phospholipid classes derived from HTC cells grown on media containing various levels of serum and lipids. The phospholipid class distribution of bovine and fetal calf serums shown for comparison was quite different from the class distribution of cells obtained during the various growth conditions. Phosphatidyletha- nolamine, absent from the serums, and phosphatidylcholine, the major phospholipid class of the serums, were the two major cellular

LIPtDS, VOL. 8, NO. 12

694 R. WOOD

FIG. 1. Thin layer chromatoplate of neutral lipids derived from minimal deviation hepatoma cells cultured on a modified Swim's 77 medium supplemented with: lane l , 20% bovine serum plus 5% fetal calf serum; lane 2, 10% bovine serum plus 5% fetal calf serum; lane 3, 5% bovine serum plus 5% fetal calf serum; lane 4, 5% fetal calf serum; lane 5, 2.5% fetal calf serum; lane 6, 5% bovine serum; lane 7, 5% lipid-free fetal calf serum; lane 8, 5% lipid-free fetal calf serum plus palmitic acid; and lane 9, 5% lipid-free fetal calf serum plus linoleic acid. Lanes 10 and 11 represent neutral lipids from fetal calf serum and bovine serum, respectively. A neutral lipid standard is represented by standard. Neutral lipid classes are: A, sterol esters; B, glyceryl ether diesters; C, triglycerides; D, free fatty acids; and E, sterols. The origin and the solvent front are marked by O and S, respectively. Each lane, except the standard, was spotted with 200 pg of sample and developed in a solvent system of hexane-diethyl ether-acetic acid 80:20:1 (v/v).

phosphol ip ids . HTC cells did n o t con t a in more than trace levels of l y so -phospha t i dy l cho l ine , while b o t h serums c o n t a i n e d relat ively h igh levels. General ly , gross d i f ferences in the phospho l ip id class d i s t r ibu t ion could no t be de- t ec t ed by visual compar i son on TLC, even for the phospho l ip ids derived f rom cells g rown in media c o n t a i n i n g lipid-free f e t a l calf serum.

TLC of Media Neutral kipids

The d i s t r i bu t ion of neu t r a l l ipid classes found in the med ia was d i f f e ren t f rom cel lular neu t ra l l ipids b u t similar to the d i s t r ibu t ion of the neutral l ipid classes f rom the serums. Sterol esters were the ma jo r l ipid class u n d e r all g rowth cond i t ions , e x c e p t when l ipid-free fetal ca l f se rum was s u p p l e m e n t e d wi th l inoleic acid. Triglycerides, a m a j o r cel lular neu t r a l lipid, were p resen t at low levels in the media . Media supplemented wi th lipid-free fetal calf se rum c o n t a i n e d the same l ip id classes as the media f rom cells grown in the presence of serum. Like the cells, the med ia did n o t c o n t a i n de t ec t ab le levels of glyceryl e t h e r diester . Distribution of


FIG. 2. Thin layer chromatoplate of phospholipids isolated from minimal deviation hepatoma cells cultured on a modified Swim's 77 medium supplemented with various amounts of serums and lipids. The numbered lanes correspond to culture conditions described in Figure 1. Phospholipid classes are: PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; SPH, sphingomyelin; and L-PC, lyso-phosphatidytcholine. The origin and solvent front are indicated by O and S, respectively. Each lane, except the standard, was spotted with 300 ug of sample and developed in a solvent system of chloroform-methanol-acetic acid - 0.9% saline, 50:25:8:4 (v/v) when the relative humid- ity was below 35%.

neu t ra l l ipid classes f rom the first ha l f incuba- t ion med ia r e sembled the last ha l f d i s t r ibu t ion .

