Int. J. Cancer: 52, 928-933 (1992) 0 1992 Wiley-Liss, Inc.
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Publication of the internalional Union Against Cancer Publication de I'Union Internationale Contre le Cancer
HUMAN PROLACTIN REGULATES TRANSFECTED MMTV LTR-DIRECTED GENE EXPRESSION IN A HUMAN BREAST-CARCINOMA CELL LINE THROUGH SYNERGISTIC INTERACTION WITH STEROID HORMONES Soichi HARAGUCHI, Robcrt A. GOOD,Robcrt W. ENGELMAN and Noorbibi K. DAY AN Children's Hospital, University of South Florida, College of Medicine, 801 Sixth Street South, St. Petersburg, FL 33701, USA. Prolactin plays a key role in the regulation and growth of mammary cells, and influences tumor promotion. W e have shown that chronic energy restriction intake depresses prolactin levels, inhibits production of MMTV proviral D N A and proto-oncogene expression in mammary glands and prevents development of mammary tumors. Since the expression and proto-oncogene activation of MMTV are regulated by promoter/ enhancer elements within its long terminal repeat (LTR), in the present study we used a chloramphenicol acetyl transferase (CAT) reporter gene system and gene transfection methods to study the effect of prolactin on MMTV LTR using a human ductal carcinoma cell line T47D stably or transiently transfected with a plasmid consisting of the LTR upstream of CAT gene. Human prolactin or dexamethasone induced, respectively, a 2-fold or 6-fold increase in CAT activity compared with background CAT activity in the absence of hormones. However, the combination of human prolactin and dexamethasone strongly enhanced (20-fold) induction of the LTR compared with the control. Human prolactin also showed a synergistic effect with progesterone on LTR induction. Both LTR and CAT genes needed to be linked for induction of CAT activity by prolactin and dexamethasone. Our results indicate that human prolactin can act synergistically with steroid hormones to regulate MMTV LTR-directed gene expression in transfected T47D cells.
o 1992 Wilqv-Liss,Inc. Prolactin, a polypeptide hormone secreted by lactotropic cells of the pituitary gland, plays an important role in the regulation of growth and differentiation of mammary cells (Buckley et al., 1988) including tumor growth. Mouse mammary tumor virus (MMTV), a major etiologic agent in the development of mammary adenocarcinoma in mice, persists in infected mammary epithelial cells as a proviral copy and induces mammary adenocarcinoma through activation of ccllular proto-oncogenes, the int genes. Activation of the protooncogenes is then directed by the enhancer and promoter elements in the long terminal repeat (LTR) of MMTV (Nusse, 1986). We have shown that dietary chronic encrgy restriction intake (CEIR) suppresses proliferation of mammary alveolar cells, prevcnts tumor development, reduces MMTV and inr gene expression and also depresses prolactin levels (Sarkar et nl., 1982; Chen et al., 1990; Hamada et nl., 1990; Engelman et al., 1991). A crucial role for prolactin in thc early expression of MMTV mRNA in vivo using C3H mice was also recently reported from our laboratory (Hamada et al., 1990). The studies were designed to alter prolactin and energy levels in order to evaluatc their effects on MMTV mRNA expression as follows. Mice on restricted diets were grafted with adenohypophyses in order to increase their prolactin levels. Alternatively, mice fed ad libitum were treated with the dopaminomimetic agent octahydrobenzo[g]quinoline to lower their prolactin levels. The results showed that adenohypophyseal grafting significantly increased prolactin levels in CEIR mice and this effect was associated with an increase in MMTV mRNA expression within thc mammary gland. A linear correlation between prolactin levels and MMTV mRNA expression was found. Conversely, elimination of the nocturnal peak of circulating prolactin by i.p. injection of the dopamine analog delayed by 8 weeks and reduced (even for as long as 25 weeks) mammary-
gland MMTV mRNA expression. Furthermore Engelman et al. (data not shown) observed that > 9 0 % of grafted mice restricted in energy intake with elevated prolactin levels developed high incidence of tumor. Similarly, when prolactin levels were suppressed, tumor incidence was markedly reduced in fully-fed mice. In the present study using CAT reporter gene and gene transfection systems, we present evidence that human prolactin regulates MMTV LTR-directed gene expression in a human breast-cancer cell line. MATERIAL AND METHODS
Cell culture T47D is a human breast-cancer cell line derived from the pleural effusion of a patient with disseminated carcinoma of the breast (Keydar et al., 1979). T47D cells contain a high concentration of prolactin receptors (Shiu, 1979). T47D cells were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO), 10 pgiml bovine insulin (Sigma, St. Louis, MO), 100 Uiml penicillin, 100 pgiml streptomycin and 2 mM L-glutamine in a humidified atmosphere of 5% COz in air.
