(CANCER RESEARCH 38, 94-102. January 1978)

Correlation Among Insulin Binding, Degradation, and BiologicalActivity in Human Breast Cancer Cells in Long-Term Tissue

Culture

C. Kent Osborne, Marie E. Monaco, Marc E. Lippman, and C. Ronald Kahn

Medicine Branch, Division of Cancer Treatment, National Cancer Institute [C. K. 0., M. E. M., M. E. L.¡,and the Diabetes Branch, National Institute ofArthritis, Metabolism, and Digestive Diseases, NIH, Bethesda, Maryland 20014 [C. R. K.]

ABSTRACT

Insulin interaction with four human breast cancer celllines in tissue culture was studied with respect to specificbinding to receptors, degradation, and biological responsiveness. All four lines bound and degraded 125l-labeled

insulin. Binding and degradation were time, temperature,and pH dependent. Unlabeled insulin competed with 12SI-labeled insulin for binding to the breast cells with half-maximal inhibition of binding being observed with lessthan 0.6 ng/ml for three of the four lines. Other peptidescompeted for insulin binding in proportion to their biological potency. Scatchard analysis of the insulin bindingdata revealed curvilinear plots consistent with negativecooperativity, and this was confirmed by kinetic studiesof dissociation. Quantitative analysis of insulin degradation revealed a similar K for all four cell lines (1.0 to 2.2x 10 M), whereas maximal velocities varied over a 7-fold range. Bacitracin, a polypeptide antibiotic, inhibitedinsulin degradation by all four lines. In one cell line,typical competitive binding data and Scatchard plots wereobtained only after inhibition of degradation with bacitra-cin. Insulin stimulated precursor incorporation into mac-romolecules and fatty acids in only two of the four celllines; with these, significant stimulation was seen withconcentrations as low as 0.6 ng/ml. No correlation wasfound between the amount of specific binding, receptorconcentration, or receptor affinity and the ability of insulinto stimulate the cells, suggesting a defect distal to thehormone-receptor interaction in the two unresponsivelines. The two responsive cell lines showed the mostinsulin degradation. These human cell lines should provide a useful tool for further study of the complex mechanisms of insulin action and for the study of factors thatregulate growth of human breast cancer.

INTRODUCTION

Peptide and steroid hormones are known to influencethe growth and development of normal and neoplastiarodent mammary glands (2, 15, 16, 34, 41, 42). The role ofthese hormones in the development of the human mammarygland is less well defined. We have shown that human

1 To whom requests for reprints should be addressed, at Department of

Medicine, University of Texas Medical School, 7703 Floyd Curl, San Antonio.Texas 78284.

* The abbreviations used are: TCA, trichloroacetic acid; HEPES, A/-2-hydroxyethylpiperazine-A/'-2-ethanesulfonic acid.

Received June 27, 1977; accepted October 6, 1977.

breast cancer cells in tissue culture provide an excellentmodel system for the study of hormone action (26). Severalof these cell lines which were initially derived from malignant effusions, possess a variety of steroid hormone receptors and biological responses (23-26). More recently, wehave demonstrated that 1 human breast cancer cell line,MCF-7, responds to physiological concentrations of insulinwith increased rates of macromolecular and fatty acidsynthesis (29, 32).

In this paper we have studied in detail insulin bindingand insulin action in 4 human breast cancer cell lines intissue culture. We have found that all 4 lines possessinsulin receptors and insulin-degrading activity. However,physiological concentrations of insulin stimulate precursorincorporation into macromolecules and fatty acids in only2 of these lines. These cell lines thereby provide a uniquesystem for the study of the relationship of insulin bindingand biological responsiveness in a human-derived tissue.

MATERIALS AND METHODS

Materials. Porcine insulin (Lot 615-D63-10; 25.4 units/mg) and porcine proinsulin were the generous gifts of Dr.M. Root and Dr. R. Chance, Lilly Research Laboratories,Indianapolis, Ind. Guinea pig insulin, epidermal growthfactor, and multiplication-stimulating activity were generously supplied by Dr. C. Yip (Banting Institute, Toronto,Ontario, Canada), Dr. R. Ladde (Hershey Medical Center,Hershey, Pa.), and Dr. M. Rechler and Dr. S. P. Nissley(NIH, Bethesda, Md.), respectively, and ovine prolactin wassupplied by the National Institute of Arthritis, Metabolism,and Digestive Diseases (NIH). Crystalline TCA2 was purchased from J. T. Baker Chemical Co. (Phillipsburg, N. J.).[14C]Thymidine (62 mCi/mmole), [3H]leucine (59 Ci/mmole),['"CJacetate (58 mCi/mmole), and 125l-labeled sodium io

dide (greater than 300 /xCi/M9) were obtained from theRadiochemical Centre (Amersham, England). ierf-Butylhydroperoxide was purchased from Lucidol, Buffalo, N. Y./V-Ethylmaleimide and phenylmethyl sulfonyl fluoride werepurchased from Sigma Chemical Co. (St. Louis, Mo.).Bacitracin was obtained from The Upjohn Co. (Kalamazoo,Mich.), and Trasylol (20,000 kallikrein inactivator units/ml)was obtained from Calbiochem (Los Angeles, Calif.).