TLC of Media Phospholipids

The d i s t r ibu t ion o f med ia phospho l ip id s was s t r ik ingly d i f fe ren t f rom the cellular p a t t e r n bu t s imilar to se rum p h o s p h o l i p i d pa t t e rns . Phosphatidylethanolamine, phosphatidylserine, and p h o s p h a t i d y l i n o s i t o l were p resen t in on ly trace amounts or absent , while p h o s p h a t i d y l - chol ine and s p h i n g o m y e t i n were the m a j o r phos p h a t i d e s present. An unidentified com- p o u n d migra t ing b e t w e e n p h o s p h a t i d y l e t h a - n o l a m i n e and phosphatidylserine did n o t conta in p h o s p h o r o u s . This c o m p o u n d appeared to increase in concentration as serum and lipid levels decreased. The d i s t r ibu t ion of med ia phospho l ip ids f rom first an d las t i n c u b a t i o n s were similar, as j u d g e d f rom TLC.

Fatty Acid Composition of Cellular Neutral Lipids

The percen tage d i s t r i bu t ion of f a t t y acids o b t a i n e d f rom the to ta l n eu t r a l l ipids of fetal calf se rum, bov ine serum, an d HTC cells grown on various se rum and l ipid levels is given in Table III. The f a t t y acids der ived f rom fetal calf and bovine se rum neu t r a l l ip id were s t r ikingly d i f ferent . Feta l ca l f se rum neu t r a l l ipids were charac te r i zed by h igh levels of 16:0, 18:1,

CULTURED t fEPATOMA CELL LIPIDS

TABLE [II

Fatty Acid Composi t ion of Total Neutral Lipids Derived f rom Serums and Minimal Deviation Hepatoma Cells Grown on Media Containing Various Amoun t s of Lipid

695

Fatty acid percentages a,b Serums and basal

medium supplement 14:0 16:0 16:1 18:0 18:1 18:2 20:1 20:2 20:4 >20:4

Serums

Fetal calf se rum T c 23.5 7.1 4.5 33.8 9.3 0,9 ~ 12.5 6.4 Bovine serum T 9.8 3.3 5.4 12.8 57.4 4.2 - - 4.1 T

Minimal deviation hepa toma

20% Bovine serum + 5% fetal calf se rum 0.5 7.4 5.8 5.7 66.4 8.1 3.7 - - 1.4 t .0 10% Bovine serum + 5% fetal calf serum 1.2 10.2 7.1 5.9 61.7 7.2 5.6 - - 1.1 T 5% Bovine serum + 5%fetal calf serum 1.4 11.6 8.8 5.3 63.3 4.1 4.4 - - 1.2 T 5% Fetal calf serum 0.8 10.6 6.9 5.8 67.9 1.6 5.0 - - 1.3 T 5% Bovine serum T 9.1 4.5 7.4 63.0 10.2 3.7 - - 3.9 T 2.5% Fetal calf serum T 8.0 8.0 5.3 65.6 4.2 5.3 - - 2.2 T 5% Lipid-free fetal calf serum 1.6 8.7 8.3 S.0 65.7 2.5 5.1 - - 0.7 T 5% Lipid-free fetal calf serum + palmitic acid d 0.5 8.4 6.0 8.7 64.3 4.6 3.9 --- 2.0 T 5% Lipid-free fetal calf s e r u m + l i n o l e i c a c i d d T 6.7 2.1 5.8 16.7 49.7 T 8.1 6.5 2.4

aDifference between the sum of the fat ty acid percentages in each row and 100% represents the sum of minor components not given in the table.

bpercentages represent the mean of duplicate analyses on a single pooled sample. CT = less than one half per cent. d37.5 /.tg per ml of medium.

