Decrease in adhesion of cells cultured in polyunsaturated fatty acids

Cell, Vol.

12, 295300,

September

1977,

Copyright

0 1977 by MIT

Decrease in Adhesion of Cells Cultured Polyunsaturated Fatty Acids Richard L. Hoover, Robert D. Lynch* and Morris J. Karnovsky Department of Pathology Harvard Medical School 25 Shattuck Street Boston, Massachusetts 02115 and * Department of Biological Sciences University of Lowell Lowell, Massachusetts 01854

Summary The addition of long chain unsaturated fatty acids (linoleic, linolenic and arachidonic acids) to BHK cells reduces the cell to substrate adhesion, causes morphological changes and alters the cellular growth properties. The new characteristics are similar to those of transformed cells. The data indicate that the effects are probably due to actual changes in the surface membrane lipids and not due to prostaglandin synthesis. Introduction In terms of the fluid mosaic model, lipids have a very important role in the structure and function of membranes. Not only do they affect the stability and permeability of the membrane, but they also affect the distribution of proteins, which in turn can influence other cellular functions. Studies have shown that changes in lipid compositions can modify enzyme activity (Engelhard et al., 1976), morphology (Ginsburg et al., 1973; Hawley and Gordon, 1976), concanavalin A agglutination (Horwitz, Hatten and Burger, 1974), differentiation (Weeks, 1976) and cell adhesion (Curtis, Chandler and Picton, 1975; R. L. Hoover, manuscript in preparation). The mechanism by which the lipids affect these cellular functions is still unclear. Two possible mechanisms are either that there is a direct change in the lipid composition of the cell surface which alters the fluidity of the membrane, or that the lipids are acting intracellularly through synthesis of prostaglandins which then alter membrane properties. In this paper, we demonstrate that when exogenous fatty acids are incorporated into tissue culture cells, functions involving the cell surface are affected and that the alterations in functions depend on the incorporation of a particular fatty acid. We also suggest procedures to help distinguish the mechanism through which the fatty acids act. Results The effects

of fatty acids

on the adhesion

of BHK

in

cells are summarized in Table 1. Since the percentage of control cells adhering ranges between 4070%, all data have been standardized based on a value of 1.00 for the control condition. Numbers >l .OO indicate that more cells are adhering to the substrate than in the control condition, and numbers
Cell 296

Table 1. Effect of Fatty Acid Concentration on Adhesion Cells to Homotypic Monolayers and to a Plastic Substrate 45 min Incubation at 37°C

Control Stearic acid 10 pg/ml

Homotypic 1 .oo (74) 1.03 + 0.20

monolayer

(57)

20 pg/ml

0.92

c 0.2 (16)

30 Fglml Linoleic acid 2.5 pglml

0.76

-t 0.2 (6)

0.89

2 0.10 (4)

5.0 pg/ml

0.62 f 0.06

(4)

10.0 pg/ml

0.49

-c 0.10 (52)

20.0 pg/ml

0.46

k 0.09 (13)

30.0 pglml

0.17

f 0.09 (6)

of BHK after a

Plastic substrate 1 .oo (13) 0.96

* 0.01

(9)

0.90

k 0.07

(11)

0.57

-t 0.04

(4)

0.41

? 0.06

(9)

The values are expressed as a fraction of the controls. The percentages of cells adhering under control conditions are: 55.9 t 1.3 for homotypic monolayers, and 43.6 -C 1.0 for plastic. The numbers in brackets represent q.

tion of linoleic acid (18:2). Linoleic acid also had effects on the growth of BHK cells. Figure 4 is a representative growth curve of cells treated with linoleic acid at 10 pg/ml, 20 pg/ml or 40 pg/ml. As the concentration of linoleic acid is increased, the maximum cell number decreases. The growth rate remains approximately the same until 40 pg/ml are added, which decreases the rate. Lipid droplets were observed in the cytoplasm but disappeared after 2-3 days. This might account for the extended lag phase and reduced growth rate. In no instances did the cells round up and come off as if in response to a toxic effect of the fatty acids. In conjunction with these growth differences, morphological differences are most obvious at the higher concentrations. Figure 5 is a phase-contrast micrograph of normal and linoleic treated cells at high concentrations of linoleic acid (40 pg/ml). The controls are typical fibroblasts exhibiting contact inhibition (Figure 5A); however, in 40 pg/ml of linoleic acid, the cells look transformed with considerable criss-crossing and piling up as if they had lost their contact inhibition properties (Figure 5B). At a low concentration of linoleic acid (10 yglml), scanning electron microscopy revealed no obvious differences in surface morphology between control and treated cells. Because the growth experiments extended for longer periods of time, there was a chance of peroxidation of the fatty acids, in which case by-products might have been responsible for the results. The addition of vitamin E (an anti-oxidant) to the medium, however, produced no observed changes. Indomethatin, when added to the growth medium with the fatty acids, also did not alter the effects on growth. Since both linoleic and arachidonic acids are precursors to prostaglandins, three experiments

