effects of free fatty acids on the growth of normal and ...of unsaturated free fatty acids and...

[CANCER RESEARCH 39, 426-435, February 1979]0008-5472/79/0039-0000$02.0O

Effects of Free Fatty Acids on the Growth of Normal and NeoplasticRat Mammary Epithelial Cells

Max S. Wicha, Lance A. Liotta, and William R. Kidwell

Laboratory of Pathophysiology, National Cancer Institute, Bethesda, Maryland 20014

ABSTRACT

To gain some understanding of the role of lipids inmammary epithelial growth and in mammary carcinogenesis, the direct effects of free fatty acids on cell growth inprimary cultures of mammary epithelial cells and 7, 12-dimethylbenz[a]anthracene-induced mammary tumors fromrats were studied. The epithelial nature of virgin mammarycultures was confirmed by light and electron microscopy,by association of cells with basement membrane, and byproduction of a-lactalbumin. fri the presence of insulin,hydrocortisone, progesterone, estrogen, prolactin, and delipidized fetal calf serum, both the normal mammary andtumor cells were stimulated in their growth by the additionof unsaturated free fatty acids and inhibited by the additionof saturated free fatty acids to the growth medium. Fornormal cells, linoleic or linolenic acids at 1.0 and 0.1 p@g/ml, respectively, and for 7,12-dimethylbenz[a]anthraceneinduced tumor cells, oleic acid or linoleic acid at 0.5 and1 .0 p.g/mI, respectively, resulted in greater than a doublingof the growth matecompared to cultures with no added freefatty acid. For both normal and tumor cells, saturated free

fatty acids inhibited growth at all concentrations tested.

The concentration optimain vitro paralleled the proportionsof essential fatty acids (free and estenified) in the normalmammary gland. A state of mammary proliferation in vivowas associated with an increase of 45% in the ratio oflinoleic plus oleic:palmitic acids in total mammary lipids.

Lipids may play some direct molein normal and neoplasticmammary cell growth.

INTRODUCTION

Despite the fact that the mammary gland of humans isencased in a matrix of fat, little is known about the role oflipids in normal mammary growth and development, although these processes are clearly affected by dietary lipids(31). There is also an involvement of lipids in mammarycancinogenesis as indicated by epidemiological analysisdemonstrating a strong positive correlation between theamount and type of fat in the diet and the age-adjustedincidence of breast cancer (14, 22, 41, 42). Studies withexperimental animals support this correlation (5-10, 12, 17,25, 34).

The mechanism of the tumor-potentiating effect of fatshas been postulated to be indirect via actions outside themammary gland (9-i 1, 25, 30), but because of the corn plexity of experiments in vivo with dietary fats, it has beendifficult to ascertain whether lipids have a more direct rolein normal mammary function and mammary tumor devel

Received July 19, 1978; accepted October 25, 1978.

opment. In vitro studies using established cell lines areopen to question because of the possibility of cell selectionin long-term culture. To overcome some of these difficulties, we have utilized a primary culture system for â€œnormalâ€•mammary epithelium and DMBA1-induced mammary tumorcells from Sprague-Dawley rats. Using this system, we havestudied the direct effects of added free fatty acids on normalmammary epithelial and DMBA-induced mammary tumorcell growth under defined lipid conditions. We report on thecharacterization of these primary cultures and the effects offree fatty acids on cell growth.

MATERIALS AND METHODS

Materials. Medium 199 with Tween 80 omitted was utilized for all culturing procedures. Collagenase, type II (150to 190 units/mg) was from Worthington Biochemical Corp.,Freehold, N. J.). Fatty acids and steroid hormones werefrom Sigma Chemical Co. , (St. Louis, Mo.), and fetal calfserum was from Grand Island Biological Co. (Grand Island,N. Y.). Ovine prolactin (NIH S 12, Bethesda, Md.) wasdissolved in 0.01 M NH4OH and diluted to 100 times finalconcentration in EBSS containing 0.i% fatty acid-free bovine serum albumin (Calbiochem, La Jolla, Calif.). Insulin(U-80) was from Eli Lilly and Co. , (Indianapolis, Ind.) andwas diluted to 100 times final concentration in EBSS plus0.1% albumin. Steroid hormones were dissolved to 100times concentration in 25% ethanol:75% EBSS. Ficoll 400

was from Phanmacia Fine Chemicals, Inc. , (Piscataway,N. J.), and perphenazine (Tnilafon, 5 mg/mI) was fromScheming Corp. (Kenilworth, N. J.).

Animals. Virgin female Sprague-Dawley rats 3 to 4months old were purchased from Taconic Farms (Germantown, N. Y.) and were given Purina rat chow (>4% fat) andwater ad libitum . They were given injections of perphenazine at 29 and 5 hr before sacrifice as described by Halloweset a!. (20) and Rutland et a!. (36). Mammary tissue obtainedfrom these animals is referred to as normal. DMBA-induced,tumor-bearing Sprague-Dawley mats were obtained fromHazelton Laboratories (Vienna, Va.). Tumors were inducedby a single intragastnic administration of DMBA to mats atabout 50 days of age. Tumors of 1 to 1.5 cm in diameterwere used.

Cell Preparations and Culturing Conditions. Alveoli andductal fragments were liberated from minces of rat mammary gland by the procedure of Malan et a!. (29), exceptthat collagenase digestion (665 units/mI) was performed inMedium 199 with 5% fetal calf serum with gentle stirringinstead of 1% serum in EBSS, and the incubation was

I The abbreviations used are: DMBA, 7,12-dimethylbenz[a]anthracene;

EBSS,Earle'sbalancedsalt solution.

