Lipid Metabolism in Cultured Cells: Growth of Tumor Cells ......essential fatty acid deficiency....

8
[CANCER RESEARCH 31, 91—97, February 19711 di-cis-decadienyl structure are apparently required for maintenance of normal growth and reproduction (16). The commonly occurring fatty acids exhibiting this structural moiety are linoleic (18 :2w6)', linolenic (1 8:3w3), and arachidonic acids (20:4co6). Since they are not synthesized de novo, they have been termed the â€oeEFA's.―2 A dietary requirement for these fatty acids has been shown to exist in all 11 species of experimental animals so far tested. Differences apparently exist, however, in the absolute requirement for EFA at the cellular level. In 1963, Ham (9) reported that in vitro cultures of Chinese hamster strain CHD-3 cells in synthetic medium require linoleic acid for normal plating efficiency. More recently, Gerschenson et aL (7) reported lack of growth and certain metabolic defects as characteristics of HeLa 53 cells cultured in a lipid-deficient medium. These effects were partially reversed by linoleic acid. In contrast to these findings, Smedley-MacLéan and Nunn (1 5), in 1941 , observed that the Walker 256 carcinoma grew as well when implanted in fat-starved rats as in those receiving a daily dose of linseed oil. In addition, a number of cell lines, including the L2071 strain of mouse fibroblast, have been adapted to a lipid-free, chemically defined medium by Evans et al. (6) since 1956. The linoleic acid content of L strain fibroblasts grown in the lipid-free synthetic medium was found to be about 6% of the total fatty acids (2). This compared with a content of about 25% linoleic acid when grown on serum-supplemented medium. It was shown by supplementing the lipid-free medium with C that the residual linoleic acid was not the result of de novo synthesis, but instead represented the operation of an efficient scavenging process by the cells for traces of linoleic acid present as contaminants in the culture environment. Geyer (8) has reported that the linoleic acid is completely depleted in rapidly growing L-cells. Laboratory mice readily display symptoms of EFA deficiency when placed on a fat-free diet. The linoleic acid content of serum lipids falls to very low levels in only a few weeks. A number of rapidly dividing tumor cell lines which grow in the ascitic form in the peritoneal cavity of mice are available. The present work was undertaken to study the growth and composition of such rapidly dividing cells in the EFA-deficient environment provided by host mice maintained on fat-free diets. SUMMARY Certain strains of cells have been grown in tissue culture in lipid-free synthetic media, whereas others appear to require essential fatty acids. Growth of two rapidly dividing ascites tumors, Ehrlich ascites carcinoma and Sarcoma 180, was therefore studied in normal mice and in mice with pronounced essential fatty acid deficiency. Essential fatty acids in blood lipids of essential fatty acid-deficient mice comprised only 2% of the total, as compared to more than 30% of the total in normal mice. The mean generation time of Ehrlich ascites carcinoma cells grown in essential fatty acid-deficient mice (1 .07 ±0.19 days) was not significantly different from that of normal cells grown in normal mice (1 .04 ±0.19 days). Comparable results were also obtained with the ascitic form of Sarcoma 180. Transplantability was unaffected by essential fatty acid deficiency, as evidenced by normal growth when deficient cells were used as inoculum. Fatty acid composition of tumor cell lipids was determined by gas-liquid chromatography. The total lipid content of the tumors was unchanged, whereas the average content of essential fatty acids, linoleic and arachidonic, was decreased from approximately 36% of the total in normal mice to less than 3% in tumor cells grown in deficient host mice. These changes were accompanied by major increases in the content of oleic acid (1 8 : 1) (from 24 to 42%) and eicosatrienoic acid (20:3) (from 0 to 14%). When deficient cells were transplanted into normal mice, the fatty acid composition returned to normal within 72 hr. Unaltered growth of cells in association with these changes in fatty acid composition suggests that oleic and eicosatrienoic acids may fulfill certain of the structural or metabolic functions of the â€oeessential― fatty acids in these two strains of tumor cells. INTRODUCTION The nutritional requirement of animal organisms for certain fats has been recognized since the late 1920's, when Evans and Burr (5) demonstrated that rats maintained on a fat-free diet developed a deficiency disease characterized by retarded growth and infertility. Later work established that polyunsaturated fatty acids with a 6,9-terminal I This work was supported by USPHS Grants HE-05062, AM-i 0082, and 5K3-16730. This is Paper 8 of a series. The preceding paper of the seriesis Reference 1. Received July 24, 1970; accepted September 23, 1970. 2 The abbreviations used are: EFA, essential fatty acid; GLC, gas-liquid chromatographic; ECL, equivalent chain length. FEBRUARY 1971 91 Lipid Metabolism in Cultured Cells: Growth of Tumor Cells Deficient in Essential Fatty Acids' J. Martyn Bailey and L. M. Dunbar Department ofBiochemistry, The George Washington University School ofMedicine, Washington, D. C. 20005 on July 20, 2021. © 1971 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Transcript of Lipid Metabolism in Cultured Cells: Growth of Tumor Cells ......essential fatty acid deficiency....