10 t (lOpg/ml)

kk

0

10

I

20

MNU Figure 1. Adhesion Function of Time

of BHK Cells

Control (0); ml) m).

acid (10 pglml)

stearic

I

30

I

I

40

50

TES to Homotypic

Monolayers

(0); and linoleic

as a

acid (10 pgl

were carried out to see whether that had any relationship to this system. Table 4 presents data in which adhesion was measured in the presence of linoleic acid and linoleic acid plus indomethacin, an inhibitor of prostaglandin synthesis. In the control samples, adhesion to both monolayers and plastic increased with the addition of indomethacin; however, in the presence of linoleic acid and indomethacin, the adhesion to monolayers was unchanged. Also, linolenic acid (18:3), which is not a precursor to prostaglandins, had a similar effect on adhesion to linoleic acid (Table 4). The addition of indomethacin did not affect the adhesion. Addition of exogenous prostaglandins produced only slight changes in the percentage of cells adhering to monolayers after 30 min (Table 5). The differences cannot account for the differences seen with the addition of linoleic acid. The adhesion assays, the apparent loss of contact inhibition and the negative prostaglandin experiments indicate that the fatty acids may be acting at the membrane level. This is substantiated by the results shown in Table 6 depicting experiments in which adhesion assays were carried out at 4°C. The effects are greater on the control and the

Fatty Acid 297

Effects

on Cultured

Cells

Table

2. Incorporation

of Linoleic

Acid

[l-14C]

%

dom Phospholipids Fatty Acids Triglycerides Cholesterol * Total

CONTROL PALMITIC STEARIC ACID ACID (16:O) IWO)

OLEIC ACID (18: I )

Figure 2. The Effects of Various Fatty Acids BHK Cells to Homotypic Monolayers at 37°C

LINOLEIC ACID l I 62)

ARACHIDONIC ACID (20.4)

on the Adhesion

of

Concentrations for all are 10 rg/ml. The values are expressed as a fraction of the controls. The percentage of cells adhering in the controls is 60.7 f 1.6; T) = 6 for all conditions.

f@ BHK

1.2 1

@

q

I o-

ii

0.8 -

-2

0.6 -

(n=52)

CHO in=61

PyBHK (n=6)

s 92

0.4 -

0.2-

CONTROL Figure 3. A Comparison BHK Ceils to Homotypic Linoleic Acid (10 fig/ml)

STEARIC ACID

LINOLEIC ACID

of the Attachment of BHK, CHO and PyMonolayers in the Presence of Stearic or at 37°C for 30 min

The percentages of cells adhering BHK, CHO and Py-BHK were 55.9 0.10, respectively.

f

in the control conditions for 1.3, 49.2 + 6.7 and 49.2 +

stearic acid-treated ceils than on the linoleic acidtreated cells. As compared to controls at 4”C, adhesion values for stearic acid remain unchanged, while they are slightly increased for linoleic acid. In all cases, the number of cells adhering decreases concomitantly with a temperature decrease, but the decrease in linoleic acid-treated cells (24%) is less than that in the controls (33%). Discussion The experimental results show that BHK cells can incorporate exogenous fatty acids which can then cause changes in growth and in both cell-to-cell and cell-to-substrate adhesion. The long chain, un-

Esters

incorporation

represents

into BHK Cells*

60,276

20.8

127,600

44.1

16,041

5.5

85,475

29.5

2.2%

of labeled

material

added.