CANCER RESEARCH VOL. 39426

on March 15, 2020. © 1979 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Fatty Acid Effects on Mammary Epithe!ium

continued for an additional 10 mm after adding 100 units ofDNase I (Sigma Chemical Co.). The fragments were thenpassed through a Nitex cloth filter (140 strands/inch),centrifuged out of suspension, washed, and resuspendedin 5 ml of 10% fetal calf serum and 1% Ficoll in EBSS (tissuefrom one animal). The ducts and alveoli in the 5-mi suspension were separated from single cells by velocity gradientsedimentation (2 to 10% Ficoll in EBSS) as described byMalan et a!. (29). Ducts and alveoli were recovered from the70- to 90-mi fraction from the top of the gradient. Tumorcell aggregates were prepared as described above, exceptthat one-half the amount of collagenase was utilized for thedigestion. The cells from approximately 0.5 to 0.75 g tumorwere separated on a single Ficoll gradient. Fractions fromseveral gradients were pooled, and the cells were recoveredby sedimentation. After washing 2 times with Medium 199(minus Tween 80), 0.5 ml cell suspension (1 to 1.5 x i0@cells) was plated onto 35-mm Corning (Corning, N. Y.)tissue culture Petni dishes. One ml of Medium 199 wassupplemented with 7.5% whole or delipidized fetal calfserum (35) was then added. Stock solutions of hormoneswere prepared, and aliquots were added to give optimumconcentrations essentially as described by HalIowes et a!.(20) and Rutland et a!. (36) (Chart 1). Gentamycin wasadded to a final concentration of 50 j.@g/ml.Fatty acids weresolubilized in 95% ethanol, and amounts were added suchthat the final concentration of ethanol in the growth medium was 1%. Control cultures received exactly the samebuffers and ethanol but with hormones or lipids omitted asindicated. Cells were propagated at 37Â°in humidified 5%CO2in air.

Analytical Procedures. Automadiogmaphicanalysis wasperformed following pulse labeling of cells for 20 hr with[3H]thymidine (3 MCi/mI at i0@ M thymidine). After labeling,cells were washed with EBSS and then fixed in absolutemethanol at 4Â°for 20 mm. Unincorporated radioactivity wasextracted by a 60-mm wash with 5% tnichloroacetic acid.Finally, after the plates were washed with water, emulsion(Kodak NTB; Eastman Kodak Co. , Rochester, N. Y.) wasadded, and the plates were exposed for 72 hr. For electronmicroscopy, ductal and alveolar tissue and tumor cellaggregates were recovered from Ficoll gradients and fixedfor 60 mm in 2% cold glutaraldehyde in phosphate-bufferedsaline (NaCi, 8 g/liter; KCI, 0.2 gluten; Na,HPO4, 1.15 g/liter; KH2PO4, 0.2 g/liter). Cell fixation and staining forbasement membrane proteins followed the procedure ofStenman and Vaheni (38). Fluorescent antibody againstbasement membrane collagen was prepared by the methodof Yaoita (43). After a 10-mm exposure to trypsin (0.25%):EDTA (0.05%), cells were detached from the dishes with arubber policeman and counted with a B/P 6300a cytograph.Rad ioimm unoassay of rat (@-lactalbum in prod uction by thecultures was performed according to the method of Qasbaand Chakrabartty (32) and Quasba and Gullino (33) utilizinglabeled and unlabeled rat t-lactalbumin and antibody provided by that laboratory.

Fatty Acid Analysis. Total tissue or serum lipids wereextracted in CHCl:@CH:@OH(2:1), 20 v/v extract, and aftersolvent evaporation, were saponified in 30% KOH in methanol at 90Â°for 2 hr. The solutions were extracted withhexane, the residue was acidified, and the fatty acids were

extracted with hexane. The hexane was then removed witha stream of nitrogen, and the residual fatty acids wereconverted to p-bmomophenacyl derivatives by crown ethercatalysis (15). The fatty acids were separated by high-pressure liquid chromatography with a gradient of acetonitnile:water (4). Quantitation was performed by assessing theamount of 254-nm absorption of the peaks emerging fromthe Waters @BondapakCi8 column using an HP 3380Aintegrator.

To quantitate free fatty acids in serum, total lipids wereextracted as described, and aliquots were applied to thinlayer plates of Silica Gel G. The plates were developed withhexane:diethyl ethem:glacial acetic acid (70:30:0.25) anddried. The free fatty acids were localized by 12vapor staining. Corresponding areas of unstained plates were scrapedfrom the thin layers, and the fatty acids were recovered byrepeated extraction with the developing solution. Afterevaporation of the extraction solvent, the fatty acids weredenivatized and separated by high-pressure liquid chromatography as described.

RESULTS

The partial collagenase digestion and Ficoll gradientsedimentation provided a means of separating epithelialelements of normal virgin (perphenazine stimulated) andDMBA-induced mammary tumors from fibmoblasts, adipocytes, and blood cells. These epithelial preparations consisted of organized cellular structures similar to thosefound in vivo.

Cell Characterization

Virgin Epithelium. The cellular aggregates isolated asdescribed from virgin rats had the appearance of alveoliand ductal fragments. Freshly isolated preparations possessed a uniform outer layer of material which stained witha fluorescent antibody specific for type IV collagen ofbasement membrane (Fig. 1). Surrounding the structuresand in between cells, there was dense periodic acid-Schiffstaining material which probably represents the glycoprotein component of basement membrane (Fig. 2). Electronmicroscopy offneshly isolated preparations (Fig. 3) revealeda layer of epithelial cells surrounded by a layer of myoepitheIial cells. The structures possess a luminal surface coyered with microvilli and an outer basement membrane.Thus, the anatomical arrangement of epithelial cells, myoepithelial cells, and basement membrane typical of thegland in vivo remains essentially intact through the isolationprocedure.