Cells and Tissue Culture Techniques. All cell lines wereinitially derived from malignant effusions of women withmetastatic breast cancer and have been in continuoustissue culture for at least 1 year. Characterization of thecells has been summarized previously (26). The ZR-75-1

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Insulin Receptors in Human Breast Cancer

line was generously supplied by Dr. Nathaniel Young andLinda Engel of the National Cancer Institute. The EVSA-Tcell line was established in this laboratory (22). The MCF-7cell line was provided by Dr. Marvin Rich of the MichiganCancer Foundation (36, 39), and MDA-231 line was providedby Dr. Ronald Herberman of the National Cancer Institute.The cells were maintained in monolayer cultures in improved Eagle's minimal essential medium supplemented

with glutamine (0.6 g/liter), penicillin, streptomycin (NIHMedia Unit), and 10% fetal calf serum (North AmericanBiologicals Inc., Miami, Fla.). Cells were cultured in ahumidified incubator with 5% CO, at 37°.All cell lines were

free of Mycoplasma contamination on multiple determinations during the period of this study. Cultured humanlymphocytes, IM-9, were maintained at 37°in Eagle's mini

mal essential medium supplemented with 10% fetal calfserum.

Precursor Incorporation Studies. Cells grown to confluence in tissue culture flasks (75 sq cm; Falcon Plastics,Oxnard, Calif.) were suspended in 0.05% trypsin:0.02%EDTA in 150 mM NaCI, added to culture medium with 10%fetal calf serum, and replicately plated in plastic multiwelltissue culture dishes (Linbro Chemical Co., New Haven,Conn.). Cell density was adjusted to give a moderatelydense subconfluent monolayer of cells. After 24 hr ofincubation, the medium was removed, and cells werewashed once with 2 ml of serum-free medium, and 2 ml offresh serum-free medium were added. After the cells hadbeen in the serum-free medium for 24 hr, insulin or fetalcalf serum was added as described in the figures. Twenty-two hr later the cells were pulsed for 2 hr by addition of 25/M!of either [14C]thymidine (0.25 /nCi/ml) or [3H]leucine (2.5

¿iCi/ml)to the 2 ml of media. The cells were then washedwith cold Dulbecco's phosphate-buffered saline (pH 7.4,without Ca2* or Mg2*), harvested by suspension with the

trypsin:EDTA solution, and collected by centrifugation (2min) at high speed in a Clay-Adams serofuge. Cell pelletswere then washed once with 1 ml of phosphate-bufferedsaline and recentrifuged. After resuspension in 1 ml ofphosphate-buffered saline, cells were disrupted sonicallyfor 3 sec in a Branson sonicator at the lowest setting.Aliquots were then taken for protein determination by themethod of Lowryef a/. (27) or for precipitation in 10%TCA.Acid-insoluble material was collected and washed with 6ml of 10% TCA on 0.45-/um Millipore type HA filters. Filterswere solubilized in Aquasol (New England Nucler, Boston,Mass.), and the radioactivity was determined in a Packardliquid scintillation counter. Rates of incorporation werecalculated as dpm per mg protein per 2 hr and expressedas percentage above control (unstimulated) values.

Acetate incorporation into fatty acids was determined ina similar manner, except that the cells were pulsed with [2-14C]acetate (1 ^Ci/ml). After harvest, the cell pellet was

collected by centrifugation and resuspended in 1 ml ofphosphate-buffered saline, and aliquots were taken for protein determination. The remaining cells were recentrifuged,and the cell pellet was incubated in 0.1 ml of 30% potassiumhydroxide in 50% methanol for 2 hr at 90°.The incubation

tubes were cooled, acidified with 0.1 ml of concentratedhydrochloric acid, and extracted once by mixing for 15 sec

with 1 ml of hexane. Aliquots (0.5 ml) of the hexane layerwere then added to scintillation vials containing Aquasol,and the radioactivity was determined. When a knownamount of labeled fatty acid was similarly processed, recoveries of the radioactivity were greater than 93%. When theextracted radioactivity of the cell incubations was chro-matographed in hexane:diethyl etherglacial acetic acid(70:30:0.25) on Silica Gel G thin-layer plates, it migratedwith an RKidentical with that observed for fatty acids.

Insulin Binding Studies. Breast cancer cells were grownto confluence in tissue culture flasks and harvested bysuspension with 150 mM NaCI:0.02% EDTA. The cells wereadded to 50 ml of culture medium with 10% fetal calfserum and centrifuged for 2 min at 600 rpm. Cell pelletswere washed once with 10 ml of HEPES binding buffer(100 mM HEPES; 120 mM NaCI; 1.2 mM MgSO,; 2.5 mMKCI; 15 mM NaC,,H.A; 10 mM glucose; 1 mM EDTA; andbovine serum albumin, Fraction V, 10 mg/ml), counted in ahemocytometer, and resuspended in buffer to give a finalconcentration of 3 x 106 cells/ml. This buffer was chosenbecause degradation of 125l-labeledinsulin in the buffer wasless than when cells were incubated in Krebs-RingerTrisor phosphate buffers (data not shown). Based on trypanblue exclusion, greater than 90% of the cells remainedviable at the end of a 4-hr incubation at 21°.

