Unsaturated Fatty Acid Requirements for Growth and Survival of a Rat Mammary Tumor Cell … · Unsaturated Fatty Acid Requirements for Growth and Survival of a Rat Mammary Tumor Cell

[CANCER RESEARCH 38, 4091-4100, November 1978]

Unsaturated Fatty Acid Requirements for Growth and Survival of a RatMammary Tumor Cell Line1

William R. Kidwell, Marie E. Monaco, Max S. Wicha, and Gilbert S. Smith

National Cancer Institute, Bethesda, Maryland 20014

Abstract

A cell line, the growth and survival of which is markedlyaffected by linoleic acid, has been established from acarcinogen-induced rat mammary tumor. The cells havebeen continuously passaged in 5% rat serum plus 10%fetal calf serum-supplemented medium. The rat serumcomponent was found to be indispensable, for when itwas omitted the growth rate rapidly declined and the cellsdied by 5 to 7 days. Removal of the rat serum from thegrowth medium also resulted in a dramatic loss of Oil RedO-positive droplets in the cells, suggesting that the lipidcomponent of rat serum might be a major growth-promot

ing principle in rat serum. This is likely since the total lipidfraction, but not the delipidized protein fraction, couldlargely supplant requirement of the cells for rat serum.Pure linoleic acid was found to be effective in maintainingthe cell growth in delipidized serum or in whole fetal calfserum-supplemented medium. Fatty acid analysis revealed a 19-fold higher amount of linoleic acid in ratserum than in fetal calf serum.

Introduction

Although many properties of cells such as morphologyand agglutination (14), adenyl and guanyl cyclase activities(10, 24), differentiation (34), membrane fluidity and cellattachment (31), amino acid transport (18), and cloningefficiency (12) are reportedly affected in specific instancesby essential fatty acids, the usual finding has been that cellsin culture show little impairment of division in the absenceof these fatty acids (for reviews see Refs. 1 and 16). Theessential fatty acids do, however, have pronounced effectson growth and development in experimental animals (13)probably at least in part via conversion to prostaglandins(32) in addition to their structural role in cellular membranes.

There are indications that mammary cells may be especially sensitive to lipids. Increased incidence of humanmammary carcinoma correlates positively with increasedconsumption of animal fat (8), and a number of studieshave shown that high unsaturated fatty acid diets increasethe incidence of mammary adenocarcinomas in rats following DMBA2 administration (2-4). Also, the growth of trans-

plantable mouse mammary adenocarcinomas is dramatically affected by dietary lipids (29). These effects have beenpostulated to be due to an elevation of serum prolactin by

1 Presented at the John E. Fogarty International Center Conference on

Hormones and Cancer, March 29 to 31, 1978, Bethesda, Md.2 The abbreviation used is: DMBA, 7,12-dimethylbenz(a)anthracene.

the dietary lipids (5) or to a suppression of immune surveillance because of a fatty acid-mediated inhibition of lymphocyte proliferation (22).

In the present report we show that a cell line (WRK-1)established from a DMBA-induced rat mammary tumor isdirectly responsive to unsaturated fatty acids, linoleic acidin particular. These results are not surprising in view of thefact that mammary epithelial cells are embedded in anadipose tissue matrix, with linoleic acid being one of themost abundant fatty acids present in the rat gland. Thesignificance of the structural relationship of the glandularcomponent and adipose tissue is highlighted by the observation that successful mammary tissue transplants requiremammary fat tissue as well as the epithelial component (7,15).

