[CANCER RESEARCH 48, 3693-3697, July 1, 1988]

Interaction of Phenol Red with Estrogenic and Antiestrogenic Action onGrowth of Human Breast Cancer Cells ZR-75-1 and T-47-DJ. F. Glover, J. T. Irwin, and Philippa D. Darbre1

Laboratory of Cellular Endocrinology, Imperial Cancer Research Fund, P. O. Box 123, Lincoln's Inn Fields, London WC2A ÃŒPX,England

ABSTRACT

Studies reported here confirm that the pH indicator, phenol red, actsas a weak estrogen and reexamine the significance of estrogenic andantiestrogenic effects on growth of the human breast cancer cell linesZR-75-1 and T-47-D in the absence of phenol red. Removal of phenolred reduces but does not immediately eliminate cell growth in the absenceof estradici. Basal cell growth can be reduced for T-47-D cells andeliminated for ZR-75-1 cells by prior growth in the absence of steroidand phenol red for 3 weeks, demonstrating that estrogens can have longlasting effects on cells in culture (termed "steroid memory") and that

there exist both cells which are responsive (T-47-D) and dependent (ZR-75-1) on i-stradini for growth. Antiestrogen action in these cell lines isaffected by at least four parameters: (a) presence of phenol red; (/>)timein culture; (c) cell density; (</) steroid memory effects. At high celldensity, antiestrogens suppress phenol red-stimulated activity but havelittle effect in the absence of phenol red. However, at low cell density inthe absence of phenol red, tamoxifen has a biphasic action: initial weakstimulation, later inhibition. rraïu-Hydroxytarnoxifen does not stimulatebut inhibition increases with time in culture. Following deprivation for 3weeks of phenol red and steroid, antiestrogen action on ZR-75-1 cells atlow density became much more inhibitory. Such responses to antiestrogens are discussed in relation to possible autocrine/paracrine growthregulation of the cells. Clinical relevance is suggested.

INTRODUCTION

It is generally agreed that steroid hormones regulate growthof both normal mammary cells (1,2) and a variety of breastcancer cell lines (3-7) in culture, although undoubtedly othercomponents can interact (8-10). Most attention has been paidto estrogen stimulation of the MCF-7 cell line (6) where differences are evident between sublines (11, 12) but it is generallyagreed that these cells are estrogen-stimulated in vivo (13).However, proliferation of ZR-75-1 (3) and T-47-D (4) cells isalso regulated by steroids. Recently, it has been shown that thepH indicator, phenol red, which is present in all culture media,can act as a weak estrogen, and that at the concentration foundin tissue culture media can cause stimulation of both cellproliferation and progesterone receptor levels in estrogen-responsive MCF-7 cells (14). These observations have causedmuch concern as to the significance of previous studies in tissueculture utilizing estrogen-responsive cell lines and in particularof conclusions about basal cell growth without added estrogens.

Antiestrogens are compounds that inhibit the effects of estrogens although they can have effects in their own right (10, 15)and are clinically important in endocrine therapy of breastcancer (16). Tamoxifen has both agonist and antagonist effectson breast cancer cell growth in vitro when studies are carriedout in the presence of phenol red (17, 18). However, it nowappears that such compounds act by antagonizing phenol red-stimulated proliferation in MCF-7 cells and have no effect inthe absence of phenol red (14, 19). To date, the only inhibitoryeffects of antiestrogens themselves on MCF-7 cell proliferation

Received 11/30/87; revised 3/21/88; accepted 3/31/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed.

in phenol red-free medium has been when growth is stimulatedby insulin or epidermal growth factor (16).

Studies reported here confirm that phenol red acts as a weakestrogen and reexamine the significance of estrogenic and antiestrogenic effects on growth of human breast cancer cell linesZR-75-1 and T-47-D in the absence of phenol red.