Page 1: Lipid Metabolism in Cultured Cells: Growth of Tumor Cells ......essential fatty acid deficiency. Essential fatty acids in blood lipids of essential fatty acid-deficient mice comprised

[CANCER RESEARCH 31, 91—97, February 19711

di-cis-decadienyl structure are apparently required formaintenance of normal growth and reproduction (16). Thecommonly occurring fatty acids exhibiting this structuralmoiety are linoleic (18 :2w6)', linolenic (1 8:3w3), andarachidonic acids (20:4co6). Since they are not synthesized denovo, they have been termed the “EFA's.―2A dietaryrequirement for these fatty acids has been shown to exist in all11 species of experimental animals so far tested.

Differences apparently exist, however, in the absoluterequirement for EFA at the cellular level. In 1963, Ham (9)reported that in vitro cultures of Chinese hamster strainCHD-3 cells in synthetic medium require linoleic acid fornormal plating efficiency. More recently, Gerschenson et aL(7) reported lack of growth and certain metabolic defects ascharacteristics of HeLa 53 cells cultured in a lipid-deficientmedium. These effects were partially reversed by linoleic acid.In contrast to these findings, Smedley-MacLéan and Nunn(1 5), in 1941 , observed that the Walker 256 carcinoma grew aswell when implanted in fat-starved rats as in those receiving adaily dose of linseed oil. In addition, a number of cell lines,including the L2071 strain of mouse fibroblast, have beenadapted to a lipid-free, chemically defined medium by Evanset al. (6) since 1956. The linoleic acid content of L strainfibroblasts grown in the lipid-free synthetic medium was foundto be about 6% of the total fatty acids (2). This comparedwith a content of about 25% linoleic acid when grown onserum-supplemented medium. It was shown by supplementingthe lipid-free medium with@ C that the residuallinoleic acid was not the result of de novo synthesis, butinstead represented the operation of an efficient scavengingprocess by the cells for traces of linoleic acid present ascontaminants in the culture environment. Geyer (8) hasreported that the linoleic acid is completely depleted inrapidly growing L-cells.

Laboratory mice readily display symptoms of EFAdeficiency when placed on a fat-free diet. The linoleic acidcontent of serum lipids falls to very low levels in only a fewweeks. A number of rapidly dividing tumor cell lines whichgrow in the ascitic form in the peritoneal cavity of mice areavailable. The present work was undertaken to study thegrowth and composition of such rapidly dividing cells in theEFA-deficient environment provided by host mice maintainedon fat-free diets.

SUMMARY

Certain strains of cells have been grown in tissue culture inlipid-free synthetic media, whereas others appear to requireessential fatty acids. Growth of two rapidly dividing ascitestumors, Ehrlich ascites carcinoma and Sarcoma 180, wastherefore studied in normal mice and in mice with pronouncedessential fatty acid deficiency. Essential fatty acids in bloodlipids of essential fatty acid-deficient mice comprised only 2%of the total, as compared to more than 30% of the total innormal mice. The mean generation time of Ehrlich ascitescarcinoma cells grown in essential fatty acid-deficient mice(1 .07 ±0.19 days) was not significantly different from that ofnormal cells grown in normal mice (1 .04 ±0.19 days).Comparable results were also obtained with the ascitic form ofSarcoma 180. Transplantability was unaffected by essentialfatty acid deficiency, as evidenced by normal growth whendeficient cells were used as inoculum.

Fatty acid composition of tumor cell lipids was determinedby gas-liquid chromatography. The total lipid content of thetumors was unchanged, whereas the average content ofessential fatty acids, linoleic and arachidonic, was decreasedfrom approximately 36% of the total in normal mice to lessthan 3% in tumor cells grown in deficient host mice. Thesechanges were accompanied by major increases in the contentof oleic acid (1 8 :1) (from 24 to 42%) and eicosatrienoic acid(20:3) (from 0 to 14%). When deficient cells were transplantedinto normal mice, the fatty acid composition returned tonormal within 72 hr. Unaltered growth of cells in associationwith these changes in fatty acid composition suggests thatoleic and eicosatrienoic acids may fulfill certain of thestructural or metabolic functions of the “essential―fatty acidsin these two strains of tumor cells.