Materials and Methods

Cell Establishment. A small mammary tumor induced ina Sprague-Dawley rat given 25 mg of DMBA at 50 days ofage was excised and minced in Eagle's minimal essential

medium containing 5% fetal calf serum and 2% collagenase(type II; Worthington Biochemical Corp., Freehold, N. J.).After incubation for 1 hr at 37Â°,the digest was filtered

through cheesecloth, and the filtrate was pelleted. Thepellet was washed with culture medium, and the cells wereresuspended and plated in Eagle's minimal essential me

dium containing 5% rat serum and 10% fetal calf serumplus penicillin, streptomycin, and glutamine (19). After analiquot of the suspension was incubated in a Retri dish for5 hr at 37Â°,the unattached cells and medium were trans

ferred to new Retri dishes and incubated for an additional72 hr. Islands of epithelial-appearing cells were localized onthe dishes. Several of these were harvested by trypsiniza-tion with stainless steel rings. A successful culture of oneof these was established; the cells were cloned twice andthen maintained in culture for about 1 year with biweeklyculture splitting. From the time of initiation of the culture,growth was maintained in Eagle's medium supplemented

with both rat and fetal calf sera.Serum Preparation. Rat serum was prepared from ma

ture, nonpregnant, female rats of the same strain, withmost preparations consisting of sera pooled from 10 to 50rats. The serum, collected from the abdominal aorta, washeated at 56Â°for 30 min, sterilized by filtration, and storedfrozen at -20Â°.A single lot of fetal calf serum (Grand Island

Biological Co., Grand Island, N. Y.) was utilized for all ofthe experiments reported here. Delipidization of sera wasperformed with ethanoliacetone (30). For testing of serumlipid fractions for growth promotion, the total serum lipids[the material soluble in acetone:ethanol (1:1)] was taken to

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W. R. Kidwell et al.

dryness by rotary evaporation and resuspended in a volumeof bovine serum albumin solution (1.4 g lipid-free albuminin phosphate-buffered saline) equivalent to the originalserum volume. (Phosphate-buffered saline was prepared bydissolving 8.0 g KCI, 1.15 g Na.HPO,, and 0.2 g KH.PO, in1 liter of water.) The lipid suspension was then dialyzed for24 hr against 400 volumes of phosphate-buffered saline (3changes in the cold). The same bovine serum albuminutilized to resuspend the lipid fraction was similarly dialyzedand utilized as a control. The protein fraction recoveredfrom the acetone:ethanol precipitate was resuspended inphosphate-buffered saline and dialyzed as described above.Protein analysis (25) indicated that 66 to 72% of the serumprotein was recovered. The total serum lipid as quantitatedby H.jSO, charring (20) was recovered in 88% yield.

The lipoprotein fraction and alipoprotein serum wereprepared by adjusting the serum density to 1.25 g/ml withsolid Kl followed by flotation centrifugation (26).

Fatty Acid Analysis. Serum lipids extracted withCHCI:i:methanol (3:1) were fractionated by thin-layer chro-matography (21), and the appropriate fractions were saponified. After acidification the liberated fatty acids were extracted with hexane. The hexane extracts (about 2 ml) weremixed with 1 ml of dimethylformamide and 20 /j.\ N,N-diisopropylethylamine. The hexane layer was removed witha stream of N,, and N. was bubbled through the dimethylformamide solution in the tube to remove residual amountsof dissolved hexane. Then 2.3 mg of a-p-dibromoaceto-phenone were added, and the mixture was heated for 15min at 65Â°.The bromphenacylated fatty acids producedwere chromatographed by high-pressure liquid chromatog-raphy on a Waters /Â¿C,*column with a gradient of 40 to100% acetonitrile-water (17). Fatty acid derivatives weredetected by their absorption at 254 nm. Mass was estimatedby integration of peak areas with an HP 3380A integrator.The mass is expressed in terms of crystalline bromphena-cyllinoleate standard. When 0.01 or 25 /J.Qof labeled palmitic acid were utilized, recoveries of label in the respectivepeak from the column were 88 and 97%, respectively. Fordetermination of the type of fatty acid synthesized in thecells, 10 /J.Qof pure fatty acid carriers were added at thetime of lipid saponification. After derivatization the sampleswere chromatographed. the peaks from the column werecollected, the solvent was evaporated off, and the radioactivity was quantitated.