Hormones Human pituitary prolactin (hPRL) was kindly provided by Dr. S. Raiti from the National Hormone and Pituitary Program, NIDDK (Baltimore, MD). Bovine pancreas insulin, dexamethasone (Dex), progesterone (Prog) and hydrocortisone (HC) were obtained from Sigma. Construction of plasmid.7 The constructs used in the present study are shown in Figure 1. Plasmid pMMTV,,,-CAT was constructed by ligating the BamHI fragment containing the neo gene, a dominant selection gene encoding resistance to the antibiotic G418 from pMAM,,, (Clontec, Palo Alto, CA) to the BamHI fragment containing the MMTV LTR-CAT fusion gene from pMSGCAT (Pharmacia LKB, Piscataway, NJ) by using T4 DNA ligase (GIBCO BRL, Gaithersburg, MD). Plasmid pMMTV,,, was constructed by ligating the BamHI fragment containing neo gene from pMAM,,,, to the BamHI fragment containing MMTV LTR gcne from pMSG (Pharmacia LKB). Plasmid pMAM,,,-CAT consisting of Rous sarcoma virus LTR enhancer upstream of MMTV LTR-CAT fusion gene was obtained from Clontech. Plasmid pCAT-control which contains SV40 promoter and enhancer sequences and a CAT gene was obtained from Promega (Madison, WI). Plasmid pCAT-basic which contains a CAT gene lacking eukaryotic promoter and cnhancer sequences was obtained from Promega. Trarisfection and hormone induction experiments Transient transfection of T47D cells was performed by the DEAE-dextranidimethyl sulfoxide (DMSO) shock procedure (Golub et nl., 1989) with slight modifications as follows. The cell suspension of T47D cells (2 x lo7) dissociated by incubaReceived: May 20. 1992 and in revised form July 28. 1992.
REGULATION OF MMTV LTR BY HUMAN PROLACTIN I
1
0
MMTV-LTR
2
3
5
4
CAT
pMMTVneo-CAT MMTV-LTR
CAT
neo
CAT
pCAT-Basic
47-
~ C A ; Control
-ClCl\S$E-
CAT
.,
6kb
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activity were monitored by 2 alternative methods as follows: ( I ) Liquid scintillation counting (LSC) assay (a phaseextraction assay) (Seed arid Sheen, 1988). The reaction products were extracted with xylene. After 2 brief back-extractions of the xylene phase with 0.25 M Tris-HCI (pH 8.0) to reduce the background, a portion of the xylene phase was mixed with scintillant (ICN, Irvine, CA) and counted in a scintillation counter (LS 3801, Beckman, Irvine, CA). (2) Thin-layer chromatography (TLC) assay. The reaction mixture described above was extracted with ethyl acetate, and then the butyryl chloramphenicol products were separated from unmodified chloramphenicol substrate by ascending TLC on silica plates (J.T. Baker, Phillipsburg. NJ) using a chloroform:methanol (97:3) solvent system. The resolved reaction products were detected by autoradiography.
