effect of insulin and estrogen on hormone binding in the ......effect of insulin and estrogen on...

[CANCER RESEARCH 37, 4641-4649, December 1977]

Effect of Insulin and Estrogen on Hormone Binding in theR3230AC Mammary Adenocarcinoma1

Samir M. Shafie,2 Scott L. Gibson, and Russell Hilf3

Department of Biochemistry Â¡S.M. S., S. L. G., R. H.], and the University oÃRochester Cancer Center [R. H.Â¡,University oÃRochester School of Medicineand Dentistry. Rochester. New York 14642

SUMMARY

When compared to dissociated mammary tumor cellsprepared from intact rats (100%), cells from diabetic animals displayed higher insulin-binding capacity (â€”135%),and those from ovariectomized rats bound more than twice(â€”240%)as much 125l-labeled insulin. The increase in insulin binding from hormonally altered hosts was detectablebetween the first and second week after tumor transplantation. Scatchard plots for insulin binding of all 3 groupswere curvilinear and roughly parallel. Resolution of thesecurves into linear components gave lines with no significantdifference in slope for the 3 groups. The dissociation ofbound 125l-labeledinsulin was enhanced in the presence of

unlabeled insulin. Average affinity profiles for each experimental group were calculated and showed no difference inthe estimated K which suggested that differences in insulinbinding reported here resulted from a change in receptornumber.

Levels of serum insulin and blood glucose from ovariectomized tumor-bearing rats were similar to those in intact(control) tumor-bearing animals; diabetic animals showedelevated blood-glucose and lowered serum insulin levels.Injection of insulin into either intact or diabetic tumor-bearing rats decreased insulin binding. Insulin binding wasalso decreased in tumor cells from ovariectomized ratstreated with estradici valerate, although the binding capacity remained significantly above control levels. Althoughthe results obtained with cells from diabetic and intact ratssupport the proposed self-modulating model for insulinreceptors, the data from ovariectomized rats suggest thatother mechanisms could be involved in regulation of insulinreceptors. Compared to tumor cells of control animals,estrogen binding was significantly reduced (25%) in cellsfrom diabetic rats but was increased 2-fold in tumors fromovariectomized rats. Treatment of the latter with estrogenreturned the estrogen-binding capacity to control levels.These results suggest a relationship between insulin andestrogen binding in the R3230AC tumor.

Initial experiments with tumor cells in short-term culture(3 to 6 days), grown with either calf or rat (tumor-bearinghosts) serum, indicated that these cells bound 2 to 3 times

' Supported by USPHS Grants CA 16660 and CA 11198 from the National

Cancer Institute, NIH.2 Recipient of NIH Fellowship, IF32CA05941-01, from the National Cancer

Institute.3 To whom requests for reprints should be addressed, at Department of

Biochemistry, Box 607, University of Rochester Medical Center, 601 Elm-wood Avenue, Rochester, N. Y. 14642.

Received April 5, 1977; accepted September 20, 1977.

more insulin than was found in freshly dissociated cells;estrogen binding was unchanged throughout. Estradiol invitro decreased insulin-binding capacity and insulin in vitroincreased estrogen-binding capacity. The data suggest thata membrane regeneration process may have occurred invitro following enzymatic dissociation of the cells, andcells in short-term culture offer an experimental system toexamine receptor regeneration and its control.

INTRODUCTION

There have been numerous studies on the effect ofestrogen, prolactin, and, more recently, insulin on thegrowth of animal and human mammary carcinomas (13,17, 25, 26, 30). Renewed interest was stimulated by theconcept that the hormone-binding capacity of breast cancers could predict the endocrine responsiveness of theneoplasm (18). It has been established that the concentration of the hormone receptor(s) may vary in pathologicalconditions (21) and under conditions in which the hormonalmilieu of the tumor-bearing host was altered (15). Thefluctuation in hormone receptor levels in response to altered conditions has been correlated to the sensitivity ofthe target tissue to the hormone(s) (12, 17, 24). Thus, byquantitatively studying hormone receptors of mammarytumors from animals with altered hormonal environments,one could obtain information regarding both the patho-physiology of mammary cancer and the regulation of hormone receptors.

The R3230AC mammary adenocarcinoma of the Fischerrat is a useful experimental model for these studies since itrepresents an autonomous (grows in hormone-deprivedhost), hormone-responsive (growth-retarded) neoplasmwith regard to estrogen, insulin, and prolactin (16). Receptors for all these hormones have been documented in thistumor (14, 28, 31). This investigation was designed toexamine and relate changes in insulin and estrogen bindingto dissociated cells of R3230AC tumors from rats that weremodified in their insulin or estrogen milieu by ablative oradditive hormone treatment.

MATERIALS AND METHODS

R3230AC Mammary Tumor. Female Fischer rats wereobtained from Charles River Breeding Laboratory, Wilmington, Mass. The mammary tumor was implanted s.c. in theaxillary region on both sides by a sterile trocar technique

DECEMBER 1977 4641

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S. M. Shafie et al.

as decribed by Hilf ef al. (16). This adenocarcinoma, whichhas been maintained by transplantation since 1963, iscomprised primarily (-95%) of epithelial cell elements.