Ultrastructure Studies. Cells grown in the presence offetal calf and rat serum were fixed in situ as monolayersafter reaching confluency. Fixation was accomplished with1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH7.2) for 30 min. After being rinsed in buffer, the monolayerswere postfixed in Dalton's chrome-osmium (6) for 1.5 hr,

dehydrated in ethanol. and infiltrated with and embeddedin a thin layer of Epon:Araldite. Following polymerization at56Â°.the epoxy plastic layer with the intact monolayer was

stripped off the surface of the Petri dish. Appropriate areasfor ultrastructural evaluation were located by light microscopy. These areas were cut out of the monolayer andaffixed to epoxy cylinders, and ultrathin sections were cuton an 1KB Ultratome. Ultrathin sections were picked up onFormvar-coated copper grids, stained with uranyl acetateand lead citrate, and examined in a Siemens Elmiscop 102

electron microscope at initial magnifications ranging from3000 to 4000 diameters. The identical procedure was followed in studies where the effects of hormones on theultrastructural appearance of cells grown for 24 hr inserum-free medium with or without added hormones.

ResultsCell Characterization. The cell line that has been isolated

has many features characteristic of secretory cells of rodentmammary glands. Electron micrographie analysis (Fig. 1)reveals intermediate tight junctions and gap junctions suchas those reported for mouse mammary glands (27, 28).Under the influence of insulin, hydrocortisone, and prolac-tin (NIH B1), the cells take on an ultrastructural appearancesimilar to mammary cells in early secretory phase (compareFigs. 2 and 3). There is a marked development of the roughendoplasmic reticulum with characteristic distended cister-nae, the cytoplasm becoming filled with lipid droplets.Hormone treatment results also in changes in surfaceactivity of cells as depicted by the increase in pinocytoticvesicles, surface blebbing, and microvillar development(Figs. 2 and 3). Compared to cells with the hormonesomitted, there is a development of an organized system ofcytoplasmic organelles which, except for the lipid dropletsand vesicles, appears to be due to a reorganization ofpreexisting cytoplasmic components. Several features thatwould make the mammary glandular epithelial origin morecertain were not found. These include the presence ofdetectable amounts of Â«-lactalbumin or casein by sensitiveradioimmune assays or desmosomes.

In 2 respects the cells were similar to secretory cells ofthe lactating gland. They accumulated massive amounts oflipid droplets as detected by Oil Red O stain and containeda hormonally responsive fatty acid synthetase. This is evident from the lipid droplet accumulation in the hormonallytreated cells (Fig. 2). These droplets are most probably theresult of de novo lipid synthesis since ['"C]acetate incorporation into fatty acids was stimulated 3- to 4-fold with insulin

Table 1Hormonal stimulation of acetate incorporation into fatty acids

Cells were grown to confluency in 60 HIM plastic dishes in fetalcalf and rat serum-supplemented medium and then changed tomedium without serum. After 6 hr the medium was again replaced(-serum). Prolactin (NIH B1, 1 /xg/ml) and/or insulin (porcine,from Lilly. 5 x 10 7 M) were added, and 30 hr later the cells werepulsed for 2 hr with [14C]acetate (1 Â¿iCi/ml; 2 x 10 5 M). The cells

were recovered from the plates by trypsinization, and lipids wereextracted, saponified, derivatized, and separated by high-pressureliquid chromatography. Controls received no hormones.

ControlFatty

acidLaurieLinolenicMyristicPalmitoleic-arachidonicLinoleicPalmiticOleicStearicTotal

dpmdpm15061.08026403.6962,1601,4648.820%1.70.0612.23.00.041.924.516.6Insulindpm72092.3525101314,9706.4623,06628,102%2.60.038.41.80.0453.323.010.9IllOUIlli

UHI UlW~lactindpm1,212185,1361,260938.55619,58410.44076,215%1.60.026.71.70.0150.625.713.7

4092 CANCER RESEARCH VOL. 38



Fatty Acid Requirements of Mammary Tumor Cells

and 9-fold with insulin plus prolactin (Table 1). [The effectof prolactin is likely due to a vasopressin contaminant (23).]Neither hormone regimen markedly altered the relativeamount of synthesis of any particular size class of fatty acidsince the percentage of distribution of radioactivity in thevarious fatty acids was essentially unchanged in thepresence or absence of hormones.

