proliferation and responsiveness to estrogen of human endometrial cancer cells under ... · were...

[CANCER RESEARCH 49, 3297-3301, June 15, 1989]

Proliferation and Responsiveness to Estrogen of Human Endometrial Cancer Cellsunder Serum-free Culture Conditions1

Christian F. Holinka,2 Yuzuru Anzai, Hiroki Hata, Nina Kimmel,3 Hiroyuki Ruminolo, and Erlio Gurpide

Department of Obstetrics, Gynecology, and Reproductive Science, Mount Sinai School of Medicine, New York, New York 10029 [C. F. H., Y. A., H. H., N. K., E. G.J,and Department of Obstetrics and Gynecology, Kitasato University, Kanagawa-Ken, Japan [H. H., H. KJ

ABSTRACT

Studies of hormonal growth regulation in cultured human endometrialcancer cells are limited by the requirement of exogenous growth factors,usually supplied by addition of serum. The present report providesevidence that estradiÂ»!can stimulate proliferation of endometrial cancercells of the Ishikawa line in the absence of serum or added growth factors.Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissueculture dishes, and in cells forming colonies in soft agar under anchorage-

independent conditions.Addition of estradiÂ»! to a mixture of serum-free, phenol red-free

Dulbecco's minimal essential medium and Ham's F-12 medium, supple

mented with L-glutamine and 4-(2-hydroxyethyl)-l-piperazineethanesul-

fonic acid [basal medium: (BM)] significantly increased the proliferationof cells attached to culture dishes. Dose-response experiments revealed

maximal estradiÂ»!stimulation at 10 IIM:significant responses were alsoobserved at 1 nM and at 100 nM concentrations. The mitogenic effect of10 nM estradiÂ»! was comparable to that of 1% charcoal-treated fetal

bovine serum and the two effects were additive. The presence of estradiciin serum-free BM resulted in a shortening of the doubling time of

exponentially proliferating cells from 38 to 29 h. From the labeling index,measured after exposure to a pulse of [3H]thymidine, and from the mitÂ»tic

index, both determined in exponentially proliferating cells, the lengths ofthe S and M phases were calculated to be 11 and 1 h, respectively. Fromthese data it was estimated that estradiÂ»! shortened the d phase byapproximately 40%, from 22 to 13 h.

Estradici doubled the colony formation efficiency of cells plated inBM containing 0.3% agar in the absence of serum as well as in thepresence of 1% charcoal-treated fetal bovine serum. The stimulation of

colony formation by estradici was influenced by medium components,since no effects were observed in minimal essential medium. The colonyformation efficiency was positively related to the serum concentrationsand remained significantly lower in minimal essential medium than inBM at comparable serum levels. The observed positive relationshipbetween colony formation efficiency and cell densities at plating suggestsa cooperative mitogenic effect, likely due to autocrine and paracrineaction of secreted growth factors.

These results define a model to evaluate hormonal growth regulationmediated by autocrine mitogens in human endometrial cancer cells in theabsence of interfering exogenous growth factors. They also call attentionto the importance of the choice of medium in the design of experimentsaiming to elucidate mechanisms of growth regulation.

INTRODUCTION

The growth-promoting effects of estrogens in vivo have beenwell documented both in human endometrii!in (1) and in theanimal uterus (2, 3). It is less clear, however, whether estrogensact as primary growth-promoting agents or stimulate otherfactors, which then, by themselves or in synergism with estrogens, increase the proliferation of uterine target cells. Observations in animals have led to the suggestion that proliferative

Received 5/17/88; revised 11/7/88, 2/28/89; accepted 3/20/89.The costs of publication of this article were defrayed in part by the payment

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

' This work was supported by Grant CA-1S648 awarded by the National

Cancer Institute, Department of Health and Human Services.2To whom requests for reprints should be addressed.3 Present address: Virology Unit, Ben Gurion University, Beer-Sheva, Israel.

responses to estrogens may in part be due to the action ofsubstances (estromedins) induced by estrogens at distant sites(4). On the other hand, evidence that estradiol stimulates IGF-1 expression in the immature rat uterus (5) supports the hypothesis that the growth-promoting effects of estrogens arelinked to the action of autocrine and paracrine mitogens. Amajor role for polypeptide growth factors as mediators ofestrogen-induced growth responses has recently been demonstrated in MCF-7 human breast cancer cells (6, 7).

