Vol. 2, 457-464, September 1991 Cell Growth & Differentiation 457

Autocrine Production of Insulin-like Growth Factor IIUsing an Inducible Expression System Results inReduced Estrogen Sensitivity of MCF-7Human Breast Cancer Cells

Roger J. Daly,’ William H. Harris, Dennis Y. Wang,and Philippa D. Darbre

Department of Pharmacology, New York University Medical Center,New York, New York 10016 [R. J. D.}; Department of ClinicalEndocrinology, Imperial Cancer Research Fund Laboratories, Guy’sHospital, London, United Kingdom [W. H. H., D. V. WI; and

Department of Biochemistry and Physiology, University of Reading,Whiteknights, Reading, United Kingdom [P. D. D.J

Abstrad

An inducible expression vedor, utilizing the metalresponse elements from the human metallothionein IIApromoter and encoding human prepro-insulin-likegrowth fador II (IGF-II), was transfected into MCF-7McG cells, an MCF-7 subline which exhibits anestrogen dependent phenotype in vitro and does notexpress detedable levels of IGF-Il mRNA. Two stablytransfeded clones, designated Ml5 and Ml7, whichexpressed IGF-II mRNA in a Zn2� regulated manner,were isolated. Clone M15 did not secrete detedablelevels of IGF-II adivity, as determined byradioimmunoassay of conditioned medium, but cloneMI7 secreted high levels of IGF-ll adivity in a Zn2’inducible fashion. Clone Ml5 and control clonestransfeded with the seledion plasmid pSV2 neo andthe control plasmid pSP65 continued to display anestrogen dependent phenotype in vitro. However,under both anchorage dependent and anchorageindependent growth conditions, clone MI7 cellsexhibited an estrogen responsive, rather thandependent, phenotype. Moreover, when grown in thepresence of inducing concentrations of Zn2�, M17 cellswere either virtually (for anchorage dependent growth)or completely (anchorage independent growth)unresponsive to estradiol. Both the basal growth rate inthe absence of metal ions and the Zn2� inducedincreases in cell proliferation could be inhibited by themonoclonal antibody a-lR3, which blocks the bindingsite of the IGF-I receptor. The antiestrogens tamoxifenand 4-hydroxytamoxifen were found to enhance thegrowth stimulation resulting from Zn2� induced IGF-IIprodudion in MI7 cells. These data confirm that IGF-IIcan fundion in an autocrine manner to stimulate breastcancer growth and suggest that IGF-lI may play a rolein loss of steroid sensitivity in breast cancer.

Introdudion

Recently, much attention has focused on the sensitivityof human breast cancer cells to polypeptide growthfactors. It has been proposed that such factors, function-ing in either an autocrine or paracrine mode, contributeboth to estrogen regulation of steroid responsive cellsand to tumor progression (1). Initial evidence for thismodel came from studies in which MCF-7 cells trans-fected with the v-Ha-ras oncogene were found to exhibitestrogen independent growth in vivo and to secreteincreased levels of TGF2-a-Iike, ICF-l-Iike, and TCF-�9activity (2, 3). However, overexpression of TCF-a inMCF-7 cells was found not to result in steroid independ-ence (4). More recently, changes in both TCF-�31 andTGF-fl2 gene expression have been correlated with lossof steroid sensitivity of human breast cancer cells in vitro(5, 6).

The insulin family of growth factors has been shownto be strongly mitogenic for both estrogen responsive (3,5, 7-1 7) and hormone independent (1 5, 16, 18) breastcancer cell lines in vitro. Such cell lines have been shownto express insulin receptors and both type I and type IIICF receptors (17). The insulin and type I ICF receptorsare both receptor tyrosine kinases capable of transmittinga mitogenic signal, although the type I receptor appearsto be more potent in this respect (19, 20). However, thetype II IGF receptor lacks intrinsic tyrosine kinase activityand has been shown to be identical to the mannose 6-phosphate receptor; the physiological relevance of thisreceptor has yet to be defined (21, 22). Although exper-iments using the monoclonal antibody a-lR3, whichblocks ligand binding to the type I IGF receptor (23),have demonstrated that the mitogenic response to bothICF-l and ICF-ll in breast cancer cells is largely mediatedvia the type I receptor (15-17), the response to insulin ismore complex. Recent work has shown that the prolif-erative response to insulin in MCF-7 cells is not blockedby a-IR3 (1 7), which indicates that insulin receptor sig-naling and/or insulin receptor-type I receptor “cross-talk”may be involved (22). Since insulin is largely producedby the beta cells of the pancreas (24), ICF-I and ICF-lIare more likely to play a role in autocrine/paracrineregulation of breast cancer growth. It has been reportedthat human breast cancer cells secrete an IGF-l likeactivity (10, 25), but the exact nature of this species isnot clear. RNase protection analyses failed to detectauthentic ICF-l mRNA in 1 1 breast cancer cell lines, and

Received 3/18/91.1 R. J. 0. is the recipient of an imperial Cancer Research Fund TravelFellowship. To whom requests for reprints should be addressed, atDepartment of Pharmacology, New York University Medical Center, 550First Avenue, New York, NY 10016.

2 The abbreviations used are: TGF, transforming growth factor; IGF,

insulin-like growth factor; hMT, human metallothionein; CAT, chloram-phenicol acetyltransferase; FCS, fetal calf serum; DC-, dextran-charcoaltreated; cDNA, complementary DNA; bp, base pair(s); kb, kilobase(s);DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate bufferedsaline.

