Role of Fatty Acids in Growth-promoting Effect of Serum Albumin on Hamster Cells In Vitro KARIN NILAUSEN Department ofAnatomy C, University of Copenhagen, 71 Raadmandsgade, 2200 Copenhagen N, Denmark

ABSTRACT

Dialyzed serum albumin had considerable growth-promoting effect on cultivated hamster cells. This effect was virtually lost on removal of the fatty acids, and it was completely restored by recombination of the fatty acid-free albumin with the isolated and purified fatty acids. The role of albumin itself appeared to be largely that of a carrier of fatty acids, protecting the cells against toxic effects of fatty acids in free solution. This conclusion was based on two observations: Fatty acids in the absence of albumin were growth-inhibitory except in extremely dilute solutions, and betalactoglobulin, a protein possessing, like albumin, the ability to bind and release fatty acids, could replace albumin in the presence of fatty acids with similar growth-promoting effect. Examination of individual molecular types of fatty acids showed that all unsaturated acids tested were growth-promoting, whereas the saturated acids were growth-inhibiting, with the exception of stearic acid in low concentrations. Although the possibility of a mitotic triggering effect was not excluded, the fatty acids presumably stimulated growth by providing substrate for cellular metabolism, since there was a direct relationship between the degree of growth stimulation and the duration of exposure of cells to the fatty acids.

Serum albumin stimulates growth of mammalian cells in synthetic and semisynthetic media (Waymouth, '56; Ham, '63a; Moskowitz a n d Schenck, '65), improves long term cultivation of diploid fibroblasts (Todaro and Green, '64), promotes the development of established cell lines from primary cultures (Matsuya and Yamane, '68, '70) and is essential for proliferation of activated lymphocytes (Polet and Spieker-Polet, '75). The growth-promoting effect may reside with the polypeptide itself, but since albumin has the ability to bind many different molecules it might be due to associated growth factors, it might be due to the binding and inactivation of growth-inhibiting metabolites (Moskowitz and Schenck, '65; Temin e t al., '72) or to the combined effect of any of these. A number of workers have shown t h a t both serum (Jenkin and Anderson, '70; Holley e t al., '74) and serum albumin (Dubin e t al., '65; Ham, '63b; Yamane e t al., '75) in the medium may be replaced by unsaturated fatty acids J. CELL. PHYSIOL. (1978)96: 1-14.

without any significant reduction in cellular growth. Since by far the major portion of free fatty acids in serum are carried by the albumin these findings suggested that fatty acids associated with albumin were responsible for the growth-promoting effect. When, however, the effect of fatty acid-free and fatty acid-associated albumin on growth of chicken spleen cells (Norkin e t al., '65) and activated human lymphocytes (Polet and Spieker-Polet, '75) were compared, the data provided no support for this suggestion, and the authors concluded that the growth-stimulating effect of albumin was due either to the binding of inhibitory metabolites or to the polypeptide itself (Norkin e t al., '65; Spieker-Polet and Polet, '76). Since these conclusions appeared difficult to reconcile with data I had previously obtained in studying the effects of albuminbound lysolecithin and fatty acids on cellular growth (Nilausen, '68), a number of additional Received Dec. 21, '76. Accepted Jan. 16, '78.

1

2

KARIN NILAUSEN

experiments were done to reexamine the question of how serum albumin promotes the proliferation of hamster cells in vitro. The present paper describes these experiments and their outcome. A preliminary report has been presented.' MATERIALS AND METHODS

Cells and medium All stock cultures were routinely cultivated in Dulbecco and Vogt's modification of Eagle's medium supplemented with 10% calf serum under a humidified atmosphere of 10%C02 in air. Chinese hamster cells, line CHEF, were obtained from Doctor M. Fraccaro EURATOM Unit for Human Radiation and Cytogenetics, University of Pavia, Pavia, Italy. The cells were transferred twice a week by trypsinization in 0.1% trypsin in phosphate buffer containing 0.01% EDTA and inoculated at a density of 2 x lo5 cells per plastic Petri plate, diameter 50 mm, in 4 ml of growth medium. The cells were tested routinely for mycoplasma infection (Barile et al., '62) and invariably found negative.

