c-3T3 on the proliferation of the human mammary cancer cell line T-47D

J. Steroid Biochem. Molec. Biol. Vol.41, No. 3-8, pp. 553-556, 1992

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EFFECT THE

OF

EMBRYONIC

PROLIFERATION CANCER

MOUSE OF

THE

CELL

CELLS

BALB/c-3T3

HUMAN

LINE

ON

MAMMARY

T-47D

G. CHETRITEand J. R. PASQUALINI* C.N.R.S. Steroid Hormone Research Unit, Foundation for Hormone Research, 26 Boulevard Brune, 75014 Paris, France Summary--In the present study, we explore the effect of the cellular extracts and culture medium of the embryonic mouse cell line BALB/c-3T3 (clone A31) on the proliferation and DNA content of the human T-47D breast cancer cell line. These effects were also studied in the presence of the potent anti-estrogen ICI 164,384. All experiments were prepared in MEM medium containing 5% fetal calf serum treated with dextran charcoal, as well as the homogenization of the BALB/c-3T3 cells to obtain the cellular extract. Aliquots of cellular extracts (2%) corresponding to 2 x 10 6 cells, or culture medium (16%), are incubated with the T-47D cells. After 9 days of culture, cellular extracts and culture medium provoke an intense proliferative effect corresponding respectively to 2 and 5 times the control value of T-47D cells. These effects on cell proliferation are correlated with DNA content. Although the antiestrogen ICI 164,384 (5 x 10 -s M) alone decreases the proliferation of T-47D cells by half, the presence of the culture medium from the BALB/c-3T3 cells abolishes this effect and, on the contrary, increases the cell proliferation 4-fold. It is concluded that mouse embryonic cells (BALB/c-3T3) contain factor(s) which stimulate very intensively the proliferation of hormone-dependent T-47D mammary cancer cells. This factor(s) is present in both the cell and the culture medium and can antagonize the anti-proliferative effect of the anti-estrogen ICI 164,384.

INTRODUCTION At present, it is very well established that growth factors are involved in the regulation of cell growth and differentiation. They bind to specific cell-surface receptors and provoke rapid biochemical and physiological changes in the cells, which ultimately lead to stimulation or inhibition of D N A replication and cell proliferation. These factors could be present during embryonic and fetal life and their actions are a function of the biological parameter considered, the species, and the period of gestation (for a recent review see Ref. [1]). Extensive studies in recent years have demonstrated that these factors (EGF, TGF-~, IGF-1 and TGF-fl) can be active in cell proliferation or cell growth inhibition of different m a m m a r y cancer cell lines [2]. These factors act by an autocrine or paracrine process and could be controlled by various hormones or anti-hormones in hormone-dependent cancers [3, 4]. Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.

*To whom correspondence should be addressed. s~41/3-~-x

In the present study, we explore the effect of the cellular extract (CE) and of the culture medium (CM) of the embryonic mouse cell line BALB/c-3T3 (clone A31) on the proliferation and D N A content of the hormone-dependent T-47D breast cancer cell line. These effects were also studied in the presence of the potent anti-estrogen ICI 164,384 [5]. EXPERIMENTAL

Chemicals

Tissue culture medium (Dulbecco's Modified Eagle's Medium; DMEM), Phosphate Buffer Saline (calcium- and magnesium-free) (PBS), fetal calf serum (FCS), L-glutamine, and HEPES were obtained from Flow Laboratories (Irvine, Scotland). Eagle's Minimal Essential Medium (MEM), H a n k ' s Buffered Saline Solution (calcium- and magnesium-free) (HBSS), penicillin-streptomycin, and trypan blue were obtained from Gibco Europe Ltd (Paisley, Scotland). 17fl-Estradiol (E2) was obtained from Steraloids (Touzart et Matignon, Vitrysur-Seine, France). ICI 164,384 IN-n-butylN-methyl- 1 1-(3,17/~-dihydroxyestra- 1,3,5(10trien-7ct-yl))undecanamide] was a gift from 553

554

G. CI-IETRITEand J. R. PASQUALINI

Dr A. E. Wakeling (ICI Ltd., Macclesfield, England).