FIGURE 1 - Diagrams of constructs. The basic features of these Long terminal repeat of constructs are described in the text. mouse mammary tumor virus (MMTV LTR); 0, chloramphenicol acetyl transferase reporter gene (CAT); 0, neo gene (neo); s LTR enhancer; m, SV40 early promoter; RESULTS , SV40 on, early promoter and enhancer; 19, Experimental strategy SV40 early splicing region plus SV40 polyadenylation. To define the influence of human prolactin on LTR, we used both transient and stable gene transfection assays. In these tioii with 0.05% trypsin/0.53 mM EDTA (GIBCO) was washed experiments, hormone-activated LTR of MMTV provides thc with suspension Tris-buffered saline (STBS; 25 mM Tris CI, regulatory elements such as promoter and enhancer for the pH 7.4, 137 mM NaCI, 5 mM KCI, 0.6 mM Na2HP04,0.7 mM expression of the bacterial chloramphenicol acetyltransferase CaCI2, 0.5 mM MgCI2) and subsequently incubated with a (CAT) reporter gene not normally expressed in T47D cells. mixture containing 10 pg of plasmid DNA and 500 yg The effects of hormones on the activation of LTR were D E E - d e x t r a n (Pharmacia) in 1 ml of STBS for 30 min at measured by the enzyme activity of the bacterial CAT. 37°C. After incubation, DMSO was added to the tube to a final concentration of 10%. After DMSO treatment for 2 min at Hiimun prolactin and daumethasone can regulate trunsietitly room temperature. the cells were washed twice with STBS and transfected MMTV LTR-CATfiision gene in T47D cells T47D cells were transiently transfected with various plasonce with RPMI 1640 medium without serum. Stable transfection of T47D cells was carried out using the Polybrene method mids and treated with hormones, i.e., they were transfected (Kawai and Nishizawa, 1984) with slight modifications as with pMMTV,,,-CAT consisting of the complete LTR upfollows. T47D cells (1.5 x lo5) were seeded in 60-mm dishes stream of the bacterial CAT gene. T47D cells were also (Costar, Cambridge, MA) and incubated for 20 hr. At the end transfected with pMMTV,,, which contains the complete LTR of the incubation period, the culture fluid was removed and 1.5 alone or pCAT-basic which contains the CAT gene alone. In ml of RPMI 1640 containing 30 yg of polybrene (Aldrich, preliminary experiments, we had observed that hPRL and Dex Milwaukee, WI) were added to the dishes. After 1 hr incuba- require confluent cell culture to induce high LTR activation. tion, 1 yg of plasmid DNA and 10 yg salmon-sperm DNA The transiently transfected T47D cells were therefore cultured (Sigma) were added to the dishes, and the cultures were in medium for 4 days and treated with hPRL, Dex and insulin incubated for 6 hr with occasional shaking. The culture fluid in the fresh medium for another 2 days, and then the CAT was then removed, and the culture was treated with 2.5 ml of activity was determined. Significant CAT activity was induced medium containing 30% DMSO at room temperature for 4 in T47D cells transfected with pMMTV,,,-CAT by the stimulamin. The culture fluid was removed, and the cells were rinsed tion of hPRL, Dex plus insulin. No detectable level of CAT with medium and then incubated with fresh medium. The activity was observed with treatment using hPRL, Dex and transfected T47D clones were selected in culture medium insulin in T47D cells transfected with LTR alone (pMMTV,,,) supplemented with 600 pg/ml G418 (GIBCO). After 2 to 4 or CAT gene alone (pCAT-Basic) (data not shown). These weeks. clones with stable transfectants appeared. The clones results indicate that both CAT and LTR genes need to be were pooled and expanded in medium containing G418. linked for induction of CAT activity by prolactin and dexaTransiently transfected T47D cells (1.2 x lo6) or T47D stable methasone. transfectant cells (6 X