INTRODUCTION

The nutritional requirement of animal organisms for certainfats has been recognized since the late 1920's, when Evans andBurr (5) demonstrated that rats maintained on a fat-free dietdeveloped a deficiency disease characterized by retardedgrowth and infertility. Later work established thatpolyunsaturated fatty acids with a 6,9-terminal

I This work was supported by USPHS Grants HE-05062, AM-i 0082,

and 5K3-16730. This is Paper 8 of a series. The preceding paper of theseriesis Reference 1.

Received July 24, 1970; accepted September 23, 1970.2 The abbreviations used are: EFA, essential fatty acid; GLC,

gas-liquid chromatographic; ECL, equivalent chain length.

FEBRUARY 1971 91

Lipid Metabolism in Cultured Cells: Growth of TumorCells Deficient in Essential Fatty Acids'

J. Martyn Bailey and L. M. Dunbar

Department ofBiochemistry, The George Washington University School ofMedicine, Washington, D. C. 20005

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J. Martyn Bailey and L. M. Dunbar

population to double. This was calculated according to theequation

T= 0.301 t/(log N/N0)

where N0 is the initial inoculum and N is the cell number attime t days following inoculation of the tumor.

Preparation and Analysis of Lipid Extracts. Lipid extractsof blood and tumor cells were prepared by extraction with 5ml or 20 volumes, whichever was greater, of a 1: 1 mixture ofethanol and diethyl ether (3). a-Tocopherol (0.2 mg) in 1:1ethanol:ether was added to prevent oxidation ofpolyunsaturated fats (2), and the sample was extractedovernight at 4°. Ethanol:ether extracts were dried overanhydrous sodium sulfate and evaporated to dryness at roomtemperature in a stream of nitrogen. The lipid residue wasredissolved in hexane. It has been shown previously that theserelatively mild extraction procedures are necessary forquantitative recovery of polyunsaturated fatty acids (2).Methyl esters of the fatty acids were prepared bytransmethylation of the lipid extracts with boron trifluoride(14%) in methanol as catalyst (1 1). The methyl esters wereanalyzed with a 1662 series gas chromatograph (HCLScientific, Inc., Chicago, Ill.) equipped with a hydrogen flameionization detector. The chromatograph was fitted with glasscolumns [6 feet x 0.25 inch (outer diameter)] , silanized, andpacked with ethylene glycol:succinate polyester (15%) on a100 to 120 mesh gas chromatograph Q. Column temperaturewas 165°,injector temperature was 268°,and the carrier gas(helium) flow rate was 75 ml/min.

In order to facilitate comparison of chromatogramsobtained over a period of several months, the retention time ofindividual fatty acids was compared to that of oleic acid(l8:1w9). Identification of peaks was by comparison ofretention ratios with those of known external standards. Peaksnot corresponding to those of available standards weretentatively identified by calculation of ECL values andcomparison with ECL values published for chemicallyidentified unsaturated fatty acids (10). Hydrogenation ofrepresentative samples of methylated lipid extracts to give thesaturated fatty acids of the corresponding chain lengths wasperformed as an additional aid in identification (4).Methylated lipid extracts were dissolved in reagent grade ethylacetate (5 ml) with palladianized charcoal (10 mg) as catalyst,and hydrogen was bubbled into the solution for 45 mm. Thehydrogenated samples were redissolved in hexane for GLCanalysis.

The relative amounts of individual fatty acids in the samplesare expressed as a percentage of the total in the range betweenpalmitic acid (16:0) and eicosatrienoic acid (22:3c@7 or 9).This was done by comparison of the areas of individual peakswith the combined areas of all peaks with retention timescorresponding to this range.

RESULTS

Demonstration of EFA Deficiency in Mice. Mice wereplaced on the deficient or control diets as weanlings weighing

MATERIALS AND METHODS

Mice of the CF, strain were used as host animals for growthof ascites tumor cells in normal and EFA-deficienenvironments. Food and water were administered ad libitum.Stock animals were divided into 2 groups as weanling micewhen approximately 3 .5 weeks old. The control group was feda normal diet of Purina laboratory chow. The experimentalgroup was maintained on a basic fat-free diet (NutritionalBiochemicals Corp., Cleveland, Ohio) for periods of up to 6months following weaning.