Cell cultures The embryonic murine cell line BALB/c-3T3 (clone A31) was obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). Cells were maintained routinely in D M E M buffered with 10mM HEPES (pH 7.6) supplemented with 2 m M L-glutamine, 100 U/ml penicillin-streptomycin and 10% (v/v) FCS, at 37°C in a humidified atmosphere of 5% CO2. BALB/c-3T3 cells were seeded at 13,000cell/cm 2 and passed every 10-12 days. The hormone-dependent human mammary cancer cell line, T-47D, was kindly provided by Drs M. E. Lippman and R. B. Dickson (Georgetown University, Washington, DC, U.S.A.). The cells were grown routinely as monolayer culture in MEM supplemented as indicated above, with the modification of 5% FCS. The cells were passed every 8-10 days and replated in 75 cm 2 flasks (C.M.L., Nemours, France) at 3 x 106 cells/flask. For the experiments, cells were grown in MEM containing 5% steroid-depleted FCS, prepared by mixing the dextran-coated charcoal (DCC, 0. I - 1 % , DCC-FCS).

Preparation of CM and CE of BALB/c-3T3 cells The preparation of CM from BALB/c-3T3 cells was obtained when confluency was achieved. At this time, medium was discarded and cells were washed with PBS and fresh complete medium, MEM + 5% DCC-FCS, was added and exposed to confluently grown BALB/c-3T3 cells for 48 h. Thereafter, these CM were harvested and centrifuged at 2000g for 15 min to remove cell debris. CM was stored at - 8 0 ° C until further use. Total CE of BALB/c-3T3 was obtained under sterile conditions. A suspension of 100 million cells in 2ml MEM + 5% D C C - F C S was homogenized by ten strokes in a glass homogenizer at 4°C, and the final volume was adjusted to 10ml with the MEM. Under these conditions, more than 95% of the cells were lysed, as determined by trypan blue exclusion under microscopic examinations. The solution was then centrifuged at 2000g for 15 min to obtain the clear soluble CE. Aliquots of 0.5 ml were tested for sterility and absence of contamination, then frozen at _ 80°C.

Cell proliferation assay For most experiments, cells were routinely grown on 24-well dishes (costar), or in 75 cm 2 flasks. T-47D cells were seeded at 50,000 cells/well on a 24-well plate containing 1 ml MEM + 5% DCC-FCS. On the following day (day 0), the medium was changed to 1 ml fresh medium, with or without various concentrations of CM or CE of BALB/c-3T3 cells. In all the experiments the proliferative effect of the BALB/c3T3 cells on the T-47D cells were tested using a CE (2% v/v) corresponding to approx. 2 x 106 cells, and for the CM was in a proportion 1/6, given a final concentration of 16% v/v. E 2 (5 x 10-SM) and ICI 164,384 (5 x 10-aM) were also tested, alone or in combination with CM; 3-4 wells were used for each sample. At the time of harvest (day 3, 6 or 9), cells were removed from the plate by treatment with PBS containing 0.05% (w/v) trypsin and 0.02% EDTA. The cell slurry was added to MEM + 5% D C C - F C S to neutralize the trypsin and centrifuged at 500 g for 5 min. The cell number was determined in triplicate cultures by counting trypan blue excluding cells in a hemocytometer. The number of cells was also correlated with the total DNA content of the cells. Three other wells were prepared in parallel in the same manner indicated above and, at the indicated time, DNA measurement was performed as described by Taylor et al. [6]. Briefly, the medium was removed and the cells stored at - 2 0 ° C until assayed. 800#1 0.5N PCA were added to each well and the plates heated at 90°C for 30 min. Standard of calf thymus DNA was made in 0.5 N PCA and treated in the same manner. After cooling at room temperature, 1.6 ml diphenylamine reagent [7] were added to each well. The optical density was measured at 595 nm after overnight incubation at room temperature. The coefficient of correlation between DNA and cell number was approx. 0.90. For experiments performed with 75cm 2 flasks, the treatment and methods were similar to those indicated above, except that DNA was measured using the Burton method [7]. RESULTS