125l-labeledporcine insulin was prepared at specific activities of 130 to 230 juCi/Mg by previously described modifications of the chloramine-T method (9, 19). The radioactivity was 95 to 98% precipitable in 5% TCA. Labeled insulin(120 pg/ml) and cells (3 x 106/ml) were incubated for 4 hrat 21°in a final volume of 0.5 ml of HEPES binding buffer,pH 7.6, in 12- x 75-mm plastic tubes (Falcon Plastics)unless otherwise noted. Unlabeled insulin or other hormones were added at concentrations noted in the charts.For minimal cell breakage incubation tubes were notshaken continuously but were gently agitated by hand athourly intervals. For determination of the amount of '25I-

labeled insulin bound to the cells, aliquots (100 to 200 ¿¿I)of the cell suspension were removed, layered onto chilledbuffer (200 /¿I)in polyethylene microfuge tubes, and centrifuged for 1 min in a Beckman microfuge. The cell-freesupernatant was aspirated and used for measurements ofinsulin degradation. The tip of each tube containing thecell pellet was excised, and the radioactivity was countedin an autogamma counter. Specific binding was defined asthe total percentage bound minus the percentage bound ina presence of excess unlabeled insulin (10 6 M) and was a

linear function of cell concentration over the range form0.5 to 10 x 10s cells/ml (data not shown).

In studies to test compounds for their effect on 125I-

labeled insulin binding and degradation, the cells werepreincubated for 10 min at 21°in HEPES binding buffer

with the test compound. Cell viability at 4 hr was greaterthan 90% in incubations with all test compounds except N-ethylmaleimide (50 to 60%) and ferf-butyl hydroperoxide(80%).

Insulin Degradation. The cell-free supernatant containing free labeled hormone obtained from the insulin-bindingassay was analyzed for degradation by measuring its TCAsolubility or, where noted, its loss of ability to bind to IM-9

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lymphocytes as previously described (10, 12). Results werecompared to appropriate control tubes prepared and incubated identically, but without cells. In general when degradation of 125l-labeled insulin was more than 10%, the rebind-

ing assay was a more sensitive method and gave significantly higher values.

Dissociation Experiments. Dissociation of 125l-labeled

insulin was measured as previously described (5). Cellswere incubated with labeled insulin as described for thebinding assay at 21°for 2 hr. Aliquots of the incubation

mixture were then diluted 50-fold in buffer alone or in

buffer with unlabeled porcine insulin (1 ¿¿g/ml)and incubated at 15°.At the times indicated duplicate tubes were

centrifuged, the cell-free supernatant was discarded, andthe cell-bound radioactivity was determined.

RESULTS

Stimulation of Macromolecular and Fatty Acid Synthesisby Insulin. Physiological concentrations of insulin stimulated precursor incorporation into macromolecules andfatty acids in 2 of the 4 human breast cancer cell linesstudied (Chart 1). As little insulin as 0.6 ng/ml (10~10 M)

stimulated leucine, thymidine, and acetate incorporation inthe MCF-7 and ZR-75-1 cell lines, and maximal stimulationwas observed with 60-ng/ml doses of insulin. The striking

insulin sensitivity of the cells was reproducible in multiple

MCF7

E

0-O

40 -

0.06 0.6 6INSULIN (ng/ml)

Chart 1. The effect of insulin on the rates of leucine, thymidine, andacetate incorporation. Insulin was added to cells previously maintained inserum-free medium for 24 hr. ["CJThymidine (0.25 nCi/ml), [3H]leucine (2.5/iCi/ml), or [14C]acetate (1 ¿¿Ci/ml)was added to the cells 22 hr after insulinfor a 2-hr pulse. After harvest, incorporation of leucine and thymidine intoTCA insoluble material and of acetate into hexane-extractable material wasdetermined as described in "Materials and Methods." Points, mean of 3

experiments, each done in triplicate.

experiments. Maximal stimulation by insulin of precursorincorporation varied from 1.5- to 4-fold. This variability is

unexplained but may represent experimental variation induced by differences in cell density, cell cycle synchronization, or other factors. No further stimulation was seenwhen higher concentrations of insulin (6 ng/m\ or 10 6 M)

were used (data not shown).The maximal rate of macromolecular synthesis produced

by insulin in the MCF-7 cells was consistently greater thanthat seen in the ZR-75-1 cells, whereas the reverse was true

for fatty acid synthesis. Stimulation of macromolecularsynthesis by insulin was accompanied by enhanced growthand reflected by an increase in the number of cells andtotal protein per dish (32). Little or no stimulation ofprecursor incorporation or growth was observed with theEVSA-T or MDA-231 cell lines (Chart 1).

Insulin cannot replace serum for long-term continuous

culture of any of the cell lines. All 4 cell lines, includingEVSA-T and MDA-231, responded with an increased rate of

macromolecular synthesis when 10% fetal calf serum wassubstituted for insulin (data not shown).