Evidence supporting the epithelial origin of the cells wasprovided by the fact that considerable cell growth occurredin medium in which L-valine was replaced by D-valine (9).After a 24-hr lag, the D-valine-supplemented cultures grewwith a doubling time only one-third greater than that ofcells plated with L-valine (Chart 1).

Although we were unable to obtain tumors by inoculationof the cells into nude mice, the cells appeared to betransformed. They piled up in culture giving clear overlapping cellular edges (Fig. 4). Additionally, as has been notedfor some DMBA-induced mammary tumors, the cells contained rat leukemia virus (about 20 RNA copies/cell) andthe associated reverse transcriptase (H. Young, NationalCancer Institute, personal communication). The cells alsohad an abnormal chromosome number as depicted in Table2, where it is seen that the modal chromosome number is80, or nearly double that of a diploid rat cell.

Rat Serum Requirement for Cell Growth and Survival. Atthe time of initiating the tumor cells in culture and continu-

Chart 1. Growth of mammary tumor cells in medium containing D- or L-valine. Cells were plated in medium with D-valine (93 mg/liter) or L-valine (46mg/liter). The rat and fetal calf sera which were at 5 and 10%concentrations,respectively, were dialyzed before use against 3 changes of Eagle's mediumwith L-valine omitted (1000 volumes/dialysis).

ously thereafter, the cells have been maintained in mediumsupplemented with 5% rat serum and 10% fetal calf serum.Under these conditions the cells are filled with refractiledroplets that stain positively with Oil Red O indicating alipophilic nature. Within 24 hr of plating of cells in mediumwith the rat serum component omitted, the lipid dropletsdisappear (cf. Figs. 5 and 6). The cells assume a moreflattened appearance, and the number of cells per dish isreduced. After 48 hr without rat serum, extensive pyknosisand vacuolization of the cytoplasm are evident (Fig. 7).After 5 days almost no viable cells are present in the dishes.

The rapid loss of lipid droplets when rat serum wasomitted suggested that the rat serum component necessaryfor growth and survival might be a lipid. Consequently, thetotal lipid fraction of rat serum and the protein fractions aswell were prepared and tested for their effects on tumor cellgrowth in cultures supplemented with 10% fetal calf serum.As shown in Chart 2, an amount of the total lipid fractionequivalent to that present in 5% rat serum markedly stimulated cell proliferation compared to control cultures platedin 10% fetal calf serum only. When both the rat serumprotein and lipid fractions were added back, the growthwas no better than with the lipid fraction alone. The totalrat serum protein fraction gave a modest stimulation of cellgrowth, but it was considerably less active than the lipidfraction (Chart 2).

With suitable adjustment of the density of the rat serumwith Kl and centrifugation. it was possible to remove thevery-low-density lipoproteins. the low-density lipoproteins,and the high-density lipoproteins from serum. This alipo-protein serum contains the free fatty acids that are boundto albumin. Each of the 2 fractions, the combined lipoproteins and the alipoprotein serum fraction, were tested forgrowth promotion as described in the last experiment. After4 days of growth, the control cultures had increased in cellnumber by about 50% (Table 3). The lipoprotein fraction,which contained about 85% of the total serum fatty acids(mono-, di-, and triglycÃ©rides and phospholipids), stimulated cell growth about 3-fold over the same time period,while the alipoprotein serum fraction enhanced cell growthrates by 8-fold. Both fractions together in amount equivalent to that present in 5% rat serum gave only a 9-foldincrement in growth rate, i.e., the removal of the lipoproteinfraction had little effect on the growth-promoting effect ofrat serum. Oil Red O staining also showed that the accumulation of lipid droplets in the cells was almost totallyassociated with the alipoprotein serum fraction.