In culture, the alveolar and ductal cells attach to the Petnidishes within 12 hr with a plating efficiency of 75 to 80%.Cells from the alveoli spread to form a monolayer andproduce an intercellular matrix of periodic acid-Schiffstaining material (not shown). While the ductal fragmentsattach to the plates, the duct cells do not completely spreadto form monolayens and were distinguishable from alveolarstructures even after 4 to 5 days of culturing. Autonadiographic analysis of the cell population indicated that onlythe alveolar cells incorporated [3H)thymidine (Fig. 4). Thiswas the case regardless of the various supplements testedfor growth-promoting activity in culture (including all lipid

FEBRUARY 1979 427



M. S. Wicha et a!.

supplements). Thus, any increase in cell number with timein culture determined by trypsinizing and counting totalcells with the cell counter represents an increase in alveolarcell number since cell division was limited to this population. The alveolar cells continue to divide in culture with anapproximately linear increase in cell number for 48 hr indelipidized serum or 96 hr in whole serum, with a subsequent reduction in growth matesthereafter. Total cell lifetime in culture was unaffected by lipid supplementation.After 72 hr in culture, theme was no detectable type Icollagen by nadioimmunoassay. On the basis of the ratio oftype lV:type I collagen produced by normal mammary cellsin vitro, there are less than 1% fibroblasts in these cultures.2

In addition to morphological features, the cultures werecharacteristic of mammary cells in that they produced a-lactalbumin in culture as detected by a specific madioimmunoassay according to the method of Qasba and Chakrabartty (32) and Quasba and Gullino (33). After 48 hr inculture, the amount of a-lactalbumin per cell increasedmore than 100% above the amount present at the time ofisolation with 18.8 Â±1.1 ng/106 cells present initially and40.4 Â±5.4 ng/106 cells present at 48 hr.

As reported by Hallowes et a!. (20), giving rats injectionsof penphenazine increased the yield of mammary epithelium2- to 4-fold, presumably through stimulation of prolactinrelease (3). This was used to reduce the number of animalsneeded, although qualitatively similar results were seenwith virgin matswithout pemphenazine treatment. Extensivetrypsinization of the duct and alveolar mixture from thegradient resulted in predominantly single-cell suspensionswhich were of low plating efficiency but were suitable forcell counting.

Tumor Cell Cultures. PartialcollagenasedigestionandFicoll gradient sedimentation of DMBA-induced mammarytumors produced small clusters of cells which were of highviability (85 to 90% plating efficiency). Unlike the normalmammary ducts and alveoli, these clusters were not associated with periodic acid-Schiff-staining material (Fig. 5).As can be seen in this figure and by electron microscopy(Fig. 6), DMBA tumors contain both epithelial- and myoepithelial-like cells. Continued digestion of these clusters withtrypsmn resulted in single-cell suspensions which were oflow plating efficiency but were usable for cell counting. Inculture, the cell aggregates spread to form monolayers witha tightly packed central population of epithelial-like cellsand a surrounding halo of stellate-shaped cells (Fig. 7).These stellate cells may represent the myoepithelial cornponent of the tumor. Autoradiognaphic analysis following[3H]thymidine labeling indicated that most of the cell division took place in the â€œepithelialâ€•-likecells, with the ratioof labeled cells in this tumor population to that in thestellate cells of approximately 8:1 (Fig. 7). This was the casewith or without lipid supplements. Thus, cell growth measured by trypsinizing and counting cells was due mostly toan increase in this epithelial cell population. The growth ofthe cells in culture was linear for approximately 48 to 72 hrwith a slow decline in growth rate thereafter.

2 L. A. Liotta, M. S. Wicha, S. Rennard, J. M. Foidart, and W. R. Kidwell.

Basement Membrane Collagen Synthesis in Primary Cultures of MammaryGland Epithelium, submitted far publication.

Effectsof FattyAcidson CellGrowth

To examine the effects of free fatty acids on the growth ofmammary epithelial cells, cultures were grown in Medium199 (minus Tween 80) supplemented with 5% delipidizedfetal calf serum plus insulin, hydrocortisone, prolactin,estradiol, and progesterone (see Chant 1). Individual fattyacids were then added to the cultures at various concentrations. Under these conditions, growth was linear for 48 hr.As shown in Chant 1A, all the unsaturated fatty acids at theirconcentration optima produced an increase in the numberof cells/dish above the level seen with no fatty acids added.Linoleic and linolenic acids were the most stimulatory,enhancing cell growth by more than 100% over the controlvalue (no added fatty acid) at their concentration optima.Oleic and anachidonic acids were less stimulatory, whilesteanic acid inhibited cell growth at all concentrationstested. The effects of palmitic acid (not shown) were yintually identical to those of steanic acid. For growth stimulation, linoleic acid had the highest concentration optimum,arachidonic and oleic acid had intermediate concentrationoptima, and linolenic acid had the lowest concentrationoptimum.

Comparative dose-response curves with the tumor cellsare depicted in Chart lB. As seen, the results were similarto those obtained with normal cells, except that oleic acidenhanced growth more effectively than did linolenic orlinoleic acids. As was the case with normal cells, steanicacid was inhibitory at all concentrations tested.

To obtain information on the 2 most likely sources of fattyacids for mammary cells in vivo, an analysis was made ofthe total long-chain fatty acids of the mammary gland andthe serum from rats on the same diet. The separation of thedenivatized fatty acids is depicted in Chart 2. The results aresummarized in Table 1. When expressed in terms of percentage of distribution, it can be seen that the long-chainfatty acid composition of the gland is very similar to that ofthe serum. A comparison of Table 1 and Chart 1A revealsthat for the unsaturated fatty acids tested in the cultures,the concentration needed for maximal growth rate of normal cells is roughly correlated with the proportion of therespective fatty acid (free and estenified) in the gland orserum. Thus, Iinolenic acid is present in the lowest amountand has the lowest concentration optimum, while linoleicacid is present in considerably higher amounts and has ahigher concentration optimum.