Ovariectomy and Induction of Diabetes. Animals wereovariectomized by standard surgical techniques 1 weekbefore tumor implantation. Diabetes was induced by 1 i.v.injection of 6 mg (50 to 60 mg/kg) streptozotocin (UpjohnCo., Kalamazoo, Mich.) dissolved in 0.9% NaCI solution,pH 4.5 (adjusted with citric acid), 2 days prior to tumorimplantation. Urinary glucose was determined with Clinistix(Ames Co., Inc., Elkhart, Ind.), blood glucose was measuredby a Glucostat procedure (Worthington Biochemical Corp.,Freehold, N. J.), and serum insulin was measured by ra-

dioimmunoassay (Immophase; Corning Scientific Instruments, Medfield, Mass.).

Treatment of Animals after Tumor Transplantation. Es-

tradiol valerate (E. R. Squibb and Sons, Inc., New York,N. Y.), 40 mg/ml, when utilized, was dissolved in sesameoil and injected s.c., at doses of 1 mg/animal for eachdose. Insulin (protamine, zinc, and lletin) suspension (EliLilly and Co., Indianapolis, Ind.), 40 IU/ml, was injected ata dose of 4 lU/injection s.c. at 45, 21, and/or 2 hr prior tosacrifice.

Preparation of Cells. Excised tumors were weighed andplaced in ice-cold 0.9% NaCI solution. After the removal of

connective tissue and necrotic areas, the tumor was finelyminced with a scalpel and minced further into 1- x 1-mm

pieces on a Mcllwain Tissue Slicer (Brinkmann Instruments,Inc., Westbury, N. Y.). Isolated cell suspensions were obtained as described earlier (14) by incubating the mincedtissue (3 g; 37Â°)in a solution of 10 ml Ca2"- and Mg2MreeHanks' balanced salt solution containing 0.1% hyaluroni-

dase (Sigma Chemical Co., St. Louis, Mo.) and 0.05%collagenase (type II; Worthington Biochemical Corp.). Finalcell viability was estimated by trypan blue exclusion; preparations that showed at least 80 to 90% viable cells wereused in these studies. Cell count was determined by standard microhematocrit procedures. This procedure yieldscells that appear to be uniformly epithelial by light microscopic examination.

Cells were also prepared for tissue culture by the use ofthe above procedure (aseptically) and were plated at 15 x106 cells/T75 Falcon flask. The cells were fed on alternate

days with Medium 199 (Grand Island Biological Co., GrandIsland, N. Y.), pH 7.6, supplemented with either fetal calfserum (Flow Laboratories, Rockville, Md.) or with rat serum(tumor-bearing host). Some cell cultures were treated witheither 17/3-estradiol or porcine insulin; hormones were

added with each additional feeding 1 day after cells wereplaced in culture, and 3 hr prior to harvesting (by scraping)on Day 6.

lodination of Insulin. Crystalline porcine insulin (Lilly)was labeled stoichiometrically with Na'25l in 0.1 M NaOH(Amersham/Searle Corp., Arlington Heights, III.) by chlora-mine-T (Eastman Kodak Co., Rochester, N. Y.), according

to the method of Freychet ef al. (7, 8). Monoiodoinsulinwas separated from sodium iodide by ion exchange chro-

matography and had a radiospecific activity of 140 to 180/j.C\/fj.g; the labeled insulin was at least 99% trichloroaceticacid precipitable.

Insulin Binding. The assay for the measurement of specific binding of 125l-labeled insulin to intact cells at 20Â°was

carried out as described previously (14). Briefly, 2 to 5 x106 cells were incubated with radioactive insulin in 1.0 mlMedium 199, pH 7.6, containing 1% BSA" (Kupits, ForkedRiver, N.J.). Each assay of 125l-labeled insulin binding was

performed (Benco Model SS30 shaking water bath) at leastin triplicate in plastic tubes, and for each determinationparallel samples were also incubated in the presence of1000-fold excess of unlabeled insulin to determine and

then correct for nonspecific binding of labeled insulin. Atthe end of the incubation period, 8 ml of chilled 0.9% NaCIsolution were added rapidly to each tube, the sampleswere quickly centrifuged (1000 x g for 3 min), and thesupernatant was discarded. Further wash steps were foundto be unnecessary.

The effect of shaking on specific binding was assessedby the comparison of the time course of binding to cells inincubation mixtures that were either shaken at 80 or 40cycles/min or not shaken at all. Specific binding washighest to cells shaken at 80 cycles/min, reached a maximum within 15 min, and remained at that level for 60 min(2.3 to 2.6 fmoles/5 x 106 cells from intact rats). Lower

binding was observed when cells were shaken at 40 cycles/min (1.8 to 2.0 fmoles/5 x 106 cells) or when cells were notshaken (1.6 to 1.8 fmoles/5 x 106 cells). Compared to the

time course seen when incubation mixtures were shaken at80 cycles/min, it required a longer time for maximumbinding to be reached when cells were shaken at 40 cycles/min (maximum reached at 30 min) or when no shaking wasused (maximum was reached at 45 to 60 min). All subsequent experiments were conducted at 20Â°with the use of a

shaking frequency of 80 cycles/min.Dissociation of the labeled ligand was examined after a

45-min binding period by resuspending the washed cellpellet with 8 ml of either Medium 199 + 1% BSA alone orwith this solution containing 10 7 M unlabeled insulin; cell

suspensions were then incubated for 5, 15, 30, or 45 min at20Â°.The radioactivity in the cell pellet obtained after centrif-

ugation was measured in a Packard Model 5220 autogammacounter with counting efficiency of 58%.

