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Regulation of breast cancer cell cycle progression by growth factors, steroids and steroid antagonists
J. Steroid Biochem. Molec. BioL Vol. 41, No. 3-8, pp. 315-321, 1992
0960-0760/92$5.00+ 0.00 Copyright© 1992PergamonPress plc
Printed in Great Britain. All rights reserved
REGULATION
OF
BREAST
PROGRESSION
BY
GROWTH
AND
STEROID
CANCER
CELL
FACTORS,
CYCLE
STEROIDS
ANTAGONISTS
ROBERTL. SUTHERLAND,*CHRISTINES. L. LEE,ROMYS. FELDMANand ELIZABETHA. MUSGROVE Cancer Biology Division, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, N.S.W. 2010, Australia Summary--The control of human breast cancer cell proliferation in vitro is known to involve complex interactions between steroid hormones, peptide hormones and growth factors. Little is known, however, of the mechanisms by which these factors, alone or in combination, control cell cycle progression and the expression of specific genes involved in cell cycle control. A pre-requisite for such studies is a cellular system in which non-proliferating or slowly proliferating cells can be maintained in a defined environment and stimulated to progress through the cell cycle by addition of hormones and growth factors. Such a system has been developed for T-47D human breast cancer cells: quiescent or slowly proliferating cells maintained in a serum-free medium can be stimulated to increase their rate of cell cycle progression upon a single addition of insulin, IGF-I, EGF, TGFet or bFGF. Oestradiol alone was ineffective but caused a significant increase in % S phase cells when added in the presence of insulin. Progestins, in the presence or absence of insulin, had a biphasic effect with an initial increase in cell cycle progression followed by cell cycle arrest. Both antioestrogens and the antiprogestin, RU 486, in the absence of oestrogen or progestin, were potent inhibitors of insulin-induced proliferation. Increases in cell cycle progression were invariably accompanied by acute increases in c-fos and c-myc mRNA levels. Induction of c-myc by oestrogen and progestin was inhibited by antioestrogens and RU 486, respectively. These data illustrate that the culture of breast cancer cells in a serum-free, chemically defined environment provides an excellent model in which to define the role of individual factors involved in breast cancer growth control. The biological data derived from this system provide a basis for identifying and characterizing genes involved in the control of cell cycle progression in human breast cancer.
INTRODUCTION The availability o f hormone-responsive h u m a n breast cancer cell lines has facilitated research directed at understanding the mechanisms o f growth control in breast cancer. Whilst early studies in h u m a n s and in animal models had identified oestrogen as the major stimulatory factor for breast cancer growth, the more detailed recent studies in vitro have demonstrated 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. Abbreviations: IGF-I, insulin-like growth factor I; EGF,
epidermal growth factor; TGF~, transforming growth factor a; bFGF, basic fibroblast growth factor; ORG 2058, Organon 2058 = 16a-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione; RU 486, mifepristone= 17//hydroxy - ! 1//- ( 4 - dimethylaminophenyl ) - 17a- ( 1 - pro pynyl)-estra-4,9-diene-3-one; ICI 164384, N-n-butyl-Nmethyl- 11- [3,17//-dihydroxyestra- 1,3,5,(10)-trien- 17a -yl]undecanamide; E2, 17lt oestradiol; FCS, foetal calf serum. SB 41/3-~-I
3 15
that the control o f breast cancer cell proliferation involves complex interactions between a number o f different steroid hormones, peptide h o r m o n e s and growth factors. The majority o f these studies, reviewed in Refs [1-5], have concentrated on the long-term growth effects in m o n o l a y e r culture and/or effects on anchorage-independent growth in soft-agar. In contrast, relatively little attention has been given to the short-term effects o f various agents on breast cancer cell cycle progression. M u c h research in this laboratory in recent years has focused on the cell cycle kinetic effects o f steroids and their antagonists in breast cancer cells [3, 4]. Initially studies were conducted in the presence o f foetal calf serum (FCS), a complex mixture o f growth stimulatory and growth inhibitory molecules. The recent demonstration by ourselves [6, 7] and others [8, 9] that cellular responses to steroids and steroid antagonists can be m o d u l a t e d by growth factors likely to be present in FCS, highlighted the need to work
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ROBERT L. SUTHERLAND et al.
under more defined conditions when attempting to delineate the effects of individual growth regulatory substances. Thus we sought to develop a totally defined, serum-free environment in which stimulation or inhibition of cell proliferation could be induced with a single agent or simple combination of agents. We have now developed such a system and have begun to define the effects of growth factors, steroids and their antagonists on breast cancer cell cycle progression in a totally defined environment. This paper describes some of our early data employing this culture system. EFFECT OF GROWTH FACTORS
The final experimental design adopted for the production of monolayer cultures of viable, but very slowly proliferating, cells was as follows. T-47D cells were first passaged from stock cultures [10] into phenol red-free medium with 10% charcoal-treated FCS, and the medium changed twice, at intervals of 1-3 days, as the cells grew to confluence over 5-7 days. This procedure depleted cells of steroids [11] while producing a population of almost confluent and therefore slowly proliferating cells. These cells were replated in phenol red-free medium containing 15% charcoal treated FCS, to allow attachment to the plastic substratum overnight. On the next two successive days the medium was aspirated and replaced with fresh serum-free medium, which was then not further replaced. These initial changes of serum-free medium served to deplete the cells of growth factors present in the serum-containing medium. The serum-free medium consisted of phenol red-free RPMI 1640 supplemented with 6mM L-glutamine, 20 mM HEPES, 14 mM sodium bicarbonate, 20~tg/ml gentamycin and 300nM human transferrin. At the completion of these pretreatments cell numbers were on average about 10% less than the plating density, cells remained attached to the substratum and showed no profound morphological changes. After an initial lag period of 1-3 days cells began to proliferate slowly in serumfree medium without further supplementation. Addition of insulin (10#g/ml) to these cells induced cell cycle progression as assessed by the percent S phase cells determined by analytical DNA flow cytometry [12]. By 12 h, the percentage of cells in S phase had begun to increase, and reached its maximum at 21-24 h (Fig. 1). The proportion of cells in G1 phase decreased
simultaneously, while the % G2 + M increased after a delay (not shown). Thus, the changes in cell cycle phase distribution were consistent with semi-synchronous entry into S phase, and the apparently cyclical variation at later times suggested that at least one further round of semi-synchronous replication occurred. Detailed studies determined a clear concentrationdependence of this effect of insulin on % S phase which was translated to changes in growth rate and cell numbers over longer time periods up to 96 h [14]. The addition of FCS to a final concentration of 10% resulted in similar changes in cell cycle phase distribution, which were of larger magnitude (Fig. 1), as expected from the higher proliferation rate observed after long term treatment. Identical experiments were undertaken to determine the temporal changes in S phase fraction, and the concentration-dependence of this effect with a number of peptide growth factors known to affect breast cancer cell proliferation. These included IGF-I, TGF~t, EGF and bFGF. All four growth factors induced increased rates of cell cycle progression with broadly similar kinetics to that shown for insulin in Fig. 1, i.e. increases in % S phase cells were first observed at about 12 h and reached a peak between 18 and 24 h. There were, however, significant differences in potency when each peptide was compared at a saturating concentration (Fig. 2). In these experiments insulin, IGF-I and bFGF induced increases in % S phase of similar magnitude, approximately 60% of that induced by FCS, while the response to TGFct was consistently more modest, ~ 70% of the response to insulin, and that of EGF less than half that of insulin (Fig. 2).