BLE

L� MAE/

//

I

/

TATA

SPIGFII �

“-.-- V

I

TpA

458 Role of IGF-ll in Loss of Steroid Sensitivity in Breast Cancer

GRE hMTIIA Promoter

/

ThMTIIA

�� �

SV4O t-IVS

�/�//� //

� � /..‘ ,/�/ ‘�,y’ ‘

r�Li

PH

pEMTIGF2

Amp’

-1 pSP65

I

Fig. 1. Construction of the expression vector p�MTl2. The map ofthe hMTIIA promoter is adapted from Ref. 30. GRE, glucocorticoid response element;BLE. basal level element; MRE, metal response element; SP, signal peptide. A, 5, P, and H, cleavage sites for the restriction enzymes Aval, Salt, Pstl, andHindlll, respectively. V. the site of processing of prepro-IGF-ll. t-IVS, small t antigen intron; TpA, large T antigen polyadenylation signal; Amp’, ampiciilinresistance gene.

in breast tissue specimens, IGF-I mRNA was only de-tected in stromal tissue and not in normal or malignantepithelial cells (26). More recently, several laboratorieshave reported the expression of IGF-il mRNA in a limitednumber of breast cancer cell lines (6, 12, 16, 27). Inparticular, T-47-D and certain MCF-7 sublines expressthis mRNA in an estrogen regulated manner (6, 12, 16)and have also been shown to secrete an IGF-Il-like activ-ity (16).

The mitogenic activity of IGF-li on human breast can-cer cells and the potential role of this polypeptide as anautocrine modulator of breast cancer growth led us toinvestigate the effect of IGF-Il expression on the steroidsensitivity of MCF-7 cells. The MCF-7 McG subline ex-

hibits a strict dependence on estradiol for growth (28)and also expresses undetectable levels of endogenousIGF-il mRNA (29). Consequently, this cell line providesan ideal model system for such a study. We report herethat expression of IGF-li in MCF-7 McG cells decreasesthe estrogen sensitivity of the cells in vitro, under bothanchorage dependent and independent growthconditions.

Results

Construction of the Inducible Expression Vectorpi�MTl2. The hMTliA promoter contains a glucocorticoidresponse element upstream of alternating basal level

enhancer elements and metal response elements (30).Since we wished to construct an expression vector whichwas inducible by metal ions and not by glucocorticoids,a 252-bp fragment of the hMTIIA promoter, situated

downstream (3’) of the glucocorticoid response element,

was utilized (Fig. 1). Upon transient transfection into ZR-

75-1, T-47-D, and MCF-7 McG breast cancer cells, thispromoter fragment was found to confer metal ion in-ducibility to a CAT reporter gene. Dose dependent in-ductions of CAT activity occurred in the concentrationrange 5 x 108 to 5 X 10� M Cd2�’ or 1 x iO� to 1 xiO-� M Zn2�, with 30-40-fold inductions being observedat the upper limit of each dose response curve (data notshown). The expression vector p�MTl2 was constructedby cloning a cDNA encoding human prepro-1GF-II (31)and SV4O processing/polyadenylation signals 3’ to thepromoter (Fig. 1).

Expression and Metal Ion Regulation of IGF-lI mRNAin Transfected Cell Lines. MCF-7 McG cells were cotrans-fected with either p�MTl2 and the selection plasmidpSV2 neo (Ml series of transfections) or pSP65 and pSV2neo (MN series), and stable G418 resistant clones wereisolated. Cytoplasmic RNA was isolated from stableclones induced with metal ions and subjected to RNaseprotection analysis using an antisense riboprobe corre-sponding to the IGF-ll cDNA utilized in p�MTI2 (6).

3684 -�-

Cell Growth & Differentiation 459

A

P 1234 5678

B

p 0 0.5 1 3 6 10 24 48h 2++Zn

IGFII -a-’ � J I I

II”!

-� S’s.,...

Fig. 2. A. identification of G418 resistant clones expressing IGF-ll mRNA. Total cytopiasmic RNA was isolated from cells induced with 5 x 10’ xi znCl2(Lane 1) or S x 10-6 M CdCl2 (Lanes 2-7) and subjected to RNase protection analysis using antisense riboprobes corresponding to IGF-ll cDNA or 36B4cDNA. Ten � of RNA were used in each protection. Lane P. 878-bp antisense riboprobe for IGF-ll, which includes 63 bp of polylinker sequence. Thepositions of the expected protected fragments for IGF-ll (815 bp) and 36B4 (220 bp) are indicated. RNA was isolated from the following clones: Lane 1,Ml5; Lane 2, MN2; Lane 3, Ml7; Lane 4, Ml8; Lane 5, Ml9; Lane 6, MIll; Lane 7, Ml12; Lane 8, tRNA control. B, time course of induction of IGF-ll mRNAin clone Ml5 cells. Cells were incubated in RPMI 1640 medium containing 5% DC-FCS for 24 h. The medium was then changed to RPMI 1640 mediumsupplemented with 5% Dc-FCS and containing 5 x 10� si zncl2. Cytoplasmic RNA was isolated from the cells after the incubation periods indicated

and subjected to RNase protection analysis using IGF-ll or 36B4 antisense riboprobes. Ten �g of RNA were used in each protection.

Screening results for a series of MN and Ml transfectantsare shown in Fig. 2A. In three clones, designated Ml5,Ml7, and Mu 1, the expected protected fragment of 815bp was observed. Clones Ml5 and Mi7, which expressedhigh levels of IGF-Il mRNA, were chosen for furtheranalysis. RNase protection studies confirmed that MN1and MN2 control clones did not express detectable levelsof iGF-ll mRNA (Fig. 2A and data not shown). Since 40bp of hMTllA 5’ noncoding sequences are present at the5’ end of RNA transcripts produced from p�MTI2, the252-bp hMTilA promoter fragment used in the construc-tion of this vector, which also contains these sequences,could be used as a probe for Northern analysis. In both

MIS and Ml7 cells, an RNA species of the expected sizefor a pL\MTI2 derived transcript (approximately 1.7 kb)was detected using this probe (data not shown). More-over, this mRNA was expressed in a metal ion induciblefashion. The kinetics of induction of IGF-ll mRNA bymetal ions was investigated in MIS cells by RNase pro-tection analysis. Upon administration of S x 1O� M Zn2�,a detectable increase in 1GF-Il mRNA could be detectedafter 1 h of induction, and maximal expression was

attained after 6-10 h (Fig. 2B).Radioimmunoassay of IGF-II Activity in Conditioned

Medium. Conditioned medium from the MCF-7 McGstock cell line, control clones MN1 and MN2, and IGF-lltransfected clones MIS and M17 was analyzed for se-creted IGF-li activity by radioimmunoassay. Since breastcancer cell lines have been shown to secrete high levels

of IGF binding proteins (32), which could interfere withthis assay, the conditioned medium was first acidified todissociate the binding proteins from IGFs and then sub-jected to chromatography on Sep-pak C3 cartridges toseparate IGF-ll activity from binding proteins. Radio-immunoassay of conditioned medium from stock MCF-7 McG cells and MNI, MN2, and MIS transfected cellsrevealed a low level of IGF-Ii immunoactivity which wasdecreased by treatment with metal ions (Fig. 3). Thisactivity probably represents residual binding proteins (33)and has been noted by other workers (16). However, a6-fold increase in the amount of IGF-il activity present inMl7 conditioned medium was observed upon treatmentwith S x iO-� M Zn2�. Thus, both IGF-lI mRNA andsecretion oflGF-Il activity were found to be Zn2� inducedin this transfected cell line.