Estradici Binding in Intact Cells. Prior to use. the 17ÃŸ-[2,4,6,7-3H]estradiol (96 Ci/mmole, New England Nuclear

Corp., Boston, Mass.) was purified by chromatography(Instant Thin Layer, SA Sheets; Gelman Instrument Co.,Ann Arbor, Mich., developed with chlorofornrmethanol(97:3).

Cells were added to scintillation vials (2 x 106 cells/vial)

in 1 ml of Medium 199, pH 7.6, as described for other cellsin culture (26). This was supplemented with the appropriateamount of purified 17ÃŸ-[2,4,6,7-3H]estradiol dissolved in

10 fjL\of absolute ethanol. A parallel set of vials received, inaddition to the 17ÃŸ-[2,4,6,7-3H]estradiol 200-fold excess

unlabeled estradici to estimate nonspecific binding. After a60-min incubation at 37Â°, the reaction was stopped by

washing with 6 volumes of iced Medium 199, and the vialswere centrifuged at 2000 rpm for 10 min at 4Â°.The radioactivity in the cell pellet was counted in 10 ml of Bray's fluor

' The abbreviation used is: BSA, bovine serum albumin.

4642 CANCER RESEARCH VOL. 37



Hormone Binding in Mammary Tumors

in an Isocap 300 liquid scintillation counter (Nuclear Chicago Searle, Chicago, III.); counting efficiency was 42%. Allassays were conducted at least in triplicate.

Presentation of Data. Data are presented as mean Â±S.E. and analyzed according to Student's f test; a probabil

ity of <0.05 was considered to be significant.

RESULTS

Insulin Binding to Tumor Cells. Insulin binding wasassayed with 125l-labeled insulin at 20Â°with tumor cells

from intact, diabetic, or ovariectomized hosts. Maximalspecific binding (total binding-nonspecific binding) wasshown to be a time-dependent process and, depending onthe shaking frequency, saturable between 15 and 60 min.Nonspecific binding, that which was not displaced by 1000-fold excess of unlabeled hormone, was related linearly tothe concentration of 125l-labeled insulin, was independent

of shaking or incubation time, and ranged from 30 to 50%of total binding (total binding by tumor cells from intactrats was 2.5 to 6.0% of added 125l-labeled insulin). Exami

nation of insulin binding as a function of time indicatedthat prolonged incubation (over 60 min at a shaking frequency of 80 cycles/min) failed to maintain a steady state;the decrease in the apparent binding capacity was probablydue to degradation of the hormone and/or degradation ofthe hormone receptor during incubation. The relativelyshort time span before which the maximal specific bindingcapacity decreased mitigated against incubation times exceeding 45 min for subsequent experiments. The timecourses of binding to tumor cells from diabetic or ovariectomized animals were similar to that seen with cells fromintact rats, the difference being that there is greater bindingat each time point for cells from diabetic rats and that thehighest binding is obtained in cells from ovariectomizedrats (specific binding for this latter group was 2 to 3 timeshigher at each time point studied). The amount of nonspecific binding was essentially identical in cells from all 3groups, which indicated that the differences in total bindingwere attributed to differences in specific binding. Degradation of the labeled ligand was also examined in incubationmixtures of cells from each group of animals. Degradationwas estimated by measurement of radioactivity in the tri-chloroacetic acid-precipitable material in the supernatantof the incubation mixture obtained after centrifugation ofthe cells and correcting for radioactivity bound to the cells(additional assessment of degradation was done with theuse of radioimmunoassay procedures). The results, whichwere the same in all 3 experimental groups, were expressedas a percentage of ligand remaining: zero time, 100; 15min, 94; 30 min, 92; 45 min, 91; and 60 min, 90. The extentof degradation reported here (incubation mixtures contained 1% BSA) was similar to that in an earlier report (14).

Saturation kinetics were examined for insulin binding totumor cells from intact, diabetic, or ovariectomized rats,and the data were analyzed according to procedure ofScatchard (24). As seen in Chart 1, this method of analysisyielded curvilinear plots that are usually seen for insulinbinding. These roughly parallel curves can be resolved intohigh- and low-affinity linear components (lines not shown).

The slopes of the former for cells from intact, diabetic, orovariectomized rats were 1.20 x 10'Â°M ', 1.03 x 10'Â°M \and 1.00 x 10'Â°M~1,respectively (no significant differences

in slopes by analysis of covariance, p = 0.954), and theslopes of the latter class of binding sites were 0.9, 1.0, and1.1 x 109 M 1, respectively. Projection of the curvilinear

Scatchard plots to intersection of the abscissa yields estimates for the number of sites, which were 1173, 1884, and2791 sites/cell for intact, diabetic, and ovariectomized rats,respectively. This relationship of insulin-binding capacityamong the 3 groups was also observed when the linearinitial portion of each curve was extrapolated to the abscissa.