A comparison of the free fatty acid profiles of rat and fetalcalf serum was made to see whether a difference in fattyacid types or amounts might explain the rat serum require-

Table 2Chromosome number of WRK-1 cells

Subconfluent cultures were treated for 5 hr with Colcemid (0.5 /Â¿g/ml). Mitotic cells dislodgedfrom the plates by shaking were pelleted, swollen in 1% sodium citrate, and fixed withmethanohacetic acid (3:1). The cells were placed on microscope slides, flamed, and stained with0.1% mÃ©thylÃ¨neblue.

No.ofchromosomesNo.

ofcells751765771781791806811822830843850861871

NOVEMBER 1978 4093




23456DAYS

Chart 2. Effect of rat serum lipid and protein fractions on cell growth. Thecells were plated in 10% fetal calf serum with additions as indicated. O.whole rat serum; â€¢¿�,rat serum lipid fraction; O. rat serum protein fraction;D, rat serum protein and lipid fractions; A, control buffer only.

Table 3Effect of lipoproteins and lipoprotein-free rat serum on cell growth

Cells were plated at 5 x 104/dish in 10% fetal calf serum-supplemented medium to which the lipoprotein fraction of ratserum or the alipoprotein serum was added. Controls receivedphosphate-buffered saline against which the 2 serum fractions hadbeen dialyzed.

Addition Cells/dish

NoneLipoproteinAlipoprotein serumLipoprotein + alipoprotein-

free serum75,957

Â±4,201"

126,735 Â±4.368437,922 Â±2,516468,472 Â±8,078

" Mean Â±S.D.

ment. As shown in Table 4, all the major fatty acids were 1-to 3-fold higher in rat serum than in fetal calf serum.However, the greatest difference was in linoleic acid whichwas 19-fold higher in rat serum.

For assessment of the effect of linoleic acid on cellgrowth and survival, cells were plated in delipidized serumand various concentrations of linoleic acid were added. Asa control either no fatty acid or palmitic acid was added.The results are presented in Table 5. Both the saturated andunsaturated fatty acids were stimulatory and had concentration optima at about 0.5 Â¿Â¿g/ml.However, linoleic acidwas 5 times as effective as palmitic acid in stimulatinggrowth. Comparisons were also made of the relative effectsof several other fatty acids which were in higher concentration in rat serum. As indicated in Table 6, linoleic acid wasmore than 2 times as effective as were oleic, arachidonic,or linolenic acids in promoting growth.

Calculations of the amount of linoleic acid that would becontributed to culture medium supplemented with 10% fetalcalf or 5% rat serum (according to the data of Table 4)indicate that the fetal calf serum supplement would contribute 0.03 /*g/ml to the medium, while rat serum wouldsupply 10 times as much. The results of addition of purelinoleic acid on the growth of the cells in medium containing 10% whole fetal calf serum (Chart 3) indicate thatcellular growth rate is markedly improved by this fatty acid.There is approximately a 4-fold increase in cells per dishwith the addition of linoleic acid. However, when whole rat

Table 4Free fatty acid concentrations of rat and fetal calf serum

ÃÂ¿g/mlserum

FattyacidLaurieLinolenicMyristicArachidonicLinoleicPalmiticOleicStearicRat4.00.77.23.65.616.412.09.0Fetalcalf2.50.35.21.90.37.25.64.9Rat/fetalcalf1.62.31.41.918.62.32.81.8

Table 5Effect of linoleic and palmitic acids on cell growth

Cells were plated in medium containing 5% delipidized rat and10% delipidized fetal calf serum. Two days later the cells wereharvested by trypsinization, and cell counts were performed. Palmitic acid at 5 Â¿Â¿g/mlwas toxic to the cells. Data are expressed interms of the percentage of change in cell number over the initialcell inoculation.