30 20
Control 0
-
0
,
12
-
,
24
-
,
36
-
i
48
•
,
60
Time (h)
Fig. l. Effect of treatment with insulin or FCS on the S phase fraction of T-47D cells. Steroid-depleted T-47D cells were maintained in serum-free medium for 4 days prior to treatment with 10#g/ml insulin ( n ) , 10% FCS ( x ) or vehicle (O) which were added directly to the medium. Cells were harvested for DNA analysis by flow cytometry (12) at intervals thereafter.
Breast cancer cell cycle progression 2"
317
40"
m .c
30-
,C_
7-
e~
20"
--~~
0
n
10"
Tm i e(h) 0
Fig. 2. Relative effects of growth factors and FCS on S phase fraction of T-47D ceils. Either 10% FCS, 10 #g/ml insulin, 10nM IGF-I, 10nM TGFct, 10nM EGF or 10 ng/ml bFGF was added to T-47D cells which had been maintained for 3 or 4 days in serum-freemedium. Increases in % S phase after 16 24 h treatment were calculated by subtraction of the % S phase of cultures maintained in serum-free medium alone, then normalized to the value obtained for insulin. EFFECT OF STEROIDS
The effects of oestrogens and progestins were also investigated in this culture system. In unsupplemented serum free medium, responses to 17fl oestradiol (E 2) were small and inconsistent in magnitude. Since it has been suggested that insulin and E2 have synergistic effects on breast cancer cell proliferation [13] we tested the effects of E 2 on T-47D cells stimulated to proliferate with insulin. Under these conditions E2 induced a concentration-dependent increase in the S phase fraction with the maximum effect seen at 0.05 nM E 2 (Fig. 3). Time--course data indicated that stimulated cells enter S phase as a semisynchronous cohort, and that the timing of entry into S phase is similar for E2- and growth factor-stimulated cells, such that the % S phase
25
A
Fig. 4. Effect of the progestin, ORG 2058, on the S phase fraction of T-47D cells growing in insulin-containing serumfree medium. Cells were maintained in serum-free medium for 2 days and then treated with 10 #g/ml insulin. Two days later ORG 2058 (11) or ethanol vehicle (/--/) was added and flasks harvested at the indicated times for DNA analysis by flow cytometry. Redrawn from Ref. [14].
begins to increase approximately 12h after addition of E2 (not shown). Treatment with progestins, in the presence or absence of insulin, resulted in a biphasic effect on cell cycle progression with an initial increase in cell cycle traverse followed by cell cycle arrest. These effects of progestin have been investigated in detail and are the subject of another publication [14]. In brief, cells treated with the synthetic progestin, O R G 2058, were initially stimulated to enter S phase in a semisynchronous manner 8 h after treatment and had reached their maximum by 12 h (Fig. 4). These cells complete S phase and mitosis as indicated by a transient increase in the G2 + M fraction (not shown). There was a concomitant decrease in the % G~ phase cells due to a progestin-induced increase in the rate of exit from G] into S phase[14]. After the passage of the stimulated cohort, entry into S phase from G t was markedly inhibited and there was a sustained decrease in the proportion of cells in S phase accompanying the long term growth inhibitory action of progestins in these cells [14, 15]. Both the initial transient increase in cell cycle progression and the subsequent decrease were progesterone receptor-mediated as assessed by steroid specificity studies and antagonism by the antiprogestin, RU 486.
A 15
EFFECT OF STEROID ANTAGONISTS io~i ..... .~ ..... .~i ...... ] .... - ; o ' " ' ~ ; o ' i o ~ o E2 concentration (nM)
Fig. 3. Effect of oestradiol in the presence of insulin on the S phase fraction of T-47D cells. Cells were maintained
in serum-free medium for 2 days and then treated with 10#g/ml insulin. Four days later F-a(A) or ethanol vehicle (A) was added; duplicate flasks were harvested for DNA analysis 21-24 h later.