2 0 : 4 , a n d t h o s e ac id s w i t h r e t e n t i o n t i m e s g r e a t e r t h a n 2 0 : 4 , w h e r e a s b o v i n e s e r u m was c h a r a c t e r i z e d by a h i g h level (ca. 60%) o f 1 8 : 2 acid. Ce l lu l a r n e u t r a l l i p id c o m p o s i t i o n d i f f e r e d f r o m e i t h e r o f t h e s e r u m s . W i t h o n e e x c e p t i o n , ce l lu l a r n e u t r a l l i p id f a t t y ac id p e r c e n t a g e s we re n o t c h a n g e d s i g n i f i c a n t l y by r e d u c e d a m o u n t s o f s e r u m a n d l ip id in t h e m e d i u m . P a l m i t i c a n d s t ea r i c ac ids r e m a i n e d r e l a t i ve l y c o n s t a n t u n d e r all g r o w t h c o n d i t i o n s , w h e r e a s 18 :1 ac id in- c r e a s e d as s e r u m a n d l ip id levels were de- c r eased . Cells , g r o w n o n m e d i u m s u p p l e m e n t e d w i t h l i p id - f r ee f e t a l ca l f s e r u m , c o n t a i n e d t h e l o w e s t level o f 18 :2 a n d 2 0 : 4 ac ids . T h e a d d i t i o n o f p a l m i t i c ac id t o m e d i u m c o n t a i n i n g l ip id - f ree f e t a l ca l f s e r u m d i d n o t e l e v a t e p a l m i t i c ac id in ce l lu l a r n e u t r a l l ip ids , wh i l e l i no l e i c a c id a d d e d t o t h i s m e d i u m i n c r e a s e d t he 18 :2 ac id level in t h i s l ip id f r a c t i o n 5- t o 10- fo ld .

Fatty Acid Composition of Cellular Phospholipids

T h e f a t t y ac id c o m p o s i t i o n o f t h e t o t a l p h o s p h o t i p i d f r a c t i o n o b t a i n e d f r o m f e t a l ca l f s e r u m , b o v i n e s e r u m , a n d H T C cells c u l t u r e d o n v a r i o u s levels o f s e r u m a n d l ip id is g iven in T a b l e IV . S e r u m p h o s p h o l i p i d f a t t y ac ids dif-

f e r e d f r o m s e r u m n e u t r a l l ip id ac ids (Tab l e I l l ) in t h a t t h e y c o n t a i n e d a h i g h e r p e r c e n t a g e o f t he l o n g e r c h a i n f a t t y ac ids . F e t a l ca l f s e r u m p h o s p h o l i p i d s c o n t a i n e d a l o w e r p e r c e n t a g e o f 18 :2 a n d a h i g h e r p e r c e n t a g e o f C-22 a n d h i g h e r f a t t y ac ids t h a n b o v i n e s e r u m p h o s p h o - l ip ids . Ce l lu l a r p h o s p h o l i p i d f a t t y ac ids s h o w e d m o r e c h a n g e w h e n m e d i a s e r u m a n d l ip id levels were v a r i e d t h a n d id t h e n e u t r a l l ip ids . P a l m i t i c a n d s t e a r i c ac ids r e m a i n e d r e l a t i ve ly c o n s t a n t u n d e r all g r o w t h c o n d i t i o n s , w h e r e a s 18:1 i n c r e a s e d as s e r u m a n d l ip id levels were de- c r e a s e d . Cel ls g r o w n o n m e d i u m s u p p l e m e n t e d w i t h l i p id - f r ee f e t a l c a l f s e r u m c o n t a i n e d t h e l o w e s t level o f 18 :2 a n d 2 0 : 4 ac ids . T h e a d d i t i o n o f p a l m i t i c ac id to m e d i u m c o n t a i n i n g l i N d - f r e e f e t a l ca l f s e r u m d id n o t e l eva t e p a l m i t i c ac id in ce l lu l a r p h o s p h o l i p i d s , b u t t h e a d d i t i o n o f l ino le ic a c id t o t h e m e d i u m c a u s e d an i n c r e a s e in 18 :2 , 2 0 : 2 , 2 0 : 4 , a n d 2 2 : 5 ac id p e r c e n t a g e s .