Response of Transfected Cells to Estradiol under An-chorage Dependent and Independent Growth Condi-tions. When maintained in 5% DC-FCS and in the ab-sence of phenol red, MCF-7 McG stock cells exhibit asteroid dependent phenotype in monolayer culture (28).MN2 control cells were also found to display this phe-notype, such that cells grown in the presence of 108 M

estradiol continued to proliferate exponentially, but noincrease in cell number above the initial plating densitywas observed for cells grown in the absence of steroid(Fig. 4A). Prolonged exposure to S x 10� M Zn2� wasfound to be weakly mitogenic, with a small increase incell number occurring after 14 days of culture under

�00

80

60

40

20

0

.c

c,’l

U)

8,0

0

U-

C.,

C

1

A io�.

�. io6.

� io�

io�

iog.c0.u0

� io5

--A

Days In culturs

1_0 20

Days In sulturs

Fig. 4. Response of MN2 (control, A) and MI7 (IGF-lI transfected, B)

cells to estradiol under anchorage dependent growth conditions. Cellswere plated in phenol red-free RPMI 1640 medium containing 5% DC-FCS and then, 24 h later, the medium was changed to phenol red-freeRPMI 1640-5% DC-FCS alone (0), or containing 10#{176}M estradiol (0), 5 xiO-� M ZnCl2 (#{149}),or 10#{176}M estradiol and 5 x 10’ M ZnCl2 (A). Points,mean cell counts for triplicate 3.5-cm dishes; bars, SE, which is not shownif too small for visual display.

460 Role of IGF-lI in Loss of Steroid Sensitivity in Breast Cancer

cr�z �z1Q� �� B

Fig. 3. Effect of metal ions on IGF-II activity in conditioned medium.Conditioned medium was isolated from cells grown in the presence (+)or absence (-) of 5 x 10� si ZnCI2, acidified, and then chromatographedon Sep-pak C,� columns. IGF-il activity was then determined by radio-immunoassay and corrected for cell number.

these conditions, but the addition of Zn2� was not mito-genic in the presence of estradiol. MNI control cells, andMIS cells, which express IGF-ll mRNA but do not secretedetectable ICF-ll activity, exhibited similar growth prop-erties (data not shown). However, M17 cells, which se-Crete ICF-lI in a metal ion regulated fashion, were foundto exhibit a steroid responsive, rather than steroid de-pendent, phenotype. When grown in the absence ofestradiol, these cells continued to proliferate at a slowrate but grew much faster upon the addition of estradiolto the culture medium (Fig. 4B). Moreover, the additionof 5 x 1o-� M Zn2�’ was strongly mitogenic for these cells,almost increasing the growth rate to that of estrogenstimulated cells. In contrast to MN2 cells, treatment withZn2�’ was also mitogenic in the presence of estradiol.

In order to investigate the effect of IGF-Il productionon growth under anchorage independent conditions,cells were grown in the presence of 1% methylcellulose(34). MN2 control cells exhibited steroid dependentgrowth under these culture conditions (Fig. SA), but MI7cells were almost steroid independent, even in the ab-sence of metal ions, and were completely unresponsiveto estradiol in the presence of 5 x i0� M Zn2” (Fig. SB).

Inhibition of MI7 Cell Proliferation by the BlockingAntibody a-IR3. Studies using the monoclonal antibodycx-1R3, which blocks the binding site ofthe IGF-I receptor(23), have demonstrated that IGF-Il stimulation of breastcancer proliferation, particularly at high concentrations,appears to be mediated by this receptor (15-17). There-fore, if the decrease in estrogen sensitivity of Ml7 cells isdue to ICF-ll functioning in an autocrine loop, thenadministration of this antibody should inhibit both thebasal proliferation of M17 cells and the Zn2�’ inducedincreases in cellular proliferation rate. Fig. 6 shows thatthis was indeed the case. Addition of a-1R3 was found torestore the estrogen dependence of M17 cell proliferationin the absence of metal ions and to inhibit the increasein cellular growth rate produced by S x 10� M Zn2’�.

0 10 20

Interadion of IGFs and Tamoxifen in Regulation ofMCF-7 McG Proliferation; Sensitivity of M17 Cells toAntiestrogens. Since increased expression of ICF-II wasfound to result in reduced estrogen sensitivity of cellproliferation, it was of interest to investigate the growthresponse of MI7 cells to the antiestrogens tamoxifen and4-hydroxytamoxifen. When grown in the absence ofphenol red and in 5% DC-FCS, MCF-7 McC stock cellswere found to exhibit a weak mitogenic response totamoxifen (10’6 M) (Fig. 7). The partial agonist effect oftamoxifen and 4-hydroxytamoxifen observed underthese growth conditions is now well documented (14,35, 36). However, tamoxifen was found to synergize witheither insulin (10 zg/mI), ICF-I (100 ng/mI), or ICF-lI (100ng/mI) in stimulation of MCF-7 McC growth, such thatthe increase in cell number observed upon addition oftamoxifen in combination with insulin/ICF was signifi-cantly greater than the sum of the increases observedwhen either agent was added alone (Fig. 7). Similarly,M17 cells responded to tamoxifen (10_6 M) or 4-hydrox-ytamoxifen (10_8 M) with a small increase in growth rate,but when either was added in combination with 5 x i0�

io6.

ia6

.C0

0

SU

Fig. 5. Response of MN2 (A) and M17 (B) cells to estradiol underanchorage independent growth conditions. Cells were suspended at adensity of 6 x 10� cells/mi in phenol red-free RPMI 1640-5% DC-FCS-1% methylcellulose alone (� , 2 or supplemented with 10#{176}M estradiol(�, U) or 5 X 10�#{176}M ZnCi, (�, U), and added in 2.5-mi aliquots to 3.5-cm dishes. Cell counts were determined after 1 5 days in culture. Points,mean cell counts for triplicate dishes; bars, SE.