MATERIALS AND METHODS

Culture of Stock Cells. The ZR-75-1 human breast cancer cells werekindly provided by Dr. M. Lippman (National Cancer Institute, Be-thesda, MD) and were of about the same passage generation as inprevious experiments (3, 17). The T-47-D human breast cancer cellswere kindly provided by the originators of the cell line to our institute(20). Stock ZR-75-1 and T-47-D cells were grown routinely as mono-layer cultures in DMEM2 supplemented with 5% PCS (Gibco-Biocult,Glasgow, Scotland) and 10 " M estradici in a humidified atmosphereof 10% carbon dioxide in air at 37°C.Cells were subcultured at weekly

intervals by suspension with trypsin (see below).The SI 15 +A mouse mammary tumor cells were an androgen/

glueoco rticoid responsive cell line (21). These cells were grown asmonolayer cultures in DMEM containing 2% PCS, 30 mM HEPESbuffer (Sigma Chemical Co., Poole, Dorset, England) and 3.5 x 10~"

M testosterone but otherwise as for the human cells above.Growth Curves in Monolayer Culture. Cells from stock plates were

washed in phosphate-buffered saline to remove all phenol red, suspended by treatment in a 0.06% trypsin/0.02% EDTA, pH 7.3, solutionlacking phenol red, and then counted on a hemocytometer. Phenol red-free media, either RPM1 1640 (special order from Gibco-Biocult) orDMEM (Sigma Chemical Co., Poole, Dorset, England) were used forall experiments. DCFCS was used for all studies, unless use of DC-treated calf serum is specified. DC treatment of serum has been described previously (3). Cells were added to the overall required volumeof phenol red-free medium containing either 5% DCFCS (ZR-75-1 andT-47-D cells) or 2% DCFCS/30 mM HEPES buffer (SI 15 +A cells) inthe absence of steroid to achieve the appropriate seeding density. Cellswere then plated onto 50-mm diameter plastic tissue culture dishes(Nunc, Roskilde, Denmark) in 5ml aliquots for 24 h. The medium wasthen changed to contain the required concentration of phenol red,DCFCS, steroid, or antiestrogen. Stock 1% phenol red (BDH Chemicals, Ltd., Poole, England) solution was diluted as appropriate. Testosterone and estradiol (Steraloids, Ltd., Croydon, England), tamoxifenand transhydroxytamoxifen (ICI Chemical Co., Macclesfield, England)were added as stock solutions in ethanol and diluted x 10,000 in theculture medium.

Growth Curves in Suspension Culture. Cells were grown as for mono-layer cultures but in 5-cm diameter plastic bacteriological dishes (Ster-ilin, Teddington, England).

Growth of Stock Cells without Phenol Red or Steroid. Growth ofstock cells in the absence of phenol red or steroid prior to assessinggrowth was achieved by growing cells in phenol red-free RPMI 1640with 5% DCFCS for the required length of time. The cells weresubcultured at weekly intervals as for normal stock cells.

Cell Counts. Cells in monolayer cultures in 5-cm diameter tissueculture dishes were washed in situ with phosphate-buffered saline andthen lysed in 2 ml 0.01 M HEPES buffer containing 1.5 mM MgCl2plus 4 drops of Zaponin (Coulter Electronics, Ltd., Harpenden, Eng-

1The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium;DC, dextran charcoal; PCS, fetal calf serum; HEPES, 4-<2-hydroxyethyl)-l-piperazineethanesulfonic acid.

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EFFECTS OF PHENOL RED ON BREAST CANCER CELL GROWTH

land) for 5 min. The nuclei released were counted in Isoton (CoulterElectronics, Ltd.) in triplicate on a Model ZB1 Coulter Counter. Thecell counts per dish were calculated as the mean of triplicate counts;each time point of a growth curve was the mean of triplicate dishes.

Cells in suspension culture were harvested in phosphate-bufferedsaline, spun down on a bench centrifuge, and washed twice. Cells werelysed and counted as for monolayers except that lysis took about l hwith gentle rocking of the cells.