The EFA status of the mice was assessed by 2 methods;these were a weekly comparison of the relative growth rate ofnormal and deficient mice and periodic assay of the fatty acidcomposition in whole blood by GLC analysis.

Blood samples for analysis of fatty acids were obtained bypuncture of the retroorbital plexus with heparinizedblood-collecting microtubes according to the method of Riley(14). Lipid extracts of blood were prepared and analyzed forfatty acid composition as described below.

A hypotetraploid sublime of the Ehrlich ascites mousecarcinoma (1) and a subline of Sarcoma 180 adapted togrowth in the ascitic form were used in these studies. Routinetransfer of tumors was carried out at 7- to 10-day intervals byinoculating groups of mice i.p. with about 10 million cells.

Quantitative measurements of tumor growth rate were madeby using the following procedure for harvesting cells. Thetumor-bearing mouse was killed by overanesthesia with diethylether. To prevent clumping of cells, 4 ml of a dilute solutionof sodium heparin (6 units/ml) in 0.9% NaC1 solution wasgiven by injection into the peritoneal cavity. The outer layerof skin was then carefully cut with scissors to expose the innerfascia. A small opening was made through the latter, and theascitic fluid was aspirated with a Pasteur pipet into acentrifuge tube. The abdominal cavity was repeatedly rinsedwith 3-mI portions of 0.9% NaC1 solution until no cells werevisible in the aspirated fluid. Cells were centrifuged andresuspended in 0.9% NaCl solution. The cell population wasdetermined by counting diluted aliquots of the cellsuspensions in a Speirs-Levy eosinophil hemacytometerchamber or by using a Coulter Model B electronic counterfitted with a glass cell with a lOO-ji aperture. Samples to becounted electronically were diluted with 0.9% NaC1 solutionwhich had been filtered through a Millipore filter to render itparticle free.

Growth of Tumor Cells in Normal and EFA-deficient Mice.Groups of normal and EFA-deficient mice were inoculatedwith portions of a suspension of freshly harvested tumor cellsin 0.9% NaCl solution. The size of the inoculum ranged from 1to 10 million cells in different experiments. Cells wereharvested from pairs of normal and EFA-deficient mice at 1-or 2-day intervals thereafter, and the total population of eachsample was determined. The remainder of the cell suspensionwas centrifuged and stored frozen for subsequent lipidanalysis. Because of the variation in the absolute weight of themice, tumor growth was expressed for comparative purposes asincrease in number of cells/g of body weight.

An additional parameter used to assess growth of cells wasthe mean generation time (1'), the time taken for the cell

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Essential Fatty Acid-deficient Tumor Cell Growth

between 11 and 14 g. Those fed the fat-free diet grew moreslowly than controls during the first 5 weeks (Chart 1). After 5weeks, EFA-deficient mice stopped growing, whereas normalmice continued to gain weight for a further 4 to 6 weeks.EFA-deficient mice, following 8 weeks on the fat-free diet,gained 45% less than controls.

EFA's in blood from mice on deficient diets were measuredbiweekly and immediately before transplantation of thetumor. The sharpest decline in linoleic acid occurred duringthe 1st month on the diet (Chart 2), during which time theamount in blood lipids dropped from 17.5% to less than 5% ofthe total fatty acid. After 3 months, levels had declinedfurther to 2%; at 6 months, levels had declined to only 0.5%,i.e. , to less than 1/30th of the normal amount.

a

TIME ON DIET (WEEKS)

Chart 2. Linoleic acid content of blood lipids of mice maintained onfat-free diet. Blood lipids were analyzed for linoleic acid content asdescribed in “Materialsand Methods.―The value at 0 time representsthe average of 6 mice. Other points are average values for at least 3 micerandomly selected after varying intervals on the experimental diet.

8

1 2 3 14

DAYS GROWTH

Chart 3. Growth of norrnal Ehrlich ascites tumor in normal andEFA-deficient mice. Tumor cells harvested from mice maintained onnormal diets were injected i.p. into 10 normal and 10 EFA-deficientmice at an inoculum level of l0@ cells/mouse. The tumor cellpopulation was determined by counting harvested cells as described in“Materialsand Methods.―Mean generation time was I .08 ±0.20 daysfor normal and 1.25 ±0.13 days for EFA-deficient cells.

Growth of Tumor Cells in Normal and EFA-deficient Mice.Ehrlich ascites carcinoma cells harvested from mice maintainedon a normal diet were injected into both normal andEFA-deficient mice. The growth of the tumors was essentiallythe same from the 3rd day on, as is shown in Chart 3, althoughthe lag phase following inoculation of the EFA-deficient cellswas somewhat longer than normal.