Antioestrogens and antiprogestins are known to be potent inhibitors of breast cancer cell proliferation in vitro and specific points of action within the cell cycle have been reported for antioestrogens [12, 16, 17]. Some studies have suggested that these compounds have potent
318
ROBERTL. SUTHERLANDet al. 30"
EFFECTS O N P R O T O - O N C O G E N E E X P R E S S I O N
Changes in the rates of cell cycle progression in a wide range of cellular systems have been shown to be associated with changes in the Q. level of expression of a number of "immediate u) 10' RU 486 early" genes which include the proto-oncogenes c-myc and c-fos [18-20]. Indeed changes in 30' the expression of these genes are amongst the I-1 earliest measurable events associated with mitogenic stimulation. To assess whether or not 20' changes in cell cycle progression induced by (/) growth factors, steroids and their antagonists 10' in our T-47D cultures were accompanied by IC1164384 changes in the level of expression of these genes, 1~9 2'4 cells were first treated with maximal stimulatory Time (h) concentrations of insulin or b F G F and the time Fig. 5. Comparison of the effectsof the antioestrogen, ICI 164384, and the antiprogestin, RU 486, on the S phase course of induction of c-fos and c-myc measured fraction of T-47D cells growing in insulin-containing, by Northern analysis. Both mitogens resulted in serum-free medium. The experimentaldesign was identical significant increases in the levels of m R N A for to that outlined in Fig. 4. Hatched line shows the mean of control, vehicle-treated samples harvested over the time both genes (not shown). Similarly, treatment of T-47D cells with steroids which initially induced course of the experiments. cell cycle progression, i.e. oestrogens and progestins, also resulted in induction of these genes. effects even in the absence of stimulation of cells Detailed studies have been completed for by oestrogen and progestin e.g. antioestrogens progestin stimulation and a representative timewill inhibit cell proliferation of MCF-7 cells course experiment is presented in Fig. 6. Both stimulated to proliferate by insulin or E G F in c-fos and c-myc mRNA were present at low the apparent absence of oestrogen [8]. Thus the levels in control cells growing in the presence development and use of defined cell systems of insulin and their expression was transiently where the effects of these compounds can be increased by O R G 2058 treatment, c-fos m R N A tested against a spectrum of individual mitogens levels reached a maximum (2- to 4-fold) at may aid understanding of their basic molecular 30 min and had returned to control levels by 2 h. mechanisms of growth inhibition. We therefore Increased expression of c-myc was first apparent tested the effects of the steroidal antioestrogen, at 15 min, was maximal (4- to 8-fold) after 1-2 h ICI 164384, and the steroidal antiprogestin, RU treatment, and returned to near control values 486, on T-47D cells that had been stimulated to by 6 h. Thus the increases in m R N A abundance proliferate by the addition of a single mitogen, for c-fos and c-myc following progestin insulin, to serum-free cultures. Both compounds led to a concentrationdependent decrease in the rate of cellular proliferation and this was accompanied by a time-dependent decrease in the S phase fraction =, 3 (Fig. 5) which was mirrored by an increase in % G~ cells (not shown). The decrease in the 2 < % S phase began after 8-10 h of treatment with z either compound and S phase reached a minio mum, approximately 50% of control values by 0 18 h. In the same experimental design MCF-7 o ~ ~ cells could be inhibited by either the steroidal Time (h) antioestrogen, ICI 164384, or the nonsteroidal Fig. 6. Effectof the progestin, ORG 2058, on the expression antioestrogen, 4-hydroxytamoxifen, with similar of c-fos and c-myc mRNA. Cells were treated with I0 nM kinetics for the changes in % S phase (not ORG 2058 and total cellular RNA extracted for Northern shown). Thus both compounds are potent inhib- analysis at the times indicated. Relative levels of c-fos (0) and c-myc (O) mRNA were determined by densitometric itors of insulin-induced cell cycle progression in scanning of autoradiographs. A representativetime-course a steroid-free environment. experiment redrawn from Ref. [14] is shown. 20'
Breast cancer cell cycle progression
stimulation of cell cycle progression follow a time-course typical of the response of these proto-oncogenes to mitogens in many cell types [18, 19] and similar to the oestrogen induction of c-fos and c-myc following oestrogen stimulation of breast cancer cells [21-23]. If the enhanced expression of these protooncogenes is involved in the accelerated cell cycle progression induced by oestrogens and progestins in breast cancer cells, one would predict that the corresponding steroid antagonists which inhibit cell cycle progression would attenuate these responses. When this was tested with c-myc, the induction of expression of this gene by E2 or ORG 2058 was entirely abrogated by simultaneous treatment with ICI 164,384 and RU 486, respectively (not shown). Together these results suggest that growth regulation of breast cancer cells by growth factors and steroids may be paralleled by regulation of the proto-oncogene, c-myc. CONCLUSIONS
The control of cell proliferation in human breast cancer cells involves the interactions of several hormones and growth factors [1-5]. However, the mechanisms by which these factors, through their various signal transduction pathways, interact to control regulatory events critical to cell cycle progression and thus cell proliferation remain unknown. In order to facilitate further studies in this area we developed a chemically defined, serum-free culture system for the hormone-responsive breast cancer cell line, T-47D. In designing this system we aimed to meet two principal criteria. Firstly the serum-free medium should maintain cells in a viable but quiescent state, to allow the greatest sensitivity to induction of cell cycle progression by mitogenic agents, with the added advantage that any stimulated cells would be likely to move through the cell cycle in a semi-synchronous manner facilitating further biochemical analysis. In addition supplementation of the basal medium, preferably with a single growth factor, should induce proliferation rates approximately those of cultures growing at maximal rates in 10% FCS, thus facilitating meaningful examination of inhibitory effects of various agents in a defined environment. Studies to date, which have been summarized here, suggest that these criteria have been essentially met. Insulin, IGF-I or bFGF alone all induce rapid proliferation rates with population doubling times only
319
slightly less than those observed in the presence of 10% FCS. Additionally the proliferation of cells stimulated to grow by a maximal concentration of insulin alone can be modulated by further addition of a single steroid or steroid antagonist. The nett result is a breast cancer cell culture system where the rates of cell proliferation can be changed at will by the addition of a single growth stimulatory or growth inhibitory agent. The data obtained to date in this experimental system have provided further insight into breast cancer cell growth control. The importance of insulin and IGF-I as potent mitogens was confirmed as was the mitogenic activity of TGF~ and EGF [13, 24]. However the latter two peptides were significantly less effective in increasing % S phase cells 18-24 h after treatment in agreement with other data demonstrating their reduced activity in long term growth assays [13, 24]. Furthermore, we noted reduced potency of EGF relative to TGF~ despite the fact that both peptides exert their effects via the EGF receptor. Such differential responses to these two growth factors have been noted in a number of other systems[25]. The largest deviation between acute effects of growth factors on cell cycle progression and longer term effects on cell proliferation rates was noted with bFGF. The data presented in Fig. 2 show that bFGF is as effective an inducer of cell cycle progression as insulin or IGF-I while in a study on growth effects bFGF induced only modest increases in cell numbers under similar culture conditions [24]. Other studies from this laboratory have shown that increases in cell number, indicative of sustained action, require higher concentrations of bFGF than do acute increases in S phase fraction (Feldman et al.; manuscript in preparation). Thus degradation of bFGF may reduce its long-term effectiveness. Oestrogen alone failed to induce significant effects but interacted synergistically with insulin to increase cell cycle progression (Fig. 3) and cellular growth rates [13]. Difficulty in demonstrating proliferative responses to E 2 has been a feature of studies into the control of replication of breast cancer cells in tissue culture which may be explained in part by the fact that oestrogen has been tested in the presence of various concentrations of insulin included routinely in culture media. Certainly the data presented here support the conclusions of van der Burg and colleagues[13] who have recently provided a mechanistic explanation for the synergism
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ROBERTL. SUTHERLANDet al.