RESULTS

Effects of Phenol Red on Steroid Regulation of Cell Growthin Culture. Growth of ZR-75-1 cells in phenol red-free DMEM(Fig. IA) (repeated four times) and phenol red-free RPMI1640medium (Fig. IB) (repeated three times) has been assessed withand without IO'* M estradici and the effects of adding back

phenol red at the normal levels present in these media (IS mg/liter DMEM-5 mg/liter RPMI 1640) have been studied. Phenolred did not affect growth in the presence of estradiol (Fig. IA,Day 7 P = 0.02; all other Ps > 0.10). In the absence of estradiol,removal of phenol red lowered base-line cell growth more inDMEM than in RPMI 1640 (but in all cases Ps of <0.001 wereobtained). This difference between the media was solely relatedto phenol red concentration because when the phenol red concentration in RPMI 1640 was trebled, it then gave similarresults to that seen in DMEM (data repeated three times butnot shown). Cell growth has been studied in many differentbatches of DCFCS and DC-stripped calf serum over a 6-monthperiod and cell growth in the absence of estradiol was alwaysreduced on removal of phenol red but on no occasion wasgrowth ever reduced to zero. Analogous experiments have beencarried out twice on growth of ZR-75-1 cells in suspensionculture with similar results (data not shown).

Similar conclusions were drawn for growth of T-47-D cellsin monolayer culture (Fig. 2). Phenol red had no effect in thepresence of estradiol (Ps > 0.10). In the absence of estradiol,phenol red had a dose-dependent effect on cell growth (Fig. 2)(all Ps < 0.001) but growth was never reduced to zero. Interestingly, the effects of removal of phenol red were also greater atlower cell densities (Fig. 2R). Data in Fig. 2,1 were reproducibleover three separate experiments.

SI 15 +A mouse mammary tumor cells are responsive toandrogen and glucocorticoid (21): phenol red had no effect ongrowth of these cells (data not shown) (all Ps > 0.01).

ZR-75-1 CELLS ZR-75-1 CELLSB RPMI 1640 (5mg/l »)

ISO 5

DAYS IN CULTURE

Fig. 1. Effects of phenol red (4>)on growth of ZR-75-1 cells in monolayerculture. Cells were grown in 5% DCFCS with or without 10"' M estradiol (£)in

either DMEM with or without 15 mg/liter phenol red (A) or RPMI 1640 mediumwith or without S mg/liter phenol red ill). Bars, SE of triplicate dishes. No barsare shown if variation was too low for visual display.

M T-47-D CELLSA RPMI 1640 <5mg/l f)

,«»«'••

T-47-D CELLSBRPMI1640 (1Smg/l »)

,f

0 5 10 15 0 5 10 15

DAYS IN CULTURE

Fig. 2. Effects of phenol red (<<>)on growth of T-47-D cells in monolayerculture at different plating densities. Cells were grown in 5% DCFCS with orwithout 10 * M estradiol (/.) in RPMI 1640 medium with or without phenol red

at 5 (A) or 15 (B) mg/liter. Bars, SE of triplicate dishes. No bars are shown ifvariation was too low for visual display.

Q

5 ««-I

A ZR-75-1 CELLSA -f-E Sweeks T-47-D CELLS

-Õ-E 3weeks

-0*E,

DAYS IN CULTURE

Fig. 3. Loss of memory effects. Growth of ZR-75-1 (A) and T-47-D (B) cellsin the absence of phenol red (0) and estradiol (/;') is lowered considerably when

the cells are grown without these compounds for 3 weeks before growth is assessed.Cells were grown first for 3 weeks as stock cultures in phenol red-free RPMI1640 medium with 5% DCFCS but no steroid, and then growth was assessed inthe same medium either with (A) or without (A) 10 * M estradiol. Bars, SE of

triplicate dishes. No bars are shown if variation was too low for visual display.