Previous studies in Ishikawa human endometrial cancer cellsrevealed significant growth-stimulatory effects of estradiol (8,9). However, these mitogenic responses were obtained in thepresence of serum, which may contain compounds that influence or mediate the action of estradiol. The present studiesdemonstrate growth-stimulatory effects of estradiol in humanendometrial cancer cells in the absence of interfering exogenousmitogens or inhibitors under two types of growth conditions,namely, in proliferating cells attached to the substratum ofmammalian tissue culture dishes and in cells growing as colonies in soft agar.

MATERIALS AND METHODS

The origin of Ishikawa human endometrial cancer cells and theirmaintenance in culture have previously been described in detail (8). Atleast two weeks before these experiments were started, the cells wereplaced into phenol red-free MEM4 (GIBCO) and 15% fetal bovine

serum (GIBCO) which had been treated with ctFBS to remove estrogens(8). The cells were kept under phenol red-free conditions to avoid thereported estrogenic effects of this indicator dye (10). Experiments wereperformed in MEM or in a basal medium in the absence of serum oradded growth factors. BM consisted of a phenol red-free mixture (1:1,v/v) of Dulbecco's minimum essential medium and Ham's F-12 medium

(prepared by Flow Laboratories), supplemented with 10 HIML-glutamine, 15 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, anda 1% antibiotic-antimycotic solution (GIBCO) to give a final concentration of 100 units/ml penicillin, 100 Mg/ml streptomycin, and 0.25Â¿ig/mlFungizone.

To evaluate proliferation rates of cells growing attached to mammalian culture dishes, cells were harvested by brief exposure to trypsin-EDTA and plated into 6-cm dishes (Falcon) in the absence of serum orin the presence of 1% ctFBS for 1 day, as indicated for each experiment.Experiments in MEM were always performed in the presence of serumat the indicated concentrations, since this medium poorly supportedgrowth under serum-free conditions.

Detailed procedures for establishing and maintaining cultures in softagarr have previously been reported (9). Briefly, bottom layers, consisting of 0.6% agar (Difco Bacto-Agar), were prepared in 6-cm culturedishes (Falcon) in serum-free BM or MEM, as required for eachexperiment. Cells suspended in media containing the specified experimental components and 0.3% agar were placed on the bottom layers.During these procedures, cells were maintained at or below 37Â°Cand

were not exposed to test compounds at concentrations exceeding thefinal levels specified for each experiment. Fresh nutrient media containing the test compounds were added twice a week. In the standard

4The abbreviations used are: MEM, minimum essential medium containingEarle's salts; BM, basal medium; ctFBS, charcoal-treated fetal bovine serum.

'' Unpublished data.

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ESTRADIOL-STIMULATED GROWTH OF ENDOMETRIAL CANCER CELLS

protocol, 50,000 cells were seeded/dish in order to evaluate the effectsof different media, of serum at different concentrations, and of estradicion colony formation.

In experiments designed to test the effects of density on colonyformation efficiency, cells were seeded at densities ranging from 10,000to 100,000/dish, as indicated. Colonies greater than 50 Â¿imin diameterwere counted under an inverted phase contrast microscope in 20 fields/dish, representing an area of 56.8 mm2. Three dishes were evaluated

for each experimental condition. The colony formation efficiency wasexpressed as the number of colonies formed/100 cells seeded. Significance was calculated by the Student's / test.

To determine the labeling index, cells maintained in MEM plus 15%ctFBS were exposed during the exponential growth phase to [3H]-thymidine (specific activity, 78.8 Ci/mmol) in serum-free MEM for 70min at 37Â°C,washed in Hanks' balanced salt solution containing 0.6

HIMthymidine, harvested by exposure to trypsin-EDTA, and spread onmicroscope slides. After drying, the cells were briefly fixed in phosphate-buffered formalin and subjected to autoradiography as previouslyreported (11). The fraction of labeled cells was determined after stainingwith safranin O. An experiment designed to compare labeling indicesobtained in cells that had been formalin fixed and processed in situ inculture dishes with cells transferred to microscope slides revealed nodifferences, indicating that no differential cell loss had occurred duringthe transfer.