between insulin a n d oestrogen [26]. Oestrogen can induce c - f o s but fails to induce genes o f the j u n family which are essential for the f o r m a t i o n o f the A P o l t r a n s c r i p t i o n complex. Insulin a n d I G F - I by c o n t r a s t are efficient inducers o f c-jun a n d act synergistically with E2 to stimulate A P - I activity a n d cell p r o l i f e r a t i o n [26]. In c o n t r a s t to the sustained effect on S phase fraction a n d cell p r o l i f e r a t i o n induced b y E2 in the presence o f insulin, progestins h a d only a transient s t i m u l a t o r y effect on cell cycle progression (Fig. 4). The increase in S phase was observed several hours before increases in S phase following g r o w t h factor s t i m u l a t i o n indicating action o f progestins at a p o i n t in Gt closer to the G~/S interface. Interestingly the decreases in S phase associated with R U 486 t r e a t m e n t occurred within a similar t i m e - f r a m e as w o u l d be expected if the i n h i b i t o r y effects o f R U 486 were m e d i a t e d via the same p a t h w a y s as the s t i m u l a t o r y effects o f progestins. P e r h a p s m o r e i m p o r t a n t was the o b s e r v a t i o n that the cell cycle kinetic changes a c c o m p a n y i n g the g r o w t h i n h i b i t o r y effects o f b o t h a n t i p r o g e s t i n s and a n t i o e s t r o g e n s were t e m p o r a l l y similar, raising the possibility that these two classes o f steroid a n t a g o n i s t s share similar m e c h a n i s m s o f g r o w t h inhibition. C e r t a i n l y the d a t a presented here (cf. Figs 4 a n d 5) w o u l d argue against a n t i p r o g e s t i n s inducing their g r o w t h i n h i b i t o r y effects via any progestin agonist properties. H a v i n g defined the responses to a spectrum o f g r o w t h r e g u l a t o r y agents, experiments were u n d e r t a k e n to d e t e r m i n e if changes in cell cycle progression were a c c o m p a n i e d by changes in the level o f expression o f two p r o t o - o n c o g e n e s k n o w n to be acutely regulated by a wide range o f agents affecting cell p r o l i f e r a t i o n a n d differe n t i a t i o n [ 1 8 - 2 0 ] . A clear association between the expression o f these genes a n d p r o l i f e r a t i o n was noted. Both genes were induced by agents stimulating p r o l i f e r a t i o n while a n t a g o n i s m o f these increases in cell cycle p r o g r e s s i o n by antioestrogens a n d a n t i p r o g e s t i n s was i n v a r i a b l y associated with inhibition o f the i n d u c t i o n o f c - m y c . W h e t h e r or n o t these changes in p r o t o oncogene expression are necessary or merely associated with the changes in cell cycle p r o g r e s s i o n requires further investigation. In s u m m a r y we have d e v e l o p e d a culture system which allows detailed analysis o f the effects o f h o r m o n e s a n d g r o w t h factors, alone or in c o m b i n a t i o n , on breast cancer cell cycle progression. Such a system should facilitate the d e v e l o p m e n t o f a deeper u n d e r s t a n d i n g o f the
m o l e c u l a r m e c h a n i s m s controlling cell cycle p r o g r e s s i o n in these cells b o t h in d e t e r m i n i n g the roles o f molecules a n d signal t r a n s d u c t i o n p a t h w a y s identified in o t h e r cell types a n d in identifying novel genes a n d m e c h a n i s m s specific to breast epithelial cells. Acknowledgements--These studies were supported by the
National Health and Medical Research Council of Australia and MLC-Life Ltd. Elizabeth Musgrove was the recipient of a Government Employees Assistance to Medical Research Fund Scholarship during the course of these studies. REFERENCES
1. Dickson R. B. and Lippman M. E.: Estrogenic regulation of growth and polypeptide growth factor secretion in human breast carcinoma. Endocrine Rev. 8 (1987) 29-43. 2. Lippman M. E. and Dickson R. B.: Mechanisms of normal and malignant breast epithelial growth regulation. J. Steroid Biochem. 34 (1989) 107-121. 3. Clarke C. L. and Sutherland R. L.: Progestin regulation of cellular proliferation. Endocrine Rev. 11 (1990) 266-302. 4. Musgrove E. A. and Sutherland R. L.: Steroids, growth factors and cell cycle controls in breast cancer. In Regulatory Mechanisms in Breast Cancer (Edited by M. Lippman and R. Dickson). Kluwer Academic, Boston, Mass. (1991) pp. 305 331. 5. Arteaga C. L. and Osborne C. K.: Growth factors as mediators of estrogen/antiestrogen action in human breast cancer ceils. In Regulatory Mechanisms in Breast Cancer (Edited by M. E. Lippman and R. B. Dickson). Kluwer Academic, Boston, Mass. (1991) pp. 289 304. 6. Koga M. and Sutherland R. L.: Epidermal growth factor partially reverses the inhibitory effects of antiestrogens on T 47D human breast cancer cell growth. Biochem. Biophys. Res. Commun. 146 (1987) 739-745. 7. Koga M., Musgrove E. A. and Sutherland R. L.: Modulation of the growth-inhibitory effects of progestins and the antiestrogen hydroxyclomiphene on human breast cancer cells by epidermal growth factor and insulin. Cancer Res. 49 (1989) 112 116. 8. Vignon F., Bouton M. M. and Rochefort H.: Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens. Biochem. Biophys. Res. Commun. 146 (1987) 15021508. 9. Sarup J. C., Rao K. V. and Fox C. F.: Decreased progesterone binding and attenuated progesterone action in cultured human breast carcinoma cells treated with epidermal growth factor. Cancer Res. 48 (1988) 5071-5078. 10. Reddel R. R., Murphy L. C. and Sutherland R. L.: Factors affecting the sensitivity of T-47D human breast cancer cells to tamoxifen. Cancer Res. 44 (1984) 2398-2405. 11. Strobl J. S. and Lippman M. E.: Prolonged retention of estradiol by human breast cancer cells in tissue culture. Cancer Res. 39 (1979) 3319-3327. 12. Musgrove E. A., Wakeling A. E. and Sutherland R. L.: Points of action of estrogen antagonists and a calmodulin antagonist within the MCF-7 human breast cancer cell cycle. Cancer Res. 49 (1989) 2398-2404. 13. van der Burg B., Rutteman G. R., Blankenstein M. A., de Laat S. W. and van Zoelen E. J. J.: Mitogenic stimulation of human breast cancer cells in a growth factor-defined medium: synergistic action of insulin and estrogen. J. Cell. Physiol. 134 (1988) 101-108.