Memory Effects. The possibility was considered that the basalgrowth seen in the absence of both phenol red and estradiol(Figs. 1 and 2) was due to a "memory effect" from previous

growth in stock cultures with phenol red and estradiol. Growthof ZR-75-1 and T-47-D cells immediately after removingphenol red and estradiol (Figs. 1 and 2) has been comparedwith their behavior after 3 weeks' deprivation (Fig. 3). Growthof ZR-75-1 cells for 3 weeks without phenol red or estradiolresulted in loss of all basal cell growth (Fig. 3/1) (repeated sixtimes). Growth of T-47-D cells for 3 weeks without phenol redor estradiol resulted in reduced basal cell growth (Fig. 3B)(repeated four times) but even after 10 weeks (data not shown)it was not lost completely as for the ZR-75-1 cells. Platingefficiencies and cell viability were unaffected by these treatments.

Effects of Phenol Red and Cell Density on Ant¡estrogenAction.Effects of the two antiestrogens tamoxifen and fra/u-hydroxy-tamoxifen on growth of ZR-75-1 (Fig. 4) and T-47-D (Fig. 5)cells in monolayer culture have been studied in the presenceand absence of phenol red. All cell culture was carried out using1% serum since higher serum levels can mask antiestrogeneffects (16). In addition, studies were performed to determine

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EFFECTS OF PHENOL RED ON BREAST CANCER CELL GROWTH

5EUa.Õ2_ju io5-o

ZR-75-1 CELLS

LOW PLATING DENSITY

A RPMI1640 (15mg/l 0)

io6-

ZR-75-1CELLSp

HIGH PLATING DENSITY

RPMI1640 (15mg/l0)m1

Fig. 4. Effects of phenol red on antiestrogen action on the growth of ZR-75-1 cells in monolayer culture at either low (A) or high (B) cell plating density.Cells were grown in RPMI 1640 medium in \% DCFCS with or without 15 mg/liter phenol red (<ì>),IO"6M tamoxifen, 10~8M fra/ií-hydroxytamoxifen as follows:Expiating density of cells; D. —phenolred-tamoxifen-fra/ii-hydroxytamoxifen; •-t-phenol red-tamoxifen-frans-hydroxytamoxifen; 0. —phenolred+tamoxifen; Q,+phenol red+tamoxifen; D. -phenol red+frani-hydroxytamoxifen; <g,+ phenolred + fran.v-hydn >\ytamox ifon.flurv. SE of triplicate dishes. No bars are shown ifvariation was too low for visual display.

to7106-3ER

DISHi8

»..IO4-T-47-D

CELLS107-LOW

PLATINGDENSITYARPM1 1640 (15mg/l0)I

CJTio8-T\io5-

ä io4-T-47-D

CELLS

HIGHPLATING-DENSITYB

RPMI1640 (15mg/l0):|

Fig. 5. Effects of phenol red (<t>)on antiestrogen action on the growth of T-47-D cells in monolayer culture at either low (A) or high (/() cell plating density.(Growth conditions and symbols are identical to those given for Fig. 4).

differences in antiestrogen action at low and high cell densities.For ZR-75-1 cells at high cell density, both tamoxifen and

frans-hydroxytamoxifen inhibited cell growth in the presence(all Ps < 0.001) but not in the absence of phenol red (Ps forfrans-hydroxytamoxifen all > 0.10; Ps showed significant stimulation for tamoxifen < 0.001) (Fig. 4B). At low cell density(Fig. 4A) in the absence of phenol red there was a biphasicresponse to tamoxifen: after 7 days, tamoxifen caused weakstimulation of cell growth (P < 0.001) but after 12 days cellnumbers declined even below control levels without phenol red(P < 0.001). This latter effect was reproducible in three separateexperiments. frans-Hydroxytamoxifen caused also inhibitoryeffects below control levels, irrespective of the presence orabsence of phenol red (all Ps < 0.001).