For examination of the transplantation characteristics ofthe EFA-deficient tumor cells, further studies were carried outin which the inoculum consisted of Ehrlich ascites carcinomacells which had been rendered deficient by growth for 10 daysin EFA-deficient hosts. The transplantation characteristics ofthe EFA-deficient cells were essentially the same as those ofthe normal cells (Chart 4), and no anomalous lag phase wasobserved.

Similar studies were performed with a 2nd type of tumor(Sarcoma 180) which had been adapted for growth in theascitic form. The lag period of the unadapted cells was againsomewhat longer in the EFA-deficient mice (Chart 5), but themean generation times, measured over the entire growth cuive,were not significantly different.

Fatty Acid Composition of Normal and EFA-deficientTumor Cells. The total lipid content of the EFA-deficientEhrlich ascites tumor (0.76 ±0.13 mg lipid/lO' cells) was notsignificantly different from that of normal cells (0.67 ±0.12mg/b7 cells), despite the markedly different composition ofthe cellular fatty acids. (See below)

Analysis of hydrogenated samples of tumor cell lipidsshowed that the major fatty acids present in both normal andEFA-deficient cells contained 16, 18, 20, and 22 carbon atoms(Chart 6). On the basis of this information, in addition to thatobtained from standards and ECL values (Table 1), a probableidentity was assigned to peaks appearing in GLC traces of lipidextracts from normal and EFA-deficient cells and blood.

Each of the experimental sources, normal and

EFA DEFICIENT DIET

DAYS ON DIET (AFTER WEANING)

Chart 1. Growth rate of mice maintained on norrnal andEFA-deficient diets. Mice of the CF, strain weaned at 3 to 4 weeks ona diet of either normal stock mouse pellets or a fat-free synthetic dietwere weighed weekly. Each point is the average of 6 or more animals.

20

15

a10

12 16 20 24

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1 2 3

Fatty acids present in lipidextractsPeakscorresponding to the fatty acids listed were routinelyobservedin

GLC traces of lipid extracts from norrnal and EFA-deficientcells.Theproba1@Jeidentity was assigned by comparison of theretentionratios

(relative to oleic acid equal to 1.0) with those ofauthenticstandardsand also by use of the linear relationship betweenlogretention

ratio and number of carbon atoms in a homologousseries.Considerationwas also given to the results ofhydrogenationexperiments

and to established metabolic pathways for elongationanddesaturationof fattyacids.Retention

Log(retentionratioratio times 10) Probableidentity0.27

0.43914:00.470.67616:00.580.76616:[email protected]:01.001.0018:[email protected]:2@..,6or20:li..,91.741.2418:3...,3,6or72.141.3320:2@,62.491.40 20:3c@,7or92.741.5522:03.251.5120:4c..,64.371.6422:3c@,7or95.51.7422:4@.,69.01.9522:5@,39.51.98 22:6w3

DAYS GROWTH

Chart 4. Growth of EFA-deficient Ehrlich ascites cells in normal andEFA-deficient mice. Tumor cells rendered EFA deficient by growth for10 days in EFA-deficient mice were transplanted into normal andEFA-deficient mice. EFA-deficient cells grew equally well in bothnormal and EFA-deficient hosts. The mean generation time was 1.04 ±0.19 days in normal and 1.07 ±0.19 days in EFA-deficient mice.

DAYS GROWTH

Chart 5. Growth of normal Sarcoma 180 in the peritoneal cavity ofnormal and EFA-deficient mice. Sarcoma 180 cells were injected at aninoculum level of 3.8 X 106 cells/mouse. Mean generation time of cellsin normal mice (0.91 ±0.15 days) was not significantly different fromthat in EFA-deficient mice (1.04 ±0.19 days).

EFA-deficient Ehrlich ascites carcinoma cells, Sarcoma 180cells, and the blood of host mice, had a characteristic fattyacid composition, values for which are given in Table 2.

In contrast to the relatively constant fatty acid compositionof cells grown for several days in a given environment, rapidchanges in fatty acid composition occurred in the first fewdays when normal cells were transplanted into EFA-deficientmice and, conversely, when EFA-defIcient cells weretransplanted back into normal mice. The followingcharacteristic changes occurred when tumor cells were grownin EFA-deficient mice : decrease in the EFA's linoleic acid(l8:2@.,6) and arachidonic acid (20:&,6), with simultaneousincreases in palmitoleic acid (16: lco7), oleic acid (18: lw9),and eicosatrienoic acid (20:3@.,7 or 9) (Chart 9). A reversal of

J. Martyn Bailey and L. M. Dunbar

these changes occurred when EFA-deficient cells weretransplanted into normal hosts, the end result being a rapidreturn to the fatty acid spectrum characteristic of normal cells(Chart 8).