One alternate interpretation of curvilinear Scatchard plotsconsiders negative cooperative site-site interactions amonga single class of receptor sites (5, 6). This possibility wasassessed by the method of De Meyts and Roth (6). Asshown in Chart 2, the dissociation curves of 125l-labeled

insulin from tumor cells prepared from either intact orovariectomized rats were obtained in the absence (dilutiononly) or in the presence of 10 7 M unlabeled insulin. For

both cell preparations, the addition of excess unlabeled

-INSULIN BOUND(pM)

Chart 1. Scatchard plot for insulin binding to R3230AC tumor cells fromintact (D). diabetic (A), or ovariectomized (A) Fischer rats. '25l-Labeledinsulin (10 " to 10 ' M) was incubated (shaken at 80 cycles/min) with 2 x106 cells at 20' for 45 min in 1 ml Medium 199 plus 1% BSA. The ratio ofbound to free '"l-labeled insulin is plotted as a function of the insulin

bound. Nonspecific binding has been subtracted.

TIME (mm)

Chart 2. Time course of dissociation of bound '"l-labeled insulin from

R3230AC tumor cells of intact and ovariectomized (OVEX) rats. The cellswere incubated for 45 min at 20Â°with 10'Â° M 125l-labeled insulin, pelleted,

resuspended in 8 ml of Medium 199, and incubated for various times at 20Data are shown as time courses of dissociation in Medium 199 (dilution)alone (â€¢)or in the presence of 10 7 M unlabeled insulin ( O); each point is

the average of triplicate samples. Data are presented as percentage of totalinsulin bound at zero time (100%).

DECEMBER 1977 4643



S. M. Shafie et al.

insulin increased the dissociation of labeled bound insulin.Since such behavior has been interpreted as supportingnegative cooperativity, it is necessary to consider othermathematical approaches for analysis of binding isotherms.With the use of the technique proposed by De Meyts andRoth, we obtained the average affinity profiles for eachbinding isotherm, ranging up to 40% occupancy. The 3profiles obtained (plots not shown) were roughly paralleland yielded an average K ranging from 2.3 to 3.1 x 109 M 1

for the 3 experimental groups. The similarity of the affinityfor insulin observed by this mathematical technique, whichconsiders negative cooperativity, and the parallel behaviorof the linear components resolved graphically togetherindicate that the affinity of insulin binding was comparablein tumor cells from intact, diabetic, or ovariectomizedanimals.

Measurement of Estrogen Receptor in Intact TumorCells. Chart 3A contains a representative plot of the saturation curve as a function of [3H]estradiol concentration for

specific 17/3-estradiol binding in tumor cells from intactrats. The cells were incubated at 37Â°for 60 min with labeled

estradiol in the absence or presence of 200-fold excess

unlabeled estradiol to account for nonspecific binding (26).These data yielded a straight line (Chart 36) when analyzedgraphically according to the Scatchard equation. The slopeof this line yielded an apparent K,, of 1 x 10 9 M, which

agrees with that obtained after incubating cytosols fromthese cells with 17ÃŸ-[2,4,6,7,3H]estradiol at 4Â°(31).

Effect of Insulin Treatment on Insulin Binding to TumorCells from Diabetic or Intact Rats. Because insulin isthought to regulate its own receptor, we first examined theamount of specific 125l-labeled insulin bound to tumor cells

from intact or diabetic rats obtained at 1, 2, 3, or 4 weeksafter tumor implantation. Specific binding to cells fromintact rats did not vary significantly (mean fmoles boundper 5 x 106 cells Â±S.E.): 1 week, 1.49 Â±0.04; 2 weeks,

1.60 Â±0.02; 3 weeks, 2.29 Â±0.40; and 4 weeks, 1.43 Â±0.04. In contrast, tumors from diabetic rats displayed con-

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Chart 3. Saturation plot and Scatchard analysis of [-'Hjestradiol specificbinding in dissociated tumor cells. Tumor cells from intact rats wereincubated at 37Â°for 60 min with increasing concentrations of 17/3-[3H)estradiol. Nonspecific binding, obtained in the presence of 200-foldunlabeled estradiol, has been subtracted. A representative plot (A) andScatchard analysis [the plot is a composite of 2 separate experiments (8)]are shown. The dissociation constant, Kd, is 1 nw, and n, the number ofbinding sites, is 6 fmoles/10* cells. Each value on the saturation curve is themean of 4 measurements Â±S.E. B/F, bound/free.