The metaphase index was estimated in exponentially growing cellsmaintained in MEM plus 15% ctFBS. Cells were exposed to colchicine(5 drops; 16 Mg/ml) in 2 ml culture medium/6-cm dish for 30 min at30Â°C.The cells were harvested by exposure to trypsin-EDTA, pelleted,

and 1 ml calf serum, followed by 5 ml of a 0.55% KC1 solution, wereadded under continuous gentle mixing. After 30 min at 37"C in the

above solution, the cells were fixed in a cold methanohacetic acidmixture at the following volume ratios, 5:1 (twice); 3:1 (twice), and 1:1(once), for 30 min each. The cells were then transferred to microscopeslides and stained with buffered Giemsa stain.

RESULTS

Proliferation of Cells Attached to Substratum of MammalianTissue Culture Dishes. As illustrated in Fig. 1, Ishikawa cellsare capable of proliferating under serum-free culture conditionsand in the absence of polypeptide growth factor supplementation. Addition of estradiol to the culture medium 1 day afterseeding, designated as day 0, significantly increased cell num-

12

o

Â§6

0 Controlâ€¢¿�Ez(IOnM)1 1% serumA E2 + 1%serum

0'-i o 369DAYS OF TREATMENT

12

Fig. 1. Effects of estradiol on Ishikawa cell proliferation. Cells (0.5 million/6-cm dish) were seeded in basal medium in the presence of 1% charcoal-treatedFBS. After 1 day in culture (day 0), medium was replaced by BM containing theindicated experimental compounds. Estradiol {/â€¢â€¢¿�!(10 UMi was added in ethanolto give a final alcohol concentration of 0.1%. Control cultures received ethanolonly. Medium was renewed every 2 days. On the indicated days, cells from 3dishes for each experimental group were counted in a hemacytometer under aninverted light microscope. Bars, SD.

bers, as determined at the indicated intervals during an 11-dayperiod after the start of treatment. The effects of estradiol (10nM) were comparable to those of 1% ctFBS. The effects ofestradiol and of serum at those concentrations were additive.

Fig. 2 presents data from an experiment designed to determine the influence of estradiol on proliferation rates of exponentially growing cells. During the period of exponential increase in cell numbers, in which all cells are considered to beproliferating, estradiol significantly decreased the intervalneeded to double the number of cells (Â¡2)relative to controls (P< 0.05) when the regression lines fitting log cell numbers at theend of days 1, 2, and 3 for each group were evaluated by theStudent's t test. The cell cycle length of exponentially prolifer

ating cells was calculated by using the relationship f2 = In 2/k,where k is the slope of In n = flj). The cell cycle length ofIshikawa cells in BM was thus estimated to be 38 h and wasshortened to 29 h in the presence of estradiol.

As shown in Fig. 3, the growth-promoting effects of estradiolwere maximal at 10 nM concentrations; significant effects werealso observed at 1 HM and at 100 nM levels. It should be

1234

DAYS OF TREATMENTFig. 2. Estrogen effects on exponential growth rates of Ishikawa cells. Cells

were seeded, maintained, and counted as described in the legend to Fig. I. Theproliferation rate of cells in the presence of estradiol (E2) was significantly greaterthan that of the control group (P< 0.05), corresponding to a decrease in the cellcycle length from 38 to 29 h. Bars, SD. (Significance of the differences of theregression lines fitting the log cell numbers on each day was evaluated by theStudent's t test.)

0 20 40 60 80 100

CELL NUMBERS (% ABOVE CONTROL)

Fig. 3. Dose-dependent stimulation of Ishikawa cell proliferation by estradiol.Cells (0.5 million/6-cm dish) were seeded in serum-free basal medium. On day 0,cells were exposed to the indicated levels of estradiol in a final ethanol concentration of 0.1%. Control cultures received ethanol only. On the indicated days, thecell numbers of 3 dishes in each treatment group were determined as described in"Materials and Methods." All values differed significantly from the control groupsby at least P < 0.05, as evaluated by the Student's / test.