For the T-47-D cells, inhibitory effects of both tamoxifenand frans-hydroxytamoxifen were found after 14 days at lowand high cell density and irrespective of the presence of phenol

red (all Ps < 0.001) (Fig. 5). Interestingly, at low density in theabsence of phenol red, the same biphasic response to tamoxifenwas found as for ZR-75-1 cells: a weak stimulation after 7 daysbut inhibition after 14 days (Fig. 5A). This biphasic responsewas reproduced in three separate experiments.

Effects of Memory on Antiestrogen Action. To test the effectof memory on antiestrogen response, ZR-75-1 cells were grown

for 3 weeks without estradici or phenol red prior to antiestrogenaddition (Fig. 6). Cells thus treated showed only inhibitoryeffects with antiestrogens at low cell density (all Ps < 0.001)and cell numbers fell even below plating density (Fig. 6/4). Thiscontrasted with the effects seen with cells tested immediatelyafter estradiol/phenol red withdrawal (Fig. 4). However, at highcell density, antiestrogens inhibited cells only in the presenceof phenol red (Ps in presence of phenol red all < 0.001; Ps inabsence of phenol red all > 0.05) and never below platingdensity (Fig. 6B).

DISCUSSION

This report has reexamined the significance of estrogenic andantiestrogenic action on growth of human breast cancer celllines ZR-75-1 and T-47-D in the absence of phenol red. Theresults support published data for MCF-7 (14, 19, 22) and ratpituitary (23) cells showing that phenol red can act as a weakestrogen and that its removal lowers cell growth in the absenceof estradiol. No evidence was found that phenol red could affectgrowth in the presence of estradiol. It is obvious that from nowon, any studies of estrogen effects on cells in culture must becarried out in medium free of phenol red. If such medium isunobtainable, then at least RPMI 1640 should be used inpreference to DMEM since the lower levels of phenol red hadnegligible effects on these cells.

The question of whether steroids affect the proliferation ofbreast tumor cells directly on their own (dependent) (9, 24) orindirectly via other agents (responsive) (12) is centered aroundthe ability of cells to grow in tissue culture without addedhormones and the effects of phenol red are of importance inthis context. Neither our data presented here (Figs. 1 and 2)nor published data for MCF-7 cells (19) suggest that phenol

iCO

offLUa.in

ZR-75-1 CELLS-0-E 3weeks

LOW PLATING DENSITY

RPM11640 (15mg/l 0) B

ZR-75-1 CELLS-0-E 3weeks

HIGH PLATING DENSITYRPMI1640 (15mg/l fi)

Fig. 6. Effects of steroid memory on antiestrogen action on growth of ZR-7S-1 cells in monolayer culture at low (.-() and high (B) cell density. Cells were grownfirst for 3 weeks as stock cultures in phenol red (./>) free RPMI 1640 mediumwith 5% DCFCS but no steroid and then antiestrogen action on cell growth wasassessed (growth conditions and symbols exactly as for Fig. 4). /•.'.estradiol.

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EFFECTS OF PHENOL RED ON BREAST CANCER CELL GROWTH

red alone can account for all the cell growth found in theabsence of added estradiol. However, we now present data toshow that this basal cell growth can be attributed, totally in thecase of ZR-75-1 cells, to steroid memory effects. Growth ofZR-75-1 cells for 2 weeks or more in the absence of phenol redand estradiol resulted in loss of all cell growth in the absenceof added steroid. The cells remained 100% viable but did notgrow at all unless estradiol was added back. Since the onlydifference between these static cells (Fig. .1-1)and stock cells(Fig. 1) was removal of phenol red and estradiol, and sinceserum at f"? remained present at all times, the effects on

residual growth must be related to steroid memory and not toany other growth factors in serum or medium. We thus concludethat ZR-75-1 cells are completely dependent on estradiol forgrowth but that in agreement with previous suggestions, estrogens can have long lasting effects on cells in culture (25) maybethrough long half-lives of regulatory factors. The same was nottrue for the T-47-D cells studied here nor for the MCF-7 cells(19) where limited cell growth remained even after 10 weeks ofculture in the absence of phenol red and estradiol. Thus, whilethese latter two cell lines are much more responsive to estradiolin the absence of phenol red, they are not totally dependent onestradiol for growth. The ability to compare steroid-responsiveand -dependent cells should aid us in our search to understandthe mechanism of steroid regulation.