DIABETICD UNIÂ« Al t D

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7 14 2l 28 28

DAYSAFTERTUMORTRANSPIANTATIONChart 4. Effect of insulin administration on insulin-binding capacity of

tumor cells from diabetic or intact rats. One-, 2-, 3-, or 4-week-old tumorswere excised from untreated or insulin-injected (4 IU) hosts that were madediabetic 2 days prior to tumor implantation. Four-week-old tumors were alsoexcised from untreated or insulin-injected intact rats. Dissociated tumorcells were incubated at 20Â°with 1 x 10 10M '"l-labeled insulin for 45 min.Specific '"l-labeled insulin binding (fmoles/5 x 10' cells) to tumor cells isplotted in relation to tumor age in untreated rats, in rats that received 1injection of insulin 2 hr prior to sacrifice, or at 21 hr prior to sacrifice.Values are means of quadruplicates Â±S.E.

sistently higher specific insulin binding and showed agradual increase in binding between the first and thirdweeks of tumor growth (Chart 4). For investigation of theeffects of administration of insulin, a single injection of 4ID insulin, at either 2 or 21 hr prior to sacrifice of animalsbearing 3- or 4-week-old tumors significantly decreasedinsulin-binding capacity (Chart 4). Similar results were

obtained in tumors of diabetic animals at 14 days aftertransplantation. The results obtained in intact animals arealso shown in Chart 4; a rapid decrease in binding ofinsulin was obtained with 2 ID insulin after 2 hr, but theeffect was no longer significant by 21 hr. None of theabove regimens normalized urinary glucose in the diabeticrats, but glucosuria was reversed when insulin was administered in 3 consecutive doses at 45, 21, and 2 hr prior tosacrifice. Specific insulin binding in tumors from animalsreceiving the 3 doses was reduced by 40 to 45%. Thechanges in specific insulin binding in the above experiments were not accompanied by changes in nonspecificbinding. These data are consistent with others in normalcells regarding the effects of insulin on subsequent bindingof insulin and provide us with an initial time course, auseful dose regimen, and a selected time after tumorimplantation to use in future studies of regulation of insulinbinding in this mammary tumor.

Insulin Binding to Tumor Cells from Ovariectomizedand Estrogen-treated Rats. Specific '"l-labeled insulin

binding to dissociated tumor cells implanted in ovariectomized rats was significantly increased in tumors obtainedat 1, 2, 3, and 4 weeks after transplantation, with thegreatest change occurring between the first and secondweeks after implantation. Tumor cells from ovariectomizedrats bound approximately 2.5-fold more labeled insulin(binding ranged from 4.42 to 6.73 fmoles/5 x 106 cells in 6

different tumor transplantations). A single s.c. injection of1 mg estradiol valerate (a long-acting estrogen) 4 daysprior to the sacrifice of ovariectomized tumor-bearing animals caused a decrease in insulin-binding capacity (Chart

5). The most dramatic effect was seen when the estrogen





was administered 2 weeks after tumor transplantation;insulin binding was reduced by -45% of that bound in

tumors of untreated ovariectomized rats. Estrogen treatment also reduced specific insulin binding in ovariectomized tumor-bearing animals treated at 3 or 4 weeks after

tumor implantation. Similar effects on insulin binding wereseen 1 day after administration of estradiol valerate whichsuggested that the response was rather rapid. Serum insulin levels in estrogen-treated rats were unchanged from

those in ovariectomized hosts, and estrogen treatment didnot influence nonspecific insulin binding.

Effect of Diabetes and Ovariectomy on Tumor and BodyWeight, Blood Glucose, and Serum Insulin. Table 1 presents data on tumor weights relative to body weights ofintact and hormonally modified hosts. To take into accountdifferences in body weight of hormonally modified hosts(diabetic rats weighed about 20% less than intact animals,

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DAYSAFTERTUMORTRANSPLANTATIONChart 5. Effect of ovariectomy and estrogen treatment on insulin-binding

capacity of tumor cells. Dissociated cells were incubated at 20Â°with 10 10M'"l-labeled insulin for 45 min. Specific 125l-labeledinsulin binding, expressedas fmoles/5 x 106cells, was measured in relation to tumor age in untreatedintact animals, ovariectomized rats, or ovariectomized rats sacrificed 4 daysafter a single s.c. injection of 1 mg estradiol valerate. Each value is themean of at least 4 measurements Â±S.E.

and ovariectomized hosts weighed 12 to 15% more thanintact rats), we calculated a tumor index. This index, whichrepresents the average tumor weight in g/100 g bodyweight, showed that tumors transplanted into diabetic ratsdemonstrated the most rapid growth, whereas those implanted in ovariectomized rats grew at about the same rateas tumors in intact rats (Table 1). As expected, diabeticrats displayed elevated blood glucose and reduced seruminsulin levels; ovariectomy did not cause a change in theseparameters compared to intact animals.

Insulin and Estrogen Binding in Tumors from Hormonally Modified Hosts. Both estradiol- and insulin-binding

capacity were measured in cells from the same tumorsimplanted in intact, diabetic, or ovariectomized rats. Acompilation of these data is presented in Table 2, in whichthe binding capacities of tumors from intact rats werearbitrarily assigned a value of 100%. The increased insulinbinding to cells from diabetic rats was accompanied by asignificant decrease in estrogen binding (25% of control).Both insulin- and estrogen-binding capacity of tumor cells

from ovariectomized rats, however, were increased abovecontrol. When ovariectomized tumor-bearing rats weregiven injections of estradiol valerate, both estrogen- andinsulin-binding capacities decreased, although the latter

remained above the levels found in intact animals. Dataalso presented in Table 2 indicate that injection of estradiolvalerate (at the dose and time studied here) into intactanimals, did not affect insulin-binding capacity of tumor

cells.Hormone Binding in Cells Grown in Vitro. The hormone

receptor measurements presented in this study were carriedout with cells prepared enzymatically by digestion of freshlyexcised tumors with a mixture of crude collagenase andhyaluronidase. These enzymes are usually contaminatedby proteolytic enzymes (8). Since the insulin receptor islocated in the plasma membrane and can be destroyed bytrypsin and other proteolytic agents (9, 20), it was importantto investigate the possible loss in insulin binding due to