3298




emphasized, however, that the net hormone concentrationsduring the 2-day interval between medium changes were likelyto be considerably lower than the initial levels, since estradiol,added to culture media of Ishikawa cells at 10 DMconcentrations, was found to be nearly completely converted to estradiolsulfate after 24 h (12).

Table 1 presents a summary of all experiments performed toexamine growth responses to 10 nM estradiol, including experiments carried out in the absence of serum even during theplating period (Table 1, A), in the presence of 1% ctFBS duringplating, followed by serum-free conditions throughout theperiod of hormone treatment (Table 1, A), and during the entireplating and culture period (Table 1, C). As evident from theseresults, the estrogen response was highly reproducible.

The cells in the experiments presented in Fig. 3 and Table 1,A, were plated in serum-free medium and were maintained inthe absence of serum throughout the period of estradiol treatment. These culture conditions minimize the possibility ofresidual serum effects on growth and hormonal responsiveness.To further exclude a possible action of long-lasting serumfactors on growth and estradiol responsiveness, we seriallysubcultured Ishikawa cells under serum-free conditions for 8passages over a period of 3 months. Estradiol, added to serum-free medium in passage 9, significantly increased cell numbers,namely, control: 3.2 x IO6;estradiol: 6.0 x IO6, after 7 days oftreatment (P < 0.05, as determined by the Student's t test).

Colony Formation in Soft Agar. Fig. 4 provides evidence fordensity-dependent increases in colony formation efficiency ofIshikawa cells in two different media. Colony formation efficiency doubled in both MEM and BM containing 1% ctFBS asthe numbers of cells seeded were increased from 50,000 to100,000. A 10-fold difference in the numbers of plated cells,that is, 10,000 versus 100,000 cells seeded/dish, tripled thecolony formation efficiency in BM.

The data presented in Fig. 5 suggest a relationship betweencolony formation efficiency and the concentration of growth-promoting factors present in serum. Colony formation efficiency in BM doubled over a serum concentration range from0 to 5%; in MEM it increased 8-fold when the serum concentration was elevated from 1 to 15%. Interestingly, MEM failedto support colony formation under serum-free conditions.

Fig. 6 documents the estrogen responsiveness of Ishikawacells under anchorage-independent conditions in BM. As illustrated in this figure, addition of estradiol doubled the numberof colonies in BM in the absence of serum as well as in thepresence of 1% ctFBS, yet no responses to estradiol wereobtained in MEM. It is noteworthy that significant differencesin colony formation efficiency were also apparent when BMand MEM were used at comparable serum concentrations, thatis, the numbers of colonies were four times greater in BM thanin MEM at 1% serum (Figs. 5 and 6).

Labeling and Metaphase Indices. When 2315 cells from randomly chosen areas of 6 slides were examined, 1011 cells werefound to have been labeled during the 70-min exposure to [3H]-

thymidine. The length of the S phase can be approximated bythe product of the fraction of labeled cells and the cell cyclelength (13), previously shown to be 27 h in MEM containing15% ctFBS (9), i.e., (1011/2315) x 27 h = 11.5h.

The fraction of cells in metaphase, determined by evaluationof randomly selected areas from 5 slides, was 103/2603 cells.The length of the M phase may then be estimated as the productof the fraction of cells in metaphase and the length of the cellcycle (13), i.e., 0.0395 x 27 h = 1.06 h.

These data provide a basis for evaluating the mitogenic effects

Table 1 Estrogen effects on Ishikawa cell growth

Control Estradiol (10 nM)Days after values (cell effects on cell

Experiment start of numbers in nos. (% ofno. treatment millions) control)Significance

ofdifference

from control(Student's t

test)A.

Cells plated in BM and cultured inBM1

692

593

2574

12357B.