that by far the major increase in cell number took place within the initial 72 hours. The choice of inoculation density was based on another series of preliminary experiments where the inoculum ranged from 1 X lo4 to 64 x lo4cells per plate. A maximal and uniform growth response was found using inocula varying from 8 X lo4 to 32 X lo4 cells per plate. For ease of counting the lowest inoculum in this range, 8 X lo4cells, was chosen for the growth experiments. To ensure that the cells counted at the end of a 72-hour growth period were in fact living cells, plating efficiency determinations were carried out in a number of experiments. In all cases the cells counted showed plating efficiencies comparable to the ones found for cells grown under routine conditions, usually around 65%. Test preparations

Fraction V (Batches no KH0270, NG0370, KFI774, TNI471, VBI370) and crystalline (Batch no KF0870) albumin prepared from bovine serum by Armour Pharmaceutical Company were used; except where specifically indicated fraction V was the albumin emEvaluation of growth ployed. Preformed free fatty acids were reExperimental cultures were inoculated moved by the methods of Goodman ('57) or with 8 X lo4 cells per plastic Petri plate, 32 Chen ('67), so that the fatty acid-albumin mm in diameter, in 1.5ml of the usual growth molar ratio was reduced to less than 0.04. Exmedium and the cells were allowed to attach cept where specifically indicated the method at 37OC. After four hours the medium was re- of Goodman was used. Following fatty acid removed and the experimental medium added. moval by either procedure the albumin was This medium, containing the various test sub- always dialyzed for at least 24 hours a t 4OC stances indicated, was serum-free but other- against 3 changes of 20 volumes of distilled wise identical with the routinely used growth water and the concentration was determined medium. The plates were then incubated for gravimetrically. Fatty acids were complexed three days. Cell numbers were determined at with albumin in the following way: Fatty the beginning (No) and at the end (N,) of this acids, dissolved in chloroform, were deposited period by counting the number of cells located in a round bottom flask by evaporating off the in five squares, each measuring 0.082 mm2, solvent under a stream of nitrogen and a n etched into the bottom of the culture plate. aqueous 10% solution of albumin a t pH 7.35 Growth was expressed as fold increase in cell was added. The mixture was then incubated at number in those five squares over three days, 37°C under nitrogen on a shaker for 2 to 24 N3/No. The precision of this procedure was hours. The completeness of fatty acid uptake evaluated by determining the variation ob- by this procedure was checked in each intained from each of the five individual squares stance by measuring the fatty acid content of of a single plate. The precision, calculated as the albumin solution before and after complex coefficient of variation, for 12 plates varied formation. In addition, the fatty acid composiwith growth: for fold increase in number of 13- tion of the albumin-bound fatty acids was 9,9-5, 5-2, and 2-1 the coefficients of variation checked by gas-liquid chromatography, to exclude changes during preparative manipula(mean standard deviation) were 4.2% tions. The fatty acid concentration was deter1.07, 6.4% 2 3.07, 7.0%* 3.05 and 11.3% 3.04, respectively. The 3-day growth period mined usually by titration following double was chosen on the basis of preliminary experi' First International Congress on Cell Biology, Boston. Massachuments in which daily cell counts had shown setts, 1976.

*

*

FATTY ACIDS, SERUM ALBUMIN AND CELL GROWTH

0 0

1

3

1.o

.5

PER CENT A L B U M I N I N M E D I U M Fig. 1 Effect of bovine serum albumin on growth of Chinese hamster cells in vitro. The albumin was divided in two portions. One was dialyzed against water, the other portion was used undialyzed. The points and the vertical bars represent average and standard error of the mean (SEM), respectively, of three experiments, each representing 18 plates.

extraction (Dole and Meinertz, '60). But in some cases the concentration was determined by gas-liquid chromatography following addition of a known amount of arachidic acid as internal standard. Standard thin-layer (Bollinger et al., '65) and gas-liquid (Johnson and Stocks, '71) chromatographic techniques were used to evaluate the composition of lipids associated with the albumin. Pure fatty acids ( 99%)were purchased from Applied Science Laboratories Incorporated, and their purity w a s confirmed by gas-liquid chromatography.

