[CANCER RESEARCH 42, 5147-5151, December 1982]0008-5472/82/0042-OOOOS02.00

Drug and Hormone Sensitivity of Estrogen Receptor-positiveand -negative Human Breast Cancer Cells in Wfro1

Gerald J. Goldenberg2 and Evelyn K. Froese

Manitoba Institute of Cell Biology [G. J. G., E. K. F.], and Department of Medicine, University of Manitoba [G. J. G.I, Winnipeg, Manitoba R3E OV9, Canada

ABSTRACT

A clonogenic assay of long-term breast cancer cell cultures

in vitro has been developed to provide a highly reproduciblemethod with which to quantitate tumor cell killing by hormonesand/or cytotoxic chemotherapeutic agents. Monolayer cultures of estrogen receptor-positive MCF-7 human breast cancer cells and of estrogen receptor-negative Evsa T cells are

harvested by treatment with 0.01% trypsin:0.02% EDTA inHanks' balanced salt solution. Cell suspensions are treatedwith drug or hormone in serum-free medium for 1 hr at 37°;

treated cells are washed, plated, and cultured for approximately 14 days; and colonies consisting of >30 cells arecounted. Compared to estrogen receptor-positive cells, estrogen receptor-negative cells were 2-fold more sensitive to mel-phalan but were conversely 1.9-fold more resistant to Adria-

mycin; these differences were statistically significant (p <0.001). Thus, response to cytotoxic chemotherapeutic agentsappeared to be independent of estrogen receptor status.

For cells treated with diethylstilbestrol, the dose of drug orhormone reducing the surviving cell fraction to 1/e (D0) forestrogen receptor-positive cells was 2.27 nmol/ml, and thatfor estrogen receptor-negative cells was 2.80 nmol/ml; thisdifference was not statistically significant. However, with ta-moxifen therapy, the D0 for estrogen receptor-positive cellswas 0.601 nmol/ml, and that for estrogen receptor-negativecells was 3.64 nmol/ml; this 6-fold greater degree of resistanceto tamoxifen of estrogen receptor-negative cells was highlysignificant (p < 0.001). Treatment of cells for 24 hr with 17/3-estradiol stimulated proliferation not only of estrogen receptor-positive cells but also of estrogen receptor-negative cells.

However, estradici at concentrations up to 200 ¡J.Mhad noapparent cytocidal activity, as measured by the clonogenicassay. Furthermore, treatment of MCF-7 cells simultaneously

with estradiol and either diethylstilbestrol or tamoxifen failed toreverse the cytocidal activity of those two agents. These findings suggest that, in the clonogenic assay described herein,diethylstilbestrol and tamoxifen may kill human breast cancercells by an independent mechanism of action and that thecytocidal activity of diethylstilbestrol and the proliferative effectof 17/S-estradiol appear to be independent of estrogen receptor

status.

INTRODUCTION

The tumors in approximately 60% of patients with breastcancer contain a cytoplasmic receptor that binds estradiol with

a high affinity and in a chemically specific manner (13, 15).The presence of ER3 correlates well not only with the biological

behavior of the tumor (6) but also with the response of thetumor to endocrine manipulation; approximately 60% of ER-

positive tumors are responsive to such therapy compared withonly 5 to 10% of ER-negative tumors (12, 15).

Lippman (9) has argued that the presence of ER may alsopredict the response of the tumor to cytotoxic chemotherapy,since ER-positive tumors are much less likely to respond thanare ER-negative tumors. Others have reported that either nosuch correlation exists (16, 19), or conversely that ER-positivetumors are more responsive to chemotherapy than are ER-

negative tumors (5). In this study, we report the developmentof a clonogenic assay of long-term breast cancer cell cultures

that provides a highly reproducible method with which to quantitate tumor cell kill by hormones and/or cytotoxic chemotherapeutic agents. The assay also provides an opportunity toinvestigate more directly the mechanism of tumor cell kill byhormones and antiestrogens.