DISCUSSION

Previous studies in tissue culture have indicated differencesin the requirement for EFA's for growth at the cellular level

Chart 6. Fatty acid cornposition of lipids from EFA-deficient Ehrlichascites carcinoma cells before and after hydrogenation. The fatty acidmethyl esters were reduced by treatment with hydrogen gas withpalladianized charcoal as catalyst. The chromatographic traces of theextracts before and after reduction assisted in identification of some ofthe unusual fatty acids present in EFA-deficient cells. The last peak toappear in the trace, after hydrogenation, is that corresponding to 22:0This demonstrates that the fatty acid peaks with longer retention timesrepresent a new pattern of polyunsaturates rather than longer-chainfatty acids.

Table 1

DIET

6

.@ BEFORE HYDROGENATION

2 3RETENTION RATIO

4

S

325

;20

@15

@ 10

5

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Distribution offatty acids occurring in lipidextractsThevalues given for EFA-deficient animals are the average percentages of composition of blood lipids of host mice following maintenanéeonthe

fat-free diet for at least 2 months. Each value for the tumor cell lipids is the average of 6 or more determinations following growthforapproximately1 week in the stated host environment.EFA-deficient

EFA-deficientBloodfrom Normal Ehrlich Normal Sarcoma Blood from EFA- Ehrlich ascites Sarcoma180Fatty

acid normal mice (%) ascites cells (%) 180 cells (%) deficient mice (%) cells (%) cells(%)16:0

23.0±13° 14.8±0.5 14.7±3.0 21.4±1.1 12.7±0.7 12.2±1.116:11.3 ±0.5 1.7 ±0.1 2.2 ±0.3 3.9 ±0.6 9.3 ±0.7 10.9 ±0.618:0

10.0±0.6 18.2±0.3 16.0±1.4 7.6±0.3 11.3±1.0 12.8±0.318:123.7 ±2.6 25.0 ±0.9 22.4 ±0.8 46.0 ±3.0 41.3 ±2.7 43.@±2.618:218.4±1.0 25.0±0.4 20.0±1.2 0.6±0.1 1.9±0.21.0±0.218:3/20:1

1.4 ±0.3 2.2 ±0.2 1.6 ±0.2 3.4 ±0.6 7.8 ±0.9 4.7 ±0.420:21.5 ±0.3 1.7 ±0.2 1.2 ±0.2 17.1 ±0.7 15.4 ±1.8 13.5 ±1.920:4

12.8 ±0.6 11.0 ±0.6 17.1 ±2.5 1.5 ±0.3 2.6 ±0.5 1.0 ±0.322:0None observed 1.6 ±02 1.8 ±0.3 1.8 ±0.8 5.4 ±0.8 1.9 ±0.4

Essential Fatty Acid-deficient Tumor Cell Growth

Table 2

a Mean ±S.E.

(1, 5—7,13). These tissue culture studies are limited both bythe limited number of cell types which have been adapted togrowth in lipid-free, chemically defined media and also by themarginal growth rates of many cell lines in the syntheticmedia. The knowledge that several lines of tumor cells growrapidly in the ascitic form in mice and that pronounced EFAdeficiency can be generated in mice by maintenance on afat-free diet prompted this investigation, the intent of whichwas to study the growth characteristics of tumor cells in EFAdeficiency. This objective required, first, that host animalsexhibit symptoms of EFA deficiency. Alteration in growthrate and in the fatty acid composition in blood tissues ofEFA-deficient mice are known to occur. These 2 factors, bothof which are easily measurable, were therefore used as a basisfor assessing deficiency in the mice.

Based on the observed decreases in growth rate and linoleicacid content of the blood, it was concluded that mice can beconsidered to exhibit marked symptoms of EFA deficiencyafter 1 month on the fat-free diet. For the present studies, theanimals had been fed the fat-free diet for a minimum of 2months from time of weaning (for as long as 2 months in someof the experiments).

The tumor lines used grow rapidly in the ascitic form andare amenable to study by quantitative cell countingtechniques. The Ehrlich ascites carcinoma is of epithelialorigin, and Sarcoma 180 represents a cell of fibroblastic origin.