Following dialysis the growth response to albumin doubled a t practically all concentrations (fig. 1). Native albumin preparations contained, therefore, besides growth-promoting, also growth-inhibiting components. Hanson and Ballard ('68) and Engel et al. ('65) showed that dialysis of bovine serum albumin eliminated mainly citrate, added originally as an anticoagulant, and also lactate, pyruvate and iron.

RESULTS

Effect of the fatty acid component

+

Effect of untreated albumin When Chinese hamster cells received medium containing neither serum nor albumin the cells showed no growth, but remained viable a s shown by plating efficiency. Addition of untreated bovine serum albumin stimulated cell proliferation, and the growth response increased with increasing concentrations of albumin in the medium (fig. 1). Since albumin in concentrations exceeding 1.5% (225 p M ) made the cells detach, growth response to concentrations above that range could not be determined by the present technique.

Effect of dialyzed albumin

Extraction of albumin with isooctane-glacia1 acetic acid by the method of Goodman ('57) removed most of the growth-promoting activity (fig. 2). Since the extraction removed virtually all of the fatty acids bound to the albumin these findings suggested that the growth-promoting activity of albumin resided largely with the associated fatty acids. To exclude the possibility that growth-stimulating factors other than the fatty acids had been removed in the process of extraction, the native fatty acid mixture removed by the Goodman procedure was recovered, purified by prepara-

4

KARIN NILAUSEN

*-*U

NEXT RAC T E D ALBUM11

o---oALBUMI N EXTRACTED A N D RECONSTITUTED WITH P U R I F I E D FATTY ACIDS

A‘

/

EXTRACTED ALBUMIN

c

L I

I

O

0

.5

1

1.oi

PER CENT ALBUMIN I N MEDIUM Fig. 2 Effect of fatty acid extraction and of fatty acid extraction plus recombination on growth-promoting property of bovine serum albumin. One portion of bovine serum albumin was extracted and another was left unextracted. The extracted native fatty acid mixture was purified by preparative thin-layer chromatography, and recombined with part of the extracted albumin, so that the fatty acid-albumin ratio was identical with that of the unextracted portion, namely 0.48. All preparations were finally dialyzed against water. The points and the vertical bars represent average and SEM, respectively, of three experiments, each representing 26 plates.

tive thin-layer chromatography, and recom- with bovine serum albumin showed them to be bined with the extracted albumin to make the a complex mixture consisting largely of 16 and same molar fatty acid-albumin ratio as that of 18 carbon chains with 0 , l and 2 double bonds the original preparation. A comparison of the (table 1). This suggested the possibility t h a t original albumin with the extracted preparation recombined with the purified fatty acids TABLE 1 showed that the growth-promoting effects of the two preparations were identical (fig. 2). Composition ofthe fatty acid mixture associated with bovine serum albumin This indicated that the loss of growth-stimulating activity following extraction was in Fatty acid Percent by weight fact due to removal of the fatty acids associ16:O ’ 17.5 ated with the albumin. 16:l 3.4 By increasing the content of the native fat18:O 27.5 ty acid mixture associated with the albumin, 18:1 30.6 keeping the concentration of albumin in the 18:2 12.9 medium constant a t 1%(150 p M ) ,it was found 18:3 2.6 that the growth response increased with inR* 5.5 creasing fatty acid concentrations up to 150 Albumin (Fraction V, lot no KH0270)was extracted by the method p M , corresponding to a molar fatty acid-alGoodman (‘57) and the fatty acids were isolated by thin-layer chrobumin ratio of 1.0 (fig. 3). Fatty acid con- of matography, methylated and analyzed by gas-liquidchromatography centrations above this, up to a molar ratio of on a diethyl glycol auccinate 115%) column with hydrogen flame detection. The individual fatty acids were identified by their relative 5.0 caused a gradually decreasing growth retention timesand by comparison with methyl estersof known fatty response. acids.