MATERIALS AND METHODS

Drugs and Chemicals. Melphalan (Alkeran) was kindly provided asa gift by Dr. J. R. MacDougal, Burroughs Wellcome and Co., Ltd.,Lachine, Quebec, Canada. Adriamycin (doxorubicin hydrochloride)was obtained from Adria Laboratories of Canada, Mississauga, Ontario,Canada. DES was purchased from Sigma Chemical Co., St. Louis, Mo.,and tamoxifen citrate [frans-(p-dimethylaminoethoxyphenyl)-1,2-di-

phenyl-but-1-ene] was kindly provided by Stuart Pharmaceuticals, Di

vision of ICI United States, Inc., Wilmington, Del.Cell Lines and Cultures. MCF-7, a cloned ER-positive cell line

originally established at the Michigan Cancer Foundation, Detroit,Mich., from a malignant pleural effusion in a female patient withmetastatic breast cancer (17), was generously provided by Dr. RobertShiu, Department of Physiology, University of Manitoba. Evsa T, anER-negative cell line which was established by Dr. Marc Lippman,National Cancer Institute, Bethesda, Md., from a malignant asciticeffusion from a female patient with metastatic breast carcinoma (10),was kindly provided by Dr. Peter Lam, Manitoba Institute of CellBiology. Both cell lines were grown in monolayer cultures in Corningtissue culture flasks in Eagle's MEM [Grand Island Biological Co.,

Grand Island, N. Y.] supplemented with glutamine (0.6 g/liter), penicillin (100 units/ml), streptomycin (100 fig/ml), and 10% FBS (GrandIsland Biological Co.). Cells were grown at 37°in a humidified atmos

phere containing 6% CO2 in air.Clonogenic Assay. Stock cultures of MCF-7 or Evsa T cells in

exponential growth phase were harvested by treatment with 0.01%crystalline trypsin (Grand Island Biological Co.):0.02% EDTA in calcium- and magnesium-free Hanks' balanced salt solution (Grand Island

Biological Co.) at 37°for 3 to 5 min. The cell suspension was centri-

1This work was supported by a grant from the National Cancer Institute of

Canada.2 To whom requests for reprints should be addressed, at 100 Olivia Street,

Winnipeg, Manitoba R3E OV9. Canada.Received April 19, 1982; accepted September 10, 1982.

3 The abbreviations used are: ER, estrogen receptor; DES, diethylstilbestrol;

FBS, fetal bovine serum; MEM, minimal essential medium; DCC, dextran-coatedcharcoal; D0, dose of drug or hormone reducing the surviving cell fraction to1/e.

DECEMBER 1982 5147

on June 13, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

G. J. Goldenberg and E. K. Froese

fuged at 1000 rpm for 5 min in a Sorvall GLC-2 centrifuge, and the cellpellets were resuspended in serum-free MEM at an approximate concentration of 2 x 105 cells/ml. Cell suspensions were treated with

either drug or hormone, which was added to the suspension in a 1:100dilution, and was incubated in a shaking water bath at 37° for 1 hr.

The drug or hormone treatment was terminated by addition of an equalvolume of ice-cold MEM, and the cells were promptly centrifuged at

1000 rpm for 5 min. The cells were washed twice with cold MEM toremove residual drug or hormone and were resuspended in MEMcontaining 10% FBS stripped of endogenous hormone by a 45-minincubation at 50° twice with DCC as described previously (14). The

cells were counted with an electronic particle counter (Model ZBiCoulter Counter; Coulter Electronics, Inc., Hialeah, Fla.) and werediluted in MEM containing DCC-stripped 10% FBS. Treated cell sus

pensions were seeded at a density ranging from 100 to 100,000 cells/well in sextuplícate in 9.6-sq cm Linbro multiwell tissue culture plates

(Flow Laboratories, Inc., Mississauga, Ontario, Canada) and wereincubated for approximately 14 days at 37°in a humidified atmosphere

containing 6% CO? in air. The colonies, which consist of >30 cells,

were fixed with absolute methanol, stained with 2% Giemsa stain(Fisher Scientific Co., Fair Lawn, N. J.), and counted over a backgroundgrid using an inverted microscope (Fig. 1).