% increase in cell no.

tion (/Â¿g/ml)0

0.050.505.00Linoleic

acid54

52203180Palmitic

acid51

5783

Table 6Effect of unsaturated fatty acids on cell growth

The experiment was performed as described in Table 5 with thefatty acids present at 1 /Â¿g/ml.

Fatty acid% increase in cell no.

above control

Linoleic acidLinolenic acidArachidonic acidOleic acid

375126141154

40

ToX

I52O

30

20

10

+ 01 UlUJÂ«t DCKÂ»r

o yoz "-z_J _1

+

Chart 3. Effect of pure linoleic acid and/or rat serum (flSi on cell growth.FCS, fetal calf serum.

serum is added, linoleic acid has no further effect on cellgrowth, indicating that most of the growth-promoting activity of rat serum is due to the linoleic acid component.

Linoleic acid was not totally capable of replacing rat





serum insofar as growth was concerned (Charts 2 and 3).One experiment performed with serum from hypophysec-tomized rats as compared to normal rats suggested thatsome hypophyseal hormone might also have an effect oncell growth. Medium supplemented with serum from intactrats gave about 30% more growth than with serum fromhypophysectomized rats.

Conclusion

All cells of the mammary gland are present in a matrixthat is very rich in lipids. These lipids appear to be importantfor glandular function and probably for growth as well,based on the results of transplant experiments (7, 15).Alterations of dietary lipid content, known to correlatepositively with the incidence of mammary cancer, may havein addition to proposed secondary roles (5, 22) a directeffect on tumor cell growth. This seems logical in view ofthe fact that the types and amounts of fatty acids of thegland are, to a first approximation, a reflection of the fattyacids of the diet. However, there are indications fromnutritional studies that essential fatty acids are considerablymore important than other classes of fatty acids in thephysiology of the normal mammary gland (13). In this studyone such essential fatty acid, linoleic acid, was found to bea major factor of rat serum for enhancing the amount ofgrowth and survival of a cell line established from a carcinogen-induced rat mammary tumor.

The WRK-1 cell line has been tested for a variety ofmarkers for identification purposes. No a-lactalbumin orcasein has been detected, nor are clearly defined desmo-somes present. However, the lack of desmosomes does notpreclude the cells as being derived from epithelial compartment since these cellular structures are known to disappearduring certain functional stages of the mammary gland (27,28). Additionally, there is present in normal glandular epithelium and in the tumors a second cell type, the myoepi-thelial cell, which does not have desmosomes. Both theepithelial and myoepithelial cells possess tight and gapjunctions as do the WRK-1 cells (27, 28). The cells grow in D-valine, a condition selective for epithelial cells (9), and theyhave an epithelial morphology. The cells respond ultra-structurally to hormones as do the mammary epithelial cellsin expiant cultures (33). The presumptive response to pro-lactin is probably due not to the prolactin molecule but tovasopressin, a contaminant in the prolactin preparation(23). Whether this indicates a physiological role of vasopressin for mammary cells remains to be seen.

Like mammary cells of lactation (11), there is a largestimulation of acetate incorporation into fatty acids byinsulin plus the prolactin preparation. The electron micro-graphic analysis presents strong evidence that hormonaltreatment produces an actual increase in fatty acid synthesis rather than affecting only [14C]acetate transport, since

the treated cells but not the controls are filled with lipiddroplets. These droplets cannot be a consequence of uptake of lipids from the growth medium because the experiments are performed in serum-free medium.