Effects of individual fatty acids Analysis of the native fatty acids associated

I Fatty acids are designated by chain length: number of double bonds. Sum of remaining trace components each constituting less than 2%of the mixture.

FATTY ACIDS, SERUM ALBUMIN AND CELL GROWTH

5

10I Y

9-

W

:

8-

3

*

7-

-I

G-

z

5-

u

2

I-

E3: -

5 2 0

?i

Fig. 3 Effect of varying the concentration of fatty acids in medium containing 1% (150 pM) albumin. Bovine serum albumin was extracted, dialyzed and recombined with increasing amounts of the extracted native fatty acid mixture purified by preparative thin-layer chromatography. The fatty acid/albumin molar ratio ranged from 0 to 5. The points and the vertical bars represent average and SEM, respectively, of four experiments, each representing 14 plates.

different molecular species might affect growth of cultured cells differently. This was indeed found to be the case. Of the six different fatty acids tested initially all four unsaturated acids stimulated growth, while the two saturated acids inhibited growth at all levels of concentration (fig. 4). In similar experiments a third saturated fatty acid, stearic acid, was found to be inhibitory at levels of 75 pmoles per liter and above, but in distinction t o palmitic and lauric acid, was growth-stimulating to a modest degree a t concentrations below this level with maximal effect at 30 pmoles per liter. As was the case with the native fatty acid mixture of bovine serum albumin (fig. 3) the maximal growth-stimulating effect of the individual unsaturated f a t t y acids was observed at about a concentration of 150 pM and a molar fatty acid-albumin ratio of 1.0. Of the unsaturated fatty acids tested the 18 carbon chains with a cis double bond at the 9 position had the greatest growth-stimulating effect. Addition of a second double bond at position 12 did not improve growth further. In other words, the essential linoleic acid did not possess any advantage

over that of the non-essential oleic acid, a phenomenon that agreed well with the general observation that most tissue culture cells grew well in the absence of essential fatty acids (Bailey and Dunbar, ’73). The cis form of the double bond at the 9 position was a t least twice as effective as was that of the trans, and it was less effective in a 16 carbon than in an 18 carbon monounsaturated fatty acid (fig. 4). The growth-promoting effect of the oleic acid-albumin complex was the same whether the albumin had been extracted by the method of Goodman (‘57) or of Chen (‘671, and whether the oleate was complexed with albumin in the form of sodium soap or undissociated free fatty acid (no data presented).

Effect of the fatty acid-free albumin When bovine serum albumin, freed of fatty acids and dialyzable impurities, was added in increasing amounts to the medium the growth response increased modestly from about 1.4 in the absence of albumin to the maximal value of 2.4 a t the highest concentration of 1%(fig. 2). Since extracted albumin contained traces of preformed fatty acids, this might explain

6

KARIN NILAUSEN

18:2 (9cis,12c is: 16 :1(9c i s ) 18:1 (9cis)

qK3--

.

-

I 1

-

-

-

O

I

18:1(9 trans)

=

=

~

~

-

-

I

015 75

-- - - - - - - -- -- - - - --

-- - - --

150

b

bJ 16:O

J

12:o 1

375

750

FATTY ACID CONCENTRATION (p MOLES per LITER) Fig. 4 Effect of chain length, number of double bonds and double bond configuration on growth-stimulating activity of albumin-bound fatty acids. The concentration of bovine serum albumin was 1% (150FM), so that the molar fatty acid/albumin ratio varied from 0 to 5. The points and the vertical bars represent average and SEM, respectively, of four experiments, each representing 62 plates. TABLE 2

Effect of albumin concentration on cellular growth response Composition of medium Oleic acid Albumin

Oleic acid p o l e s p e r liter

15 75 195

Albumin Molar ratio

75 75 75

5.0 1.0 0.5

Fold increase in cell numbei

'