An increase in dividing time occurred in the initial stagesof growth when both Ehrlich ascites and normal Sarcoma 180cells were transplanted into deficient mice. This slower growthfollowing transplantation of normal cells into deficient hostsmay reflect an extension of the lag phase related to somenonspecific adaptation of the cells to the differentenvironment provided by EFA-deficient mice. The fact thatthe growth rate returned to normal after the first 24 to 48 hrsuggests that the deficient environment did not represent anymajor metabolic stress. This conclusion is also supported bythe apparently normal transplantation characteristics of thedeficient cells in both normal and deficient hosts. Theincreased lag phase was not observed during transplantation ofdeficient cells, and the mean generation time was essentiallythe same as that of normal cells over the whole of the growth

curve (Chart 4).transplantability wereSarcoma 180 (Chartrequirement for EFAdifferent cell types.

The inability of the host animals to synthesize linoleic,linolenic, and arachidonic acids leads to changes in thecomposition of blood and tissue lipids when fats are excludedfrom the diet. These changes were reflected in alterations inthe fatty acid composition of the tumor cells grown in themice (Charts 7 to 10). The predominant alterations observedwere large decreases in the EFA's and increases in thepolyunsaturates such as 20:3 and 22:3 which can besynthesized de novo. Small amounts of fatty acids tentativelyidentified as 22 :4, 22 :5, and 22 :6 were present in normal cellsand blood samples in addition to the fatty acids listed in Table

RETENTION TIME RATIO

Chart 7. Fatty acid composition of lipids from Ehrlich ascitescarcinoma cells. Fatty acid rnethyl esters were chromatographed asdescribed in “Materialsand Methods.―The chromatographic tracesreproduced here illustrate the principal differences in the lipidcomposition. Note particularly the marked decrease in 18:2 and 20:4and their replacement by 16:1, 18:1, and 20:3. Ofparticular interest isthe appearance of significant quantities of eicosatrienoic acid 20:3, afatty acid not present in detectable quantities in normal cells andcharacteristic of pronounced EFA deficiency in mammals.

Essentially normal growth andalso found for the ascitic form of5). This indicates that lack of a

may well be a characteristic of many

EFA DEFICIENT

2

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J. Martyn Bailey and L. M. Dunbar

2. Because the former are metabolically derived from 18:2and/or 18 :3 , they are technically a part of the EFAcomplement of the cell lipids. They were not found in theEFA-deficient samples.

Alterations in mitochondrial function, skin permeability,and erythrocyte fragility in rats and mice with fat deficiencyhave been cited by MacMillan and Sinclair (12) as evidence forstructural importance of the EFA's. Gershenson et al. (7)

Chart 8. Changes in fatty acid composition of EFA-deficient Ehrlichascites tumor cells following transplantation into normal mice.Following transplantation into normal mice, the fatty acid compositionof EFA-deficient tumor cells returned to normal Within 3 days. Theprincipal changes occurring, as shown here, were increases in linoleicacid 18:2 from less than 1% to approximately 25% of the total andcorresponding decreases in oleic acid 18 :1 and eicosatrienoic 20 :3 from50 and 10% of the total to 24 and 0%, respectively.

DAYS GROWTH

Chart 9. Changes in fatty acid composition of Sarcoma 180 cellsgrown in EFA-deficient mice. Rapid changes in fatty acid compositionoccurred within 24 hr of transplantation, including decrease in 18:2and 20:4 and replacement by 20:3, 16:1 and 18:1, which were similarto those observed with the Ehrlich ascites tumor cells. A morecomprehensive comparison of the fatty acid composition of the normaland deficient Sarcoma 180 cells after 7 days of growth is given in Chart10. The fatty acid composition rapidly returned to normal when theywere transplanted back into normal mice.

— NORMAL

EFA DEFICIENT

I 3 4 5 b 7

60

RETENTION TIME RATIO

Chart 10. Comparative fatty acid composition of lipids fromSarcoma 180 cells grown in normal and EFA-deficient mice. Thepercentage of composition of the fatty acids from Sarcoma 180 cellsgrown for 7 days in the normal or EFA-deficient host is displayed onthe basis of the retention time ratio on GLC traces relative to oleic acid(18:1) equal to 1.0. The retention times of 18:3 and 20:1 were tooclose to enable them to be distinguished, and the percentages for these2 fatty acids have therefore been combined.

likewise suggested that altered mitochondrial function in HeLacells cultured in lipid-free medium may result from disruptionof the membrane integrity in the absence of the EFA's. Also,these cells failed to grow and did not exhibit increases in 20:3.The evidence presented here showing relatively normal growthof cells in association with the appearance of considerablequantities of eicosatrienoic acid (20:3) in the cells indicatesthat this fatty acid can substitute functionally in these tumorcells for the EFA's.