Demonstration of Insulin Binding and Degradation. Although only 2 of the 4 human breast cancer cell linesshowed a significant biological response to insulin, alllines bound '"l-labeled insulin (Chart 2). The degree ofinsulin binding was variable. The ZR-75-1 and EVSA-T cell

lines consistently showed the highest binding, whereas theMCF-7 (cells most responsive to insulin) and the MDA-231

lines showed the least. There was no apparent correlation

QZR EVSA MCF MDA75-1 T 7 231

Chart 2. Lett, effect of duration of incubation on binding of 12Sl-labeledinsulin to breast cancer cell lines. Breast cancer cells (3 •10' cells/ml)were incubated at 21°with 125l-labeledinsulin (120 pg/ml) without ( ) and

with ( ) an excess of unlabeled porcine insulin (6 ^g/m\). At the timesindicated, duplicate 200-»ilaliquots were removed from the incubationmedium, layered onto 200 ¿ilof chilled buffer in plastic microfuge tubes,and centrifuged for 1 min. The supernatants from tubes with labeled insulinonly were saved for measurements of degradation. The tip of each tube thatcontained a cell pellet was excised, and the radioactivity was counted. Theradioactivity bound to the cells is expressed as the percentage of the totalradioactivity in the system. The radioactivity bound to the cells in thepresence of labeled insulin alone is referred to as "total binding," and thatbound in the presence of excess unlabeled insulin is referred to as "nonspecific binding." Specific binding is the difference between total and nonspecific binding. Right, degradation of '"l-labeled insulin. Supernatants fromthe 4-hr time points used for the determination of total binding in theexperiments described above were used to measure degradation. Degradation was determined by failure of the labeled insulin to precipitate in 5%TCA (see "Materials and Methods"). '"l-Labeled insulin in control tubes

(cells omitted) was 95 to 98% precipitated by 5% TCA.

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between the amount of binding and the ability of insulin toinduce a biological response.

Similarly, all 4 cell lines degraded insulin to a variabledegree (Chart 2). With cells that had been washed once,degradation, measured by TCA precipitation, ranged from0 to 10% in the MDA-231 cell line to 30 to 40% in the MCF-7cell line after 4 hr at 21°.Elimination of the single cell wash

step prior to incubation increased the degradation by theMCF-7 cells by 20%; however, additional washes did notreduce degradation further. When MCF-7 cells were incu

bated in buffer for 4 hr and removed by centrifugation andwhen labeled insulin was added to the cell-free supernatant,

more than 60% of the total degrading capacity remained,indicating that the degrading activity had been releasedfrom the cells into the medium in this cell line. In addition,when labeled insulin was added to MCF-7 cells in mono-

layer culture under otherwise similar conditions, and themedia were assayed for degradation of the insulin by TCAprecipitation, nearly 50% of the tracer had been degraded.These data together with the time and temperature dependence of degradation (Chart 4; below) suggest that therelease of degrading activity was not an artifact of cellhandling. No correlation was observed between the amountof binding and the extent of degradation of '"l-labeledinsulin. The MCF-7 (the most insulin responsive) and MDA-

231 cell lines, both relatively low insulin binders, had thehighest and lowest levels of degradation, respectively. However, the 2-insulin-responsive cell lines, MCF-7 and ZR-75-

1, were the most active insulin degraders.Effects of Time, Temperature, and pH on Insulin Binding

and Degradation. Binding of '-"'l-labeled insulin was time

and temperature dependent; this is shown for the ZR-75-1and MDA-231 cells (highest and lowest binders, respectively) in Chart 3. Specific binding at 37°was rapid and

maximal by 45 to 60 min. A steady state was apparent forthe MDA-231 line, whereas binding decreased in the ZR-75-

1 line as incubation time increased, probably reflecting themore extensive insulin degradation by the ZR-75-1 cells.

With both cell lines, binding was highest at lower temperatures and maximal at 15°,although the time required to

achieve a steady state was prolonged. Increased insulinbinding at lower temperatures has also been seen with allother tissues studied, including cultured human lympho-

A. TCA Precipitation B Rebinding

Chart 3. Effect of duration of incubation and temperature on the specificbinding of '"l-labeled insulin to ZR-75-1 (left) and MDA-231 (right) humanbreast cancer cell lines. Cells (3 x I0'/ml) were incubated with '"l-labeledinsulin (120 pg/ml) with and without excess unlabeled insulin. At the timesindicated the amount of radioactivity bound to the cells was determined andexpressed here as specifically bound as defined in the legend to Chart 2.

100

§ 80HZ

2 60

S 40ff

# 20

2468 02468

HOURS

Chart 4. Effect of duration of incubation and temperature on the degradation of '"{-labeled insulin by MCF-7 human breast cancer cells MCF-7cells (3 x 106/ml) were incubated with'"l-labeled insulin (120 pg/ml). At thetimes shown aliquots were taken, and the supernatants were isolated bycentrifugation. Integrity of the labeled insulin was determined by TCAprecipitation (A) or by rebinding to IM-9 lymphocytes (B) as previouslydescribed (10, 12). Values are expressed as percentage '"l-labeled insulinintact as a function of time.

cytes (12) and rat liver plasma membranes (10). At 21°,

specific binding in the breast cancer cells was half-maximal

by 1 hr and had reached a steady state at approximately 4hr.