The plating efficiency for untreated MCF-7 cells ranged from 75 to

95%, and that for Evsa T cells ranged from 25 to 50%. The cloningefficiency of treated cells was determined at each drug concentrationand the surviving cell fraction was calculated. Linear regression analysis of each dose-survival curve was obtained, and the D0 was derived

from the negative reciprocal of the regression slope as describedpreviously (1).

ER Assay. The ER content of MCF-7 and Evsa T cells was monitoredperiodically using minor modifications of the DCC assay (7, 8). Cellsprepared for binding studies were grown as noted above but wereincubated in serum-free medium overnight before harvesting in order

to deplete estrogen receptors of endogenously bound steroid. Theseassays were performed in the laboratories of Dr. Lome J. Brandes and

Dr. Peter Lam, Manitoba Institute of Cell Biology. Over a period of 1year considerable variability in ER content was noted, particularly inMCF-7 cells; however, the distinction between ER-positive" and ER-negative" status was maintained. ER levels for MCF-7 cells ranged

from 35 to 173 fmol/mg of cytosol protein, and that for Evsa T cellsvaried from 2 to 8 fmol/mg of cytosol protein.

RESULTS

Dose-Survival Curves of ER-positive and -negative HumanBreast Cancer Cells Treated with Melphalan. Dose-survivalcurves of ER-positive and -negative cells treated with mel-

phalan are shown in Chart 1. The D0 for Evsa T cells treatedwith melphalan was 2.02 nmol/ml, and that for MCF-7 cellswas 4.16 nmol/ml. ER-negative Evsa T cells were 2-fold moresensitive to the cytocidal action of melphalan than were ER-positive MCF-7 cells.

Dose-Survival Curves of ER-positive and -negative HumanBreast Cancer Cells Treated with Adriamycin. Dose-survival

curves of cells treated with Adriamycin are presented in Chart2. The Do for Evsa T cells treated with Adriamycin was 0.365nmol/ml, and that for MCF-7 cells was 0.194 nmol/ml. Contrary to the finding with melphalan, Evsa T cells were 1.9-foldmore resistant to Adriamycin than were MCF-7 cells.

Dose-Survival Curves of ER-positive and -negative HumanBreast Cancer Cells Treated with DES. The survival of MCF-

7 cells and of Evsa T cells treated with DES for 1 hr is shownin Chart 3. The D0 for ER-positive cells was 2.27 nmol/ml, andthat for ER-negative cells was 2.80 nmol/ml. The difference

was not statistically significant.Dose-Survival Curves of ER-positive and -negative Human

Breast Cancer Cells Treated with Tamoxifen. Dose-survivalcurves of MCF-7 cells and of Evsa T cells exposed to tamoxifenfor 1 hr are illustrated in Chart 4. The D0 for MCF-7 cells was

Fig. 1. A photograph of 1 of the 6 wells in a Linbro multiwell tissue cultureplate illustrating macroscopic colonies of MCF-7 human breast cancer cells after12 days in culture. The colonies consisting of 30 or more cells were fixed withabsolute methanol, stained with 2% Giemsa, and counted as described above.The actual area of the well is 9.6 sq cm.

O

idoo

1.0

0.1

0.01

0.001

O 10 20

[MELPHALAN]>jMChart 1. Dose-survival curves of MCF-7 human breast cancer cells (O) and

Evsa T cells O treated with melphalan for 1 hr at 37°,as described in the text.

Each point represents the mean of 12 to 18 determinations; the confidenceintervals were too small to be illustrated The linear regression equation for MCF-7 cells was

logey = -0.240X + 3.95 x 10 3

with a correlation coefficient of —¿�0.989,and that for Evsa T cells was

log„y = -0.495* - 0.202

with a correlation coefficient of —¿�0.957.A t test comparing the significance ofthe difference of slopes was highly significant (p < 0.001).