Fatty Acid Composition of Media Neutral Lipids

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C U L T U R E D H E P A T O M A C E L L LIPIDS

T A B L E V

C o m p a r i s o n of Tota l Neu t ra l Lipid F a t t y Acid C o m p o s i t i o n of Se rums and Media af ter HTC Cells were Cu l tu red unde r Various G r o w t h Cond i t ions

697

I n c u b a t i o n Se rums and basal per iod media

m e d i u m s u p p l e m e n t were co l lec ted

Fa t ty acid percen tagesa , b

16:0 16:1 18:0 18:1 18:2 20:1 2 0 : 4 > 2 0 : 4

Se rums

Fetal calf s e r u m - - 23 .5 "7.1 4.5 33.8 9.3 0.9 12.5 6.4

Bovine s e rum - - 9.8 3.3 5.4 12.8 57.4 4.2 4.1 T c

Media

20% Bovine s e r u m + 5% fetal calf s e r u m Tota l 9.0 3.4 1.6 12.4 67.5 3.8 3.0 - -

10% Bovine s e rum + 5% fetal calf s e rum Last ha l f 9.5 3.9 1.5 12.2 64.5 4.0 4.0 T

5% Bovine se~um + 5% fetal calf s e ru m Last ha l f 10.0 4.4 1.2 12.8 62.1 4.0 4.8 1.5

5% Fetal calf s e r u m Last half 24 .4 10.3 3.4 31.4 11.2 0.7 9.8 3.4

5% Bovine s e rum Last half 8.6 3.6 3.0 16.8 61.2 4.4 2.4

2.5% Fetal calf s e rum First ha l f 21.5 8.9 4.6 33.6 16.6 1.1 8.3 2.1

2 .5% Fetal calf s e rum Last half 17.2 4.3 7.1 5 t . 0 6.8 - - "7.8 5.9

5% Lipid-free fetal calf s e rum First ha l f 23.5 10.5 7.1 34.2 10.4 1.0 6.9 5.0

5% Lipid-free fetal calf s e rum Last half 14.4 11.2 7.9 53.9 4.1 1.2 3.6 0.7

5% Lipid-free fetal calf s e rum + pa lmi t ic acid d Last ha l f 22.9 9.6 7.5 42.3 7.7 T 6.7 2.5

5% Lipid-free fetal calf s e rum + linoleic acid d Last ha l f 10.1 3.7 12.2 27.8 40.1 - - 2.5 T

aDi f fe rence b e t w e e n the s u m of the f a t ty acid pe rcen tages in each r o w and 100% represen t s the s u m of minor c o m p o n e n t s no t given in the table .

bpe rcen tages r ep resen t the mean of dupl ica te analyses on a single poo led sample .

CT : de tec tab le but less than one ha l f per cent .

d37 .5 I-tg per ml o f media .

tions. The percentage distribution of fatty acids from bovine and fetal calf serum neutral tipids also are given for comparison. When bovine serum at any concentration was used to supplement the media, t8:2 acid represented more than 60% of the neutral lipid fatty acids after incubation. Medium from cells grown on lipid- free fetal calf serum, plus linoleic, contained a high level of linoleic acid but not as high as when bovine serum was used in the culture media. The addition of palmitic acid to medium, supplemented with lipid-free fetal calf serum, did not elevate the concentration of palmitic in the medium after incubation. When either whole fetal calf serum or lipid-free fetal calf serum was used to supplement the medium, the second half incubation media contained higher levels of 18:1 acid and lower 18:2 acid concentrations than did the first half incubation media.