Effect of the embryonic mouse BALB/c-3T3 (CM and CE) on the proliferation and DNA content of T-47D breast cancer cells CM of BALB/c-3T3 cells is obtained when these embryonic cells are at confluence. The

Effect of embryonic T-47D breast cancer cells addition of CM at a final concentration of 16% (v/v) on a monolayer culture of T-47D hormone-dependent breast cancer cells provokes a rapid and important increase of the proliferation of T-47D cells and their D N A content. After 9 days of culture the growth of T-47D cells and their D N A increase to about 5 times those of the non-treated cells [Fig. 1 (A and B)]. The proliferative effect is particularly intense between 6 and 9 days of culture. In another series of experiments, CE of the embryonic BALB/c-3T3 cells was added at a final concentration of 2% (v/v) and cultivated with T-47D breast cancer cells. This homogenate of the BALB/c-3T3 cells represents approx. 2 x 10 6 cells. After 9 days of culture, the T-47D cells and the D N A content increase more than twice in relation to the values of the non-treated cells [Fig. 1 (A and B)]. As for the CM of BALB/c-3T3 cells, this effect is more intense after 6 days of culture.

Effect of the embryonic BALB/c-3T3 CM on the growth of T-47D breast cancer cells in the presence of the anti-estrogen ICI 164,384 In recent years, anti-estrogens have been used extensively in the treatment of breast cancer. One of these most frequently used is tamoxifen, due to its lack of toxicity [8, 9]. Recently, ICI Pharmaceuticals (Macclesfield, England) syn-

CELLSx 106 ,o

~

~ 1 I~

,.,T,. ÷cE I

BE

30 2O

10 0 mg DNA

0.6 ~

0.5 0.4 0,3 0,2 0.1 0

CELLSx 106 2030i ~

o~

555

[ 4' Control 1 [~ +(ICI+CM) 0 A +CM

A[~

0 00

[

I

3[

6I DAYSOF CULTURE

I

9I

Fig. 2. Effect of ICI 164,384 (ICI) and E2, in the presence of BALB/c-3T3 CM, on the proliferation (A) and DNA content (B) of the hormone-dependentT-47D human breast cancer cells. T-47D cells were plated in triplicate in MEM+5% DCC-FCS and at 0 time ICI 164,384 (5 x 10-s M) and E2 (5 x 10-s M) were added, alone or associated with BALB/c-3T3CM (16%, v/v). Cellular proliferation and DNA content were determined in each flask after 3, 6, or 9 days of culture. Data shown are the mean of 2-3 experiments.

thesized an estradiol derivative, ICI 164,384, with full anti-estrogenic properties [5, 10]. In previous studies in this laboratory, we found that ICI 164,384 (5 × 10 -s M) provokes an inhibitory effect on the growth of T-47D cells of about 40% in relation to the non-treated cells [11]. Figure 2 (A and B) shows that the CM of the BALB/c-3T3 cells can not only block the inhibitory effect provoked by ICI 164,384 on T-47D growth, but can also have a significant stimulatory action 3-4 times that in the non-treated cells. A similar effect is observed on the D N A content. It was also observed that the CM of BALB/c3T3 cells can increase the stimulatory proliferative effect of E 2 (5 × 10 -s M) on T-47D cells [Fig. 2 (A and B)]. DISCUSSION

0

3 6 DAYSOF CULTURE

9

Fig. 1. Effectof embryonicmousecellsBALB/c-3T3CM

and CE on the proliferation (A) and DNA content (B) of the hormone-dependent T-47D human breast cancer cells. CE and CM were obtained as described in Experimental. T-47D cells were plated in triplicate in MEM+5% DCC-FCS and at 0 time CE (2%, v/v) or CM (16%, v/v) were added. Cellular proliferation and DNA content were determined in each flask after 3, 6, or 9 days of culture. Data shown are the mean +__SD (bars) of 2-3 experiments.