A B

.c0

0

.U

Fig. 6. Inhibition of M17 monolayer growth by a-IR3 blocking antibody.M17 cells were plated into 24-well plates in phenol red-free RPMI 1640medium containing 5% DC-FCS. Twenty-four h later, the medium waschanged to phenol red-free RPMI 1640-5% DC-FCS medium containingeither 5 �g/ml mouse IgG (0), 5 dig/mI a-IR3 (al, 10� M estradiol ( ), 5x iO-� M ZnCI, (D), or 5 X 10’s M ZnCl, and 5 �g/ml a-1R3 (U). The initialplating density was 3.85 ± 0.34 x 10’ cells/well, and cells were countedafter 7 days in culture. Columns, mean cell counts for quadruplicate wells;bars, SE.

Cell Growth & Differentiation 461

M Zn2�’, the two treatments appeared to interact in anadditive rather than synergistic manner to further stimu-late M17 proliferation (Fig. 8). Consequently, tamoxifenwas found to enhance both the mitogenic response ofMCF-7 McG cells to exogenous IGF-ll and that of Ml7cells to IGF-II produced in an autocrine manner.

Discussion

This paper describes, in terms of modulation of estrogensensitivity, the effects of introducing an expression vectorencoding human ICF-lI into the estrogen dependentMCF-7 McC subline. In the MCF-7 McG transfectantsMIS and MI7, and also in a parallel series of clonesisolated from the T-47-D breast cancer cell line (data notshown), the expression vector pi.�MTl2 was found toproduce ICF-ll transcripts in a metal ion regulated man-ner. The constitutive expression observed by other work-ers using hMTIIA based vectors (4) may be due to differ-ences in the cell lines used in the studies, or due to theinclusion of regulatory elements not present in the 252-bp hMTIIA promoter fragment utilized in p�MTl2. Al-though clones MIS and M17 were found to express highlevels of ICF-ll mRNA in the presence of metal ions, onlyclone Ml7 secreted detectable ICF-lI activity under theseconditions, as determined by radioimmunoassay of con-ditioned medium from the cells. At present, it is notknown whether the block in ICF-II production in MIScells is at the level of protein translation or secretion. Itshould be noted that the cDNA utilized in p�MTI2 isderived from human liver mRNA (31). Differential initia-tion of transcription can occur within the ICF-II gene,resulting in a series of mRNA species which exhibit tissueand developmental specific expression (37). The pre-dominant ICF-lI mRNA detected in human breast cancercells is 6.0 kb in length (12), whereas in adult humanliver, the major transcript is 5.3 kb in length. The effectof the different 5’ untranslated regions present in thesemRNAs on mRNA stability and/or translatability has yetto be determined.

Under both anchorage dependent and independentgrowth conditions, clone M17 cells were found to exhibit

an estrogen responsive phenotype in the absence ofmetal ions, and the addition of S x i0� M Zn2” up-regulated the growth rate so that the proliferative re-sponse to estradiol was either greatly reduced (for an-chorage dependent growth) or abolished (anchorage in-dependent growth). Although the proliferation exhibitedby M17 cells in the absence of Zn2� was inhibited by theblocking antibody a-lR3, it could be argued that thisbasal growth represents a response to serum borne IGFsrather than ICF-Il produced in an autocrine manner. Thiswould require that M17 cells possess a greatly increasedsensitivity to ICFs compared to McC stock cells andcontrol cell lines since the MCF-7 McG cell line retains adependence on estradiol for growth even in the presenceof 20% DC-FCS (28), and the basal growth rate of Ml7cells and the effect of a-1R3 were found to be similar in1 and 5% DC-FCS (data not shown). Also, although thelevel of ICF-Il activity in conditioned medium from un-induced Ml7 cells was not significantly higher than thatfound in conditioned medium from MCF-7 McG stockcells or control transfected cells, it is possible that a lowbasal production of ICF-II may be obscured by IGFbinding proteins secreted by the cells (32), or by rapidreceptor binding and internalization. However, an induc-ible expression vector was chosen for this study so thatincreasing levels of ICF-II production could be inducedwithin an individual clone of cells, thereby circumventingclonal variation problems encountered in comparing dif-ferent clones of cells. The advantage of this system isparticularly evident in the M17 growth experiments undermonolayer conditions. Here, the addition of Zn2” re-suIted in an approximately 7-fold increase in cell growthin the absence of estradiol. Two lines of evidence supportthe hypothesis that this up-regulation of cell growth isdue to stimulation by ICF-ll functioning in an autocrineloop. First, Zn2’� induced M17 cells secrete levels of IGF-II activity comparable to those shown to be mitogenicfor MCF-7 McG stock cells, and second, this increase incell growth could be inhibited by the blocking antibodya-1R3, which blocks the binding site of the ICF-I receptor

io6

.C0

�DolO

S

U

io�

Fig. 7. Interaction of lGFs and tamoxifen in regulation of MCF-7 McGgrowth. MCF-7 McG cells were plated in phenol red-free RPMI 1640medium containing 5% DC-FCS, and then, 24 h later, the medium waschanged to phenol red-free RPMI 1640-5% DC-FCS alone (0), or con-taming 10-#{176}M estradiol (0), 10#{176}M tamoxifen (�t. 10 �tg/ml insulin (�),10 �tg/ml insulin and 10’ M tamoxifen (n), 100 ng/ml IGF-l (�). 100 ng/ml IGF-I and 10_6 M tamoxifen (U), 100 ng/ml IGF-ll (0), or 100 ng/mlIGF-ll and 10’ si tamoxifen (U). The initial plating density was 0.59 ±0.04 x i0� cells/dish, and cells were counted after 7 days in culture.Columns, mean cell counts for triplicate dishes; bars, SE.