saturated fatty acids (linoleic, linolenic and arachidonic) decrease adhesion, while, in contrast, long chain saturated acids (oleic, palmitic and stearic) have little or no effect. Linoleic acid also caused an apparent loss of contact inhibition in the cells. How the fatty acids affected these changes is unclear. One possible mechanism was through the synthesis of prostaglandins, since both linoleic and arachidonic acids are prostaglandin precursors and since both fatty acids have an effect on adhesion. This has been suggested by Horwitz et al. (1974) who found that Swiss 3T3 cells undergo a morphological change with the addition of linoleic acid to the medium. Mertin and Hunt (1976) have also implicated prostaglandins in a regulatory role. They found that when linoelic acid is given orally or subcutaneously to mice, the survival of allografts is prolonged. Holley, Baldwin and Kiernan (1974) showed that linoleic acid can initiate DNA synthesis and growth in quiescent mouse myeloma XS 63.5 cells in culture and suggested that prostaglandins are the actual factors involved. Finally, Weiss (1973) found decreased adhesion in Ehrlich-Lettre hyperdiploid ascites carcinoma cells with the direct addition of prostaglandin F*(Y and PGE,, and no effect with PGF,P. L929 fibroblasts showed no effect with any of the prostaglandins, unless the cells had first been trypsinized. Our results indicate however, that prostaglandin synthesis cannot account for the results seen for several reasons: indomethacin, an inhibitor of prostaglandin synthesis, does not inhibit the action of linoleic acid: prostaglandin synthesis does not inhibit the action of linoleic acid; prostaglandins added externally do not mimic the effects of linoleic acid: and linolenic acid, a long chain unsaturated fatty acid, and not a precursor to prostaglandin, produces results similar to linoleic acid. Because of these reasons, another mechanism must be suggested for the effects of the fatty acids. Possibly there are changes in the membrane composition with the addition of fatty acids. This is a particularly attractive hypothesis because we have shown that the fatty acids are incorporated into the cellular phospholipids, but it also should be noted that at this time, we do not know whether the phospholipids are actually in the surface membrane. The possibility of this is strongly indicated because the effects observed

Cell 298

Table 18x2

3. Percentage

18:2

of 18:2 incorporated

into the Phospholipids

of BHK Cells When

Incubated

in Increasing

Concentrations

Control 9.9

10 pg/ml 12.5

20 pglml 21.9

30 pg/ml 22.27

40 pg/ml 33.4

Control

IO pg/ml

20 pglml

30 pg/ml

40 fig/ml

0.98

0.99

1.32

1.67

2.32

1.06

0.68

0.92

1.47

0.22

0.38

0.83

2.78

of Exogenous

16:O 18:2 18:O 18:2 18:l

3.03

18:2

2.50

20:4 Incorporation concentration

periods varied between of 18:2 is increased.

3-4 days. The ratios

of 18:2 to 16:0, 18:0,

HOURS

Figure 4. Typical Growth Curves Concentrations of Linoleic Acid Each point pg/ml (A);

represents 2 replicates. and 40 pg/ml (m).

for BHK Cells Control

(0);

Grown

in Various

IO pg/ml

(0); 20

(adhesion, growth and contact inhibition) are surface membrane-mediated. Weeks (1976) has recently shown that incorporation of polyunsaturated fatty acids into the slime mold Dictyostelium discoideum impairs cell-cell contacts, which in turn affects differentiation. He suggested that the effects may be due to alterations in membrane fluidity, or that the fatty acids are affecting the molecules by changing either the conformation or composition of the receptors. Curtis, Chandler and Picton (1975) found that adhesion of embryonic chick neural retinal cells was altered depending on the type of fatty acid incorporated and explained this in terms of changes of membrane fluidity, or changes in intramembrane van der Waals London forces. In this scheme, a change in the acyl chains of the phospholipids could alter the charge on the surface. This is based on the length of the fatty acid chain and on its rigidity (saturation)-both of which might change the thickness of the membrane and likewise the van

18:l

and 20:4 represent

fatty

acids

in phospholipids

as the

der Waals forces. Our data substantiate other findings that fatty acid alterations in a cell can influence cellular functions, but they do not say clearly what is causing these changes. At present, we are doing freeze fracture studies of the cells to determine whether the number and distribution of intramembranous particles in the surface membrane change with fatty acid alterations. Kleeman, Grant and McConnell (1974) have shown that the distribution of particles in the plasma membrane of E. coli is dependent on temperature and on the fatty acid addition, and Ueda et al. (1976) recently showed that the adhesion of BHK cells is temperature-dependent, which may be due to the physical state of the membrane lipids and their fluidity properties. Our experiments might indicate if surface lipids are involved and whether this can account for fluidity changes. Preliminary data suggest that there are no obvious differences between control and treated cells, but it might be necessary to perform the fractures at various temperatures to detect differences in fluidity due to phase transitions. This system may also be interesting to look at using a method devised to measure lateral transport of molecules in the surface membrane, such as that of Schlessinger et al. (1976), who monitored mobility of Con A receptors in myoblasts by a “fluorescence photobleaching recovery” method. Experimental