Cells plated in BM+5

258116

2468107

258118

123457109

3712C

Cells plated in BM+10

2581111

2581112

246g103.26.82.87.81.02.03.70.50.81.63.75.4166128121128110160130139159164Â»145195*<0.005<0.05NSÂ°<0.02NS<0.02<0.05NS<0.01<0.01<0.005<0.001l%ctFBS

and cultured inBM0.8r1.9e3.0e1.8e0.61.42.23.12.60.82.02.43.00.7"1.1"1

7"2.03.43.28.21.85.16.1100142158244157157138158152116145158173114127155155150163141122161136l%ctFBS

and cultured in BM+0.82.55.43.90.83.14.86.00.82.03.44.63.6111180167172156165150182100126160170290NS<0.05<0.05<0.01<0.01<0.01<0.05<0.05<0.01NS<0.05<0.005<0.02NSNS<0.005<0.02<0.005<0.02<0.05NS<0.01<0.02I%ctFBSNS<0.01<0.01<0.02<0.02<0.005<0.05<0.01NSNS<0.01<0.01<0.01

Â°NS, not significant.* These values also appear in Fig. 3.' These values also appear in Fig. 1.Â¿These values also appear in Fig. 2. The regression lines fitting log cell

numbers at the end of days 1, 2. and 3 were significantly different for the controland estradiol groups (P< 0.05; Student's t test).

of estradiol at the level of the cell cycle. By using the abovevalues, namely, 11 h and l h for the length of the S and Mphase, respectively, and an estimated 3 h for the length of theG: phase (14), the length of the (., phase in exponentiallygrowing cells can be calculated from the relationship

Gy = Ã•T- (is + Ã•M+ Ã•GÂ¡)

3299



ESTRADIOL-STIMULATED GROWTH OF ENDOMETR1AL CANCER CELLS

50 100 10 50 100

CELLS PLATED (thousands)Fig. 4. Effects of cell density on colony formation efficiency of Ishikawa cells.

Cells were seeded into 6-cm culture dishes at the indicated densities in MEM orBM containing 0.3% agar in the presence of 1% ctFBS, as described in "Materialsand Methods." Bars, SD. The asterisks indicate significant differences relative tothe next lower density. *, P< 0.05; ",P< 0.01. The colony formation efficienciesbetween BM and MEM were significantly different at P < 0.01 for 50,000 cellsand P < 0.001 for 100,000 cells.

c/> 5000 hLuâ€¢¿�z.

3 4000oo

0 3000rr1 2000r>

1000

o

MEM BM

_L

EH

O I 5 15 O I 5 15% SERUM

Fig. 5. Effects of serum concentration on colony formation of Ishikawa cells.Cells were seeded as described in "Materials and Methods" at a density of 50,000/6-cm dish in MEM or BM containing 0.3% agar and ctFBS at the indicatedconcentrations. Bars, SD. The asterisks denote significant differences relative tothe next lower serum concentration. **,P< 0.01; ***,/>< 0.001. The differencesbetween BM and MEM were significant (/' < 0.01) in cultures containing 1%ctFBS and in cultures containing 5% ctFBS.

where iT is the total period of the cell cycle, estimated to be 29h for cells in the presence of estradiol and 38 h for controlcultures, as described above. The appropriate calculations indicate that addition of estradiol to exponentially proliferatingcultures of Ishikawa cells reduced the d phase by approximately 40%, from 22 to 13 h. In these calculations it is assumedthat during exponential, nonsynchronous proliferation cells arerandomly distributed in the mitotic (M), postmitotic (GÃ¬),synthetic (S), and postsynthetic (Gj) phases of the cycle and thatthe number of quiescent cells arrested in Go is negligible. It isfurther assumed that the likelihood of a cell to be at a givenphase of the cell cycle is directly related to the length of that

6000-

y socoz

0 4000oÂ° 3000LU

1 2000

1000

0

MEM BM

I Control

JE2(IOnM)

15 0% SERUM

Fig. 6. Stimulation by estradici (Â£2)of colony formation of Ishikawa cells.Cells were seeded as described in "Materials and Methods" at a density of 50,000/6-cm dish in MEM or BM containing 0.3% agar in the presence of the indicatedserum concentrations. Estradiol was added in ethanol to give a final concentrationof 10 nM estradici and 0.1% vehicle. Control cultures received ethanol only. Bars,SD. *** Significantly different from control values at P < 0.001. The controlcultures in BM, 1% ctFBS differed from those in MEM, 1% ctFBS at P < 0.01.

phase, that the S, 62, and M phases are relatively constant, andthat changes in proliferation rates are primarily due to alterations in the Gr phase of the cell cycle (2, 13).