Insulin degradation was also time and temperature dependent, as shown for the MCF-7 cells, which demonstratedthe most extensive degradation (Chart 4). At 0°most of the

labeled hormone remained intact for up to 8 hr whetherdegradation was measured by the TCA or lymphocyte-re-

binding methods. At higher temperatures, more extensivedegradation was observed, especially when measured bythe more sensitive rebinding method. At 37°no labeled

hormone remained intact by the rebinding method, while30% remained TCA precipitable at 3 hr. All subsequentstudies were performed at 21°for 4 hr since insulin binding

was relatively rapid (steady state at 4 hr) and near-maximaland since degradation was significantly less than at 37°.

Insulin binding and degradation were also pH dependentbut had different optima. Specific binding showed a relatively sharp pH optimum of 7.6 to7.8 (Chart 5>A),similar tothat reported for other tissues (3, 9, 12). Degradation,however, increased as a function of increasing pH and wasmaximal at pH 8.5 (the highest pH tested), except in theMDA-231 cells in which the small amount of degradation

seen was not pH dependent (Chart 5B).Effects of Enzyme Inhibitors on Insulin Degradation.

Since degradation of labeled insulin at pH 7.6 by thesehuman breast cancer cells was considerable, especiallywith the MCF-7 cell line, we examined the effect of several

agents that previously had been shown to inhibit insulindegradation by human granulocytes (11) (Table 1). Phenyl-

methyl sulfonyl fluoride, a serine protease inhibitor thatstrongly inhibited degradation by the granulocyte (11), hadno effect on binding or degradation by the MCF-7 cells.ferf-Butyl hydroperoxide, which depletes the cell of gluta-

thione and also inhibited degradation by granulocytes (11),had no effect on degradation by the breast cancer cells butreduced binding by 26%. Trasylol, a protease and kallikreininhibitor, inhibited both binding and degradation onlyslightly. /V-Ethylmaleimide, a sulfhydryl group blocker,

completely inhibited degradation, significantly reduced

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C. K. Osborne et al.

binding, and was toxic to the cells. Bacitracin, a peptideantibiotic with protease inhibitor activity (28), nearly abolished degradation in all 4 cell lines (Chart 6; Table 1) andwas not toxic to the cells as determined by trypan blueexclusion. All subsequent experiments were performedboth with and without bacitracin (70 units/ml) to determine

16

QZ

Dinz

SEVSAT

8.5

Chart 5. Effect of pH on binding (A) and degradation (B) of '"l-labeledinsulin in human breast cancer cell lines. Cells (3 x 10*/ml) were incubatedat 21°in HEPES binding buffer at varying pH's for 4 hr. Then aliquots were

taken, the cells were centrifuged, and specific binding was determined asdescribed in the legend to Chart 2. Degradation of '"l-labeled insulin in thesupernatant was determined by TCA precipitation as described in the legendto Chart 2.

its effect on insulin binding and degradation in more detail.Analysis of Insulin Binding and Degradation. The per

centage of labeled insulin degraded by the cells was afunction of unlabeled insulin concentration (Chart 6). Degradation of the labeled insulin was significantly inhibitedby the addition of high concentrations of unlabeled insulin.When these data were analyzed by the Eadie-Hofstee

method (6), straight lines were obtained for each of the 4cell lines. The Km's for insulin degradation for all 4 lines

ranged between 1.0 to 2.2 x 10 7 M (Table 2), in close

agreement with the Km for insulin degradation obtained inother tissues (1, 10). Maximal velocities of insulin degradation varied over a 7-fold range in the 4 cell lines. Degrada

tion was inhibited in all cell lines by bacitracin to such lowlevels that quantitative analysis could not be performed(Chart 6).

50r

EO

30

_<2 20

# 10

MCF-7

0.6 6 60

INSULIN (ng/ml)

600 6000

Chart 6. Percentage of '"(-labeled insulin degraded as a function oftotal unlabeled insulin concentration. Cells (3 x I0'/ml) were incubatedwith '"{-labeled insulin (120 pg/ml) for 4 hr at 21°in binding buffer, pH 7.6.

with increasing concentrations of unlabeled porcine insulin without ( )or with ( ) bacitracin (70 units/ml). Degradation of labeled insulin wasthen determined by TCA precipitation as previously described. •,O. MCF-7; A, A, ZR-75-1, •d, EVSA-T; ». O. MDA-231. Values, represent themean of 3 separate experiments.

Table 1The effect of inhibitors on '"/-labeled insulin binding and degradation

MCF-7 cells (4 x 106 cells/ml) were incubated with '"l-labeled insulin for 4 hr at 21°with or without a 10-min preincubation with inhibitors at the indicated concentrations.Specific binding was determined as described in the legend to Chart 2. Degradation wasmeasured by TCA precipitation and, where indicated, by rebinding to IM-9 lymphocytesas described in the legend to Chart 4.

% inhibition of

Degradation

TCAInhibitorPhenylmethyl

sulfonylfluorideter(-Butyl

hydroperoxideTrasylolA/-EthylmaleimideBacitracinConcentration

precipitation5x 10-•M2

x 10 3M4000units/ml1

x 10 3M0.07unit/ml0.7unit/ml3.5units/ml7units/ml35units/ml70

units/ml0026100008166084Rebinding66664399Binding0263888009365356

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Table 2Physical constants " of the insulin degrading system

CelllineZR-75-1

EVSA-TMCF-7MDA-231Vmax

(ng/3 x 10«

cells/hr)76

±2062 ±2

166 ±10225 ±21K™

(10 7M)1.5

±0.61.5 ±0.22.2 ±1.11.0 ±0.8

" Mean of 3 experiments ±S.E.: calculated from Eadie-Hofstee

plots (6) of the data shown in Chart 6.