Procedures

BHK-Cl3 and Py-BHK cells were grown as monolayer cultures in Dulbecco’s modified minimal essential medium (DMEM) containing 10,000 units per ml penicillin and 10,000 fig/ml streptomycin supplemented with 10% fetal calf serum. Chinese hamster ovary cells &HO) were grown in Ham’s FIO nutrient medium containing 10,000 units of penicillin and 10,000 pg/ml streptomycin supplemented with 10% fetal calf serum. Delipidated serum was prepared essentially by the methods of Horwitz, Hatten and Burger (1974). Fetal calf serum (up to 100 ml) was added to a chilled (- 25°C) mixture of ethanol, diethyl ether

Fatty 299

Acid

Figure

Effects

5. Phase

on Cultured

Cells

Micrographs

of Control

Table 4. Effects of lndomethacin BHK Cells to Homotypic Monolayers Homotypic Control Linoleic

(2 PM)* on the and Plastic Monolayer

1 .oo (12)

Acid

Adhesion

(40 pg/ml)-Treated

of

Table 5. The Effects of the Addition Adhesion of BHK Cells to Homotypic of Incubation at 37°C

of Prostaglandins Monolayers after

on the 30 min

1 .OO (6)

Control

1 .oo

0.65 f 0.10

(12)

0.52 k 0.07 (10)

0.54

+ 0.09

(6)

0.74 k 0.06 (4)

1.13 2 0.09

(6)

1 .16 * 0.07 (6)

0.65 k 0.11

(12)

0.61

0.56 i 0.06

(6)

0.69 k 0.08

1.05 i 0.01 (6)

5 pglml 10 pg/ml

Acid

Control + lndomethacin

Linolenic Acid lndomethacin

(B)

PGE,

(10 w3hnl)

Linoleic Acid lndomethacin

Cells

Plastic

Acid

(10 cL9h-d) Linolenic

(A) and Linoleic

+ + 0.06 (10)

0.85

5 rglml

0.93 2 0.01 (6)

10 rglml

0.97 k 0.06 (6)

PGFze 5 pg/ml

+ (4)

* lndomethacin was added 3 hr before addition of fatty acids and was present during the assay. Values are expressed as a fraction of the controls. The percentages of untreated cells adhering to homotypic monolayers and to plastic are 60.5 -t 4.2 and 47.7 ? 2.4, respectively. and distilled water (900:300:7) and stirred for 2 hr. The solution was centrifuged and reextracted with ethanol, ether and water (900:300:62) for 2 hr. The mixture was centrifuged and the pellets washed twice with cold diethyl ether. After the final wash, the pellets were extracted with diethyl ether overnight in the cold (-20°C). The solution was centrifuged and the pellets washed twice more with diethyl ether. Finally, the pellets were dried under avacuum and dissolved in 0.9% NaCl at a concentration of 80 mgl ml. The delipidated serum solution was sterilized through a series of Millipore filters of decreasing pore size (1.2, 0.6, 0.45 and

2 0.01 (6)

PGE2

1.04 * 0.01

The values 5.2%).

(6)

1.01 ? 0.06 (6)

10 pg/ml are expressed

0.22 p) and stored

at -20°C

as a fraction

until

ready

of the

controls

(74.9

+

for use.

Growth studies were carried out on BHK cells. They were plated at a concentration of 25,000 cells per ml in DMEM + 10% delipidated serum. Various concentrations of fatty acids, dissolved in ethanol, were added to the growth medium, and the cells were plated in Costar (X3524) multiwell dishes, two wells for each time point. At particular time intervals, the cellular monolayers were washed twice with a CaMg-free Hank’s solution (CMF), and single cells were obtained for counting by treating the monolayers for 2 min with 0.025% trypsin per 0.5 mM EDTA. Adhesion measurements were made using the monolayer collection assay of Walther, Ohman and Roseman (1973). The cells were grown as monolayers, either in multiwell dishes for collect-

Cell 300

Table 6. Effect of Temperature on Adhesion of BHK Cells to Homotypic Monolayers in the Presence of Stearic (IO pg/ml) or Linoleic (10 pg/ml) Acids

Control Stearic Linoleic

Acid Acid

37-x

4°C

1 .OO (6)

1 .OO (6)

1.06 t 0.14 (6)

1.07 ? 0.10

(6)

0.67’