Lipid and Hormone Effects on Cell Growth

In the previous experiments, the effects of fatty acids oncell growth were examined in medium containing addedhormones. In Table 2, the growth-promoting effects oflinoleic acid were examined with and without added honmones. Cultures of normal cells (Table 2A) showed noappreciable growth in the absence of added hormoneswhether linoleic acid was present or absent. With hormonesonly, the cells had a doubling time of 58 hn, while with bothhormones and linoleic acid, the doubling time was 34 hr.The inhibitory effect on growth with steanic acid shown inChant 1A is also seen here. In a second experiment,[3H]thymidine inconponation into acid-precipitable material

428 CANCER RESEARCH VOL. 39




1.5 Lauric

Linolenic2.2

3.0

5.5

Myristic

Arachidanic/Palmitoleic

Linoleic

Palmitic

Oleic

29.2

CONCENTRATIONFREE FATTY ACID A9/mI 23.231.3

â€˜U

z-aâ€˜UU

z

â€˜U

zIU

â€˜U

z

â€˜UU

zâ€˜U

z4IU

Stearic

Chart 2. High-pressure liquid chromatography of total fatty acids tram ratmammarygland. Numbersin parentheses,percentageof the total of majorfatty acids. See Materials and Methodsâ€•tar description of technique.

0.2 0.5 1 2

CONCENTRATIONFREEFATTY ACID pg/mI

Chart 1. Effects of varying concentrations of free fatty acids an growth ofnormal mammary epithelium (A) and DMBA-induced tumor cells (B). Cellswere plated in 5% delipidized fetal calf serum plus hydrocartisone (0.5 @.tg/ml), Insulin (0.1 @tg/mI),17-f3-estradial (1.0 ng/mI). progesterone (1.0 ng/mI),andovineprolactin(0.3 @g/ml).At 18 hr in culture,mediumwaschangedto5% delipidized fetal calf serum plus hormones as above plus indicatedconcentrations of free fatty acid. Cells were counted 24 to 30 hr after mediumchange. Charts show difference in cell number between dishes with mdicated free fatty acid and those with no added free fatty acid as a function atfree fatty acid concentration. In A, linoleic acid at 1 gtg/mI gave 125%stimulation; B, Ilnoleic acid at 1 @@g/mIgave 100% stimulation, while stearicacid at 1 @.tg/mlinhibited growth 40% in A and 45% in B aver no added treefatty acid. Far the total number of cells per dish, multiply by 25. U. stearicacid; L@,arachidonic acid; A. oleic acid; â€¢,linalenic acid; 0, linaleic acid.Bars,rangeat duplicateswith experimentsrepeatedat least2 times.

during a 2-hr pulse was used to verify the above hormoneand lipid effects, and the results were qualitatively the same(Table 2A).

Examination of the effects of hormones and lipids ontumor cell growth and [3H]thymidine incorporation (Table

FEBRUARY 1979 429

2B) indicated that maximal growth stimulation requiredboth hormones and linoleic acid. The tumor cells differedfrom the normal cells in that there was less stimulation ofgrowth by added hormones alone but a greaten response tolinoleic acid with hormonal support present. These effectswere not due to a change in plating efficiency by addedhormones or lipids since these varied by no more than 5 to10% with any of the supplements.

Fatty Acid Supplements to Growth Medium ContainingWholeFetalCalf Serum

An analysis was performed of the free fatty acids in whole(not delipidized) fetal calf serum to determine what contnibution the whole serum at the 5% concentration wouldmake to the growth medium . These results are presented inTable 3. From the growth optima values obtained in Fig. 1and the amounts of free fatty acid supplied by whole fetalcalf serum, one would predict that linoleic acid in particularwould be deficient in the culture media containing 5%whole fetal calf serum. Chart 3 shows the effects of addedlinoleic and steanic acids (1 @.tg/mI)on growth of normal



Total lang-chain fatty acids (>C,) were analyzed in pooledserafrom10 rats or from the whole mammary glands of 2 separate

animals. Animals were on standard diets as indicated in â€˜â€˜MaterialsandMethods.â€•Mammary

glandâ€•Freefattyacid

Serumâ€• RatlRat2Launic<1.0 1.51.5Linolenic

2.0 2.22.0Myristic3.1 3.03.0Arachidonic:palmitoleic8.4 5.5â€•5.3Linoleic

29.4 29.229.5Palmitic17.7 23.221.1Oleic33.7 31 .333.6Stearic

5.6 4.1 4.1

Fetal calf serum was the same lot as used throughoutallexperiments.Free fatty acids were isolated by thin-layer chroma

tography and then derivatized and separated byhigh-pressureliquidchromatography.5%

fetal calf SeFree fatty acid rum (j.@g/ml) %optimumâ€•Oleic

0.2855Linoleic0.022Linolenic0.0216Arachidonic0.0919Stearic0.24IiPaimitic0.36 /â€˜

Effects of hormone and free fatty acid addition on growthandDNAsynthesis in normal mammary epithelium andDMBA-inducedtumor

epitheliumNormal(A) or DMBA-induced tumor cells (B) were plated in5%delipidized

fetal calf serum. Free fatty acids were addedwhereindicatedat 1 @g/ml.Cell growth was measuredover 48 hr,andcell

doubling time was calculated based on maximum growth rate.[3H]Thymidineincorporation was measured during a 2-hrpulseafter

18 hr in culture as in Chart 4. Plating efficiencies varied bynomarethan 10% as determined by cell count at 18hr.[3H]ThymidineincorporationDoubling

(10@xcpm/Additionstime (hr)cell)A.