Unlabeled insulin also competed with 125l-labeled insulin

for binding (Chart 7). For 3 of the 4 cell lines, the bindingof labeled insulin was inhibited by low concentrations ofunlabeled porcine insulin; half-maximal inhibition of binding was observed with insulin concentrations of less than0.6 ng/ml (ICT10M) for the ZR-75-1, EVSA-T, and MDA-231

cell lines. Inhibition of degradation with bacitracin in thesecell lines had no effect on maximal binding but tended toshift the competition curves downward and to the left.

Surprisingly, with the MCF-7 cells, the most biologicallyresponsive to insulin, unlabeled insulin competed onlypoorly for labeled insulin binding in the absence of bacitracin. With this cell line, bacitracin caused a decrease inmaximal binding by more than 50% and an increase inbinding sensitivity. Half-maximal displacement of bindingwas reduced from about 300 to 0.6 ng of unlabeled insulinper ml. The decrease in total binding observed in thepresence of bacitracin suggests that bacitracin itself directly affects receptor binding or that the MCF-7 cells areable to rebind or incorporate labeled fragments after degradation in the absence of the inhibitor.

Scatchard plots (37) of the competitive binding data werestrikingly curvilinear for the ZR-75-1, EVSA-T, and MDA-231 cells (Chart 8). Bound versus free values decreaseddramatically with small increments in the amount of 125I-labeled insulin bound. However, for the MCF-7 cells astraight-line Scatchard plot was obtained in the absence ofinhibitors of degradation with an apparent equilibrium constant of 2 x 107 M"1. Bacitracin did not dramatically alter

the curvilinear plots observed in the other 3 cell lines. Withthe MCF-7 cells, however, bacitracin blocked degradation,reduced binding, and induced a curvilinear Scatchardtypical of insulin binding data in most other tissues.

The total receptor concentrations (fi,,) determined fromthe abscissa intercepts of the Scatchard plots were similarfor all cell types except for the MCF-7 cell line in whichconcentrations were 2- to 3-fold higher (Table 3). Inhibitionof degradation with bacitracin caused a decrease in receptor concentration in all 4 lines by about 50 to 70% (Chart 8;Table 3).

The curvilinear Scatchard plot is subject to several interpretations (20, 21). De Meyts ef al. (5) have presentedevidence that for insulin receptors the curvilinear Scatchardplot is due, at least in part, to site-site interactions of thenegatively cooperative type. Analysis of the Scatchard binding data and calculation of the "empty-sites" affinity (Ke)by

the method of De Meyts and Roth (4) yielded the equilibriumbinding constants shown in Table 3. The K,, varied over a10-fold range among the cell lines and in every case was

increased when bacitracin was added to the incubationmixture.

Graphic representation of the average affinity, K, as afunction of fractional receptor occupancy, /, with the useof the average affinity profile (4) is shown in Chart 9. Theaverage affinity profiles demonstrate that the average affinity of the insulin receptors fell strikingly as fractional occupancy increased from 0.1 to 10% with the ZR-75-1, MDA-231, and EVSA-T cell lines. At fractional occupancy greaterthan 10%, no significant further reduction was noted in K.With the MCF-7 cells in the absence of bacitracin, nochange in K was observed as Y increased consistent withthe linear Scatchard plot. In the presence of bacitracin, the

600 6000 0.06 0.6

INSULIN Ing ml)

Chart 7. Percentage of '"l-labeled insulin bound as a function of totalunlabeled insulin concentration. Cells (3 x I0*/ml) were incubated with 12SI-labeled insulin (120 pg/ml) for 4 hr at 21°,pH 7.6, with increasing concentrations of unlabeled porcine insulin without or with bacitracin. Radioactivitybound to the cells was determined as described in Chart 2. Points, mean ±S.E. for 3 experiments, each done in duplicate, U. units.

ZR-75-1

No Bacitracin

Bacitracin 170 U ml)

EVSA-T

MDA-231

1000 2000 3000 4000 0 200

BOUND [fmole ml)

400 600 800 1000

Chart 8. Scatchard plots of the insulin-competitive binding data in 4human breast cancer cell lines. Plot of bound versus free '"l-labeled insulinas a function of insulin bound. Data from a representative experiment inChart 7 were replotted according to the method of Scatchard (37). All datawere corrected for nonspecific binding.

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Table 3Equilibrium binding constants

ño"(pmoles/3 x10"cells)Cell

lineZR-75-1

EVSA-TMCF-7MDA-231-

Bacitracin1.8

0.85.41.4±

0.1"±0.2±

1.3±0.3+

Bacitracin-0.5

±0.4 ±1.3 ±0.4 ±0.1

0.10.20.12

200K.*(108M

')Bacitracin

+.27

±.13 ±.19±.44

±0.69

0.620.010.118

4.01Bacitracin.90

93.27.85±

0.89±0.77±0.08±0.20Biological

response"1+

" Receptor concentration (not corrected for differences in cell surface area).b "Empty sites" affinity calculated by the method of De Meyts and Roth (4).' Presence (+ ) of or absence (-) of stimulation by insulin of DMA, protein, and fatty

acid synthesis (see Chart 1).d Mean ±S.E. of 3 separate Scatchard plot analyses for each cell line.

