[CANCER RESEARCH 41, 5004-5009, December 1981]0008-5472/81 70041-OOOOS02.00

Effects of 17/f-Estradiol on Progesterone Receptors and the Uptake ofThymidine in Human Breast Cancer Cell Line CAMA-11

Winston C. Y. Yu, Benjamin S. Leung,2 and Y. L. Gao

Hormone Research Laboratory, Department of Obstetrics and Gynecology, University of Minnesota. Minneapolis, Minnesota 55455

ABSTRACT

Two sublines, CAMA-1 R and CAMA-1 N, were derived fromthe original CAMA-1 cells after continuous passages in our

laboratory. 1R cells were supplemented in culture with estradioland retained the properties of the original cell line, whereas 1Ncells, not supplemented with estradiol, had a partial loss ofestrogenic responsiveness. The doubling times for 1R and 1Ncells were 2 and 2.4, respectively. These two sublines showeddifferent effects on thymidine uptake after estradiol stimulation.Cells of 1R had a 2-fold greater thymidine incorporation than

did the control without estradiol. In contrast, no difference ofthymidine uptake was demonstrated in cells of 1N in thepresence or absence of estradiol. Both cell lines containedcytoplasmic progesterone receptors with a Kd of 0.56 nw anda sedimentation coefficient of 6 to 7S on a 5 to 20% sucrosegradient. The progesterone receptor level in 1N was about 8-fold higher than in 1R cells. Both sublines exhibited a dose-

dependent increase in progesterone receptor levels in response to estradiol with the maximal stimulation (8-fold increase) reached by using 5 nmol of estradiol. The rate ofincrease for estradiol-induced progesterone receptor appears

to be the same for both sublines; a linear increase was demonstrated between Days 3 and 7 following plating. Our resultsshow that estrogen regulation of thymidine uptake and progesterone receptor induction may be mediated through two separate mechanisms. Thus, following a chronically deficient supplyof estradiol to 1N cells, there is apparent loss of responsivenessto estradiol-induced thymidine uptake, but the ability of these

cells to respond to estradiol induction of progesterone receptorwas retained.

INTRODUCTION

Breast cancer-derived cell lines are plausible model systems

for studying the mechanisms of steroid action in tumors because of a more homogeneous cell population and a betterbiochemically and hormonally defined culture condition. Several cell lines from human breast tumors have been derived forthis purpose (5, 9, 11, 21, 25, 29). Among these cell lines, afew contain estradiol receptors (3, 9, 12, 18) and have beenshown to be estradiol responsive (9, 18). These cell lines areMCF-7, ZR-75-1, ZR-75-27, and ZR-75-30. In addition, estradiol can induce PRC3 in MCF-7 cells (14).

Received April 9. 1981; accepted September 10. 1981.1This investigation was supported by Grant 1RO1-CA 25998 and BRSG DC

3842 0685-5202, awarded by the National Cancer Institute, Department ofHealth, Education, and Welfare. A preliminary report of this work was presented(30).

2 To whom requests for reprints should be addressed, at Box 395, Mayo

Building, Department of Obstetrics and Gynecology. University of Minnesota,Minneapolis, Minn. 55455.

3 The abbreviations used are: PRC, cytoplasmic progesterone receptor; FBS.

fetal bovine serum; MEM. minimal essential medium; DC, dextran-coated char-

We have adopted an established cell line, CAMA-1, as our

model for the investigation of hormonal action and interactionin breast cancers (17). CAMA-1 is a non-Hela-contaminated

cell line originated from pleural effusion of a postmenopausalwhite female with adenocarcinoma of the breast (10). Thesecells appear morphologically as epithelial-like cells. Karyotypestudy revealed a chromosome number of 78.4 CAMA-1 cells

are also hormone dependent and show increased incorporationof thymidine, uridine, and leucine into cells upon estradiolstimulation (17). Following continuous passage in the laboratory, the original cell population lost its ability to respond toestradiol-induced thymidine uptake. An early frozen passage

(passage 35) was reestablished in the laboratory and cultured2 ways. One subline, cultured in the original condition, lost itsability to respond to estradiol when thymidine uptake waschecked 22 passages later and is designated as CAMA-1 N;the other subline, cultured identically except with the additionof 1 nM estradiol to the medium, retains the original CAMA-1cell properties and is designated as CAMA-1 R. In this report,

we described the responses of these 2 sublines to estradiolstimulation on thymidine uptake and PRC levels.