NormalepitheliumNoaddition a 6.7 Â±1.2@+

hormonesâ€• 58 Â± 5 20.8 Â±0.5+

linoleic acid a 5.8 Â±0.5+

linaleic acid + hormones 34 Â± 4 30.0 Â±1.6+

steanic acid + hormones 76 Â± 8 15.5 Â±0.7B.

DMBA-induced tumor epithehumNa

addition 132Â±12 33.9 Â±1.1+hormones89Â±8 60.1Â±1.5+

hormones + linoleic acid 35 Â± 5 90.1 Â±1.8+

hormones + stearic 210 Â± 40 NDd

M. S. Wichaeta!.

Table 1Totallong-chain fatty acid composition of rat seraand mammary

glands

Table 3

Concentration of free fatty acids in 5% fetal calf serum and thepercentage optimum for normal cell growth

â€œ Percentage optimum based on concentration optimum for

each freefattyacidfrom Fig.1.ii Stearic and palmitic acids inhibited cell growth at all concen

trations tested.

U)

.iâ€˜UU

zâ€˜UU)

â€˜U

Uz

DAYS IN CULTURE

Chart 3. Effects of added linoleic and stearic acids on normal A andDMBA-induced tumor cell (B) growth in 5% fetal calf serum. Normal epithehum (A) or DMBA-induced tumor cells (B) were plated in 5% whole fetal calfserum plus hormones (as in Chart 1). After 18 hr in culture, medium waschanged to 5% whole fetal calf serum plus hormones (as above). 0, noadded free fatty acid; C, linoleic acid (1 @.&g/mI);A, stearic acid (1 @.tg/mI).Bars, range of duplicates with experiments repeated at least 2 times.

[3H]thymidine in media containing delipidized fetal calfserum indicated that free fatty acids affected incorporationof this labeled compound into acid-precipitable material.For both cells, hinoleic acid stimulated [3H]thymidmneincorponation and steanic acid inhibited [3H]thymidine incorponation within 1 to 3 hr after lipid addition (Chart 4). Also

â€œExpressed as a percentage of the total of these major free fatty

acids.h Gas-liquid chromatography utilizing a 5% DEGS-PS column

demonstrates that palmitoleic acid constitutes 2.1% of major fattyacids in rat mammary gland. By subtraction of this value from thecombined amount determined from high-pressure liquid chromatography analysis, arachidonic acid is calculated to be 3.4% of themajor long-chain fatty acids.

Table 2

a Na detectable cell growth.

b Mean Â± SE.

C@ hormones, concentrations as in Chart 1.

d ND, not done.

and DMBA-induced tumor cells, respectively, plated inwhole 5% fetal calf serum. A stimulation or inhibition of cellgrowth was obtained by adding linoleic on steanic acids,respectively, to either normal on tumor cells, and thiscontinued up to 72 hr in culture.

Early Effects of Fatty Acids on [3HjThymidine Incorporation

Pulse labeling of tumor and normal cells with

430 CANCERRESEARCHVOL. 39



Effects of perphenazine on fatty acid ratios in rat mammaryglandAs

described in Materials and Methods,â€• perphenazine was injected at 29 and 5 hrprior to sacrifice into rats on diet, and total mammary fatty acids wereanalyzed.Linoleic

:palmitic Oleic :palmiticUn:aturat@i:Experi-

Experi- Experi- Experi- Experi- Experiment 1â€• ment 2 ment 1 mont 2 ment 1 mont2Normal

(untreated) 1 .39 1 .47 1 .57 1 .60 2.77 2.81Perphenazine 2.01 2.13 2.30 2.16 3.533.40â€œ

Experiments 1 and 2 represent separate experiments.

Fatty Acid Effects on Mammary Epithelium

shown is the fact that inhibition of thymidine incorporationby the saturated fatty acid occurred in the presence orabsence of unsaturated fatty acid but that the inhibition wasrelieved by the unsaturated fatty acid.

Effects of Perphenazine on Total Free Fatty Acid Ratios InMammaryFat

As indicated in Table 4, perphenazine injection causedan increase in the ratio of total mammary hinoleic plus oleic:palmitic acids and unsaturated:saturated fatty acids overnontreated controls.

DISCUSSION

This report describes the characteristics of a primaryculture system adapted from the method of Malan et a!. (29)for culture of epithelial â€œorganoidsâ€•from normal (perphenazine stimulated) rat mammary glands and DMBA-inducedmammary tumors. This isolation procedure effectively memoves connective tissue cells but maintains much of thestructural orientation of epithelial elements found in vivo.The epithelial nature of the â€œductsâ€•and â€œalveoliâ€•is supported by: (a) structural analysis by light and electronmicroscopy; (b) detection of basement membrane proteinsby fluorescent antibody and periodic acid-Schift-stainingtechniques; (c) synthesis of a-Iactalbumin in culture; and(d) enhanced yield of cells by pretreatment with a drug(perphenazine) which increases circulating pnolactin levels.The morphology of the centrally located, DMBA-inducedtumor cells is also epithehial in nature. At the time ofisolation and in culture, these epithelial tumor cells aresurrounded by a morphologically distinct population charactenized by little cell division, which we think may mepresent the myoepithelial component of the tumor.

These primary cultures, with fat and connective tissueelements removed , were used to assess the effects of freefatty acids on normal mammary and tumor epithelial cell

10 growth under defined lipid conditions. In the presence of

hormones, the growth of both normal and tumor cells wasstimulated by unsaturated free fatty acids and inhibited bysaturated fatty acids. For normal mammary epithelium,linoleic and Iinolenic acids were the most stimulatory; whilefor tumor cells, oleic and linoleic acids were best.

With few exceptions (18, 44), established cells are unaffected in their growth by addition of free fatty acid to thegrowth medium (1, 39). This lack of growth stimulationmight be due to cell selection by passaging in medium witha low lipid content or to an inherent difference in the lipidrequirement of these cells from normal mammary andtumor cells. Since cell plating efficiency of our primarymammary cultures was 80 to 90%, there could be littleinitial selection of cell populations.