Breast cancer cell cycle progression 14. Musgrove E. A., Lee C. S. L. and Sutherland R. L.: Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor a, epidermal growth factor receptor, c-fos and c-myc genes. Molec. Cell. Biol. 11, (1991) 5032-5043. 15. Sutherland R. U, Hall R. E., Pang G. Y. N., Musgrove E. A. and Clarke C. L.: Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells. Cancer Res. 48 (1988) 5084-5091. 16. Sutherland R. L., Hall R. E. and Taylor I. W.: Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells. Cancer Res. 43 (1983) 3998-4006. 17. Taylor I. W., Hodson P. J., Green M. D. and Sutherland R. L.: Effects of tamoxifen on cell cycle progression of synchronous MCF-7 human mammary carcinoma cells. Cancer Res. 43 (1983) 4007-4010. 18. Kelly K., Cochran B. H., Stiles C. D. and Leder P.: Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35 (1983) 603-610. 19. Greenbcrg M. E. and Ziff E. B.: Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature, Lond. 311 (1984) 433-438.
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20. Kruijer W., Cooper J. A., Hunter T. and Verma I. M.: Platelet.derived growth factor induces rapid but transient expression of the c-los gene and protein. Nature, Lond. 312 (1984) 711-716. 21. Dubik D., Dembinski T. C. and Shiu R. P. C.: Stimulation of c-myc oncogene expression associated with estrogen-induced proliferation of human breast cancer ceils. Cancer Res. 47 (1987) 6517-6521. 22. Wilding G., Lippman M. E. and Gelmann E. P.: Effects of steroid hormones and peptide growth factors on protooncogene c-fos expression in human breast cancer cells. Cancer Res. 48 (1988) 802-805. 23. van der Burg B., van Selm-Miltenburg A. J. P., de Laat S. W. and van Zoelen E. J. J.: Direct effects of estrogen on c-fos and c-myc protooncogene expression and cellular proliferation in human breast cancer cells. Molec. Cell. Endocr. 64 (1989) 223-228. 24. Karey K. P. and Sirbasku D. A.: Differential responsiveness of human breast cancer cell lines MCF-7 and T-47D to growth factors and 17]/-estradiol. Cancer Res. 48 (1988) 4083-4092. 25. Derynck R.: Transforming growth factor ~. Cell 54 (1988) 593-595. 26. van der Burg B., de Groot R. P., Isbrucker L., Kruijer W. and de Laat S. W.: Stimulation of TPA-responsive element activity by a cooperative action of insulin and estrogen in human breast cancer cells. Molec. Endocr. 4 (1990) 1720-1726.
0960-0760/92$5.00+ 0.00 Copyright© 1992PergamonPress plc
Printed in Great Britain. All rights reserved
REGULATION
OF
BREAST
PROGRESSION
BY
GROWTH
AND
STEROID
CANCER
CELL
FACTORS,
CYCLE
STEROIDS
ANTAGONISTS
ROBERTL. SUTHERLAND,*CHRISTINES. L. LEE,ROMYS. FELDMANand ELIZABETHA. MUSGROVE Cancer Biology Division, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, N.S.W. 2010, Australia Summary--The control of human breast cancer cell proliferation in vitro is known to involve complex interactions between steroid hormones, peptide hormones and growth factors. Little is known, however, of the mechanisms by which these factors, alone or in combination, control cell cycle progression and the expression of specific genes involved in cell cycle control. A pre-requisite for such studies is a cellular system in which non-proliferating or slowly proliferating cells can be maintained in a defined environment and stimulated to progress through the cell cycle by addition of hormones and growth factors. Such a system has been developed for T-47D human breast cancer cells: quiescent or slowly proliferating cells maintained in a serum-free medium can be stimulated to increase their rate of cell cycle progression upon a single addition of insulin, IGF-I, EGF, TGFet or bFGF. Oestradiol alone was ineffective but caused a significant increase in % S phase cells when added in the presence of insulin. Progestins, in the presence or absence of insulin, had a biphasic effect with an initial increase in cell cycle progression followed by cell cycle arrest. Both antioestrogens and the antiprogestin, RU 486, in the absence of oestrogen or progestin, were potent inhibitors of insulin-induced proliferation. Increases in cell cycle progression were invariably accompanied by acute increases in c-fos and c-myc mRNA levels. Induction of c-myc by oestrogen and progestin was inhibited by antioestrogens and RU 486, respectively. These data illustrate that the culture of breast cancer cells in a serum-free, chemically defined environment provides an excellent model in which to define the role of individual factors involved in breast cancer growth control. The biological data derived from this system provide a basis for identifying and characterizing genes involved in the control of cell cycle progression in human breast cancer.