.c

0

�00

S

U

Fig. 8. Effect of antiestrogens on Ml7 cell proliferation. Ml7 cells wereplated in phenol red-free RPMI 1640 medium containing 5% DC-FCS,and, 24 h later, the medium was changed to phenol red-free RPMI 1640-5% DC-FCS alone (0) or containing 10#{176}M estradiol (a), 10_6 M tamoxifen(�), 10#{176}M 4-hydroxytamoxifen (a), 5 x 10’ M znci2 (0), 5 x 10’ xi

ZnCl, and 106 M tamoxifen (c), or 5 x 10� xi ZnCI2 and 10#{176}M 4-hydroxytamoxifen (U). The initial plating density was 0.56 ± 0.01 x 10�/dish, and cells were counted after 14 days in culture. Columns, mean cellcounts for triplicate dishes; bars, SE, which is not shown if too small forvisual display.

462 Role of IGF-ll in Loss of Steroid Sensitivity in Breast Cancer

and has been shown to inhibit IGF-il stimulated prolif-eration of breast cancer cells (16, 17).

Consequently, these findings represent the first timethat an autocrine loop involving a single polypeptidegrowth factor has been shown to up-regulate the basalgrowth rate of a steroid responsive breast cancer cell lineand thereby reduce the proliferative response of the cellsto estradiol. Interestingly, this effect was not observedwhen MCF-7 cells were programmed to overproduceTGF-a, another growth factor proposed to play an auto-crine role in regulation of breast cancer growth (4). Since

IGF-II is produced by a limited number of breast cancercell lines, it cannot function as a general mediator ofestrogen regulated growth; instead, in agreement with amodel in which the pathways for loss of estrogen sensi-tivity may not be those involved in steroid regulation ofresponsive cells (38), up-regulation of CF-li productionpresents a potential mechanism for progression to steroidinsensitivity. However, since it has been demonstratedthat MCF-7 sublines unresponsive to growth stimulationby estrogens in vitro may still exhibit an estrogen respon-sive phenotype in vivo (39), it will be necessary to deter-mine the effects of overproduction of 1GF-Il on MCF-7tumor formation in nude mice before extrapolating theseconclusions to tumor growth in vivo.

The interaction between insulin/IGFs and antiestro-gens in regulation of breast cancer growth appears to becomplex, and conflicting results have been presented forthe effect of 4-hydroxytamoxifen on insulin stimulatedMCF-7 cell proliferation. Although Vignon et a!. (18)reported that 4-hydroxytamoxifen could inhibit the mi-togenic effect of insulin on MCF-7 cells, Wakeling et a!.(14) found that this compound inhibited the growthresponse to IGF-I in MCF-7 cells but exhibited a syner-gism with insulin in stimulation of MCF-7 proliferation.In the studies presented here, tamoxifen was found tosynergize not only with insulin but also with IGF-I andCF-lI in stimulating MCF-7 McG growth, and the agonist

effect of tamoxifen or hydroxytamoxifen was also found

to enhance the mitogenic action of IGF-ll in Zn2� inducedMI7 cells. The reason for these discrepancies probablyresides in the different MCF-7 sublines used in thesestudies, and in particular, their sensitivity to certaingrowth factors. Since tamoxifen has been shown to in-crease production of growth inhibitory TGF-fl (40), andTGF-f3 can inhibit IGF-l stimulated breast cancer prolif-eration (41), differences in sensitivity to TGF-fl may beresponsible for the contrasting results obtained. MCF-7sublines have been shown to vary in terms of TCF-flreceptor number (41). A potential mechanism for theobserved synergism between tamoxifen and IGFs is pro-vided by the recent observation that estradiol up-regu-lates ICF-I receptor levels in MCF-7 cells (42); sincetamoxifen and hydroxytamoxifen possess estrogenic ac-tivity, a similar, albeit weaker, effect may also be ob-served with these compounds. It is tempting to speculatethat this synergism may act to enhance tamoxifen in-duced tumor flare (43).

Materials and Methods

Cell Lines. MCF-7 McG cells (passage no. 390) weregenerously provided by Dr. Kent Osborne (44). Stockcells were grown as monolayer cultures in DMEM (con-taming phenol red) supplemented with 5% FCS (FlowLaboratories, Irvine, Scotland), 108 M estradiol, and 10zg/ml insulin in a humidified atmosphere of 10% carbondioxide in air at 37#{176}C.Cells were subcultured at weeklyintervals by suspension with 0.06% trypsin-0.02% EDTA(pH 7.3).

Plasmid Construds. The expression plasmid p�MTI2was constructed as follows. The hMTIIA promoter con-tains a unique Aval site situated 212 bp upstream fromthe cap site. Cleavage at this site separates the metalresponse elements from the glucocorticoid response ele-ment (positioned at approximately -250 bp with respectto the cap site) (45). A 252-bp Aval-SalI fragment, con-taming hMTIIA promoter sequences spanning from -212to +40 with respect to the cap site, was cloned into the

Cell Growth & Differentiation 463

corresponding sites in the polylmnker of the plasmidpSP6S (Promega). An 847-bp Xholl-BamHl fragment de-rived from the vector pSV2 CAT (46) and containing theSV4O small t antigen intron and SV4O large T antigenpolyadenylation signal, was cloned into the HindIIl siteof the polylinker using HindIII linkers (New England Bio-labs). The expression plasmid was completed by cloningthe 81 S-bp Pstl fragment from phigf2 (31), which includesthe entire coding region for prepro-IGF-Il, into the Pstlsite of the polylinker.

Transfedion Procedure. MCF-7 McC cells were platedat a density of 7.5 x 10� cells/14-cm dish in stock me-dium. Six days later, cells were transfected for 6 h withSO �zg of pz�MTl2, S �ag pSV2 neo (44), or SO �zg pSP6S, Sfag pSV2 neo (control transfections) using the calciumphosphate coprecipitation method (47). The cells werethen washed three times with DMEM, treated with 25%glycerol in DMEM for 1 mm, and then incubated over-night in stock medium. Two days later, the medium waschanged to stock medium containing 200 zg/mI C418,and 3 days later, the concentration of C418 was in-creased to 1 mg/mI. Selection was then continued at thisconcentration of G418 until individual clones could beisolated using cloning rings. Transfected clones wereroutinely cultured in stock medium containing 500 �zg/ml G418.