MATERIALS AND METHODS

Chemicals. [6,7-3H]Progesterone (4-pregnene-3,20-dione; 55.7 Ci/mmol), [meffty/-3H]thymidine (20 Ci/mmol), and unlabeled R5020

(17,21 -dimethyl-10-nor-4,9-pregnadiene-3,20-dione) were obtainedfrom New England Nuclear (Boston, Mass.). The purity of [3H]proges-

terone was checked by using thin-layer chromatography on silica gel.Impure [3H]steroids were further purified on Celile and Sephadex LH-

20 chromatographies. 17/8-Estradiol, progesterone, cortisol (4-preg-nene-11/3,17a,21-triol-3,20-dione), dihydrotestosterone (5a-andros-tan-17/3-ol-3-one), 11/}-hydroxyprogesterone (4-pregnene-11/8-ol-3,20-dione), and 20/8-hydroxy-4-pregnene-3-one were purchased fromSteraloids, Inc. (Wilton, N. H.). Medroxyprogesterone acetate (4-preg-nene-3,20-dione-17-(acetyloxy)-6o-methyl) was bought from The Upjohn Company (Kalamazoo, Mich.). Tris-(hydroxymethyl)aminomethanehydrochloride, dithiothreitol, activated charcoal, diethylstilbestrol, an-drostenedione (4-androstene-3,17-dione), and triamcinolone aceto-nide were purchased from Sigma Chemical Co. (St. Louis, Mo.). Aldo-sterone (4-pregnene-11/},21-diol-3,18,20-trione) was obtained from

Ikapharm (Ramat Gan, Israel). EDTA was bought from Fisher ScientificCo. (Pittsburgh, Pa.). Dextran T-70 was purchased from Pharmacia

Fine Chemicals (Uppsala, Sweden), and sucrose was obtained fromMallinckrodt Chemical Works (St. Louis, Mo.). Culture medium, FBS,trypsin, L-glutamine, and antibiotics were purchased from Grand Island

Biological Company (Grand Island, N. Y.).Cell Culture. CAMA-1 is a human breast cancer cell line which was

kindly supplied to us in living culture at passage 32 by Dr. J. Fogh ofSloan-Kettering Institute for Cancer Research. CAMA-1 cell line, tested

to be free of Mycoplasma, has been maintained and cultivated as

coal; DCFBS. dextran charcoal-treated fetal bovine serum; TCA, trichloroaceticacid.

* J. Fogh. personal communication.

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monolayers in MEM with Earle's salts and supplemented with 2 mM L-

glutamine, penicillin (100 units/ml), streptomycin (100 /ig/ml), and25% FBS. Cells were plated in MEM at a density of 106 cells in a T-75

flask without further change of medium for 7 days and were incubatedin a humidified atmosphere of 95% air-5% CO2 at 37°.At the end of 7

days, cells were lifted from the surface of the flask by trypsin-EDTA(0.025%) in Hanks' balanced salt solution without CA2+ and Mg2+.

Two sublines of cells, designated as CAMA-1N and CAMA-1R, werederived from the original CAMA-1 cells. Both sublines were cultivated

as monolayers in the same growth medium as described. In addition,a supplement of 1 UM estradici was added during plating to 1R cells ineach passage. In experiments involving cell responsiveness to estradici, free steroids were removed from FBS by DC. This is done routinelyby incubating FBS with DC (0.5% Norit A charcoal plus 0.05% DextranT-70) for 60 min at 50°.A trace amount of [3H]estradiol was added to

the serum to monitor the effectiveness of the procedure. Normally,after 2 incubations with DC, radioactivity remaining in the serum wasnegligible.