The mechanism of agonist/antagonist activity of antiestro-gens in vitro remains a subject of much debate and the role ofphenol red in this context is of vital importance. Recently, ithas been suggested that when phenol red is removed fromculture media, antiestrogens are ineffective (14). Our data agreewith these conclusions but only when considering cells at highdensity at early 1-week time points. However, we now suggestthat antiestrogen action is affected not only by the presence ofphenol red but also by three other parameters: (a) time inculture; (¿>)cell density; (c) steroid memory effects. As forMCF-7 cells (14), antiestrogen effects on ZR-75-1 cells at highcell density in the presence of phenol red can be accounted forby the suppression of phenol red-stimulated activity and noinhibitory effects were seen in the absence of phenol red. However, the story is different at low cell density. Under theseconditions in the absence of phenol red, tamoxifen showed abiphasic action: initial weak stimulation but later inhibitoryeffects. irans-Hydroxytamoxifen did not have the stimulatoryeffects but inhibitory action was much greater as time in cultureincreased. Interestingly, these effects at low cell density occurred with both ZR-75-1 and T-47-D cells. These cell densityeffects could be due to differential antiestrogen action on exponentially growing and plateau phase cells (4, 26). However,they could also be explained by antiestrogen antagonism ofgrowth stimulating factors (16). At low cell density, the localautocrine growth factor milieu will be lower resulting instronger inhibitory effects of antiestrogens. Published data forMCF-7 cells refer to antiestrogen effects at fairly high celldensities (14) but it may be that since MCF-7 cells producerelatively more growth factors than do ZR-75-1 or T-47-D cells(27) that the high levels of autocrine stimulation from thesecells render antiestrogens less effective. It is interesting to notealso that in the absence of any steroid memory effects, antiestrogens become much more inhibitory to ZR-75-1 cells, actuallycausing cell death.

Finally, these data carry clinical implications: (a) the effectsof cell density on antiestrogen action in culture suggest thatthese agents may be more effective in vivo on tumors of lowcellularity and that areas within a tumor packed densely with

malignant cells may be difficult to attack with antiestrogensalone; (b) the biphasic effects of tamoxifen at IO"6 M with time

in culture may explain the response sometimes seen in vivo ofan early rise in tumor size before regression. This phenomenonof tamoxifen tumor flare is seen in both MCF-7 cells grown innude mice (28) and in the clinic (29-31). It has been shownpreviously that tamoxifen is weakly stimulatory at low concentration and inhibitory at high concentration (17, 18) but this isthe first report of biphasic effects at a single high concentration.Previous data suggested that tumor flare could result from lowblood levels of tamoxifen at the start of therapy while remissionresults from a higher concentration of tamoxifen achieved afterprolonged treatment (18). The data presented here imply thatthe phenomenon of tumor flare could also occur with time at asingle high tamoxifen concentration. However, whether theinitial stimulation is due to the fact that tamoxifen effects haveto accumulate before they become inhibitory or to an interactionwith "estrogen memory" (in culture, tamoxifen is more inhibi

tory to ZR-75-1 cells in the absence of steroid memory) remainsspeculatory.

ACKNOWLEDGMENTS

We thank Dr. R. J. B. King (Imperial Cancer Research Fund) forhis advice and for his critical reading of this manuscript. We are verygrateful to Dr. R. Hallowes (Imperial Cancer Research Fund) for hishelp in obtaining a good and reliable source of phenol red-free medium.

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