6.8'0.72 5.6? 0.83 2.8r0.49

p< 0.05 p< 0.05

Extracted bovine serum albumin was combined with oleic acid as described in the methods section. The growth response in each experiment was determined from nine plates, three plates for each level of albumin concentration in the medium. ' Mean 2 SEM of five experiments. p value determined according to Student's t-test by paired comparison.

the modest growth response. In a n attempt to avoid this difficulty the effect of increasing albumin concentrations was examined in the presence of a constant amount of oleic acid. The data showed that the growth response decreased with increasing concentrations of albumin (table 2). This experiment thus provided no evidence that albumin per se stimulated growth. But the data did show that for a given concentration of oleic acid the growth

response increased with increasing fatty acidalbumin ratios.

Effect of fatty acids in the absence of albumin Addition of individual fatty acids, dissolved in a small amount of ethanol, to medium containing no albumin affected growth in a way qualitatively similar to t h a t observed when albumin was present. Thus oleic and linoleic acids stimulated growth, palmitic acid inhib-

FATTY ACIDS, SERUM ALBUMIN AND CELL GROWTH

I

0

I

7

I

10 20 30 Lb 50 GO 70 FATTY ACID CONCENTRATION p MOLES per LITER

80 90 100

IN

MEDIUM

Fig. 5 Effect of individual fatty acids in albumin-free medium. The fatty acids were added to each individual plate, containing medium without albumin and serum, in 10 pl of ethanol. The plates receiving no fatty acids also had 10 pl of ethanol added to them. The points and vertical bars, respectively, represent average and SEM of eight experiments, each representing 60 plates. Control experiments showed a slight growth-inhibitory effect of ethanol under the present conditions: Fold increase in cell number, N3/No,was 0.81 2 0.18 and 1.09 C 0.27 (average 2 SD, n = 8 ) with and without ethanol (p < 0.05),respectively.

ited growth (fig. 5) and elaidic acid (no data presented) had a n effect intermediate between that of oleic/linoleic acids and palmitic acid. But the maximal growth stimulating effect obtained was (1) much lower than that observed in the presence of albumin, and was (2)obtained a t much lower fatty acid concentrations.

Comparison of P-lactoglobulin and albumin If the main function of albumin were to serve as a carrier of fatty acids, then other proteins capable of binding and releasing fatty acids would be expected to produce growthstimulating effects similar to those observed with albumin. Preformed fatty acids were removed from P-lactoglobulin (Schwarz/Mann. 3 X crystallized. Lot no 3148) by the procedure described by Chen (‘67) for the removal of fatty acids from albumin. This procedure apparently did not modify the protein as judged by its electrophoretic mobility and its capability for association with fatty acids (fig. 6). A comparison of the effects of equimolar concentrations of P-lactoglobulin and albumin complexed with different fatty acids is shown in figure 7. The pattern of growth pro-

motion and growth inhibition of the different fatty acids was similar in the presence of the two different proteins, both quantitatively and qualitatively. Although the corresponding values in fold increase were somewhat different, due to differences in the basal effects of the two proteins in the absence of fatty acids, the net effects of the different fatty acids were similar.

Comparison of crystalline serum albumin and fraction V Thin-layer chromatography of total lipid extracts of the two albumin preparations revealed a compound associated with the crystalline product, which was not present in fraction V (fig. 8). This unknown and unexpected component, which was present in greater amounts than the free fatty acids (3.8 mg versus 3.2 mg per g crystalline albumin, Batch no KF0870),was tentatively identified as decanol by thin-layer and gas chromatography. As it turned out the decanol had been added by the manufacturer since Cohn et al. (‘47) recommended the addition of decanol in their procedure for crystallization of albumin. Following extraction by the method of Goodman (‘57) neither of the albumin preparations