Fatty Acid Composition of Media Phospholipids

The fatty acid composit ion of the phospholipid fractions, derived from the last half

incubation media obtained from the several different growth conditions, is given in Table VI. Media phospholipid fatty acids were dis- tinctly different from media neutral lipid fatty acids (Table V). Stearic and 18:2 acid levels were noticeably higher when the media contained bovine serum, whereas those media supplemented with fetal calf serum or lipid-free fetal calf serum contained less stearate and higher percentages of 18:1 acid. Media, supplemented with only fetal calf serum or lipid-free fetal calf serum, contained relatively higher percentages of 16:1 acid, but bovine serum supplemented media did not contain measur- able levels of this acid. The addition of palmitic acid and linoleic acid to media containing lipid-free fetal calf serum led to an increase in the concentrations of stearic and 18:2 acids, respectively, in media phosp~olipids after cell cultivation. Generally, the fatty acid composition of first and last half media phospholipids were similar.

DISCUSSI ON

A number of investigators (12,24-31) has


698 R. WOOD

TABLE VI

Comparison of Total Phospholipid Fat ty Acid Composition of Serums and Media after Minimal Deviation Hepatoma Cells were Cultured under Various Growth Condi t ions

Fat ty acid percentagesa, b

22:4 Serums and basal +

medium supplement 16:0 16:1 18:0 18:1 18:2 20:3 20:4 24:0 24:1 22:5

Serums

Fetal calf serum 20.9 0.8 20.2 19.8 1.5 2.5 8.4 2.0 6.3 6.5 Bovine serum 18.3 0.8 25.5 16.0 12.7 5.9 6.9 1.2 3.2 3.3

Media c 20% Bovine serum + 5% fetal calf serum 23.1 - - 29.4 20.5 16.1 4.5 6.5 - - T d T 10% Bovine serum + 5% fetal calf serum 22.4 - - 31.4 21.4 12.2 5.2 6.5 - - T T 5% Bovine serum + 5% fetal calf serum 26.8 - - 30.3 22.4 10.3 4.2 5.9 - - T T 5% Fetal calf serum 20.7 8.0 15.7 43.4 2.4 - - 4.6 T T T 59~o Bovine serum 15.8 - - 26.3 21.4 13.4 6.1 7.6 T T 1.8 2,5% Fetal calf serum 20.8 5.4 18.1 34.6 3.4 1.3 7.0 0.6 1.3 2.4 5% Lipid-free fetal calf serum 21.4 4.9 16.7 36.5 2.0 - - 4.6 1.8 5.7 2.1 5% Lipid-free fetal calf s e r u m + palmit icacid e 22.3 3.4 22.7 31.4 2.6 1.2 6.3 2.7 4.0 2.0 5% Lipid-free fetal calf s e r u m + l i n o l e i c a c i d e 20.5 3.5 17.2 17.4 21.4 - - 9.0 1.9 2.6 3.2

aDifference between the sum of the fatty acid percentages in each row and 100% represents the sum of components not given in the table.

bpercentages represent the mean of duplicate analyses on a single pooled sample. CMedia was f rom the last half of the incubation in all cases except when the medium was supple-

mented with 20% bovine serum plus 5% fetal calf serum, which was media for the total incubation time.

dT = detectable but less than one half per cent. e37.5 /~g per ml o f medium.

s h o w n , w i t h a v a r i e t y o f c u l t u r e d cel ls , t h a t , w h e n s e r u m l ip ids we re p r e s e n t in t h e m e d i a , l i t t l e ce l lu l a r l i p id s c a m e f r o m de n o v o b i o s y n - t h e s i s a n d m o s t we re d e r i v e d f r o m t h e s e r u m . T h e r e f o r e , t h i s i n v e s t i g a t i o n was i n i t i a t e d to d e t e r m i n e t h e m i n i m u m level o f s e r u m l ip ids H T C cel ls r e q u i r e f o r g r o w t h a n d to d e t e r m i n e w h a t l ip id b i o s y n t h e s i s t h i s cel l is c a p a b l e o f c a r r y i n g o u t in t h e a b s e n c e o f o r a t l o w levels o f e x o g e n o u s s e r u m l ip id . Cell Growth