INTRODUCTION The availability o f hormone-responsive h u m a n breast cancer cell lines has facilitated research directed at understanding the mechanisms o f growth control in breast cancer. Whilst early studies in h u m a n s and in animal models had identified oestrogen as the major stimulatory factor for breast cancer growth, the more detailed recent studies in vitro have demonstrated 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. Abbreviations: IGF-I, insulin-like growth factor I; EGF,
epidermal growth factor; TGF~, transforming growth factor a; bFGF, basic fibroblast growth factor; ORG 2058, Organon 2058 = 16a-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione; RU 486, mifepristone= 17//hydroxy - ! 1//- ( 4 - dimethylaminophenyl ) - 17a- ( 1 - pro pynyl)-estra-4,9-diene-3-one; ICI 164384, N-n-butyl-Nmethyl- 11- [3,17//-dihydroxyestra- 1,3,5,(10)-trien- 17a -yl]undecanamide; E2, 17lt oestradiol; FCS, foetal calf serum. SB 41/3-~-I
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that the control o f breast cancer cell proliferation involves complex interactions between a number o f different steroid hormones, peptide h o r m o n e s and growth factors. The majority o f these studies, reviewed in Refs [1-5], have concentrated on the long-term growth effects in m o n o l a y e r culture and/or effects on anchorage-independent growth in soft-agar. In contrast, relatively little attention has been given to the short-term effects o f various agents on breast cancer cell cycle progression. M u c h research in this laboratory in recent years has focused on the cell cycle kinetic effects o f steroids and their antagonists in breast cancer cells [3, 4]. Initially studies were conducted in the presence o f foetal calf serum (FCS), a complex mixture o f growth stimulatory and growth inhibitory molecules. The recent demonstration by ourselves [6, 7] and others [8, 9] that cellular responses to steroids and steroid antagonists can be m o d u l a t e d by growth factors likely to be present in FCS, highlighted the need to work
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under more defined conditions when attempting to delineate the effects of individual growth regulatory substances. Thus we sought to develop a totally defined, serum-free environment in which stimulation or inhibition of cell proliferation could be induced with a single agent or simple combination of agents. We have now developed such a system and have begun to define the effects of growth factors, steroids and their antagonists on breast cancer cell cycle progression in a totally defined environment. This paper describes some of our early data employing this culture system. EFFECT OF GROWTH FACTORS
The final experimental design adopted for the production of monolayer cultures of viable, but very slowly proliferating, cells was as follows. T-47D cells were first passaged from stock cultures [10] into phenol red-free medium with 10% charcoal-treated FCS, and the medium changed twice, at intervals of 1-3 days, as the cells grew to confluence over 5-7 days. This procedure depleted cells of steroids [11] while producing a population of almost confluent and therefore slowly proliferating cells. These cells were replated in phenol red-free medium containing 15% charcoal treated FCS, to allow attachment to the plastic substratum overnight. On the next two successive days the medium was aspirated and replaced with fresh serum-free medium, which was then not further replaced. These initial changes of serum-free medium served to deplete the cells of growth factors present in the serum-containing medium. The serum-free medium consisted of phenol red-free RPMI 1640 supplemented with 6mM L-glutamine, 20 mM HEPES, 14 mM sodium bicarbonate, 20~tg/ml gentamycin and 300nM human transferrin. At the completion of these pretreatments cell numbers were on average about 10% less than the plating density, cells remained attached to the substratum and showed no profound morphological changes. After an initial lag period of 1-3 days cells began to proliferate slowly in serumfree medium without further supplementation. Addition of insulin (10#g/ml) to these cells induced cell cycle progression as assessed by the percent S phase cells determined by analytical DNA flow cytometry [12]. By 12 h, the percentage of cells in S phase had begun to increase, and reached its maximum at 21-24 h (Fig. 1). The proportion of cells in G1 phase decreased
simultaneously, while the % G2 + M increased after a delay (not shown). Thus, the changes in cell cycle phase distribution were consistent with semi-synchronous entry into S phase, and the apparently cyclical variation at later times suggested that at least one further round of semi-synchronous replication occurred. Detailed studies determined a clear concentrationdependence of this effect of insulin on % S phase which was translated to changes in growth rate and cell numbers over longer time periods up to 96 h [14]. The addition of FCS to a final concentration of 10% resulted in similar changes in cell cycle phase distribution, which were of larger magnitude (Fig. 1), as expected from the higher proliferation rate observed after long term treatment. Identical experiments were undertaken to determine the temporal changes in S phase fraction, and the concentration-dependence of this effect with a number of peptide growth factors known to affect breast cancer cell proliferation. These included IGF-I, TGF~t, EGF and bFGF. All four growth factors induced increased rates of cell cycle progression with broadly similar kinetics to that shown for insulin in Fig. 1, i.e. increases in % S phase cells were first observed at about 12 h and reached a peak between 18 and 24 h. There were, however, significant differences in potency when each peptide was compared at a saturating concentration (Fig. 2). In these experiments insulin, IGF-I and bFGF induced increases in % S phase of similar magnitude, approximately 60% of that induced by FCS, while the response to TGFct was consistently more modest, ~ 70% of the response to insulin, and that of EGF less than half that of insulin (Fig. 2).
30 20
Control 0
-
0
,
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,
24
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,
36
-
i
48
•
,
60
Time (h)
Fig. l. Effect of treatment with insulin or FCS on the S phase fraction of T-47D cells. Steroid-depleted T-47D cells were maintained in serum-free medium for 4 days prior to treatment with 10#g/ml insulin ( n ) , 10% FCS ( x ) or vehicle (O) which were added directly to the medium. Cells were harvested for DNA analysis by flow cytometry (12) at intervals thereafter.
Breast cancer cell cycle progression 2"
317
40"
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30-
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20"
--~~
0
n
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Tm i e(h) 0
Fig. 2. Relative effects of growth factors and FCS on S phase fraction of T-47D ceils. Either 10% FCS, 10 #g/ml insulin, 10nM IGF-I, 10nM TGFct, 10nM EGF or 10 ng/ml bFGF was added to T-47D cells which had been maintained for 3 or 4 days in serum-freemedium. Increases in % S phase after 16 24 h treatment were calculated by subtraction of the % S phase of cultures maintained in serum-free medium alone, then normalized to the value obtained for insulin. EFFECT OF STEROIDS
The effects of oestrogens and progestins were also investigated in this culture system. In unsupplemented serum free medium, responses to 17fl oestradiol (E 2) were small and inconsistent in magnitude. Since it has been suggested that insulin and E2 have synergistic effects on breast cancer cell proliferation [13] we tested the effects of E 2 on T-47D cells stimulated to proliferate with insulin. Under these conditions E2 induced a concentration-dependent increase in the S phase fraction with the maximum effect seen at 0.05 nM E 2 (Fig. 3). Time--course data indicated that stimulated cells enter S phase as a semisynchronous cohort, and that the timing of entry into S phase is similar for E2- and growth factor-stimulated cells, such that the % S phase
25
A
Fig. 4. Effect of the progestin, ORG 2058, on the S phase fraction of T-47D cells growing in insulin-containing serumfree medium. Cells were maintained in serum-free medium for 2 days and then treated with 10 #g/ml insulin. Two days later ORG 2058 (11) or ethanol vehicle (/--/) was added and flasks harvested at the indicated times for DNA analysis by flow cytometry. Redrawn from Ref. [14].
begins to increase approximately 12h after addition of E2 (not shown). Treatment with progestins, in the presence or absence of insulin, resulted in a biphasic effect on cell cycle progression with an initial increase in cell cycle traverse followed by cell cycle arrest. These effects of progestin have been investigated in detail and are the subject of another publication [14]. In brief, cells treated with the synthetic progestin, O R G 2058, were initially stimulated to enter S phase in a semisynchronous manner 8 h after treatment and had reached their maximum by 12 h (Fig. 4). These cells complete S phase and mitosis as indicated by a transient increase in the G2 + M fraction (not shown). There was a concomitant decrease in the % G~ phase cells due to a progestin-induced increase in the rate of exit from G] into S phase[14]. After the passage of the stimulated cohort, entry into S phase from G t was markedly inhibited and there was a sustained decrease in the proportion of cells in S phase accompanying the long term growth inhibitory action of progestins in these cells [14, 15]. Both the initial transient increase in cell cycle progression and the subsequent decrease were progesterone receptor-mediated as assessed by steroid specificity studies and antagonism by the antiprogestin, RU 486.