Thymidine Uptake. Cells growing in log phase were plated in T-25flasks at a density of 5 x 10" cells per 5 ml of medium per flask in

15% DCFBS. Various concentrations of estradici in MEM were addedat the time of plating. After 48 hr of growth, cells were pulsed with[3H]thymidine (0.5juCi/flask) for 2 hr. Cells were then rinsed twice with

NaCI solution (0.15 M), harvested by trypsinization onto glass fiberfilters (Reeve Angel 934AH; Whatman, Inc., Clifton, N. J.), and washedwith another 10 ml of 0.15 M NaCI solution, and the radioactivity wasdetermined. [3H]Thymidine uptake in these cells was also measured

following the method of Allegra and Lippman (1). In this procedure,after harvesting, the pulsed cells were centrifuged, resuspended inwater, and lysed by sonication (15 sec at medium setting; Artek SonicDismembrator; Artek Systems Corp., Farmingdale, N. Y.). Proteins andnucleic acids were then precipitated by using 10% TCA. The acid-

insoluble materials were collected and washed on glass fiber filters asdescribed for the previous method. Radioactivity in either whole cellsor TCA-precipitated materials was counted in plasmasol (28) by a

Packard scintillation counter with an overall efficiency of 29% fortritium.

DC Assay. For the determination of PRC, cells were harvested byscraping into Hanks' balanced salt solution, resuspended in 10 mM

Tris-1 mM EDTA-1 mw dithiothreitol-10% glycerol (pH 7.4) buffer, andkept at 0-4° for the remaining procedure. They were then homogenized

in a glass-Teflon Dounce homogenizer (Kontes) until more than 90% of

the cells were shown by phase microscope to be disrupted. Cytosolwas collected after centrifugation at 110,000 x g for 60 min andadjusted to a protein concentration of about 4 mg/ml. To obtain totalbinding, aliquots of cytosol were incubated with 2.5 nM [3H]progester-one-250 nw cortisol at 4°for 4 hr. Radioinert progesterone (250 nM)

was used as competitor to assess nonspecific binding. Bound and free[3H]progesterone was separated by the addition of 0.5 ml of DCsuspension and incubated for 10 min at 4°.Free steroids bound to DC

were removed by centrifugation for 10 min at 3200 rpm with a BeckmanJ-6 centrifuge. Specific binding was calculated by subtracting nonspe

cific binding from total binding. Protein content in cytosol was determined by the method of Lowry ef al. (19).

Sucrose Density Gradient Sedimentation. Cytosol, containing steroid-receptor complexes obtained by the DC procedure, was transferred to the top of a 5 to 20% sucrose density gradient in 10 mM Tris-1 mM EDTA-1 mM dithiothreitol-10% glycerol (pH 7.4). '"C-labeled

bovine serum albumin, which sedimented at about 4.6S, was used asan internal marker. Centrifugation was done in an SW60 rotor with aBeckman L2 658 ultracentrifuge at the speed of 53,000 rpm for 17 hr.At the end of the run, tubes were pierced at the bottom, and thegradient was collected into fractions by gravity. Radioactivity of eachfraction was then determined.

Scatchard Analysis. Aliquots of cytosol were incubated with increasing amounts of [3H]progesterone (0.5 to 5.0 nM) and cortisol to assesstotal binding. An additional set of aliquots with [3H]progesterone was

Estradici Effect on CAMA-1 Cells

incubated with 100-fold excess of unlabeled progesterone to determine

the nonspecific binding. The results were calculated according to themethod of Scatchard as modified by Edsall and Wyman (6).