8

KARIN NILAUSEN

Fig. 6 Agarose gel electrophoresis of P-lactoglobulin preparations together with radiochromatoscans of t h e electrophoretic strips. 50 p g of P-lactoglobulin (1 p1 of a 5%solution) was applied to agarose gel (1%)in sodium barbital buffer (0.08 M, pH 8.6) and electrophoresed for 50 minutes at 20 V/cm. The strips were stained w i t h Coomassie Brilliant Blue R-250 (Johansson, '72). The P-lactoglobulin preparations were: (1) untreated, (3) following extraction by method of Chen ('67) whereby the molar fatty acidlP-lactoglobulin ratio was reduced from 0.27 to 0.09. (2) a n d (4) same as (1) and (3), respectively, following combination with "C-oleic acid. The points of application are located at the upper edge of the numbered labels. The radiochromatoscans, done on a Packard Instrument Radiochromatogram Scanner Model 7200, show the "C-label to be associated completely with both t h e untreated (11, and the extracted (4), preparations, each containing approximately 200,000 dpm of "C-oleic acid.

p-LACTOGLOBULI N

ALBUMIN

T

NONE l8:l CIS

18:l 16:O

trans

NONE 18:l 18:l 16:O cis trans

ADDITI 0 N S Fig. 7 Comparison of albumin and P-lactoglobulin a s carriers of fatty acids in growth medium. Preformed fatty acids were removed from both proteins by t h e extraction method of Chen ('67). The molar f a t t y acid-protein ratio obtained after extraction was 0.04 for albumin and 0.09 in case of P-lactoglobulin. Oleic (18:l cis), elaidic (18:l trans) and palmitic (16:O) acid were complexed to the proteins as described in the methods section, to yield a molar fatty acid-protein ratio of 1.The extracted proteins both with and without added fatty acids, were added t o g r o w t h medium in a final concentration of 125 pM, corresponding to 0.8%albumin and 0.5%P-lactoglobulin. The columns and vertical bars, respectively, represent average and SEM of six experiments, each representing 16 plates. The s h a d e d portions of t h e columns represent the net growth effect of t h e added fatty acids.

contained decanol or fatty acids in amounts detectable on the thin-layer plate (fig. 8). In figure 9 is shown a comparison of the effects of crystalline albumin and fraction V, both with and without oleic acid and decanol. Crystalline albumin, without any additions, produced nearly double the growth response of

fraction V at the lower concentrations. I n the presence of oleic acid, whether or not decanol had been added, the crystalline preparation was superior to fraction V a t all levels of concentration. The presence of decanol, added to the preparation in quantities to m a k e the molar de-

9

FATTY ACIDS, SERUM ALBUMIN AND CELL GROWTH

Fig. 8 Thin-layer chromatogram of total lipids from fraction V and crystalline albumin. Albumin was extracted with chloroform-methanol (Folch e t al., '57).The rectified extract from 50 mg of albumin was applied to a silica gel G plate, which was then developed in petroleum ether, diethyl ether, glacial acetic acid (60:40:1);the components were made visible by charring after spraying with 50% sulfuric acid. The numbers on the plate represent: Fraction V, before (1)and after (5)Goodman extraction (Goodman, '57);crystalline albumin, before (2) and after (6) Goodman extraction. Numbers (3)and (4) are decanol and oleic acid standards, respectively.

I

I

FRACTION V

CRYSTALLINE

10K W

0-0

.-.

9-

m

fz

-

G-

cn

a w

K

OECANOL

+

OLElC ACID

*DECANOL f

7-

w

+

0 0

OLElC ACID

T

2 W Z

NO ADDITION

A-A

54-

0

5 2

E

3-

21-

0'

.

I

01

.5

1.0

1.5

0 1

.5

1.0

1.5

PER CENT ALBUMIN I N MEDIUM Fig. 9 Comparison of crystalline albumin and fraction V extracted by the method of Goodman ('57). To the extracted and dialyzed alhumin preparations were added decanol, oleic acid and decanol plus oleic acid to make molar ratios of decanol/albumin and oleic acidialbumin of 1.58and 0.5,respectively. The points and vertical bars represent, respectively, average and SEM of four experiments, each representing 64 plates.