Table 1Change of body and tumor weights of intact, diabetic, and ovariectomized rats after

tumor transplantation

IntactDiabeticOvariectomizedWk

aftertrans-

plantation012301230123Bodywt(g)106

Â±1.17Â°120

Â±0.83131Â±1.91145Â±2.2698

Â±0.9696Â±4.40109Â±4.63119Â±5.23104

Â±1.34122Â±2.30151Â±1.50163Â±3.05Tumor

index(g)"0.251.833.440.312.654.960.322.133.86Blood

glucose (mg/100ml)6936578216224178658265Serum

insulin (M x

10^10)63.03.03.0<1.0<1.0<1.03.13.03.2

' Average tumor weight/100 g body weight.' Average of 2 values.' Weight means of 4 rats Â±S.E.

DECEMBER 1977 4645



S. M. Shafie et al.

thÃ©dissociation procedure. For this purpose, measurementof insulin binding was performed on cells grown in culturefor 3, 4, or 6 days, and these results were compared todata obtained with freshly dissociated cells (Table 3). Compared to freshly dissociated cells, cells grown for 3 days inthe presence of 10% fetal calf serum bound twice as much125l-labeled insulin; cells grown in the presence of 20% ratserum bound 3 times more '25l-labeled insulin than did

freshly dissociated cells. The increase in binding was notdue to a change in nonspecific binding, since nonspecificbinding was the same in all 3 cases. On the other hand, theintracellular estrogen-binding capacity was similar infreshly dissociated cells and cultured cells (Table 3). Nofurther increase in specific '"l-labeled insulin binding wasobserved in cells grown for 4 or 6 days in vitro; estrogen-binding capacity remained the same as that seen in freshlydissociated cells or in cells after 3 days in vitro.

Cultured cells exposed to 10 8 M 17ÃŸ-estradioladded to

the medium on Day 1 and, 3 hr before the cells were

Table 2

Relationship between insulin and estrogen binding in freshlydissociated tumor cells from intact, diabetic, and ovariectomized

ratsTumors were excised from animals 3 weeks postimplantation;

values presented are average from 2 to 6 experiments.

AnimalsIntactIntact

+estradicivalerateDiabeticOvariectomizedOvariectomized

+estradiolvaler

ate%

specificInsulin100"89135242169bindingEstrogen100"25196110

" Specific insulin binding was 0.4 fmole/106 cells.6 Specific estrogen binding was 6 fmoles/106 cells.

harvested, on Day 6, showed an inhibition of growth and asignificant decrease ( 20%) in the insulin binding per cell(Table 3). The addition of insulin (5 //g/ml) to the culturemedium (Medium 199 + 10% calf serum) produced anincrease ( 23%) in the 17/i-estradiol receptor content (Table 3). These data indicate that effects of estrogen andinsulin are demonstrable in vitro.

DISCUSSION

Specific insulin binding to isolated mammary tumor cellswas shown to be rapid, saturable, and reversible at 20r and

was somewhat increased by continuous shaking of theincubation mixture (80 cycles/min). The enhanced insulinbinding observed with shaking was probably due to themaintenance of a more uniform cell suspension that rendered plasma membranes more accessible to insulin. Theapparent decrease of insulin binding after 45 min could bedue to insulin and/or receptor degradation. In fact, it hasbeen shown (8) that all preparations that contain insulin-specific receptors also possess system(s) that degrade(s)insulin.

The specific insulin binding obtained with tumor cellsfrom intact, diabetic, or ovariectomized rats yielded 3 curvilinear Scatchard plots. These experiments reproducedthe results of Harmon and Hilf (15), who showed that tumorcells from diabetic rats bound more insulin than did cellsfrom intact hosts. Tumor cells obtained at 7, 14, 21, or 28days after implantation into ovariectomized rats, however,possess a specific insulin-binding capacity even higherthan those from diabetic hosts and at a level that wasgreater than twice that from intact rats. This apparentincrease in insulin binding in hormonally modified hosts isof considerable interest since it suggests a possible regulation of insulin receptors by other hormones, i.e., estrogenand progesterone. Regulation of insulin receptors by avariety of metabolic states, stages of development, and

Table 3

Insulin and estrogen binding in freshly dissociated and cultured tumor cells from intactrats

Days incultureFreshly

dissociatedcells334666Media10%

calf seruminMedium19910%


rat seruminMedium19910%



calf seruminMedium199+10

6 M170-es-tradiol10%

calf seruminMedium199+insulin,

5 pg/m\Cells

inT75

flask (x106)15212433522438Specific

binding (fmoles/106cells)Insulin

Estrogen0.46Â±0.024" 5.8 Â±0.421.00

Â±0.019 5.4 Â±0.391.42

Â±0.020 6.2 Â±0.281.04

Â±0.015 5.8 Â±0.330.98

Â±0.017 6.0 Â±0.180.80

Â±0.0217.4

Â±0.53

" Mean Â±S.E. (4 experiments).