B

z0I-

20Uzâ€˜Uz

>.IIâ€”

â€˜U

zI0

HOURS

Chart 4. Effects of free fatty acid additions on [3H]thymidine incorporationin normal (A) and DMBA-induced (B) epithelium. Normal (A) or DMBAinduced tumor cells (B) were plated in 5% delipidized fetal calf serum plushormones (as in Chart 1). At 18 hr. medium was changed to medium with 5%delipidized fetal calf serum plus hormones (as above) plus indicated freefatty acids. Cultures were pulsed for 2-hr periods with rHjthymidine (3 @Ci/ml, 20 Ci/mmol). Reaction was terminated with 10 mM thymidine, andmaterial was precipitated with 5% trichloroacetic acid at 4' for 30 mm.Precipitates were collected on trichloroacetic acid-soaked Whatman No. 3MM filter discs, washed with cold trichloroacetic acid, dried, oxidized with aPackard Tri-Carb oxidizer, and counted with a Beckman L-355 scintillationcounter. Results are given as ratio of @H]thymidmneincorporation per cell inthe presence of free acid fatty addition to [3H]thymidmneper cell in theabsence of free fatty acid addition as a function of time after addition.- - - -, no added tree fatty acid; â€¢, linoleic acid (2 @g/ml); A, stearic acid (2

pg/mI); 0, linoleic acid plus stearic acid (2 pg/mI each). Results indicaterange at duplicates with experiments repeated at least 2 times.

Table 4

FEBRUARY 1979 431



M. S. Wicha et a!.

The response of normal mammary cells to various fattyacids in culture may reflect physiological requirements ofthese cells in vivo . The concentration optima for unsaturated free fatty acids in culture roughly parallel the percentage of distribution of these fatty acids (free plus estenified)in the mammary gland and serum. A comparison of freefatty acid effects in culture with total fatty acid content ofthe mammary gland on serum was performed because cellsin culture preferentially utilize fatty acids in their unestenified form (27) while in vivo mammary gland fatty aciduptake and release is mediated by hormonally sensitivelipases (21). We also found that when the mammary glandis induced to proliferate with perphenazine there was aselective increase in the ratio of unsaturated to saturatedfatty acids (free plus estemified) in the gland. This is consistent with the observation that in vitro , unsaturated fattyacids stimulated growth while saturated fatty acids wereinhibitory. The importance of the fat pad in mammaryepithelial growth and development is supported by theclose proximity of epithelial cells and adipocytes in thegland (31), and suggested by the mammary fat pad transplantation experiments of DeOme and Faulkin (13) andHoshino (26) and by the effects of essential fatty aciddeficiency on mammary gland development and lactation(23, 24).

The effects of fatty acids on tumor cell growth in primaryculture may relate to the well-documented, selective effectsof unsaturated fatty acids on enhanced mammary cancinogenesis in experimental animals (5-10, 12, 25, 34) as well asto the epidemiologicahly defined role of dietary lipids inhuman mammary cancer (2, 14, 22, 41, 42). Oleic andhinoleic acids, which maximally stimulated mammary tumorcell growth in primary culture, are the same fatty acidswhich are reported to be most efficient in increasing mammary carcinogenesis in rats (6, 12). If these correlations aremeaningful, a direct effect of fatty acids in the diet on tumorcell growth is implicated in addition to the indirect effectsthat have been proposed (9-11, 25, 30). This conclusion issupported by the observation of Rao and Abraham (34) thatboth the fatty acid composition and growth in vivo of atransplantable mammary tumor could be altered by dietarylipid modifications.

Mechanistically, it is possible that fatty acid effects on thegrowth of normal mammary and tumor epithelium are theresult of changes in cell membrane fluidity (16, 19, 37) and/or transport (28, 40). Additionally, since cell growth inculture, especially for normal epithehium, required hormonal supplementation, lipid effects of cell growth mightbe mediated by modifications of membrane-bound hormone receptors. The in vitro primary culture system shouldprove valuable for studying these phenomena as well asother aspects of growth and differentiation of mammarycells.

ACKNOWLEDGMENTS

We thank Dr. Douglas Janss for details of the gradient separation technique, Dr. Pradman Qasba for supplying a-lactalbumin protein and antibody.and Kenneth Burdette for excellent technical assistance. Antibody to Type IVcollagen was a gift from Dr. J. M. Foidart.

REFERENCES

1. Bailey, M. J., and Dunbar, L. M. Essential Fatty Acid Requirements of

Cells in Tissue Culture: A Review. Exptl. and Mal. Pathal., 18: 142-161,1973.

2. Basu, T. K., and Williams, D. C. Plasma and Body Lipids in Patients withCarcinoma of the Breast. Oncology, 31: 172-176, 1975.

3. Ben-David, M. Mechanism of Induction of Mammary Differentiation inSprague-Dawley Female Rats by Perphenazine. Endocrinology, 83:1217-1223, 1968.

4. Barch, A. F. Separation of Lang Chain Fatty Acids as Phenacyl Esters byHigh Pressure Liquid Chromatography. Anal. Chem., 47: 2437-2440,1975.

5. Carroll, K. K. Experimental Evidence at Dietary Factors and Hormonedependent Cancers. Cancer Res., 35: 3374-3383, 1975.

6. Carroll, K. K., Gammal, E. B., and Plunkett, E. R. Dietary Fat andMammary Cancer. Can. Med. Assac. J., 98: 590-594, 1968.

7. Carroll, K. K., and Khor, H. T. Effects of Level and Type of Dietary Fat onIncidence of Mammary Tumors in Female Sprague-Dawley Rats by 7,12-Dimethylbenz[a]anthracene. Lipids, 6: 415â€”420,1971.