10

KARIN NILAUSEN

100-

80-

60-

40: 20

/' i

Fig. 10 Growth response versus time of exposure to oleic acid-albumin. The experimental cultures were incubated with oleic acid-albumin for varying periods and then changed to fresh medium containing fatty acid-free albumin. The control cultures were likewise incubated with oleic acid-albumin for corresponding periods and then changed to fresh medium again containing oleic acid-albumin. The molar oleic acid/albumin ratio was 1.0, and the albumin concentration in the medium 1% (150 pMf. The points and vertical bars represent, respectively, average and SEM of eight experiments, each representing 18 plates.

canol/albumin ratio similar to that of the commercial crystalline albumin, caused in most cases inhibition of growth, whether oleic acid was present or not. An exception was that of the lowest albumin concentration (0.1%) in which case decanol stimulated growth both in the presence and absence of oleic acid (fig. 9).

DISCUSSION

The growth-promoting effect of bovine serum albumin on cultivated hamster cells was due largely to the associated fatty acids. This conclusion was based on two observations. Firstly, removal of the fatty acids eliminated most of the growth-stimulating effect, and, secondly, recombination of albumin with the Time effects on growth response to previously extracted and subsequently purioleic acid-albumin fied fatty acids returned the growth-promotExperimental cultures were incubated for ing activity to its original level. Concerning varying periods of time with oleic acid-al- the first observation, the objection might be bumin medium and then changed to fatty raised that other lipids capable of growth acid-free albumin medium. The control cul- stimulation might have been removed durtures, incubated with oleic acid-albumin ing extraction. Lysolecithin, for instance, is throughout the 72 hours, received fresh me- known to be associated with serum albumin dium a t the same time as did the experimental (Switzer and Eder, '65) and albumin-bound lyplates. The data (fig. 10) showed that the solecithin has been shown to stimulate cellugrowth response increased directly with the lar growth (Nilausen, '68).But the extraction amount of time that the oleic acid-albumin procedures used in the present experiments was available. Although the interpretation of were found by thin-layer chromatography to this observation is uncertain it appeared to extract only fatty acids (and decanol in case of indicate that the growth stimulation was due crystalline albumin). As for the second obserlargely to a substrate rather than a trigger ef- vation it might be argued that a growth factor existed which stayed with the fatty acids fect of oleic acid.

FATTY ACIDS, SERUM ALBUMIN AND CELL GROWTH

throughout extraction and purification without itself being a fatty acid. The likelihood of this being the case appeared remote because individual, highly purified fatty acids prepared commercially from various sources stimulated growth to a degree similar to that observed with fatty acids derived from bovine serum albumin. In a recent paper Yamane et al. (‘75) reported findings supporting the notion that the growth-stimulating effect of serum albumin was due largely to the associated lipids. Whether the albumin polypeptide per se promoted growth remained uncertain. The limited residual growth-promoting activity of fatty acid-extracted albumin might be due to the polypeptide, but i t could also be due to remaining traces of fatty acids and to lysolecithin, both of which are known to be present and the two have been shown to act synergistically (Nilausen, ’68). The finding that fatty acid-free albumin stimulated growth to a greater degree than did fatty acid-free P-lactoglobulin suggested a growth-promoting effect of the polypeptide, but a direct comparison of the two protein preparations is complicated by the finding (unpublished) that Plactoglobulin, following the removal of fatty acids, retained a number of fatty esters which by themselves might affect cellular proliferation. That serum albumin per se in some cases may be an important stimulator of growth was suggested by the recent findings of Polet and Spieker-Polet (‘75) and Spieker-Polet and Polet (‘76). They showed that albumin stimulated incorporation of labeled thymidine into DNA of mitogen-stimulated human peripheral lymphocytes, and that this effect was independent of the presence or absence of fatty acids in the albumin. They furthermore attempted, in an extensive series of experiments, to separate the stimulating agent from the albumin polypeptide. Since this proved impossible and since only pepsin degradation of t h e albumin eliminated the stimulatory effect, they concluded that the polypeptide itself was responsible for the increased thymidine incorporation. Whether the separation procedures employed would eliminate such growth-promoting molecules as lysolecithin and even decanol (in low concentrations), presumably present in the crystalline albumin studied, was not entirely clear. Recently, Yamane et al. (‘76) concluded from similar ex-