REFERENCES

1. Bailey, J. M., Clough, J., and Lohaus, A. Influence of LDH Viruseson Growth of Ehrlich Ascites Tumor in Mice. Proc. Soc. Exptl.Biol.Med.,119:1200—1204,1965.

2. Bailey, J. M., and Menter, J. Lipid Metabolism in Cultured Cells.VII. Linoleic Acid Content of Cells Grown on Lipid-free SyntheticMedium. Proc. Soc. Exptl. Biol. Med., 125: 101—105,1967.

3. Bloor, W. R. A Method for the Determination of Fat in SrnallAmounts of Blood. J. Biol. Chern., 1 7: 377—384, 1914.

4. English, J., and Cassidy, H. G. Principles of Organic Chemistry, Ed.3, pp. 64—69.New York: McGraw-Hill Book Co., 1961.

5. Evans, H. M., and Burr, G. 0. New Dietary Deficiency with HighlyPurified Diets. Proc. Soc. Exptl. Biol. Med., 25: 390—394, 1928.

6. Evans, V. J., Bryant, J. C., Fioramonti, M. C., Sanford, K. K.,McQuilkin, W. T., and Earle, W. R. Studies on Nutrient Media forTissue Culture Cells in Vitro. I. A Protein-free Chernically DefinedMedium for Cultivation of Strain L Cells. Cancer Res., 16: 77—82,1956.

7. Gerschenson, L. E., Mead, J. G., Harary, I., and Haggerty, D. F.Studies on the Effects of Essential Fatty-Acids on Growth Rate,Fatty-Acid Composition, Oxidative Phosphorylation andRespiratory Control of HeLa Cells in Culture. Biochim. Biophys.Acta, 131: 42—49,1967.

8. Geyer, R. P. Uptake and Retention of Fatty Acids by TissueCulture Cells. In: G. Rothblat and D. Kritchevsky (eds.), LipidMetabolism in Tissue Culture Cells, pp. 33—47.Philadelphia, WistarPress Monograph, 1967.

8.2

4 5 6 7

DAYS GROWTH

96 CANCER RESEARCH VOL. 31

on July 20, 2021. © 1971 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Page 7: Lipid Metabolism in Cultured Cells: Growth of Tumor Cells ......essential fatty acid deficiency. Essential fatty acids in blood lipids of essential fatty acid-deficient mice comprised

Essential Fatty Acid-deficient Tumor Cell Growth

9. Ham, R. G. Albumin Replacements by Fatty-Acids in ClonalGrowth of Mammalian Cells. Science, 140: 802—806,1963.

10. Hofstetter, H. H., Sen, N., and Holman, R. T. Characterization ofUnsaturated Fatty Acids by Gas-Liquid Chromatography. J. Am.Oil Chemists Soc., 42: 537—540,1965.

11. Hyun, S. A., Vahouny, G. V., and Treadwell, C. R. QuantitativePreparation of Methyl Esters of Short-Chain and Long-Chain FattyAcids for Gas Chromatographic Analysis. Anal. Biochem., 10:193—196,1965.

12. MacMillan, A. L., and Sinclair, H. M. The Structural Function ofEssential Fatty Acids. In: H. M. Sinclair (ed.), Essential FattyAcids, pp. 208—211. Washington, D. C.: Butterworth Inc., 1958.

13. Paul, J. Cell and Tissue Culture, pp. 49—54. Baltimore: TheWilliams & Wilkins Co., 1965.

14. Riley, V. Adaptation of Orbital Bleeding Technique to Rapid SerialBlood Studies. Proc. Soc. Exptl. Biol. Med., 104: 75 1—754,1960.

15. Smedley-MacLean, I., and Nunn, L. C. A. Fat Deficiency Disease ofRats. The Relation of the Essential Unsaturated Acids to TumorFormation in the Albino Rat on Normal Diet. Biochem. J., 35:996—1000, 1941.

16. Thornasson, H. J. Intern. Z. Vitaminforsch., 25: 62—66, 1953.Cited in A. F. Wagner and D. Folkers. The Essential Fatty AcidGroups. In: Vitamins and Coenzymes, Chap. 19, pp. 391—397.New York: John Wiley and Sons, Inc., 1964.

97FEBRUARY 1971

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1971;31:91-97. Cancer Res   J. Martyn Bailey and L. M. Dunbar  Deficient in Essential Fatty AcidsLipid Metabolism in Cultured Cells: Growth of Tumor Cells

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