RESULTS

CAMA-1 N and CAMA-1 R cells showed different growth rates

under their cultured conditions (Chart 1). Both sublines of cellswere in log phase of growth for 10 days. When cultured in 25%FBS, CAMA-1 R cells exhibited a faster growth rate than that ofthe CAMA-1 N cells. The cell-doubling times for 1R and 1N

cells were 2 and 2.4 days, respectively. Morphologically, the2 cell lines were basically alike with the exception that 1N cellswere more spherical in appearance and were trypsinized morereadily (data not shown).

A striking difference in [3H]thymidine incorporation in re

sponse to estradiol stimulation was observed between the 2sublines. In CAMA-1 R cells, 0.1 nw of estradiol stimulated an86% increase of [3H]thymidine uptake into whole cells over

controls without exogenous estradiol (Chart 2). This increasereached 138% when 10 nM of estradiol was supplemented tothe 1R cells. The estradiol stimulatory effect, however, was notdetected in CAMA-1 N cells under the same experimental con

ditions. Higher concentrations of estradiol had an adverseeffect on [3H]thymidine uptake and cell growth in both cell lines

(data not shown). When 10 ¡IMof estradiol were added to themedium, both lines of cells appeared sick, and most of themdied. The actual [3H]thymidine incorporation into macromole-

cules for these 2 cell lines was also detected by the TCAprecipitation method (Chart 3). Significant stimulation by estradiol was exhibited by 1R cells but not by 1N cells.

PRC. The PRC level was determined to further assess theestradiol responses of these 2 cell lines. Chart 4 shows thetime course of PRC appearance in CAMA-1 N cells in thepresence or absence of 5 nwi estradiol. During the first 2 daysof plating, PRC content decreased slightly. Without the additionof estradiol, levels of PRC remained at a low level throughoutthe next 7 days. However, estradiol caused a linear increase in

200

6Days

10

Chart 1. Cell growth rate of CAMA-1 N and CAMA-1 R cells. Cells were platedin T-25 flasks in the regular growth medium containing 25% FBS. Cells weretrypsinized and harvested each day, and the cell numbers were determined by aCoulter Counter. •,CAMA-1 R cell line supplemented with 1 nM estradiol; O,CAMA-1 N cell line. Points, mean of 3 determinations. The linear regressioncoefficients for the rate of growth were calculated to be 1.5 and 1.2 for 1R and1N cells, respectively.

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W. C. Y. Yu et al.

8Ü5?<S=

6T~

4zx-CAMA-1Ni-=t-i~rgE3-QC

.0CL1'S-rrO

8^CAMA-IRz

?L^,JCOC<o6Q

(0•-s:s'-r-'n¿'04_-X

3 --T-i«0t__

10°

E2,MChart 2. Effect of estradici (E,) on [3H]thymidine ([3H]TDRìuptake in CAMA-

1N and 1R cells. CAMA-1N or CAMA-1R cells were plated in T-25 flasks inmedium containing 15% DCFBS. Different amounts of estradici were also addedduring plating. After 48 hr of growth, cells were pulsed with [3HJthymidine for 2

hr, harvested by trypsinization, and washed twice with 0.15 M NaCI solution, andthe radioactivity was determined. Each value represents the mean of 3 determinations; bars, S.E.

Chart 3. Effect of estradici (E2) on [3H]thymidine uptake on CAMA-1N and

CAMA-1R cells using 2 different techniques in estimating incorporation. In A, thesame procedure as that described in Chart 2 was used; in B. after the cells wereharvested, they were resuspended in water and then lysed by sonication. Theacid-insoluble (10% TCA) materials were collected onto filters, and the radioactivity was determined. Each value represents the mean of 3 determinations; bars,S.E. Stimulation by estradici for 1R cells was statistically significant ( p < 0.05).

the PRC level from Days 3 to 7. Based on this result, the rateof increase was approximately 40 fmol per mg protein per dayor 1.2 fmol per 106 cells per day. Since the maximal estradiol

response on PRC was reached on the seventh day, all subsequent experiments were carried out with 7-day-old cells.