other hormones has been proposed recently by Baref al. (1).As 1 approach to the examination of the difference in

insulin binding to tumor cells, saturation experiments wereconducted, and the results were analyzed by the method ofScatchard (24). As has been seen by others for insulinbinding analysis, a curvilinear plot was obtained for cellsfrom each experimental group; the 3 curves were roughlyparallel. Such behavior could result from more than 1 classof binding sites, which has been suggested earlier. It hasalso been proposed that Scatchard plots that demonstratedupward concave deviations from linearity could be explained by negative cooperative interactions among a singleclass of binding sites (6). Support for this explanation hascome from the studies of De Meyts ef al. (5, 6), in whichthe enhanced dissociation of bound ligand in the presenceof excess unlabeled hormone would result from the decreased affinity (increased dissociation rate constant) induced by increased site occupancy. Although the datapresented here show that excess insulin enhanced thedissociation of bound ligand from tumor cells of intact orovariectomized rats and would therefore be compatiblewith the interpretation of negative cooperativity, this interpretation has been seriously challenged in a recent paperby Rollet era/. (23), who in recent experiments with culturedlymphocytes, observed that the dissociation rate of labeledinsulin was largely independent of binding site occupancyover a 100-fold range (a range that spanned the physiological range of insulin and exceeded the physiological rangeby 1 order of magnitude), and further that enhanced dissociation could be shown under conditions in which binding-site occupancy was actually decreased. Thus, they conclude that enhanced dissociation of bound hormone byexcess hormone does not provide a demonstration of negative cooperativity for hormone-receptor systems (see alsoadditional citations in Ref. 23). While the above considerations are important for accurate assessment of bindingaffinity constants, our studies were conducted to ascertainwhether differences in insulin binding resulting from removal of the ovaries of the host were due to alterations inreceptor affinity (dissociation constant) or differences inreceptor number. We suggest from our analysis of thesaturation kinetics obtained, regardless of which hypothesis is used to explain the curvilinear behavior of the Scatchard representation, that the enhanced specific bindingof insulin to cells from ovariectomized rats was probablydue to an increase in the number of binding sites. The factthat the amount of nonspecific binding of insulin wassimilar in all 3 groups, as was the extent of ligand degradation measured during the incubation period, also rules outdifferences in binding that could be attributed to alterationsin these 2 parameters, which require consideration for theestimation of specific binding.

Our findings indicated that hypoinsulinemia (diabetes)resulted in an increased number of receptor sites availablefor insulin binding. Treatment of diabetic rats with insulin(3 injections) decreased urinary glucose excretion to normal and reduced the insulin-binding capacity of their tumors to control levels (cells from intact rats); treatment ofdiabetic or intact rats with a single dose of insulin (hyper-insulinemia) also decreased the apparent insulin-binding

capacity in their tumor cells. Such observations couldsuggest that endogenous insulin was masking availablereceptor sites (10). However, these results are more usuallyconsistent with the proposal that there is an inverse relationship between circulating insulin levels and insulin binding (3, 10). Since the insulin-binding measurements wereperformed with cells prepared by enzymatic dissociation(see "Materials and Methods"), it seemed unlikely that any

endogenously bound insulin could resist the cell isolationprocedure and, hence, remain bound to membrane receptors, i.e., masking. Certainly, it is also reasonable to expectthat insulin would dissociate from its receptor by the combined effects of 90-min shaking of the tissue in the enzymesolution at 37Â°,which rapidly reaches a pH of 6.8, followed

by several washes with 0.9% NaCI solution at room temperature.5 Insulin degradation can also take place under theseconditions by whatever mechanism(s) is involved. Previousstudies in cell culture (10) have suggested that the regulation of the insulin receptor level was insulin dependent. Onthe bases of the assumption that insulin was not translocated to an intracellular site and the reasoning presentedabove, our results seem to be consistent with a physiologically self-modulating insulin receptor model.

More than twice as much insulin was bound by tumorcells prepared from ovariectomized rats. The data presented indicate that the increased binding of insulin wasnot due to decreased nonspecific binding or to a change inthe binding sites affinity, as indicated by the similarity ofthe results obtained by analysis of the Scatchard representations. No alteration in plasma levels of insulin was observed in ovariectomized animals, which suggests an absence of the close relationship between the circulatinginsulin concentration and the estimated insulin receptorlevel (3,10). Thus, a simple self-modulating insulin receptormodel is less than adequate to explain fully the hormonalregulation of the insulin receptor.