B o v i n e s e r u m c o n t a i n s ca. f o u r t i m e s m o r e l i p i d / m l t h a n f e t a l c a l f s e r u m . T h e r e f o r e , t h e a m o u n t o f l ip id in t h e m e d i a was r e d u c e d m o r e t h a n 90% f r o m t h e level o f t h e s t o c k c u l t u r e s w h e n i t was d e t e r m i n e d t h a t t h e m o d i f i e d S w i m ' s 77 m e d i u m s u p p l e m e n t e d w i t h 5% fe ta l ca l f s e r u m s u p p o r t e d g o o d g r o w t h . T h i s level o f l ip id ( 3 8 . 5 / a g / m l m e d i u m ) was ca. t he s a m e as t h a t u s e d by W a t s o n ( 1 2 ) in g r o w i n g s u s p e n s i o n c u l t u r e s o f H T C cells. M e d i u m c o n t a i n i n g 2 . 5 % fe ta l c a l f s e r u m did s h o w s o m e g r o w t h b u t

p r o b a b l y w o u l d n o t have s u p p o r t e d g r o w t h fo r a n y e x t e n d e d p e r i o d o f t i m e . S t o c k c u l t u r e s g r o w n on 25% s e r u m a n d u s e d as s t a r t e r c u l t u r e s in all e x p e r i m e n t s p r o b a b l y c o n t a i n e d e n o u g h s t o r e d l ip ids , g r o w t h f a c t o r s , e t c . , t o p e r m i t o n e o r t w o cell d iv i s ions . M e d i u m s u p p l e m e n t e d w i t h 5% l ip id - f r ee f e t a l c a l f s e r u m o r s u p p l e m e n t e d w i t h l i p id - f r ee f e t a l ca l f s e r u m , p l u s e i t h e r l i no l e i c o r p a l m i t i c ac ids , gave p o o r g r o w t h o f H T C cells in ro l le r cul - t u re s . H o w e v e r , t h e level o f f ree a c id s u s e d was p r o b a b l y i n h i b i t o r y r a t h e r t h a n g r o w t h p ro - m o t i n g . R e c e n t e v i d e n c e f r o m t h i s l a b o r a t o r y ( u n p u b l i s h e d ) ha s s h o w n t h a t l o w e r levels o f l i no l e i c ac id s h o w s o m e g r o w t h s t i m u l a t i n g ac t i v i t y , wh i l e t h e h i g h e r levels p r e v e n t g r o w t h . G e r s c h e n s o n , e t al. , ( 3 2 ) h a s s h o w n t h a t l o w levels o f l i no l e i c a d d e d to H e L a cel ls g r o w i n g in l i p i d - d e f i c i e n t m e d i u m s t i m u l a t e d cell g r o w t h . H a m ( 3 3 ) a lso h a s s u g g e s t e d t h a t l i no le i c ac id was a g r o w t h f a c t o r f o r c u l t u r e d C h i n e s e h a m s t e r cells.


C U L T U R E D H E P A T O M A C E L L LIPIDS 699

Extraction of Serum Lipids

Described procedures for reducing the amount of lipid in serum (12,34-36) either required too much time, low temperatures, and purification of the proteins precipitated with organic solvents, or failed to remove all the phospholipids. In search of a simple and quick method that would remove all the lipids, we tried chloroform-methanol (2:1) extraction of lyophilized serum. Surprisingly, the dry chloroform-methanol insoluble material dissolved completely in either distilled water or Swim's 77 medium and showed little denatured protein, as judged by filtration (0.2 p pore size) and polyacrylamide gel electrophoresis (a single run). The lipids extracted from the serum with chloroform-methanol were chromato- graphically (GLC and TLC) identical to the lipids extracted from lyophilized fetal calf serum by the Bligh and Dyer procedure (19).