EVSA-T

2

* 1.6r

0.001 0.01 0.1 1.0 0.001 0.01 01

06-

Chart 9. Average affinity as a function of fractional receptor occupancy.Competitive binding data from an experiment shown in Chart 7 werereplotted by the method of De Meyts and Roth (4) where the averageaffinity, K, is ((BIF)I(R,, - 6)) and fractional occupancy, V, is (B/Ra).

MCF-7 cells showed an average affinity profile similar tothose of the other 3 lines. With all cell lines, bacitracinincreased K at low levels of fractional occupancy (empty-

sites affinity, K,,) as noted in Table 3. The values of Ke areprobably an underestimate since a plateau has not yetbeen reached at low fractional occupancy. For achievementof an even smaller /, however, a prohibitively small amountof labeled insulin would have been required during thebinding incubation.

No consistent correlation was found between insulinresponsiveness and the receptor concentration or affinity(Table 3). Of the 2 insulin-responsive cell lines, the MCF-7

had the highest receptor concentration but the lowest K,,,whereas the ZR-75-1 had the highest «<,.

Site-site interactions of the negatively cooperative typewere also suggested by the studies of dissociation of 125I-

labeled insulin from the cells following dilution of thehormone-receptor complexes in the presence and absence

of excess unlabeled insulin (Chart 10) (5). In each cell linethe rate of dissociation was enhanced when the number ofoccupied receptors was increased by the addition of unlabeled insulin. Also, the MCF-7 and MDA-231 cell lines,

which had the lowest Ke and the least specific binding, hadthe most rapid spontaneous dissociation rates, whereas

EVSA-T

0--0 Dilution + 1.7 x 107M Insulin

2 0 1

TIME (hours)

Chart 10. Kinetics of dissociation of '"l-labeled insulin. Cells (3 x 10'/ml) were incubated with 125l-labeled insulin (120 pg/ml) for 2 hr at 21°.Aliquots were then diluted 50-fold in buffer alone (•)or in buffer withporcine insulin (1 M9/rnl; 1.7 x 10~7 M) (O) and incubated at 15°.At thetimes indicated duplicate tubes were centrifugea, and the cell-bound radioactivity was determined. Data were plotted as percentage of initial 1MI-labeled insulin bound at time 0 as a function of duration of incubation.

the ZR-75-1 and EVSA-T cells, which had relatively high

affinities, had slower rates of dissociation.Specificity of Insulin Binding. 125l-Labeled insulin binding

to these breast cancer cells was inhibited by porcine insulin, other insulin analogs, and unrelated peptides to adegree roughly proportional to their biological potency inthe fat cell bioassay (Chart 11) (8). Although porcine proin-sulin has usually been more potent than guinea pig insulinin inhibiting binding and stimulating glucose oxidation (5),the lot of proinsulin used in these studies was less potentboth as an inhibitor of binding and as a stimulator ofglucose oxidation than was the lot of guinea pig insulin (K.Baird and C. R. Kahn, unpublished data). Multiplication-stimulating activity, a growth factor found in conditionedmedium from Buffalo rat liver cells that possesses insulin-like activity (7, 38), also inhibited '"l-labeled insulin binding

but was less potent than proinsulin. Epidermal growthfactor and ovine prolactin did not significantly compete forinsulin binding. Thus, in 4 tissue culture cell lines derivedfrom human breast cancers, insulin receptors have beenidentified that demonstrate the high affinity, specificity, and

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Insulin Receptors in Human Breast Cancer

MCF 7 l + Racilracin 70 U'mll

EGF p

60 600 6000 06

PEPTIOE tng/mll

60 600 6000

Chart 11. Specificity of 125l-labeledinsulin binding. Cells (3 x 10ecells/ml) were incubated with '"l-labeled insulin (120 pg/ml) for 4 hr at 21°in thepresence of the indicated concentrations of polypeptide hormones: porcineinsulin (/); guinea pig insulin (GP); porcine proinsulin (PRO), multiplication-stimulating activity (MSA); epidermal growth factor (EGF); and ovine prolac-tin (P). Bacitracin (70 units/ml) was used to inhibit degradation in the MCF-7 cells only. The bound radioactivity was determined as described in thelegend to Chart 2 and plotted as percentage of '"l-labeled insulin bound asa function of peptide concentration in ng/ml.

other binding characteristics observed for insulin receptorsin other tissues.

DISCUSSION

Although insulin is known to control in part normalmammary gland growth and differentiation and to influencegrowth of certain rodent mammary tumors (2, 15, 16, 34,41, 42), there is a paucity of data on the binding of insulinto normal or neoplastic mammary gland cells (14, 17, 18,30). Most of these studies have utilized heterogeneous cellpreparations and have not fully characterized binding withrespect to saturability, affinity, or specificity.