RNA Extradion. Cytoplasmic RNA was prepared aspreviously described (48).

RNase Protedion Analyses. Riboprobes for detectionof IGF-Il and 36B4 mRNA and RNase protection meth-odology were as previously described (6).

Preparation of Conditioned Medium. Confluentmonolayers of cells were first incubated for 24 h inphenol red-free RPMI 1640 medium containing 1% DC-FCS, in either the presence or absence of S x 10’� M

ZnCI2. Dextran-charcoal treatment of FCS was as de-scribed previously (49). The monolayers were thenwashed in phenol red-free RPMI 1640 medium, and themedium was changed to phenol red-free RPMI 1640medium containing 10 mt�i 4-(2-hydroxyethyl)-1-pipera-zineethanesulfonic acid (pH 7.2), 5 zg/mI transferrin(Sigma), 0.75 zg/ml fibronectin (Sigma), 1 mI/liter traceelements (GIBCO), and, for Zn2� induced cells, S x i0�M ZnCI2. After 24 h, the conditioned medium was har-vested and centrifuged briefly to remove cell debris.Aprotinin (Sigma) was then added to a final concentrationof 0.23 trypsin inhibitor unit/mI (1% v/v), and the condi-tioned medium was stored at -70#{176}C.Cell numbers onthe dishes were determined using a model ZB1 CoulterCounter (Coulter Electronics) as previously described (6).

Radioimmunoassay of IGF-lI Adivity in ConditionedMedium. Each sample of conditioned medium was firstdialyzed for 24 h at 4#{176}Cagainst three changes of distilledwater to remove metal ions. In order to remove IGFbinding proteins, a modified version of a Sep-pak C18extraction protocol was used (33). Each sample was acid-ified to 4% acetic acid and then incubated at roomtemperature for 1 h. The acidified medium was thenloaded onto a Sep-pak C18 cartridge (Waters Associates!Millipore) prewashed with 4% acetic acid. After washingwith 4% acetic acid, the IGF activity was eluted with87.5% acetonitrile-12.S% 1 M HCI. The samples werethen dried on a centrifugal evaporator and resuspendedin assay buffer (0.1% bovine serum albumin-0.1% Tritonx-100 in PBS).

The ICF-ll radioimmunoassay was performed using apolyclonal rabbit anti-ICF-lI antiserum (Mediagnost, Tu-bingen, Federal Republic of Cermany), raised against apeptide consisting of amino acids 33-40 of human ICF-II (SO). Cross-reactivities against human ICF-I and humaninsulin were both less than 0.05%.

Cell Growth Experiments. Growth curves under mono-layer conditions were performed as follows. Cells weresuspended from stock plates by treatment with phenolred-free 0.06% trypsin-0.02% EDTA (pH 7.3), added toan equal volume of phenol red-free RPMI 1640 contain-ing 5% DC-FCS, and counted on a hemocytometer. Cellswere then resuspended in phenol red-free RPMI 1640medium containing 5% DC-FCS at a concentration of0.2-0.8 x i0� cells/mI and plated in 2.5-mI aliquots into3.5-cm plastic tissue culture dishes, except for experi-ments involving a-1R3, in which cells were plated in 0.5-ml aliquots into 24-well plates (Falcon). After 24 h, themedium was changed to phenol red-free RPMI 1640medium supplemented with 5% DC-FCS and containingthe appropriate concentration of steroid, growth factor,or antibody. Culture medium was changed routinelyevery 3-4 days. Cell counts were performed using amodel ZB1 Coulter Counter, as described previously (6).

Growth curves under anchorage independent condi-tions were performed in RPMI 1640 medium supple-mented with 5% DC-FCS and containing a final concen-tration of 1% (w/v) methylcellulose (34). Cell suspensionscontaining 6 x i0� cells/mI were added in 2.5-mI aliquotsto 3.5-cm plastic bacteriology dishes (Sterilin). Afterwashing the cell pellet three to four times in PBS toremove the methylcellulose, cell numbers were deter-mined using a Coulter Counter. IGF-I and IGF-ll (Boeh-ringer) were dissolved as 500x concentrates in PBS.Insulin (Sigma) was dissolved as a 1000x concentrate in6 m�i HCI. 1 7fl-Estradiol, tamoxifen, and 4-hydroxyta-moxifen were dissolved as 10,000x concentrates inethanol. In growth experiments using a-lR3, mouse IgG(Sigma) was used as a control. Both a-1R3 (OncogeneScience) and mouse lgG were dissolved in phenol red-free RPMI 1640 medium as 200x concentrates.

Acknowledgments

We thank Dr. J. Scott (Clinical Research Centre, Harrow, Middlesex,United Kingdom) for his gift of IGF-ll cDNA and Professor P. Chambon(Centre National de Ia Recherche Scientifique, Strasbourg, France) for hisgift of 36B4 cDNA. We thank Dr. Roger King (Imperial Cancer ResearchFund Breast Biology Group, University of Surrey) for continued adviceand encouragement.

References1. Lippman, M. E., Dickson, R. B., Gelmann, E. P., Rosen, N., Knabbe,C., Bates, S., Bronzert, D., Huff, K., and Kasid, A. Growth regulation ofhuman breast carcinoma occurs through regulated growth factor secre-tion. J. Cell. Biochem., 35: 1-16, 1987.

2. Kasid A., Lippman, M. E., Papageorge, A. G., Lowy, D. R., andGelmann, E. P. Transfection of v�rasH DNA into MCF-7 human breastcancer cells bypasses dependence on estrogen for tumorigenicity. Sci-ence (Washington DC), 228: 725-728, 1985.

3. Dickson, R. B., Kasid, A., Huff, K. K., Bates, S. E., Knabbe, C., Bronzert,D., Gelmann, E. P., and Lippman, M. E. Activation of growth factorsecretion in tumorigenic states of breast cancer induced by 1 7�-estradioIor v-Ha-ras oncogene. Proc. NatI. Acad. Sci. USA, 84: 837-841, 1987.