While there is some uncertainty as to the origin of ourcell line, the probability is that it represents a tumor cell typethat became established in culture because its high linoleic

acid requirement was met by the rat serum supplement inthe culture medium. High concentrations of essential fattyacids may be a general requirement of mammary cells of alltypes, since we have recently demonstrated that primarycultures of mammary gland alveolar cells and those fromDMBA-induced rat tumors show a marked stimulation ofgrowth upon the addition of linoleic acid to the growthmedium. Additionally, we have demonstrated that the mammary gland selectively alters the ratio of linoleic acid relativeto palmitic acid when the gland is induced to proliferate byperphenazine administration.3 These results point to a need

for a great effort to understand growth and differentiationas consequences of the coordinated and integrated response of various cell types of the mammary gland tohormones.

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

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13. Holman, R. T. Bilogical Activities of and Requirements for Polyunsatu-rated Acids. Progr. Chem. Fats Other Lipids, 9: 611. 1970.

14. Horwitz, A. F., Hatten, M. E., and Burger, M. M. Membrane Fatty AcidReplacements and Their Effect on Growth and Lectin Induced Aggluti-nability. Proc. Nati. Acad. Sei. U. S., 71: 3115-3119, 1974.

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18. Kaduce.T. L., Awad, A. B.. Fontanelle, L. J., and Spector, A. J. Effect ofFatty Acid Saturation on a-Aminoisobutyric Acid Transport in EhrlichAscites Cells. J. Biol. Chem..252: 6624-6630, 1977.

19. Â«Â¡dwell,W. R. Fidelity of DNA Replication in Isolated L-Cell Nuclei.Biochim. Biophys. Acta, 269: 51-61, 1972.

20. Kritchevsky, D.. Davidson, L., Kim, H., and Malhorta, S. QuantitÃ¤ten ofSerum Lipids by a Simple TLC-charring Method. Clin. Chim. Acta, 46:63-68, 1973.

21. Mangold, H. K. In: R. J. Jones (ed.). Evolution of the AtheroscleroticPlaque, p. 85-108. Chicago: University of Chicago Press, 1963.

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

3 M. S. Wicha, L. A. Liotta, and W. R. Kidwell. Effects of Free Fatty Acidson the Growth of Normal and Neoplastic Rat Mammary Epithelial Cells,submitted to Cancer Research.

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â€¢¿�'mm.

Fig. 1. Junctional complexes of the eelIs. Upper arrow, gap junction; center arrow, area of intermediate and tight (unctions, x 35.000. Inset, tight junctionbetween apposing cells, x 140,000

NOVEMBER 1978 4097




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Fig. 2. Hormonal effects on cell ultrastructure. Cells treated for 24 hr with insulin (5 x 10 7M), prolactin (B1,1 n9/m|). and hydrocortisone (5 x 10"" M) in

medium with serum omitted. R, distended rough endoplasmic reticulum; P, pinocytotic vesicles; L, lipid droplets; M, mitochondria, x 14,000.





i-â€¢¿�â€¢$Â£*â€¢>

..â€¢^"T.^

Fig. 3. Ultrastructure of cells in the absence of hormones. Note the absence of lipid droplets, the relative scarcity of mitochondria, and the lack ofdistension of the cisternae of the rough endoplasmi reticulum.

NOVEMBER 1978 4099




Fig. 4. Abnormal piling up of the cells in culture. Note the prominent overlapping of cellular edges.

Fig. 5. Accumulation of lipid droplets in cells grown in the presence of both rat and fetal calf serum. The lipid droplets appear as discrete foci throughoutthe cytoplasm. Oil Red 0.

Fig. 6 Absence of Oil Red O-staining material in cells grown for 24 hr with the rat serum component omitted.

Fig. 7. Extensive pyknosis and vacuolization of cells 48 h r after plating in medium with rat serum omitted.




1978;38:4091-4100. Cancer Res William R. Kidwell, Marie E. Monaco, Max S. Wicha, et al. of a Rat Mammary Tumor Cell LineUnsaturated Fatty Acid Requirements for Growth and Survival

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Unsaturated Fatty Acid Requirements for Growth and Survival of a Rat Mammary Tumor Cell … · Unsaturated Fatty Acid Requirements for Growth and Survival of a Rat Mammary Tumor Cell