Cloned tissue culture cell lines have facilitated the studyof hormone action. Our recent demonstration that a humanbreast cancer cell line, MCF-7, responds to low levels ofinsulin with increased DNA, RNA, protein, and fatty acidsynthesis suggests that some human breast cancers (atleast in tissue culture) may be insulin dependent andpresumably may possess insulin receptors (29, 32). RNA,protein, and fatty acid synthesis were stimulated within 30min of insulin addition, whereas enhanced DNA synthesiswas not seen for 10 hr. Furthermore, these effects were notthe result of increased glucose availability (11). In thisstudy we have demonstrated that 4 human breast cancercell lines grown in long-term tissue culture have specific,high-affinity insulin receptors.

Characterization of these receptors reveals that bindingis saturable, reversible, and time, temperature, and pHdependent in a manner similar to other insulin receptorsfrom human as well as nonhuman tissues (11-13, 31, 33,35). The receptors of 3 of the human breast cell linesdemonstrate high affinity with half-maximal displacementof about 0.6 ng/ml (10 10M) and K, that ranges from 0.44to 2.27 x 10" M '. In addition, only insulin-related peptides

compete for insulin binding, and in a manner roughlyproportional to their biological potency.

These cells were also active degraders of insulin, and inthe MCF-7 cell line, the most insulin-responsive, high-affinity, and binding specificity could be demonstrated satisfactorily only when bacitracin was used to inhibit degradation.MCF-7 cells degraded 100% of the tracer insulin by 3 hr at37°,whereas insulin stimulation of thymidine incorporationis delayed for 10 to 12 hr and remains near-maximal for 24hr at the same temperature (32). Several possible explanations may exist for these findings: (a) insulin stimulation ofthymidine incorporation does not require prolonged insulin-receptor interaction, but the initial binding initiates anirreversible pathway eventually leading to the biologicalresponse; (b) degraded insulin fragments are capable ofeliciting a response; or (c) as has been shown for livermembranes (8), the bound insulin may be protected fromdegradation, and only a small number of undissociatedand intact insulin-receptor complexes are required for abiological response.

The Km for the insulin-degrading system (1.0 to 2.2 x10 7 M) in this study was in close agreement with that

obtained with liver plasma membranes (8). Furthermore,the concentration of unlabeled insulin required for theinhibition of degradation by 50% was about 2000 timesgreater than the concentration of insulin required for theinhibition of binding by 50%. Since most of the degradingactivity of these cells was released into the medium andremained after removal of the cells, in this system it appearsthat most of the degradation does not require receptorbinding. The higher level of degradation seen in the biologically responsive cell lines suggests a possible relationshipbetween these 2 processes. Whether the receptor-boundinsulin can also be a substrate for degradation, as hasbeen suggested by Terris and Steiner (40) for hepatocytes,is not known.

In rat muscle (1), liver membranes (8), human granulo-cytes (11), and the breast cancer cells studied here, degradation is partially inhibited by sulfhydryl and proteaseinhibitors. The most effective inhibitor of the insulin-degrading activity of these breast cancer cells is bacitracin.Our data also suggest that bacitracin affects the plasmamembrane, since equilibrium binding data were somewhataltered in a cell line with minimal degradation (MDA-231).

The cell lines examined here also allow us to study thequalitative correlation between insulin binding and degradation and to study biological responsiveness. However,since the binding and degradation and the biological response studies were performed under different experimental conditions for the optimization of each event, precisequantitative interpretations are difficult. Nevertheless, totalinsulin binding, receptor affinity, and receptor concentration correlated poorly with the ability of insulin to stimulatemacromolecular and fatty acid synthesis. The most insulin-responsive line had the lowest specific binding and bindingaffinity and the most degradation. Furthermore, 2 cell lineswith specific high-affinity receptors failed to respond toinsulin but did respond to fetal calf serum which impliedthat these lines were not totally autonomous. In the 2insulin-responsive cell lines, the concentration of insulin

JANUARY 1978 101

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C. K. Osborne et al.

that caused half-maximal displacement of binding wassimilar to that which caused half-maximal stimulation ofprecursor incorporation into macromolecules and fattyacids (0.6 ng/ml or 10 10M) which suggests that receptor

binding is coupled to the biological response. The lack ofresponse in 2 cell lines that have insulin receptor implies adefect in transmission of the signal distal to the receptor.Whether this represents actual uncoupling of the receptor-biological response pathway or merely the loss of a normalcontrol mechanism in 2 of these malignant cell lines remains to be defined. It is hoped that further study of thedifferences between the insulin-responsive and -unresponsive breast cancer cell lines may help explain the complexmechanisms of insulin action and clarify the factors thatregulate the growth of human breast cancer. This modelsystem offers potentially unique advantages for the studyof insulin action, including that the cells are human derived,that some are extremely sensitive to insulin whereas othersdo not respond at all, that they possess a panoply ofresponses that allows investigation of both the metabolicand growth-stimulating properties of insulin, and finallythe presence of receptors and biological responses to otherhormones facilitates study of hormone interaction at themolecular level.

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12. Gavin, J. R., Gorden, P., Roth, J., Archer, J. A., and Buell, D. N.Characteristics of the Human Lymphocyte Insulin Receptor. J. Biol.Chem.,248: 2202-2207, 1973.

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1978;38:94-102. Cancer Res   C. Kent Osborne, Marie E. Monaco, Marc E. Lippman, et al.   CultureActivity in Human Breast Cancer Cells in Long-Term Tissue Correlation Among Insulin Binding, Degradation, and Biological

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