4. Clarke, R. B., Brunner, N., Katz, D., Glanz, P., Dickson, R. B., Lippman,M. E., and Kern, F. G. The effects of a constitutive expression of trans-forming growth factor a on the growth of MCF-7 human breast cancercells in vitro and in vivo. Mol. Endocrinol., 3: 372-380, 1989.

464 Role of IGF-ll in Loss of Steroid Sensitivity in Breast Cancer

5. Daly, R. J., King, R. J. B., and Darbre, P. D. Interaction of growthfactors during progression towards steroid independence in T-47-D hu-man breast cancer cells. j. Cell. Biochem., 43: 199-211, 1990.

6. Daly, R. J., and Darbre, P. D. Cellular and molecular events in loss ofestrogen sensitivity in ZR-75-1 and T-47-D human breast cancer cells.Cancer Res., 50: 5868-5875, 1990.

7. Osborne, C. K., Bolan, G., Monaco, M. E., and Lippman, M. E.Hormone responsive human breast cancer in long term tissue culture:effect of insulin. Proc. Nati. Acad. Sci. USA, 73: 4536-4540, 1976.

8. Furlanetto, R. W., and Dicarlo, J. N. Somatomedin receptors andgrowth effects in human breast cells maintained in long term tissueculture. Cancer Res., 44: 2122-2128, 1984.

9. Myal, Y., Shiu, P. C., Bhaumick, B., and Bala, M. Receptor binding andgrowth promoting activity of insulin-like growth factors in human breastcancer cells (T-47-D) in culture. Cancer Res., 44: 5486-5490, 1984.

10. Huff, K. K., Kaufman, D., Gabbay, K. H., Spencer, E. M., Lippman,M. E., and Dickson, R. B. Secretion of insulin-like growth factor I-relatedprotein by human breast cancer cells. Cancer Res., 46: 4613-4619, 1986.

1 1 . Van der burg, B., Rutteman, G. R., Blankenstein, M. A., De Laat, S.W., and Van Zoelen, E. J. J. Mitogenic stimulation of human breast cancercells in a growth factor defined medium: synergistic action of insulin andestrogen. J. Cell. Physiol., 134: 101-108, 1988.

12. Yee, D., Culien, K. I., Paik, S., Perdue, J. F., Hampton, B., Shwartz,A., Lippman, M. E., and Rosen, N. Insulin-like growth factor Ii mRNAexpression in human breast cancer. Cancer Res., 48: 6691-6696, 1988.

13. Karey, K. P., and Sirbasku, D. A. Differential responsiveness of humanbreast cancer cell lines MCF-7 and T47D to growth factors and 17�-estradiol. Cancer Res., 4B: 4083-4092, 1988.

14. Wakeling, A. E., Newboult, E., and Peters, S. W. Effects of antiestro-gens on the proliferation of MCF-7 human breast cancer cells. J. Mol.Endocrinol., 2: 225-234, 1989.

15. Arteaga, C. L., and Osborne, C. K. Growth inhibition of human breastcancer cells in vitro with an antibody against the type I somatomedinreceptor. Cancer Res., 49: 6237-6241, 1989.

16. Osborne, C. K., Coronado, E. B., Kitten, L. J., Arteaga, C. I., Fuqua,S. A. W., Ramasharma, K., Marshall, M., and Li, C. H. Insulin like growthfactor II: a potential autocrine/paracrine growth factor for human breastcancer acting via the IGF-l receptor. Mol. Endocrinol., 3: 1701-1709,1989.

17. Cullen, K. J., Yee, D., Sly, W. S., Perdue, J., Hampton, B., Lippman,M. E., and Rosen, N. Insulin like growth factor receptor expression andfunction in human breast cancer. Cancer Res., 50: 48-53, 1990.

18. Vignon, F., Bouton, M. M., and Rochefort, H. Antiestrogens inhibitthe mitogenic effect of growth factors on breast cancer cells in the totalabsence of estrogens. Biochem. Biophys. Res. Commun., 146: 1502-1508, 1987.

19. Flier, J. S., Usher, P., and Moses, A. C. Monoclonal antibody to thetype I insulin like growth factor (1GF-l) receptor blocks IGF-l receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms ofIGF-l and insulin in human skin fibroblasts. Proc. NatI. Acad. Sci. USA,83:664-668, 1986.

20. Lammers, R., Gray, A., Schlessinger, J., and Ullrich, A. Differentialsignalling potential of insulin and IGF-l receptor cytoplasmic domains.EMBOJ., 8: 1369-1375, 1989.

21. Roth, R. A. Structure ofthe receptor for insulin like growth factor II:the puzzle amplified. Science (Washington DC), 239: 1269-1271, 1988.

22. Czech, M. P. Signal transmission by the insulin like growth factors.Cell, 59: 235-238, 1989.

23. Kuil, F. C., Jacobs, S., Su, Y-F., Svoboda, M. E., van Wyk, J. J., andCuatrecasas, P. Monoclonal antibodies to receptor for insulin and soma-tomedin-C. J. Biol. Chem., 258: 6561-6566, 1983.

24. Cryer, P. E. Glucose homeostasis and hypoglycemia. In: I. 0. Wilsonand D. W. Foster (eds.), Williams Textbook of Endocrinology. Philadel-phia: W. B. Saunders Co., 1985.

25. Huff, K. K., Knabbe, C., Lindsey, R., Kaufman, D., Bronzert, D.,Lippman, M. E., and Dickson, R. B. Multihormonal regulation of insulinlike growth factor-I-related protein in MCF-7 human breast cancer cells.Mol. Endocrinol., 2: 200-208, 1988.

26. Yee, D., Paik, S., Lebovic, G. S., Marcus, R. R., Favoni, R. E., Cullen,K. J., Lippman, M. E., and Rosen, N. Analysis of insulin like growth factorI gene expression in malignancy: evidence for a paracrine role in humanbreast cancer. Mol. Endocrinol., 3: 509-517, 1989.