5148 CANCER RESEARCH VOL. 42

on June 13, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Sensitivity of Human Breast Cancer Cells in Vitro

1.0

<

o0.01

0.001

1.50 0.5 1.0

[ADRIAMYCIN]>jM

Chart 2. Dose-survival curves of MCF-7 cells (O) and Evsa T cells O treatedwith Adriamycin for 1 hr at 37°,as described in the text. Each point represents

the mean of at least 6 determinations; the confidence intervals were too small tobe illustrated. The linear regression equation for MCF-7 cells was

log. y - -5.17x + 0.286

with a correlation coefficient of -0.985, and that for Evsa T cells was

log. y - -2.74X + 0.138

with a correlation coefficient of —¿�0.980.A ( test comparing the significance ofthe difference of slopes was highly significant (p < 0.001).

0.0001 •¿�

40

[DES]pM

Chart 3. Dose-survival curves of MCF-7 cells (O) and Evsa T cells O treatedwith DES for 1 hr at 37°. as described in the text. Each point represents the

mean of at least 6 determinations; bars, S.E.; at some points, the confidenceintervals were too small to be illustrated. The linear regression equation for MCF-7 cells was

log. y = -0.440* + 8.18

with a correlation coefficient of -0.966. and that for Evsa T cells was

log. y = -0.358X + 4.61

with a correlation coefficient of —¿�0.959.A ( test comparing the significance ofthe difference of the slopes was not statistically significant.

0.601 nmol/ml, and that for ER-negative cells was 3.64 nmol/ml. Unlike the response to DES, ER-negative cells were 6-foldmore resistant to tamoxifen than were ER-positive cells, andthe difference was highly significant (p < 0.001 ).

Effect of Estradici on MCF-7 Cells. Treatment of MCF-7

10 20

[TAMOXIFEN] pM

Chart 4. Dose-survival curves of MCF-7 (O) and Evsa T cells O treated withtamoxifen for 1 hr at 37°, as described in the text. Each point represents the

mean of at least 6 determinations; oars, S.E.; the confidence intervals were toosmall to be illustrated. The linear regression equation for MCF-7 cells was

log»y = -1.67X + 8.74

with a correlation coefficient of -0.991, and that for Evsa T cells was

log. y - -0.275X -I- 3.42

with a correlation coefficient of -0.979. A f test comparing the significance of

the difference of slopes was highly significant (p < 0.001).

cells for 1 to 24 hr with 17/S-estradiol at concentrations of

£200 /iM had no apparent cytocidal effect as measured by theclonogenic assay. An increase in the number of MCF-7 cells

was noted 24 hr after treatment at various concentrations ofestradici (Table 1); this apparent stimulation of cell proliferationby estradiol has been reported previously for ER-positive cell

lines (10). However, apparent stimulation of proliferation of theER-negative Evsa T cells was also observed 24 hr after expo

sure to estradiol (Table 1).Using estradiol, attempts were undertaken to rescue MCF-7

cells from the clonogenic-inhibitory effects of DES and tamox

ifen. Cells were treated simultaneously either with DES andestradiol or with tamoxifen and estradiol, and dose-survival

curves were compared to those obtained for cells treated withDES or tamoxifen alone. The survival curves of MCF-7 cells

treated with DES alone or DES and estradiol were identical(Chart 5/4); similarly, no difference was noted in the survival ofcells treated with tamoxifen alone compared to those treatedwith tamoxifen and estradiol (Chart 5ß).

DISCUSSION

This study describes clonal growth of long-standing culturesof human breast cancer cell lines using a single-layer cell

culture technique. A high cloning efficiency varying from 75 to95% for MCF-7 cells and from 25 to 50% for Evsa T cells has

been attained. Using this procedure, a quantitative assay hasbeen developed to investigate the mechanism of cytocidalactivity of drugs and hormones on ER-positive and -negative

human breast cancer cell lines.In a retrospective study, Lippman ef al. (9) evaluated the

DECEMBER 1982 5149

on June 13, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

G. J. Goldenberg and E. K. Froese

Table 1Stimulatory effect of 17-ß-estradiol on the growth of MCF-7 and Evsa T human

breast cancer cells in vitroMCF-7 or Evsa T cells were plated in 75-sq cm Corning tissue culture flasks

in MEM containing 10% FBS and were allowed to attach overnight. Estradiol wasadded in fresh MEM containing either 0.5 or 10% FBS, and the cells werecultured for an additional 24 hr. Cell number was determined using a Coulterelectronic particle counter.