When PRC of CAMA-1 N cells cultured in DCFBS were ex

amined in a 5 to 20% sucrose density gradient, the majority ofradioactivity was found close to the top of the gradient, suggesting a lower-molecular-weight form of PRC (Chart 5). This

PRC form was competitively displaced by unlabeled progesterone. When cultured in the presence of 5 nM estradiol, a discrete6 to 7S component was also observed. The PRC could besaturated by increased levels of progesterone (Chart 6, inset),and maximal binding was reached at 2.5 nM [3H]progesterone.

A Kd of 0.56 nM was calculated by the Scatchard analysis(Chart 6).

The competition by other steroids for PRC in CAMA-1 cells

is reported in Table 1. All the compounds with progestin effect,

eDAYS

Chart 4. Daily PRC (PWc) values during a 9-day growth period. Cells culturedin 15% DCFBS from 2 T-150 flasks were harvested on each of the assigneddays for each group. Cytosol was collected, and the levels of PRC were determined as described in 'Materials and Methods." •.CAMA-1 N cells plated with

the presence of 5 nM estradiol (£2).Medium was changed to 15% DCFBS withoutestradiol 48 hr before harvest time. O, CAMA-1 N cells plated in 15% DCFBSthroughout the period. Bars, S.E.

600 -

500 •

400 -

Q.O

Tolol Binding,Total Binding, CAMA-INNon-specific Binding CAMA-IN

300 -

200 •

FractionsCharts. Sucrose density gradient profile of PRC in CAMA-1 N cells. Cells

cultured in 15% DCFBS for 7 days from 4 T-150 flasks were harvested, and theirPRC sucrose density gradients were done according to "Materials and Methods. ' '

•,CAMA-1 N cells plated in 15% DCFBS containing 5 nM estradiol (L,) Cytosolfrom these cells was incubated with 2.5 nM [3H]progesterone and 250 nM cortisol.

O, CAMA-1 N cells plated in 15% DCFBS. Aliquots of cytosol from these cellswere incubated with 2.5 nM [3H]progesterone and 250 nM cortisol. •cytosolfrom CAMA-1 N cells incubated with 2.5 nM [3H]progesterone, 250 nM cortisol,

and 250 nM progesterone. BSA. bovine serum albumin.

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Estradiol Effect on CAMA-1 Cells

for example, R5020, progesterone, and medroxyprogesteroneacetate, displaced 100% [3H]progesterone. Similarly, 2 of theprogesterone metabolites, 11 /?-hydroxyprogesterone and 20/6-hydroxy-4-pregnene-3-one, were also inhibitory. In contrast,aldosterone, cortisol, diethylstilbestrol, dihydrotestosterone,and 17/S-estradiol only displaced [3H]progesterone to a limited

degree.Effect of Estradiol on PRC Levels in CAMA-1 N and CAMA-

1R. CAMA-1 N and CAMA-1 R cells are shown to possessdifferent amounts of PRC (Chart 7). After growing for 7 days in15% DCFBS, 1N cells contained 79.8 ± 11.5 fmol PRC permg of cytosol protein, whereas the 1R cells had only 10 ±2.5fmol (Chart 7). Both cell lines exhibited a dose-related increase

of PRC in response to estradiol stimulation, and both were

.16

.14

.04 O6Bound nM

O8 IO

Chart 6. Scatchard plot and the saturation curve of [3H]progesterone bindingin CAMA-1N cell cytosol. Estradiol-stimulated cells were harvested after 7 daysof growth. Cytosol was collected, and the bound steroids and ratio of bound tofree were calculated as described in "Materials and Methods." Inset, specificbound [3H]progesterone C[3H/-P)plotted against the total tracer used in each

incubation.