27. Peres, R., Betzholtz, C., Westermark, B., and Heldin, C-H. Frequentexpression of growth factors for mesenchymal cells in human mammarycarcinoma cell lines. Cancer Res., 47: 3425-3429, 1987.

28. Darbre, P. D., and Daly, R. J. Effects of oestrogen on human breastcancer cells in culture. Proc. R. Soc. Edin. B, 95: 119-132, 1989.

29. Darbre, P. D., and Daly, R. J. Transition of human breast cancer cellsfrom an oestrogen responsive to unresponsive state. J. Steroid Biochem.Mol. Biol., 37: 753-763, 1990.

30. Karin, M., Haslinger, A., Heguy, A., Dietlin, T., and Cooke, T. Metalresponsive elements act as positive modulators of human metallothioneinhA enhancer activity. MoI. Cell. Biol., 7: 606-613, 1987.

31 . Bell, G. I., Merryweather, J. P., Sanchez-Pescador, R., Stempien, M.M., Priestley, L., Scott, J., and RaIl, L. B. Sequence of a cDNA cloneencoding human prepro insulin-like growth factor II. Nature (Lond.), 310:775-777, 1984.

32. De Leon, D. D., Wilson, D. M., Bakker, B., Lamsom, G., Hintz, R. L.,and Rosenfeld, R. G. Characterization of insulin like growth factor bindingproteins from human breast cancer cells. Mol. Endocrinol., 3: 567-574,1989.

33. Hsu, C-J., and Hammond, J. M. Gonadotropins and estradiol stimu-late immunoreactive insulin like growth factor I production by porcinegranulosa cells in vitro. Endocrinology, 120: 198-207, 1987.

34. Harkonen, P. L., Laaksonen, E. I. G., Valve, E. M., Solic, N., andDarbre, P. D. Temperature sensitive mutants for steroid sensitive growthofSll5 mouse mammarytumor cells. Exp. Cell Res., 186: 288-298, 1990.

35. Berthois, Y., Katzenellenbogen, I. A., and Katzenellenbogen, B. S.Phenol red in tissue culture media is a weak estrogen: implicationsconcerning the study of estrogen responsive cells in culture. Proc. NatI.Acad. Sci. USA, 83: 2496-2500, 1986.

36. Giover, J. F., Irwin, I. T., and Darbre, P. D. Interaction of phenol redwith estrogenic and antiestrogenic action on growth of human breastcancer cells ZR-75-1 and T-47-D. Cancer Res., 48: 3693-3697, 1988.

37. de Pagter-Holthuizen, P., Jansen, M., van der Kammen, R. A., VanSchaik, F. M. A., and Sussenbach, J. S. Differential expression of theinsulin like growth factor II gene: characterization of the IGF-lI mRNAsand an mRNA encoding a putative IGF-Il associated protein. Biochim.Biophys. Acta, 950: 282-295, 1988.

38. King, R. J. B., Wang, D. Y., Daly, R. J., and Darbre, P. D. Approachesto studying the role of growth factors in the progression of breast tumoursfrom the steroid sensitive to insensitive state. J. Steroid Biochem., 34:133-138, 1989.

39. Clarke, R., Brunner, N., Katzenellenbogen, B. S., Thompson, E. W.,Norman, M. J., Koppi, C., Paik, S., Lippman, M. E., and Dickson, R. B.Progression of human breast cancer cells from hormone-dependent tohormone-independent growth both in vitro and in vivo. Proc. Natl. Acad.Sci. USA, 86: 3649-3653, 1989.

40. Knabbe, C., Lippman, M. E., Wakefield, L. M., Flanders, K. C., Kasid,A., Derynck, R., and Dickson, R. B. Evidence that transforming growthfactor $ is a hormonally regulated negative growth factor in human breastcancer cells. Cell, 48: 417-428, 1987.

41. Zugmaier, G., Ennis, B. W., Deschauer, B., Katz, D., Knabbe, C.,Wilding, G., Daly, P., Lippman, M. E., and Dickson, R. B. Transforminggrowth factors type j�1 and �92 are equipotent growth inhibitors of humanbreast cancer cell lines. J. Cell. Physiol., 141: 353-361, 1989.

42. Stewart, A. I., Johnson, M. 0., May, F. E. B., and Westley, B. R. Roleof insulin-like growth factors and the type I insulin-like growth factorreceptor in the estrogen-stimulated proliferation of human breast cancercells. J. Biol. Chem., 265: 21172-21178, 1990.

43. Reddel, R. R., and Sutherland, R. 1. Tamoxifen stimulation of humanbreast cancer cell proliferation in vitro: a possible model for tamoxifentumor flare. Eur. J. Cancer Clin. Oncol., 20: 1419-1424, 1984.

44. Osborne, C. K., Hobbs, K., and Trent, J. M. Biological differencesamong MCF-7 human breast cancer cell lines from different laboratories.BreastCancerRes., 9: 111-121, 1987.

45. Karin, M., Haslinger, A., Hoitgreve, H., Richards, R. I., Krauter, P.,Westphal, H. M., and Beato, M. Characterization of DNA sequencesthrough which cadmium and glucocorticoid hormones induce humanmetallothionein IlA gene. Nature (Lond.), 308: 513-519, 1984.

46. Gorman, C. High efficiency gene transfer into mammalian cells. In:D. M. Glover (ed), DNA Cloning, Vol. II. Oxford: IRL, 1985.

47. Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G., and

Chasm, L. DNA mediated transfer of the adenine phosphoribosyltrans-ferase locus into mammalian cells. Proc. NatI. Acad. Sci. USA, 76: 1373-1376, 1979.

48. Darbre, P. D., and King, R. J. B. Progression to steroid autonomy in5115 mouse mammary tumour cells: role of DNA methylation. J. Cell Biol.,99: 1410-1415, 1984.

49. Darbre, P., Yates, J., Curtis, S., and King, R. J. B. Effect of estradiolon human breast cancer cells in culture. Cancer Res., 43: 349-354, 1983.

50. Bium, W. F., Ranke, M. B., and Bierich, I. R. A specific radioimmuno-assay for IGF-ll: the interference of IGF binding proteins can be blockedby excess IGF-l. Acta Endocrinoi., 1 18: 374-380, 1988.