0.49

0.55 f 0.12

(6)

0.76’

* These values represent ing to monolayers after The values are expressed particular temperature. adhering under control respectively.

f 0.06 (6)

0.67’

a ratio of the percentages of cells adher45 min at 4 and 37°C. as a fraction of the controls Ht that At 4 and 37”C, the percentages of cells conditions are 34.0 t 2.5 and 50.6 * 1.5,

procedure for extraction and chromatography was the same as above, except that iodine vapors were not used. The phospholipid spot was removed and analyzed for fatty acid composition by gas chromatography. Acknowledgments We thank Mary Mauri for her assistance in the preparation of this manuscript and Robert Rubin for his technical assistance in preparation of photographic material. We also would like to thank Dr. Eveline Schneeberger for her helpful discussions and Dr. John Pike of the Upjohn Company (Kalamazoo, Michigan) who kindly provided the prostaglandins. These experiments were supported by a grant from the NCI. Received

April

29, 1977;

revised

May 3, 1977

References ing lawns, or in tissue culture flasks for cell labeling, for 2 days. The cells were labeled with 10 ~1 of 3H-leucine (59.8 Ci/mmole; New England Nuclear) per 5 ml Hank’s HEPES buffer solution. Single cells were obtained by washing twice in CMF and once with trypsin/EDTA. Trypsin action was stopped with the addition of growth media plus 10% delipidated serum. The cells were centrifuged, resuspended in CMF and dissociated by titration with a Pasteur pipette. The suspension was recentrifuged, and the cells were resuspended at a concentration of 100,000 cells per ml in Hank’s balanced salt solution supplemented with 15 mM HEPES buffer (pH 7.4). These cells were then used for the adhesion assay. Fatty acids, dissolved in ethanol at a concentration of IO sg/ ml, were added to the cells at various concentrations. ATP (12.5 mole/l) and coenzyme A (5 mole/l) were added according to the methods of Curtis and Buultjens (1973) based on the work of Fischer et al. (1967). The cells plus fatty acids were incubated at room temperature for 20 min before addition to the collecting lawns. Viability was measured by trypan blue exclusion which revealed that at least 90% of the cells were viable. The monolayers used as collecting lawns were washed 3 times with Hank’s HEPES buffer (HH) and allowed to stand for 20 min. The final wash was removed, and 1 ml of the suspended cells was added to each well. At various time intervals, the medium was removed and the monolayer washed once with HH. The monolayers plus adhering cells were dissolved in 1 N NH*OH and added to 10 ml of scintillation fluid. Radioactive counts were made on a Beckman liquid scintillation counter, Model LS 333. The percentage of cells attaching to the monolayers was based on counts received on 1 ml of cell suspension. To test whether the cells incorporated the fatty acids into their phospholipids after the 20 min of incubation, 50 pg of linoleic acid [(l-14C) spec. act. 50.6 mCi/mmole; New England Nuclear] were added with unlabeled linoleic acid to 5 ml of cell suspension (567,000 cells per ml) at a final concentration of 20 wg/ml. Labeled linoleic acid with a radiochemical purity > 99% was taken from sealed ampules, freshly opened for each experiment. After a 20 min incubation, the lipids were extracted. The cells were centrifuged and washed twice with CMF. The final pellet was suspended in 0.8 ml H,O, to which were added 2 ml of chloroform and 1 ml of methanol. The solution was agitated on a vortex mixer and allowed to stand at 4°C. After 30 min, 1 ml Hz0 and I ml of chloroform were added to the solution and it was again agitated on a vortex mixer. The solution was centrifuged at 4°C to separate the layers. The bottom chloroform layer was removed and stored under Nz, redissolved in 2-3 drops of chloroform/methanol (l:l), spotted on a silica gel H thin-layer chromatography plate, and developed in a hexane-ether diethyl-acetic acid (60:39:1) solvent system. The spots were visualized with iodine vapor (1% in methanol), scraped off, added to scintillation fluid and counted. Analysis of the extracted phospholipids was also made. BHK cells were incubated in various concentrations of linoleic acid until the cultures just reached confluency, usually 3-4 days. The

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and Sym-

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Ueda, M. J., Ito, T., Okada, T. S., and Ohnishi, S. (1976). A correlation between membrane fluidity and the critical temperature for cell adhesion. J. Cell Biol. 77, 670-674. Walther, B. T., Ohman, assay for intercellular 1569-1573.

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adhesion in tissue culture. and cyclic nucleotides.