No. of cells x 1oVflask

MCF-7 Evsa Ttstradioi concen

tration(flM)0

022

2020010%

FBS16.2

27.827.124.825.60.5%

FBS13.3

27.629.418.712.910%

FBS5.52

8.678.71

10.608.100.5%

FBS3.54

5.854.556.177.63

1.0

0.01

0.001

10 20 30

[DES]>iM

« 7 8 9

[TAMOXFEN].uM

Chart 5. A, dose-survival curves of MCF-7 cells treated with DES alone (O) orwith DES and 40 JIM 17/5-estradiol (•)for 1 hrat 37°.Each point represents the

mean of 6 determinations; confidence intervals were too small to be illustrated.The linear regression equation for cells treated with DES alone was

log. y = -0.262* + 2.31

with a correlation coefficient of -0.942, and that for cells treated with DES +estradiol was

log. y = -0.245X + 1.61

with a correlation coefficient of -0.986. A f test comparing the significance ofthe difference of the slopes of regression lines was not statistically significant. B,dose-survival curves of MCF-7 cells treated with tamoxifen alone (O) or withtamoxifen and 10 ;uM17/î-estradiol(•),for 1 hr at 37°.Each point represents the

mean of 6 determinations; confidence intervals were too small to be illustrated.The linear regression equation for cells treated with tamoxifen alone was

log. y = -2.38x -I- 12.49

with a correlation coefficient of -0.998, and that for cells treated with tamoxifen

+ estradiol was

log.y- -2.37X+ 12.26

with a correlation coefficient of -0.946. A t test comparing the significance of

the difference of the slopes of the regression lines was not statistically significant.

relationship between ER status and the response rate to cyto-

toxic chemotherapy in 70 patients with metastatic breast cancer. The study concluded that ER status was an importantpredictor of chemotherapeutic response since 34 of 45 ER-

negative patients responded to chemotherapy, whereas only 3of 25 ER-positive patients had objective remissions. However,

in a study of 143 patients with advanced breast cancer at the

University of Minnesota, the response rate to chemotherapywas 86% in ER-rich tumors, which was significantly higher thanthe response rate of 36% in ER-poor tumors (5). In this study,the ER-negative Evsa T cell line was significantly more sensitiveto melphalan than was the ER-positive MCF-7 cell line (Chart1), but conversely, Evsa T cells were more resistant to Adria-mycin than was their ER-positive counterpart (Chart 2). Thisfinding suggested that response to cytotoxic chemotherapy asdetermined by the clonogenic assay appeared to be independent of ER status. Furthermore, a review of several clinicalreports in the literature indicated that 53% of ER-positive and54% of ER-negative subjects responded favorably to chemo

therapy (Table 2).The sensitivity of ER-positive and -negative breast cancer

cells to DES was identical (Chart 3). This observation togetherwith the inability of equimolar concentrations of estradiol toreverse or protect cells from the growth-inhibitory effect of DESsuggest at least 2 possible interpretations: (a) that the mechanism of cell kill of DES as measured by the clonogenic assayin this study is ER independent; and (£>)that the observedcytotoxicity is "nonspecific" and of questionable biological

relevance.Although the data do not permit a clear choice between

these 2 options, we favor the first interpretation. Quantitatively,the cytocidal effect of DES appears to be pharmacologicallyrelevant. With 1 hr of DES treatment, the D0 was 2.27 nmol/mlfor MCF-7 cells and 2.80 nmol/ml for Evsa T cells. Plasma

levels of DES, as determined by radioimmunoassay, in patientswith prostatic cancer receiving 1 mg DES 3 times/day, rangedfrom 0.15 to 6 ng/ml or 0.56 to 22 nM (4). Patients with breastcancer have been treated with as much as 15 to 20 mg DESdaily (2), so that plasma DES levels of 3.7 to 149 mvi would bea realistic expectation. In evaluating the pharmacological significance of these dose-survival studies, the product of drug

concentration x treatment time is particularly pertinent. If oneextrapolates and theoretically extends the treatment time from1 to 24 hr, then the "corrected" D0 for DES treatment of MCF-

7 and Evsa T cells would be 0.095 and 0.117 nmol/ml,respectively (or 95 and 117 nw). Thus, it would be reasonableto assume that such plasma levels would be clinically attainablein patients receiving 15 to 20 mg DES daily.