In this context it is relevant to emphasize similar findings inthe mouse uterus, which revealed that estrogen treatment ofimmature or ovariectomized animals greatly shortened the generation time of epithelial cells, an effect which the authorsdemonstrated to be principally due to a significant reduction inthe length of the d phase of the cell cycle (2).

DISCUSSION

Addition of serum or specific growth factors to the mediumis usually required for the proliferation of cultured cells. Thisreport presents a model for evaluating growth responses tohormonal stimulation in human endometrial cancer cells without the interference of serum mitogens or added growth factors.Our data provide evidence for rapid exponential growth ofIshikawa cells in a serum-free basal medium and demonstratethat colony formation in soft agar in BM does not requireexogenous mitogens.

In this completely defined medium Ishikawa cells exhibitedsignificant responses to estradici, both when attached to culturedishes and under anchorage-independent conditions in softagar. These data, as well as our observations of a positiverelationship between density of cells after plating and colonyformation efficiency, support the hypothesis that the cells secrete growth factors which may act as autocrine and paracrinemitogens, and that growth factor secretion is stimulated byestrogen.

The increases in cell numbers over control levels producedby addition of estradici were comparable to those achieved by1% serum, although the putative autocrine mitogens secretedby Ishikawa cells under the influence of estradiol are likely todiffer quantitatively and qualitatively from those present inserum. Direct measurements of secreted proteins are likely toreveal one or several compounds with growth-promoting activity, as have been demonstrated in response to estrogens inMCF-7 cells (15, 16).

The mitogenic effects of estrogen on Ishikawa cells under3300




serum-free culture conditions contrast with those reported forthe human breast cancer cell line MCF-7, where the hormone

has been considered to promote proliferation by neutralizingone or several inhibitory factors present in serum, but has noeffect on cells cultured in serum-free media (17). Such differences in growth regulation among different cell lines are likelyto reflect distinct phenotypes of cells transformed from estrogen-responsive target tissues.

A surprising observation emerging from these studies concerns the influence of the culture medium on growth responsesto estrogens as well as to serum. This point is illustrated by thefinding that MEM failed to support colony formation in theabsence of serum, while in the presence of comparable serumconcentrations the colony formation efficiency was substantially lower in MEM than in BM. Furthermore, estradiol didnot affect the colony formation efficiency of cells maintainedin MEM, regardless of serum concentration, whereas the hormone caused a significant increase in the number of colonies inBM with or without serum. In this context it is also relevantthat cells growing attached to culture dishes attained markedlyhigher densities in BM than in MEM in the presence of comparable serum concentrations.5 Stimulatory effects of hydrox-

ytamoxifen on Ishikawa cell proliferation have also been foundto be different in MEM and BM. In phenol red-free MEM,

hydroxytamoxifen acted as a classical antiestrogen when addedto cultures in the presence of estradiol but did not exhibitindependent growth-promoting effects, whereas in BM it stim

ulated cell growth to a greater extent than did estradiol butfailed to act as an antiestrogen (18). Although specific mediumfactors that mediate the responsiveness to hormones and toserum mitogens remain to be identified, our data call attentionto the importance of medium components in establishing anadequate research model to evaluate hormone-regulated mech

anisms of growth control.The maximum colony formation efficiency, achieved at a

lower serum concentration in BM than in MEM, did not exceed10%. This observation suggests the presence of a finite subpop-ulation endowed with colony-forming properties in the parent

cell population. The colony formation efficiency was increasedto 22% when dispersed cells from colonies were further subcul-tured in soft agar for two passages.5 In contrast, the proportion

of cells attaching to culture dishes and subsequently proliferating was usually 80%. The biological properties of cells activelyproliferating when attached to the substratum of mammalian

tissue culture dishes, but not capable of forming colonies underanchorage-independent conditions remain to be elucidated.