Table 1

Competition of PRC binding sites by various steroids

Steroid Inhibition (%)

ProgesteroneR502011/?-Hydroxyprogesterone20/3-Hydroxy-4-pregnene-3-oneMedroxyprogesterone acetateCortisolDexamethasoneAldosteroneDiethylstilbestrol17/J-EstradiolDihydrotestosteroneAndrostenedioneTriamcinolone acetonide

1001009268

100107

122

1539

079

C

Ä"o

¿"5IOo>N

Oo>

_o>'O

PR

CAM A-INCAM A-IR

ru WhIO'11 5X10-9

[EJM

IO'8

a All these steroids were supplied at concentrations of 100-fold excess (250

Chart 7. Dose effect of estradiol (£2)induction of PRC (PRC) levels in CAMA-1N and CAMA-1 R cells. Estradiol-stimulated CAMA-1 N and CAMA-1 R cells wereharvested after 7 days of growth. PRC levels in cytosol were measured accordingto the DC method as described in "Materials and Methods." Each value repre

sents the mean of 3 determinations; bars, S.E.

maximally stimulated at 5 nM estradiol. At this estradiol concentration, PRC levels in 1N and 1R cells were 614 ±15 fmoland 79.8 ±11 fmol, respectively, an increase of approximately8-fold over the levels without estradiol. Higher concentration of

estradiol had a cytotoxic effect resulting in lower levels of PRC(data not shown).

DISCUSSION

Estrogen regulates growth and cellular events of some breastcancers in animals and humans. In the last 2 decades, mucheffort has been spent in understanding its action in cancers.Since the hormonal environment in vivo is complicated, the useof established cell lines cultured in chemically defined conditionis appealing. Several breast cancer cell lines have been established for this purpose (5, 9, 11, 21, 25, 29). We reportedpreviously that CAMA-1 cells responded to estradiol, antiestra-

diol, progesterone, and prolactin in the uptake of precursorsfor proteins, RNA, and DMA (17). This report shows 2 sublinesof CAMA-1 ; each exhibits a distinct growth rate and a different

response to estradiol stimulation.CAMA-1 N cells, under the present experimental conditions,

were not stimulated by estradiol in [3H]thymidine uptake. Partial

restoration of this response to estradiol was achieved followingcontinued culture in 1 nM estradiol-supplemented medium for

3 consecutive passages (data not shown). 1R cells, however,showed a dose-related estradiol stimulation of [3H]thymidine

uptake. Maximal stimulation was achieved from 1 to 10 nMestradiol similar to results reported previously for the originalCAMA-1 cells (17). Similar results were also reported for MCF-

7 cells by other investigators (18).[3H]Thymidine uptake into whole cells may be due to one or

a combination of events such as increased influx of [3H]thymi-

dine, increase in thymidine kinase activity, increased incorporation of [3H]thymidine into DNA, increase in S-phase cell

population, or increase in cell division. From both whole-celland TCA precipitation experiments, the estradiol-induced increase of [3H]thymidine appears to involve direct incorporation

into DNA molecules. From other experiments in our laboratory,we also observed an estradiol-induced increase in cell growth

(data not shown). Since all these experiments were performed

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W. C. Y. Yu et al.

in the presence of serum, the possibility that estradiol-inducedcell growth or[3H]thymidine incorporation into DMA is mediated

through serum growth factors cannot be ruled out (23, 24).Both sublines contained PRC which was specific for proges-

tins and was not competitively inhibited by androgen, gluco-

corticoid, and estradiol for progesterone binding capacity. Allthese characteristics are common to all PRC in other targettissues. PRC content in CAMA-1 cells is lower than that reported for MCF-7 and T47D cells (15). Similar to MCF-7 cells,CAMA-1 cells also contain estradiol receptors (17), androgen,and glucocorticoid receptors.5