Media Lipids

The accumulation of both neutral lipids and phospholipids in the media under all growth conditions (Table II) was unexpected and ap- parently is the first report of lipid excretion by HTC cells. Hruban, et al., (37) has reported the formation and excretion of lipoproteins by a number of other hepatomas based upon histo- logical examinations. The accumulation of lipids in the media of all growth conditions is of interest since most cultured cells studied thus far obtain a high percentage of their lipids from the media. The effect of exogenous lipids on lipid metabolism of cultured cells has been reviewed by Rothblat (38) and Bailey, et al. (39). Media neutral lipid and phospholipid profiles do not indicate a predominance of any one lipid class under any growth condition. Contrarily, the major lipid components from lipid-free serum supplemented medium, as well as the other media, were sterol esters, free fatty acids, free sterol, sphingomyelin, phosphatidylcholine, and an unidentified compound. The excretion of free sterols has been shown to occur in L-cells and MBIII cells by Bailey (40) who also reported data to suggest the excretion may be under the influence of serum globulins. Cultured L5178Y cells have been shown to increase the release of free sterol when phospholipids were added to the medium containing either delipidized serum protein or serum albu- min (41). The excretion of other lipid classes by cultured cells does not appear to have been reported previously.

The similarity between the media neutral lipid and phospholipid class profiles and the corresponding pattern of the serums suggest that HTC cells may excrete into the media

lipids with the same class composition as serum. Phosphatidylethanolamine and trigiycerides are present in only trace amounts in serums, and they are also absent from all media even though the cells contain high levels of these two lipid classes. The presence of lipid classes in the cell, but not in the medium in which the cells were grown, is important. First, it rules against the possibility that the lipids, assumed to be excreted by the cells, did not arise from dead cells; and secondly, it suggests that the cellular lipids are not in equilibrium with media lipids. The reasons are numerous as to why the HTC cells may excrete lipids into the media of the same lipid class composition as serum. The most logical reason seems to be that these cells are performing functions similar to that carried out by normal liver cells. Another reason may be that serum lipids induce the synthesis of only the lipid classes present in the serum. A third possibility is that these cells may continue to secrete lipids into the medium in the presence of serum, because these cancer cells may have lost their feedback control. The loss of feedback control of cholesterol biosynthesis in almost all tumors is well known and has been reviewed recently by Sabine (42).

When low levels of fetal calf serum or lipid-free fetal calf serum were used to supplement the medium, significant differences in the fatty acid composition between first and last half media neutral lipids were observed. The composition of the media lipids from the last half of the incubation probably more nearly represented the composition of the fatty acids the cells excreted under the particular nutritional regime, because any cellular stores of lipid accumulated during the growth of the starter cultures would have been depleted or removed in the first half incubation media. This conclusion is supported by the fact that higher levels of 18:1 acid and lower levels of palmitic acid from the last half incubation media ap- proach the percentage composition of these acids found in the cells. The difference in the fatty acid composition of the serum neutral lipids and phospholipids from media neutral lipids and phospholipids is significant. The higher percentages of monoenoic acids in the media, than were present originally in the serum lipid fractions, demonstrate that acyl moieties were excreted. The increased level of media 18:2 acid in the phospholipid fraction from cells grown on lipid-free fetal calf serum, plus linoleic acid, indicated the ability of the cells to take up free fatty acids, incorporate them into phospholipids, and excrete them into the medium. The ability of the HTC cell to carry out the above mentioned sequence of

LIPIDS, V O L . 8, NO. 12

700 R. WOOD

events has been es tabl ished i n d e p e n d e n t l y in this labora tory using labeled palmit ic acid.

Cellular kipids

Cellular neutra l l ipid and phospho l ip id pat- terns (Figs. 1 and 2) were dis t inct f rom bo th serum and media lipid pat terns . Al though some differences were observed, a visual compar i son of cellular lipid classes does no t reveal the dramatic a l tera t ion in lipid pa t te rns one might have expec ted as a result o f the ex t remes in g rowth condi t ions . This suggests that exogenous serum lipids may have less inf luence on HTC cell l ipid b iosynthes is than previously r epo r t ed for most other cultured cells. Triglyc- eride b iosynthes is appeared to be inhib i ted by the absence of fetal calf serum or the presence of only bovine serum in the med ium. Sterol ester synthesis appeared to be reduced by high levels o f free fa t ty acids. The decreased level o f tr iglycerides and sterol esters i l lustrates that cellular lipid tu rnover occurred.