A 15
EFFECT OF STEROID ANTAGONISTS io~i ..... .~ ..... .~i ...... ] .... - ; o ' " ' ~ ; o ' i o ~ o E2 concentration (nM)
Fig. 3. Effect of oestradiol in the presence of insulin on the S phase fraction of T-47D cells. Cells were maintained
in serum-free medium for 2 days and then treated with 10#g/ml insulin. Four days later F-a(A) or ethanol vehicle (A) was added; duplicate flasks were harvested for DNA analysis 21-24 h later.
Antioestrogens and antiprogestins are known to be potent inhibitors of breast cancer cell proliferation in vitro and specific points of action within the cell cycle have been reported for antioestrogens [12, 16, 17]. Some studies have suggested that these compounds have potent
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EFFECTS O N P R O T O - O N C O G E N E E X P R E S S I O N
Changes in the rates of cell cycle progression in a wide range of cellular systems have been shown to be associated with changes in the Q. level of expression of a number of "immediate u) 10' RU 486 early" genes which include the proto-oncogenes c-myc and c-fos [18-20]. Indeed changes in 30' the expression of these genes are amongst the I-1 earliest measurable events associated with mitogenic stimulation. To assess whether or not 20' changes in cell cycle progression induced by (/) growth factors, steroids and their antagonists 10' in our T-47D cultures were accompanied by IC1164384 changes in the level of expression of these genes, 1~9 2'4 cells were first treated with maximal stimulatory Time (h) concentrations of insulin or b F G F and the time Fig. 5. Comparison of the effectsof the antioestrogen, ICI 164384, and the antiprogestin, RU 486, on the S phase course of induction of c-fos and c-myc measured fraction of T-47D cells growing in insulin-containing, by Northern analysis. Both mitogens resulted in serum-free medium. The experimentaldesign was identical significant increases in the levels of m R N A for to that outlined in Fig. 4. Hatched line shows the mean of control, vehicle-treated samples harvested over the time both genes (not shown). Similarly, treatment of T-47D cells with steroids which initially induced course of the experiments. cell cycle progression, i.e. oestrogens and progestins, also resulted in induction of these genes. effects even in the absence of stimulation of cells Detailed studies have been completed for by oestrogen and progestin e.g. antioestrogens progestin stimulation and a representative timewill inhibit cell proliferation of MCF-7 cells course experiment is presented in Fig. 6. Both stimulated to proliferate by insulin or E G F in c-fos and c-myc mRNA were present at low the apparent absence of oestrogen [8]. Thus the levels in control cells growing in the presence development and use of defined cell systems of insulin and their expression was transiently where the effects of these compounds can be increased by O R G 2058 treatment, c-fos m R N A tested against a spectrum of individual mitogens levels reached a maximum (2- to 4-fold) at may aid understanding of their basic molecular 30 min and had returned to control levels by 2 h. mechanisms of growth inhibition. We therefore Increased expression of c-myc was first apparent tested the effects of the steroidal antioestrogen, at 15 min, was maximal (4- to 8-fold) after 1-2 h ICI 164384, and the steroidal antiprogestin, RU treatment, and returned to near control values 486, on T-47D cells that had been stimulated to by 6 h. Thus the increases in m R N A abundance proliferate by the addition of a single mitogen, for c-fos and c-myc following progestin insulin, to serum-free cultures. Both compounds led to a concentrationdependent decrease in the rate of cellular proliferation and this was accompanied by a time-dependent decrease in the S phase fraction =, 3 (Fig. 5) which was mirrored by an increase in % G~ cells (not shown). The decrease in the 2 < % S phase began after 8-10 h of treatment with z either compound and S phase reached a minio mum, approximately 50% of control values by 0 18 h. In the same experimental design MCF-7 o ~ ~ cells could be inhibited by either the steroidal Time (h) antioestrogen, ICI 164384, or the nonsteroidal Fig. 6. Effectof the progestin, ORG 2058, on the expression antioestrogen, 4-hydroxytamoxifen, with similar of c-fos and c-myc mRNA. Cells were treated with I0 nM kinetics for the changes in % S phase (not ORG 2058 and total cellular RNA extracted for Northern shown). Thus both compounds are potent inhib- analysis at the times indicated. Relative levels of c-fos (0) and c-myc (O) mRNA were determined by densitometric itors of insulin-induced cell cycle progression in scanning of autoradiographs. A representativetime-course a steroid-free environment. experiment redrawn from Ref. [14] is shown. 20'
Breast cancer cell cycle progression
stimulation of cell cycle progression follow a time-course typical of the response of these proto-oncogenes to mitogens in many cell types [18, 19] and similar to the oestrogen induction of c-fos and c-myc following oestrogen stimulation of breast cancer cells [21-23]. If the enhanced expression of these protooncogenes is involved in the accelerated cell cycle progression induced by oestrogens and progestins in breast cancer cells, one would predict that the corresponding steroid antagonists which inhibit cell cycle progression would attenuate these responses. When this was tested with c-myc, the induction of expression of this gene by E2 or ORG 2058 was entirely abrogated by simultaneous treatment with ICI 164,384 and RU 486, respectively (not shown). Together these results suggest that growth regulation of breast cancer cells by growth factors and steroids may be paralleled by regulation of the proto-oncogene, c-myc. CONCLUSIONS
The control of cell proliferation in human breast cancer cells involves the interactions of several hormones and growth factors [1-5]. However, the mechanisms by which these factors, through their various signal transduction pathways, interact to control regulatory events critical to cell cycle progression and thus cell proliferation remain unknown. In order to facilitate further studies in this area we developed a chemically defined, serum-free culture system for the hormone-responsive breast cancer cell line, T-47D. In designing this system we aimed to meet two principal criteria. Firstly the serum-free medium should maintain cells in a viable but quiescent state, to allow the greatest sensitivity to induction of cell cycle progression by mitogenic agents, with the added advantage that any stimulated cells would be likely to move through the cell cycle in a semi-synchronous manner facilitating further biochemical analysis. In addition supplementation of the basal medium, preferably with a single growth factor, should induce proliferation rates approximately those of cultures growing at maximal rates in 10% FCS, thus facilitating meaningful examination of inhibitory effects of various agents in a defined environment. Studies to date, which have been summarized here, suggest that these criteria have been essentially met. Insulin, IGF-I or bFGF alone all induce rapid proliferation rates with population doubling times only