11

periments with activated peripheral lymphocytes that the effect of serum albumin was largely caused by the fatty acids associated with the polypeptide, because the fatty acidfree albumin had only limited effect, and 70% of the original effect could be achieved by replacing the native fatty acid mixture with oleic and linoleic acids. Fatty acids, in the absence of albumin, supported growth but to a very limited degree and the effective concentration range was narrow and much lower than that observed in the presence of albumin. These findings agree well with the view of albumin as a carrier and store of f a t t y acids which are gradually made available to cells in suitably dilute, non-toxic concentrations (Spector, ’68, ’75).This view of albumin as a relatively inert carrier of fatty acids was supported by the results obtained with P-lactoglobulin. Using this protein, which also had the capacity to function as a fatty acid carrier because of its ability to bind and release fatty acids, the different individual fatty acids had the same net effects on cellular growth as they did in the corresponding albumin-containing media. Thus serum albumin may promote cellular proliferation and DNA synthesis either by providing required fatty acids, as suggested by the present report, or through an effect of the polypeptide itself, as suggested by the work of Spieker-Polet and Polet (‘76), or by inactivation of growth-inhibiting substances (Moskowitz and Schenck, ‘65; Temin e t al., ’72). How albumin affected growth in the experiments of Dubin et al. (’65)and Norkin e t al. (‘65) with embryonic spleen macrophages remained uncertain. In some cases i t appeared that the fatty acids were responsible since the albumin could be replaced by linoleic acid, while in others the fatty acid-free albumin was superior to fatty acid-associated albumin in promoting growth. The mechanism by which fatty acids stimulated growth remained undetermined. Unpublished experiments with labeled fatty acids showed that they were incorporated into both phospholipids and triglycerides, and that a substantial amount (15%)of the oleic acid in the medium was assimilated by the cells under optimal conditions yielding a 10-fold increase in cell number during 72 hours of growth. Thus the fatty acids taken up from the medium probably provided important building materials for synthesis of cellular

12

KARIN NILAUSEN

membranes and other structures. The demonstration that the magnitude of the effect was directly related to the length of time that the fatty acids were available to the cells was in agreement with the suggestion that the fatty acids served as substrate for cellular metabolism, but it did not rule out the possibility of a mitotic triggering effect. Why saturated fatty acids inhibited growth remained uncertain. A number of cell lines, such HeLa (Gerschenson e t al., '67) and L (Cowen and Heydrick, '72) cells were unable to desaturate fatty acids. If the hamster cells were similarly deficient this could explain their inability to grow on saturated fatty acids, because i t is inconceivable that a mammalian cell containing only saturated fatty acids would be viable. Another explanation might be that the saturated fatty acids inhibited synthesis of phospholipids as was recently shown in rat liver cells (Sundler e t al., '74). Although the native mixture of fatty acids associated with bovine serum albumin contained about 40% saturated fatty acids it nevertheless supported growth quite efficiently. The explanation appeared to be that by far the major saturated component was stearic acid, which stimulated growth in concentrations up to about 70 pM. Maximal growth stimulation was obtained at fatty acid concentrations of about 150-200 p M in case of the native albumin-associated fatty acid mixture, corresponding to a stearic acid concentration of about 40-70 pM. Only a limited number of experiments have been done with simple twocomponent f a t t y acid mixtures, but the data obtained so far indicate an additive effect when both growth-promoting and growth-inhibiting fatty acids were present. ACKNOWLEDGMENTS

This work was supported by the Danish Medical Research Council and by the Carlsberg Foundation. I wish to thank Ms. Inge Mdler for her diligent technical assistance. LITERATURE CITED Bailey, J. M., and L. M. Dunbar 1973 Essential fatty acid requirements of cells in tissue culture: A review. Exp. Mol. Path., 18: 142.161. Barile, M. F., W. F. Malizia and D. B. Riggs 1962 Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol., 84: 130.136.

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