8. Carroll, K. K. , and Khor, H. T. Dietary Fat in Relation to Tumarigenesis.Pragr. Biachem. Pharmacal., 10: 308-353, 1975.

9. Chan, P., and Cohen, L. A. Effect of Dietary Fat, Antiestragen, andAntiprolactin on the Development of Mammary Tumors in Rats. J. NatI.Cancer Inst., 52: 25-30, 1974.

10. Chan, P., and Cohen, L. A. Dietary Fat and Growth Promotion of RatMammary Tumors. Cancer Res., 35: 3384-3386, 1975.

11. Daa, T. L., Bock, F. G., and Crouch, S. Level at 3-Methylcholanthrene inMammary Glands of Rats after Intragastric Instillation of Carcinogen.Proc. Sac. Exptl. Bial. Med., 102: 635-638, 1959.

12. Dayton, S.. Hashimato, S., and Wollman, J. Effect of High-Oleic andHigh-Linoleic Safflower Oils on Mammary Tumors Induced in Rats by7,12-Dimethylbenz(a)anthracene. J. Nutr., 107: 1353â€”1360,1977.

13. DeOme, K. B., and Faulkin, L. J., Jr. Mammary Tumor Developmentfrom Hyperplastic Alvealar Nodules and Normal Lobules Transplantedinto Mammary Gland-free Fat Pads and in the Dorsal Subcutis of FemaleC3H Mice. Proc. Am. Assac. Cancer Res., 3: 16-17, 1959.

14. DeWaard, F. The Epidemiology of Breast Cancer: Review and Prospects.J. Cancer, 4: 577-583, 1969.

15. Durst, H. D., Milano, M., Kikta, E. J., Jr., Connelly, S. A., and Grushka,E. Phenacyl Esters of Fatty Acids via Crown Ether Catalysis tar EnhancedUltraviolet Detection in Liquid Chromatography. Anal. Chem., 47: 1797-1801, 1975.

16. Edidin, M. Rotational and Translational Diffusion in Membranes. Ann.Rev. Biaphys. Bioeng., 3: 199-201 , 1974.

17. Gammal, E. B. , Carroll, K. K., and Plunkett, E. R. Effects of Dietary Faton Mammary Carcmnogenesis by 7,12-Dimethylbenz(a)anthracene inRats. Cancer Res., 27: 1737-1742, 1967.

18. Gerschenson, L. E., Mead, J. G., Harary, I., and Haggerty, D. F. Studieson the Effects of Essential Fatty Acids on Growth Rate, Fatty AcidComposition, Oxidative Phosphorylatian, and Respiratory Control ofHeLa Cells in Culture. Biachim. Biophys. Acta, 131: 42-49, 1967.

19. Gitler, C. Plasticity of Biological Membranes. Ann. Rev. Biophys.Bioeng., 1: 51-102. 1972.

20. Hallowes, R. C., Rudland, P. 5., Hawkins, R. A., Lewis, D. J., Bennett,D., and Durbin, H. Comparison of the Effects of Hormones on DNASynthesis in Cell Cultures of Nonneoplastic and Neaplastic MammaryEpithelium from Rats. Cancer Res., 37: 2492-2504, 1977.

21. Hamosh, M., Clary, T. R. , Chernick, S. S., and Scow, R. 0. LipaproteinLipase Activity at Adipose and Mammary Tissue and Plasma Triglyceridein Pregnant and Lactation Rats. Biachim. Biophys. Acta, 201: 473â€”482,1970.

22. Hems, G. Epidemialogical Characteristics of Breast Cancer in Middleand Late Age. Brit. J. Cancer, 24: 226-234, 1970.

23. Holman, R. T. Essential Fatty Acid Deficiency, In: R. Holman (ed),Progress in the Chemistry at Fats and Other Lipids, Vol. 9, p. 279.Oxford, England: Pergamon Press, 1968.

24. Holman, R. T. Biological Activities of and Requirements for Polyunsaturated Acids. In: R. Holman (ed), Progress in the Chemistry of Fats andOther Lipids, Vol. 9, p. 611. Oxford, England: Pergaman Press, 1970.

25. Hopkins, G. J. , and West, C. E. Possible Roles at Dietary Fats inCarcinagenesis. Life Sci., 19: 1103-1116, 1976.

26. Hashino, K. Morphagenesis and Growth Potentiality at Mammary Glandsin Mice. Transplantability and Growth Potentiality at Mammary Tissue ofVirgin Mice. J. NatI. Cancer Inst., 29: 835-851 , 1962.

27. Howard, B. V., and Howard, W. J. Lipid Metabolism in Cultured Cells.Advan. Lipid Res. 120: 51-95, 1974.

28. Kaduce, T. L., Awad, A. B., Fontenelle, L. J., and Spector, A. A. Effect atFatty Acid Saturation an @-AminoisabutyricAcid Transport in EhrlichAscites Cells. J. Biol. Chem., 252: 6624-6630, 1977.

29. Malan, L. B. . Janss, D., Hellman, E. H., and Ben, T. L. Development of aSystem far the Isolation and Growth of Rat Mammary Epithelial Cells inCulture. Endocrinology, in press, 1978.

30. Mertmn,J., Shenton, B., and Field, E. Unsaturated Fatty Acids in MultipleSclerosis. Brit. Med. J., 2: 777-778, 1973.

31. Patton, S., and Jensen, R. Lipid Metabolism and Membrane Function atthe Mammary Gland. In: R. Halman (ed), Progress in the Chemistry at




Fatty Acid Effects on Mammary Epithelium

38. Stenman, S. , and Vaheri, A. Distribution of a Major Connective TissueProtein, Fibronectin, in Normal Human Tissues. J. Exptl. Med., 147:1054-1064, 1978.