In this study, it is unclear why the cytocidal action of DES onbreast cancer cells should be ER independent, particularlysince the correlation between ER positivity and response toendocrine manipulation is clinically well established (12, 13,15).

Unlike the response to DES, the ER-positive MCF-7 cell linewas 6-fold more sensitive to tamoxifen than were Evsa T cells(Chart 4). Although this finding suggests a possible correlation

Table 2Response of patients with ER-positive and -negative breast cancer to cytotoxic

chemotherapy

ReferenceLJppman

et al. (9)Jonat and Maass (3)Kiang ef al. (5)Webster et al. (19)Samai ef al.(16)TotalsResponders/no.

ofpatientsER-positive

ER-negative3/25

34/456/14 20/28

24/28 13/3612/20 17/4512/2015/2957/107(53%)

99/183(54%)

5150 CANCER RESEARCH VOL. 42

on June 13, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Sensitivity of Human Breast Cancer Cells in Vitro

between ER positivity and tamoxifen sensitivity in human breastcancer cells, the inability of estradici to block tamoxifen-related

cytotoxicity suggests an independent mechanism of action fortamoxifen and estrogen analogs. The identical response of ER-positive and -negative cell lines to DES, compared to the 6-fold

difference in response to tamoxifen, also suggests separatemechanisms of action. Evidence for separate receptors forestrogen and tamoxifen in MCF-7 breast cancer cells has beenpresented recently by comparing properties of a tamoxifen-

resistant variant, R27, with those of the parent cell line (14).The concentration range of tamoxifen used in the dose-

survival curves was similar to that used by others in studies ofhormone effect on cell proliferation and thymidine incorporation(14). However, the treatment time in our study was restrictedto 1 hr, whereas others have exposed cells to tamoxifen fortime intervals as long as 15 days (10, 14).

Finally, estradiol-induced stimulation of cell proliferation ofboth ER-positive and ER-negative breast cancer cells was

observed 24 hr after treatment (Table 1). Although this phenomenon has been described for MCF-7 cells (10, 11, 14), ithas not been observed previously with ER-negative cells suchas MDA-231 (18). Of interest was the observation that estra-

diol, at concentrations up to 200 JUMwith 24 hr of treatment,had no apparent antitumor activity on either cell line as measured by the clonogenic assay. This contrasts with the"nonspecific" inhibition of thymidine incorporation into DMA of

breast cancer cells reported by others with /ÕMconcentrationsof estradiol (10).

ACKNOWLEDGMENTS

The authors thank Drs. Lome J. Brandes and Peter H. Y. Lam for helpfuldiscussions and Dorothy Faulkner for typing the manuscript.

REFERENCES

1. Goldenberg, G. J., Lyons, R. M., Lepp, J. A., and Vanstone, C. L. Sensitivityto nitrogen mustard as a function of transport activity and proliferative ratein L5178Ylymphoblasts. Cancer Res., 37. 1616-1619, 1971.

2. Ingle, J. N., Ahmann, D. L., Green, S. J., Edmonson, J. H., Bisel, H. F.,Kvols, L. K., Nichols, W. C., Creagan. E. T., Hahn, R. G.. Rubin. J., andFrytak, S. Randomized clinical trial of diethylstilbestrol versus tamoxifen inpostmenopausal women with advanced breast cancer. N. Engl. J. Med.,

304. 16-20, 1981.3. Jonat. W.. and Maass, H. Some comments on the necessity of receptor

determination in human breast cancer Cancer Res., 38. 4305-4306. 1978.4. Kemp, H. A., Read, G. F., Riad-Fahmy, D., Pike. A. W.. Gaskell, S. J.,

Queen. K., Harper, M. E., and Griffiths, K. Measurement of diethylstilbestrolin plasma from patients with cancer of the prostate. Cancer Res.. 41: 4693-4697, 1981.