In the studies presented with ovariectomized rats, removal of gonadal steroids was probably responsible for thesignificant increase in insulin-binding capacity in the carcinoma. Along the same lines, treatment of tumor-bearingovariectomized rats with estradiol valerate brought about adecrease in insulin binding, although the amount of insulinbound after this treatment was still significantly higherthan that of control (intact rats) tumor cells. Thus, inaddition to the probability that the selected dose of estrogen did not duplicate the endogenous level, a possibleexplanation for these results is that, after ovariectomy, amore complex hormonal change occurred, and estrogenalone could not completely reverse this effect; e.g., progesterone was also removed by ovariectomy. In fact, the mostsignificant increase in insulin binding occurred in ovariectomized rats sometime between the first and second weeksafter tumor implantation; no further change was notedafter 2 weeks. This observation suggests that the increase

5 Cells were brought to steady-state binding with 12sl-labeled insulin at20Â°.Cells were pelleted and resuspended in 1 ml of the enzyme solutionused to dissociate the tumor tissue. After a 60-min incubation at 37Â°,>90%of specifically bound '"l-labeled insulin was lost from the cells. Otherexperiments involved the injection of labeled insulin into tumor-bearinganimals prior to sacrifice. Tumor cells obtained by the methods used herehad a level of radioactivity slightly above background (less than 1% recovery).

DECEMBER 1977 4647



S. M. ShafÃ¬eet al.

in insulin binding might have been a reflection of theadjustment of the tumor tu the altered hormonal milieu ofthe host.

The comparison of specific insulin and estrogen bindingin intact and hormonally modifk 1 hosts (Table 2) revealeda possible relationship between receptor levels for the 2hormones. Removal of endogenous hormone resulted inthe elevated binding capacity of insulin in diabetic rats andof estrogen in ovariectomized rats. The latter response isprobably due to binding site vacancy since estrogen-binding capacity returned to control levels after estrogen treatment. However, although decreased, the insulin receptorlevels did not completely return to control values aftercomparable treatment with estrogen in ovariectomized rats.Plasma insulin levels were not altered by the estrogentreatment used here. This would implicate factors otherthan insulin or estrogen alone in regulating the insulinreceptor level. Another consequence of ovariectomy wasseen as an increase in overall body weight and a modestincrease in tumor weight, whereas diabetes caused a decrease in body weight but an increase in tumor weight.Thus, the change in the insulin receptor level in both casesmay be brought about by different mechanisms.

The estrogen-insulin relationships presented in thesestudies have been noted by others who studied mammarycarcinomas and other tissues. Shafie and Brooks (25) haveshown that estradiol augmented the insulin stimulatoryeffect on cell division of the human cell line, MCF-7.Estrogen-binding capacity in 7,12-dimethylbenz(a)anthra-cene-induced mammary tumors was significantly reducedin regressing and static tumors from diabetic rats (11).Furthermore, Cohen and Hilf (2) reported that estrogenand insulin, injected simultaneously, had an additive effectto retard growth of the R3230AC tumor. Treatment of ratswith estrogen increased muscle sensitivity to insulin-stimulated glucose uptake (27). It would appear that furtherinvestigation of insulin-estrogen interactions is warrantedto elucidate the role of both hormones on mammary tumorgrowth and metabolism.

Finally, results of preliminary experiments with short-term tissue culture were presented, and these indicatedthat the estrogen-binding capacity of tumor cells grown ineither calf or rat serum was unchanged. Insulin binding, onthe other hand, was significantly increased (2- to 3-fold),under in vitro culture conditions. Estrogen receptor hasbeen described as an intracellular component (19), whileinsulin is known to bind to its receptor at the surface of thetarget cell (4). The observation that insulin binding, andnot estrogen binding, was increased in cultured cells suggests that a cell membrane regeneration process occurredfollowing the enzymatic dissociation procedure. In fact,similar findings of regeneration of membrane antigen components in mouse mammary epithelial cells in culture havebeen reported (29). However, another possible interpretation of these results is that the increase in binding could bedue to differences in binding at different stages of the cellcycle. Under our tissue culture conditions, we observedthat both the estrogen receptor level and insulin-bindingcapacity were changed by addition of insulin or estrogen,respectively. These responses seen in vitro were in the

same direction as the hormonal effects observed in vivo.Since the conditions in vitro allow for a study of directhormone effects, the findings reported here m the animalare supported by the data from short-term tissue cultureexperiments with the tumor cells. However, the magnitudeof the changes in vitro was less than those m vivo and thismight be due, at least in part, to a lack of establishment ofthe proper conditions for the full hormonal effect to bemanifest. Once the proper conditions m vitro are established, the short-term tissue culture system could serve asa most useful model for the exploration of these hormonalrelationships and their regulatory mechanisms; such studies are currently in progress.

ACKNOWLEDGMENTS

The authors wish to acknowledge the competent technical assistance ofM. Terranova. J. Cech. D. Vossler. and M. Harrison and the continuingcooperation of W. Swanson and H. Oche), Animai Tumor Research Facility,University of Rochester Cancer Center (CA-11198). We wish to thank Dr W.Dulin, Upjohn Co.. for the supply of streptozotocin; Dr. M. Root. Eli LillyCo., for the gift of porcine insulin; and A. Kharroubi, Corning MedicalDiagnostics, for the gift of the Immophase radioimmunoassay kit for insulinassays. The continuing interest and advice of Dr. M. Feldstein. Departmentof Biostatistics. is greatly appreciated We wish to thank Dr J. N. Livingston,Department of Medicine, for his helpful comments.

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1977;37:4641-4649. Cancer Res Samir M. Shafie, Scott L. Gibson and Russell Hilf R3230AC Mammary AdenocarcinomaEffect of Insulin and Estrogen on Hormone Binding in the

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