39. Takaata, T., and Katsuta, H. Lang Term Cultivation of Mammalian CellStrains in Protein- and Lipid-Free Chemically Defined Synthetic Media.Exptl Cell Res., 67: 295-304, 1971.

40. Van Deenen, L. L. M. Permeability and Topography at Membranes.Chem. Phys. Lipids, 8: 366-373, 1972.

41. Wynder, E. L. Current Concepts of the Aetialagy at Breast Cancer. In:Forrest A. , and Kunkler P. (eds.), Prognostic Factors in Breast Cancer,Proceedings of the First Tenavus Symposium, pp. 32-49, Edinburgh:Livingstane, 1968.

42. Wynder, E. L. Identification at Women at High Risk from Breast Cancer.Cancer,24: 1235â€”1240,1969.

43. Yaoita, H., Faidart,J.M., and Katz, 5. I. Localization of the CallagenausComponent in 5km Basement Membrane. J. Invest. Derm., 70: 191-193,1978.

44. Yavin, E., Yavin, Z., and Menkes, J. H. Effect at Essential Fatty Acids anProliferation of Two Neural Tumor Lines. Neurobiology, 5: 214-220,1975.

Fats and Other Lipids, Vol. 2, pp. 137-163. Oxford, England: PergamonPress,1973.

32. Qasba, P. K., and Chakrabartty, P. K. Purification and Properties of TwoFarms at Rat a-Lactalbumin. J. Bial. Chem., 253: 1167-1173, 1978.

33. Qasba, P. K. , and Gullina, P. M. a-Lactalbumin Content of Rat MammaryCarcinomas and the Effects of Pituitary Stimulation. Cancer Res., 37:3792-3795, 1977.

34. Raa, G. A., and Abraham, 5. Enhanced Growth Rate at TransplantedMammary Adenocarcinoma Induced in C3H Mice by Dietary Linoleate. J.NatI. Cancer Inst., 56: 431-432, 1976.

35. Rothblat, G. H., Arbogast, L. V., Ouellette, L., and Howard, B. V.Preparation at Delipidized Serum Protein far Use in Cell Culture Systems. In Vitro, 12: 554â€”557,1976.

36. Rudland, P. 5., Hallowes, R. C., Durbin, H., and Lewis, 0. MitogenicActivity at Pituitary Hormones on Cell Cultures at Normal and Carcinogen-induced Tumor Epithelium tram Rat Mammary Glands. J. Cell Biol.,73: 561-577, 1977.

37. Spector, A., and Sabartf, J. M. Utilization at Free Fatty Acids Camplexedto Human Plasma Lipoprateins by Mammalian Cell Suspensions. J . LipidRes., 12: 545-552, 1971.

FEBRUARY1979 433



t@ 1'@ f

0'.

.4a

pI'@@ j0'@

0


M. S. Wichaeta!.

/â€˜ â€˜@ . â€¢.: @a@@ . @i .,,@ @,:%. I â€¢@@ #e@-d@ Jr'

. 1â€”@;%,@@@ ,@â€˜:â€¢@

, . ,...,â€œ..%f â€˜

-.. . . I,, @4 â€˜ @.@@@ #:@@

. .â€˜ @.@

-â€˜ ; -. ,â€˜. ,â€˜ @: â€˜@@ .@ â€˜@

@ â€” ,@ â€”,â€˜ â€” 0 @â€˜

4 !Z@r@# â€˜@. . 4 .â€” . , ., I@ â€˜

i','', . -@ dli@@ i''@ -,-.@ 4 ,@.:; â€˜;@â€˜â€”â€˜.@â€˜@

i;;:@ rel â€¢

â€¢r@ .%:â€˜@@1 ,.

I@

â€.̃ . . .@@ ,

; : .@@ â€¢@@

@., .. .â€˜.;.@, ..â€œ@,

F ,@

Fig. 1. Fluorescent antibody stain against basement membrane type IV collagen (see â€œMaterialsand Methodsâ€•).Antibody against antiovalbumin gave nofluorescence (not shown). Duct. x 50.

Fig. 2. Periodic acid-Schiff stain of section of organoids from normal rat isolated on Ficoll gradient (see â€˜Materialsand Methodsâ€•). BM, basementmembrane. x 500.

Fig. 3. Electron micrograph of normal organoid isolated on Ficoll gradient. E, epithelial cell; M, myoepithelial cell; D, desmosome. Glutaraldehyde fixed(see â€œMaterialsand Methodsâ€•).x 6000.

Fig. 4. Autaradiagraph of normal primary culture (see â€œMaterialsand Methodsâ€•), labeled from 24 to 46 hr with [3H]thymidine, fixed with methanol at 46 hrin culture. A, alveoli; D, duct; B, basement membrane remnant. Unstained, x 130.

â€”




5@

p.

.â€˜@.

a

\ a 1Fig. 5. Section of DMBA-induced mammary tumor isolated on Ficoll gradient. H & E, x 500.Fig. 6. Electron micrograph of DMBA-induced mammary tumor. M, myoepithelial cell; E, epithelial cell; BM, basement membrane. Glutaraldehyde fixed,

x 3550.

Fig. 7. Autoradiograph of DMBA-induced mammary tumor culture, labeled as in Fig. 4 from 24 to 26 hr in culture. Unstained, x 220.

FEBRUARY 1979 435

.:....:::4@? @L

.



1979;39:426-435. Cancer Res Max S. Wicha, Lance A. Liotta and William R. Kidwell Neoplastic Rat Mammary Epithelial CellsEffects of Free Fatty Acids on the Growth of Normal and

Updated version

http://cancerres.aacrjournals.org/content/39/2_Part_1/426

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/39/2_Part_1/426To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



effects of free fatty acids on the growth of normal and ...of unsaturated free fatty acids and...

Documents