The interesting aspect of the present data is the intense proliferative effect in the mammary hormone-dependent cell line T-47D using the CE and CM of the embryonic BALB/c-3T3 cells. We suggest that this effect is due to growth factors which are present in these cells and, as a very strong effect is also provoked by the CM, it is believed that this factor is secreted by the cells to the medium. Similar data, but with a less

G. CHETRITEand J. R. PASQUALINI

556

intense effect were f o u n d in M C F - 7 cells, a n o t h e r h o r m o n e - d e p e n d e n t m a m m a r y cancer cell line ( d a t a n o t shown). This p r e l i m i n a r y study leaves m a n y p r o b l e m s to be solved: (1) the c h a r a c t e r i z a t i o n o f this factor(s); (2) the effect in function o f the dose; a n d (3) the utilization o f the other, including h o r m o n e - i n d e p e n d e n t , m a m m a r y cancer cell lines. These studies are in progress. A n o t h e r interesting aspect o f the present results is the proliferative effect in c o m b i n e d experiments o f the C M a n d the p o t e n t antiestrogen I C I 164,384. This c o m p o u n d , which is considered as a p u r e anti-estrogen, was shown to inhibit p r o l i f e r a t i o n o f m o s t o f the h o r m o n e d e p e n d e n t cell lines [11-13]. It was also d e m o n strated that the p o t e n c y o f this anti-estrogen is 100 times that o b t a i n e d with tamoxifen. The fact that in the present d a t a the C M o f these cells b l o c k the anti-proliferative effect o f I C I 164,384, a n d also that a s t i m u l a t o r y effect is observed, indicates that the action o f the stimul a t o r y factors o f B A L B / c - 3 T 3 is very stong. The question is: W h a t could be the m e c h a n i s m o f this process? W e k n o w that in general antiestrogens can block the estrogenic effect by binding to estrogen receptor, b u t the interrelationship o f o t h e r factor(s), as is the situation o f this report, which are involved in the m e c h a n i s m o f the anti-estrogen process needs to be explored.

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REFERENCES

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tors. In Hormones and Fetal Pathophysiology (Edited by J. R. Pasqualini and R. Scholler). Dekker Inc., New York (1992). In press. Lippman M. E. and Dickson R. B.: Mechanisms of normal and malignant breast epithelial growth regulation. J. Steroid Biochem. (Proc.) 34 (1989) 107-121. Dickson R. B. and Lippman M. E.: Estrogenic regulation of growth and polypeptide growth factor secretion in human breast carcinoma. Endocrine Rev. g (1987) 29-43. Sporn M. B. and Roberts A. B.: Autocrine, paracrine, and endocrine mechanisms of growth control. Cancer Surv. 4 (1985) 627~532. Wakeling A. E. and Bowler J.: Steroidal pure antiestrogens. J. Endocr. 112 (1987) R7-RI0. Taylor C. H., Blanchard B. and Zava D. T.: A simple method to determine whole cell uptake of radiolabelled oestrogen and progesterone and their subcellular localization in breast cancer cell lines in monolayer culture. J. Steroid Biochem. 20 (1984) 1083-1088. Burton K.: A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62 (1956) 315-323. Cole M. P., Jones C. T. A. and Todd I. D. H.: A new anti-estrogenic agent in late breast cancer. An early clinical appraisal of ICI 46,474. Br. J. Cancer 25 (1971) 270-275. Ward H. W. C.: Anti-estrogenic therapy for breast cancer: a trial of tamoxifen at two dose levels. Br. Med. J. 1 (1973) 13-14. Wakeling A. E. and Bowler J.: Novel anti-estrogens without partial agonist activity. J. Steroid Biochem. 31 (1988) 645-653. Pasqualini J. R. and Gelly C.: Biological response of the anti-estrogen ICI 164,384 in human hormonedependent and hormone-independent mammary cancer cell lines. Cancer Lett. 50 (1990) 133-139. Wakeling A. E.: Therapeutic potential of pure antioestrogens in the treatment of breast cancer. J. Steroid Biochem. Molec. Biol. 37 (1990) 771-775. Poulin R., Merand Y., Poirier D., Levesque C., Dufour J. M. and Labrie F.: Antiestrogenic properties of keoxifene, trans-4-hydroxytamoxifen, and ICI 164,384, a new steroidal anti-estrogen, in ZR-75-1 human breast cancer ¢¢!1s. Brca~t Canc'er Res, Treat. 14 (1989) 65-76