PRC has been considered as an end product of estradiolaction in many target tissues such as the chick oviduct (22),hamster uterus (16), human endometrium (2), and breast cancer cells (14). We also demonstrated that PRC levels in bothsublines were stimulated by estradiol. In the absence of estradiol, the majority of radioactivity is in the lower-molecular-

weight form. However, besides an induction of the lower molecular species, estrogen markedly stimulates an increase ofthe 6 to 7S PRC. It cannot be determined from these resultswhether or not estrogen stimulation of PRC represents a denovo biosynthesis of these macromolecules. Our data suggestthat, besides the above-mentioned induction, estrogen mightbe responsible for inducing a non-steroid binding component

of the 6 to 7S receptor. Alternatively, estrogen may induce acoupling enzyme needed for PRC subunits assembly. In orderto ascertain that this observation is not a technical artifact dueto a possible increase of protease activity in the cytosol whencells are not cultured with estrogen, 20 nM molybdate wereincluded in th.e PRC assay system to stabilize the high-molecular-weight receptor molecule (results not shown). The result

was no different, at least qualitatively, from that assayed without molybdate.

Although 1N cells have lost responsiveness to estradiol-induced [3H]thymidine uptake under our present experimental

conditions, this subline contained about 8-fold-higher levels ofPRC than did 1R cells. Nevertheless, estradiol effected thesame rate and dose-related increase of PRC in both sublines.Thus, at maximal stimulation with 5 nM estradiol, an almost 8-

fold increase over control level was noted in both 1N and 1Rcells. In experiments with 1N cells, following the initial decreasein the first 3 days, estradiol induced a linear increase ofapproximately 40 fmol of PRC per mg protein per day or 1.2fmol per 106 cells per day from Days 3 to 7 following plating.

The reason for the long lag time in PRC induction in responseto estradiol in vitro cannot be explained from our experimentalresults. Possibly, PRC induction may be mediated by a differentmechanism from that of the normal estradiol-induced proteinsynthesis, which is mediated through transcriptional events(20). A longer lag time may also be observed if PRC synthesiswas modulated by an inducer which is regulated by estradiol.Using MCF-7 cells as a model, Horwitz and McGuire (14)

indicated that PRC induction is related to estradiol receptortranslocation to the nucleus and its subsequent processing.Experiments are in progress to determine if such a relationshipexists in CAMA-1 cells.

The partial loss of estradiol responsiveness in CAMA-1 Ncells was also reported in other model systems. For example,in some breast tumors induced by dimethylbenzanthracene in

1B. S. Leung and W. C. Y. Yu, unpublished results.

rats, sustained growth of tumors following castration was observed; PRC contents in these postcastration tumors, however,were markedly reduced (13). In MCF-7 cells, the estradiol-stimulated [3H]thymidine uptake observed by some investiga

tors (18, 26) was not noted by others (8). The loss of estradiol-

regulated cell growth in these tumor cells did not jeopardizethe responsiveness of the cells to estradiol induction of PRC(14) or lactate dehydrogenase (4). From our own data and thatof others, it appears that 2 different estradiol-modulated mechanisms are operating to induce PRC and [3H]thymidine uptake.

In conclusion, we have presented results demonstrating thatCAMA-1 cells are estradiol responsive in PRC induction and[3H]thymidine uptake. CAMA-1 R cells retained both responses,

but 1N cells exhibited an apparent loss of responsiveness inestradiol-induced [3H]thymidine uptake. A chronically deficient

supply of estradiol to estradiol-dependent cell populations may

eventually lead to a partial and temporal loss of estradiolresponsiveness. Our results also suggest that PRC inductionand [3H]thymidine uptake may be regulated by estradiol via 2

different mechanisms.

ACKNOWLEDGMENTS

We thank Ann Stolee and Ofrey Deleon for their skillful technical assistance.We also thank Dr. J. Fogh of Sloan-Kettering Institute of Cancer Research for hissupply of the CAMA-1 cell line.

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-Estradiol on Progesterone Receptors and theβEffects of 17

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