ACKNOWLEDGMENTS

We wish to thank Drs. Beth Schachter and Fred Gilbert for valuableadvice on autoradiographic methods and on procedures to determinethe labeling and mitotic indices.

REFERENCES

1. Dallenbach-Hellweg, G. Histopathology of the Endometrium, pp. 55-86.New York: Springer-Verlag, 1987.

2. Quarmby, V. E., and Korach, K. S. Influence of 17/S-estradiol on patterns ofcell division in the uterus. Endocrinology, 114: 694-702, 1984.

3. Walters, M. R. Steroid hormone receptors and the nucleus. Endocr. Rev., 6:512-543, 1985.

4. Ikeda, T., and Sirbasku, D. A. Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus. J.Biol. Chem., 259: 4049-4061, 1984.

5. Murphy, L. J., Murphy, L. C., and Friesen, H. G. Estrogen induces insulin-like growth factor-1 expression in the rat uterus. Mol. Endocrinol., Â¡:445-450, 1987.

6. Dickson, R. B., and Lippman, M. E. Estrogenic regulation of growth andpolypeptide growth factor secretion in human breast carcinoma. Endocr.Rev.,Â«:29-43, 1987.

7. Rochefort, H., Capony, F., Garcia, M., Cavailles, V., Freiss, G., ChambÃ³n,M., Morisset, M., and Vignon, F. Estrogen-induced lysosomal proteasessecreted by breast cancer cells: a role in carcinogenesis? J. Cell. Biochem.,55:17-29, 1987.

8. Holinka, C. F., Hata, H., Kuramoto, H., and Gurpide, E. Responses toestradiol in a human endometrial adenocarcinoma cell line (Ishikawa). J.Steroid Biochem., 24: 85-89, 1986.

9. Holinka, C. F., Hata, H., Gravanis, A., Kuramoto, II.. and Gurpide, E.Effects of estradiol on proliferation of endometrial adenocarcinoma cells(Ishikawa line). J. Steroid Biochem., 25: 781-786, 1986.

10. Berthois, v.. Katzenellenbogen, J. A., and Katzenellenbogen, B. S. Phenolred in tissue culture media is a weak estrogen: implications concerning thestudy of estrogen-responsive cells in culture. Proc. Nati. Acad. Sci. USA, 83:2496-2500, 1986.

11. Holinka, C. F., and Gurpide, E. Ornithine decarboxylase activity in humanendometrium and endometrial cancer cells. In Vitro (Rockville), 21: 697-706, 1985.

12. Hata, H., Holinka, C. F., Pahuja, S. L., Hochberg, R. B., Kuramoto, H., andGurpide, E. Estradiol metabolism in Ishikawa endometrial cancer cells. J.Steroid Biochem., 26:699-704, 1987.

13. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D.Molecular Biology of the Cell, pp. 613-616. New York: Garland Publishing,Inc., 1983.

14. Baserga, R. The Biology of Cell Reproduction, p. 18. Cambridge, MA:Harvard University Press, 1985.

15. Huff, K. K., Kaufman, D., Gabbay, K. H., Spencer, E. M., Lippman, M. E.,and Dickson, R. B. Secretion of an insulin-like growth factor-1-relatedprotein by human breast cancer cells. Cancer Res., Â¥6:4613-4619, 1986.

16. Dickson, R. B., Huff, K. K., Spencer, E. M., and Lippman, M. E. Inductionof epidermal growth factor-related polypeptides by 17/3-estradiol in MCF-7human breast cancer cells. Endocrinology, 118: 138-142, 1986.

17. Soto, A. M., and Sonnenschein, C. The role of estrogens on the proliferationof human breast tumor cells (MCF-7). J. Steroid Biochem., 23:87-94, 1985.

18. Anzai, Y.. Holinka, C. F., Kuramoto, H., and Gurpide, E. Stimulatory effectsof 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells (Ishikawa line). Cancer Res., 49: 2362-2365, 1989.

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1989;49:3297-3301. Cancer Res Christian F. Holinka, Yuzuru Anzai, Hiroki Hata, et al. Endometrial Cancer Cells under Serum-free Culture ConditionsProliferation and Responsiveness to Estrogen of Human

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