Detectable quant i t ies of glyceryl e the r diesters were n o t observed in the HTC ceils or media ob ta ined f rom the various growth conditions. These data demons t r a t e that the presence of glyceryl e ther diesters is n o t a character is t ic o f all neop lasms and, therefore , is n o t a good cr i ter ion for iden t i fy ing neoplasms, as has been suggested previously (43,44). The phospholipids o f HTC cells also con ta in low levels o f plasmalogens, as judged by the small size of the a ldehyde peaks when the me thy l esters f rom the phospho l ip id f rac t ion were analyzed.

The increased level of 20:2 and 20:4 acids indicates that the HTC cell can elongate and desaturate dienoic acids. Harary, e t al., (45) has shown tha t HeLa cells and hear t cells cultured for long per iods of t ime show p o o r conversion of linoleic acid to arachidonic acid, whereas fresh hear t cells p e r f o r m e d this conversion readily.

The di f ferences in the class compos i t ions of the cells and the serums indicate tha t HTC cells are capable of synthes iz ing triglycerides, phospha t idy le thano lamine , phospha t idy l se r ine , and phospha t idy l inos i to l . The class compos i t i on of the cells grown on m e d i u m s u p p l e m e n t e d with l ipid-free fetal calf serum fur ther suggests that these cells also can synthes ize sterol esters, free fa t ty acids, sterols, phospha t idy lcho l ine , and sphingomyelin. Results f rom this labora tory , that will appear in a separate publ ica t ion, show tha t radioactive labeled fa t ty acids are incorpo- ra ted in to individual phosphol ip ids and acyl conta in ing neutra l lipid classes by these cells. These data suggest that HTC cells are capable of synthes iz ing most , if n o t all, of their s tructural neutral lipids and phosphoglycer ides .

ACKNOWLEDGMENTS

This work was supported by a grant from the National Cancer Institute (USPH 5-RO1 CA 12973). J. Falch and R. Zaun provided technical assistance.

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Haggerty, Jr., Biochim. Biophys. Acta 131:42


CULTURED HEPATOMA CELL LIPIDS 701

(1967). 33. Ham, R.G., Science 140:802 (1963). 34. Albutt, E.C., J. Med. Lab. Tech., 23:61 (1966). 35. Williams, C.D., and J. Avigan, Biochim. Biophys.

Acta, 260:413 (1972). 36. Avigan, J., J. Biol. Chem. 226:957 (1957). 3"/. Hruban, Z., Y. Mochizuke, Mor~'is, H.P. and A.

Slesers, Lab. Invest. 26:86 (1972). 38. Rothblat, G.H., Advan. Lipid Res. 7:135 (1969). 39. Bailey, J.M., B.V. Howard, L.M. Dunbar and S.F.

Tillman, Lipids 7:125 (1972). 40. Bailey, J.M., in "Lipid Metabolism in Tissue

Culture Cells," Edited by G.H. Rothblat and D. Kritchevsky, Wistar Institute Symposium Mono- graph, 6, 1967, p. 85.

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44. Snyder, F., and R. Wood, Ibid. 28:972 (1968). 45. Harary, I., LE. Gerschenson, D.F. Haggerty, Jr.,

W. Desmond and J.F. Mead, in "Lipid Metabolism in Tissue Culture Cells," Edited by G.H. Rothblat and D. Kritchevsky, Wistar Institute Symposium Monograph 6, 1967, p. 17.

[Received June 14, 1973]

L1PIDS, VOL. 8, NO. 12

lipids of cultured hepatoma cells: i. effect of serum lipid levels on cell and media lipids

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