5. Kiang, D. T.. Frenning, D. H., Goldman. A. I., Ascensao, V. F., and Kennedy,B. J. Estrogen receptors and responses to chemotherapy and hormonaltherapy in advanced breast cancer. N. Engl. J. Med., 299. 1330-1334,1978.

6. Knight. W. A., Livingston, R. B., Gregory. E. J.. and McGuire, W. L. Estrogenreceptor as an independent prognostic factor for early recurrence in breastcancer. Cancer Res., 37 4669-4671. 1977.

7. Korenman. S G. Comparative binding affinity of estrogens and its relationto estrogenic potency. Steroids, 13: 163-177, 1969.

8. Korenman, S. G.. and Dukes, B. A. Specific estrogen binding by thecytoplasm of human breast carcinoma. J. Clin. Endocrinol. Metab., 30. 639-645, 1970.

9. Lippman. M. E.. Allegra. J. C., Thompson, E. B.. Simon, R., Barlock. A..Green, L., Huff, K. K., Do, H. M. T., Aitken, S. C.. and Warren, R. Therelation between estrogen receptors and response rate to cytotoxic chemotherapy in metastatic breast cancer. N. Engl. J. Med., 298: 1223-1228,1978.

10. Lippman, M. E., Bolán. G., and Huff. K. The effects of estrogens andantiestrogens on hormone-responsive human breast cancer in long-termtissue culture. Cancer Res., 36. 4595-4601, 1976.

11. Lippman, M., Bolán, G., and Huff, K. Interactions of antiestrogens withhuman breast cancer in long-term tissue culture. Cancer Treat. Rep.. 60.1421-1429, 1976.

12. McGuire, W. L. Steroid receptors in human breast cancer. Cancer Res., 38.4289-4291, 1978.

13. McGuire, W. L., Carbone, P. P.. Sears, M. E.. and Escher, G. C. Estrogenreceptors in human breast cancer: an overview. In: W. L. McGuire, P. P.Carbone, and E. P. Vollmer (eds.), Estrogen Receptors in Human BreastCancer, pp. 1-7. New York: Raven Press. 1975.

14. Nawata, H., Bronzert, D., and Lippman, M. E. Isolation and characterizationof a tamoxifen-resistant cell line derived from MCF-7 human breast cancercells. J. Biol. Chem., 256. 5016-5021, 1981.

15. Osborne, C. K., Knight, W. A., Yochmowitz, M. G.. and McGuire. W. L.Estrogen receptor and prognosis in breast cancer, In: W. L. McGuire (ed.).Breast Cancer: Advances in Research and Treatment, Vol. 4, pp. 33-49.

New York: Plenum Publishing Corp., 1981.16. Samal, B.. Singhakowinta, A., Brooks, S. C.. and Vaitkevicius, V. K. Estrogen

receptors and response of breast cancer to chemotherapy. N. Engl. J. Med..299. 604, 1978.

17. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A humancell line from a pleural effusion derived from a breast carcinoma. J. Nati.Cancer Inst.. 5Õ 1409-1416, 1973.

18. Strobl, J. S.. and Lippman, M. E. Studies of steroid hormone effects onhuman breast cancer cells in long-term tissue culture, tn: W. L. McGuire(ed.). Hormones, Receptors and Breast Cancer, pp. 85-106. New York:

Raven Press, 1978.19. Webster, D. J. T., Bronn, D. G., and Minton. J. P. Estrogen receptors and

response of breast cancer to chemotherapy. N. Engl. J. Med., 299 604,1978.

DECEMBER 1982 5151

on June 13, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1982;42:5147-5151. Cancer Res   Gerald J. Goldenberg and Evelyn K. Froese 

in Vitroand -negative Human Breast Cancer Cells Drug and Hormone Sensitivity of Estrogen Receptor-positive

  Updated version

  http://cancerres.aacrjournals.org/content/42/12/5147

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/42/12/5147To request permission to re-use all or part of this article, use this link

on June 13, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from