319
slightly less than those observed in the presence of 10% FCS. Additionally the proliferation of cells stimulated to grow by a maximal concentration of insulin alone can be modulated by further addition of a single steroid or steroid antagonist. The nett result is a breast cancer cell culture system where the rates of cell proliferation can be changed at will by the addition of a single growth stimulatory or growth inhibitory agent. The data obtained to date in this experimental system have provided further insight into breast cancer cell growth control. The importance of insulin and IGF-I as potent mitogens was confirmed as was the mitogenic activity of TGF~ and EGF [13, 24]. However the latter two peptides were significantly less effective in increasing % S phase cells 18-24 h after treatment in agreement with other data demonstrating their reduced activity in long term growth assays [13, 24]. Furthermore, we noted reduced potency of EGF relative to TGF~ despite the fact that both peptides exert their effects via the EGF receptor. Such differential responses to these two growth factors have been noted in a number of other systems[25]. The largest deviation between acute effects of growth factors on cell cycle progression and longer term effects on cell proliferation rates was noted with bFGF. The data presented in Fig. 2 show that bFGF is as effective an inducer of cell cycle progression as insulin or IGF-I while in a study on growth effects bFGF induced only modest increases in cell numbers under similar culture conditions [24]. Other studies from this laboratory have shown that increases in cell number, indicative of sustained action, require higher concentrations of bFGF than do acute increases in S phase fraction (Feldman et al.; manuscript in preparation). Thus degradation of bFGF may reduce its long-term effectiveness. Oestrogen alone failed to induce significant effects but interacted synergistically with insulin to increase cell cycle progression (Fig. 3) and cellular growth rates [13]. Difficulty in demonstrating proliferative responses to E 2 has been a feature of studies into the control of replication of breast cancer cells in tissue culture which may be explained in part by the fact that oestrogen has been tested in the presence of various concentrations of insulin included routinely in culture media. Certainly the data presented here support the conclusions of van der Burg and colleagues[13] who have recently provided a mechanistic explanation for the synergism
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between insulin a n d oestrogen [26]. Oestrogen can induce c - f o s but fails to induce genes o f the j u n family which are essential for the f o r m a t i o n o f the A P o l t r a n s c r i p t i o n complex. Insulin a n d I G F - I by c o n t r a s t are efficient inducers o f c-jun a n d act synergistically with E2 to stimulate A P - I activity a n d cell p r o l i f e r a t i o n [26]. In c o n t r a s t to the sustained effect on S phase fraction a n d cell p r o l i f e r a t i o n induced b y E2 in the presence o f insulin, progestins h a d only a transient s t i m u l a t o r y effect on cell cycle progression (Fig. 4). The increase in S phase was observed several hours before increases in S phase following g r o w t h factor s t i m u l a t i o n indicating action o f progestins at a p o i n t in Gt closer to the G~/S interface. Interestingly the decreases in S phase associated with R U 486 t r e a t m e n t occurred within a similar t i m e - f r a m e as w o u l d be expected if the i n h i b i t o r y effects o f R U 486 were m e d i a t e d via the same p a t h w a y s as the s t i m u l a t o r y effects o f progestins. P e r h a p s m o r e i m p o r t a n t was the o b s e r v a t i o n that the cell cycle kinetic changes a c c o m p a n y i n g the g r o w t h i n h i b i t o r y effects o f b o t h a n t i p r o g e s t i n s and a n t i o e s t r o g e n s were t e m p o r a l l y similar, raising the possibility that these two classes o f steroid a n t a g o n i s t s share similar m e c h a n i s m s o f g r o w t h inhibition. C e r t a i n l y the d a t a presented here (cf. Figs 4 a n d 5) w o u l d argue against a n t i p r o g e s t i n s inducing their g r o w t h i n h i b i t o r y effects via any progestin agonist properties. H a v i n g defined the responses to a spectrum o f g r o w t h r e g u l a t o r y agents, experiments were u n d e r t a k e n to d e t e r m i n e if changes in cell cycle progression were a c c o m p a n i e d by changes in the level o f expression o f two p r o t o - o n c o g e n e s k n o w n to be acutely regulated by a wide range o f agents affecting cell p r o l i f e r a t i o n a n d differe n t i a t i o n [ 1 8 - 2 0 ] . A clear association between the expression o f these genes a n d p r o l i f e r a t i o n was noted. Both genes were induced by agents stimulating p r o l i f e r a t i o n while a n t a g o n i s m o f these increases in cell cycle p r o g r e s s i o n by antioestrogens a n d a n t i p r o g e s t i n s was i n v a r i a b l y associated with inhibition o f the i n d u c t i o n o f c - m y c . W h e t h e r or n o t these changes in p r o t o oncogene expression are necessary or merely associated with the changes in cell cycle p r o g r e s s i o n requires further investigation. In s u m m a r y we have d e v e l o p e d a culture system which allows detailed analysis o f the effects o f h o r m o n e s a n d g r o w t h factors, alone or in c o m b i n a t i o n , on breast cancer cell cycle progression. Such a system should facilitate the d e v e l o p m e n t o f a deeper u n d e r s t a n d i n g o f the
m o l e c u l a r m e c h a n i s m s controlling cell cycle p r o g r e s s i o n in these cells b o t h in d e t e r m i n i n g the roles o f molecules a n d signal t r a n s d u c t i o n p a t h w a y s identified in o t h e r cell types a n d in identifying novel genes a n d m e c h a n i s m s specific to breast epithelial cells. Acknowledgements--These studies were supported by the
National Health and Medical Research Council of Australia and MLC-Life Ltd. Elizabeth Musgrove was the recipient of a Government Employees Assistance to Medical Research Fund Scholarship during the course of these studies. REFERENCES
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