lo5) were cultured in media in a 60-mm To examine the specificity of the hormonal response, T47D cell-culture dish (Costar) for 4 days. The appropriate hor- cells were transiently transfected with pCAT-control consistmones were then added after medium change and cultured for ing of SV40 promoter and enhancer sequences upstream of an additional 2 days. CAT activity was assayed as described CAT gene. CAT activity was comparable with that of hPRL, below. Dex and insulin or insulin alone (data not shown). The above results clearly indicate that hPRL and Dex can act to regulate CA T assay the promoter and/or enhancer elements in the LTR in human CAT assay was performed according to the method dc- breast-cancer cells, whereas hPRL and Dex have no effect on scribed by Gorman et al. (1982) with a slight modification. The SV40 promoter and/or enhancer. These control experiments cells were disrupted 3 times by freezing in a dry-iceiethanol were performed in transiently transfected cells, because pCATbath and thawing at 37°C. The crude extracts were taken for basic and pCAT-control plasmids do not contain neo gene to protein determinations using a protein assay kit (Bio-Rad, select stable transfectants. Richmond, CA) and assayed for CAT activity to convert [I4C] chloramphenicol to the acetylated form. Typically, CAT en- Human prolactin can replate stably transfected MMTV zyme activities were determined in a reaction containing 300 LTR-CATfusion gene 111 T47D cells pg of proteins with 0.1 pCi [ ' T I chloramphenicol (55.3 Following the above transient transfection experiments, a mCi/mM, NEN, Boston, MA), 25 yg n-butyryl Coenzyme A stable transfection assay was conducted to further analyze the (Sigma) in 125 pl of 0.25 M Tris-HCI (pH 8.0). Reactions were influence of hPRL on the activation of LTR. The recombinant allowed to proceed for 120 min at 37°C. Quantitations of CAT plasmid pMMTV,,,-CAT was transfected into T47D cells.
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HARAGUCHI E T A L . TABLE I -SYNERGISTIC EFFECTS OF HUMAN PROLACTIN WITH STEROID HORMONES ON MMTV LTR INDUCTION Hormone
Construct
pMMTV,,,-CAT'
CAT' activity
hPRL
Dex
+ + + + -
+
Prog
Insulin
-
+ + + ++
(cpm)
Induction ratio. -fold
8,188 23.46 7,114 20.56 855 2.45 799 2.29 + 2,573 7.37 +2,317 6.64 335 0.96 349 1.oo pMAM,,,-CATI ++ 147,000 49.00 ++ 35,000 11.67 3,000 1.00 pMMTV,,,,-CAT2 + + 25,810 77.28 +885 2.65 + 9.908 29.66 334 1.oo 'T47D cells stably transfected with pMMTV,,,-CAT or pMAM,,,-CAT construct were cultured in medium for 4 days and then incubated with 1 pgiml human prolactin (hPRL), 1 pM dexamethasone (Dex). and/or 500 ng/ml insulin in the fresh medium for another 2 days.-*T47D cells were transiently transfected with PMMTV,,,-CAT, cultured in medium for 4 days, and treated with 1 pgiml human prolactin (hPRL) and/or 10 nM progesterone (Prog) in the fresh medium for another 2 day~.-~Crudecell extracts were tested for CAT activity using LSC assay. The data represent CAT activity in cellular extract containing 300 pg protein. Results are expressed as the mean of duplicate cultures. The variability of duplicate determinations in the CAT assay is less than 10%. Similar results were obtained in 2 additional experiments.
+-
Stably transfected T47D cells were selected on the basis of their resistance to the antibiotic G418. The stably transfected T47D cells were cultured in medium for 4 days and treated with hormones in the fresh medium for another 2 days, and then CAT activity was determined. Table I (representative of 3 separate experiments) shows the effects of various combinations of hPRL, Dex and insulin on the induction of LTR. Addition of hPRL or Dex alone caused approx. 3-fold or approx. 7-fold induction above the basal CAT activity detected in T47D cells cultured in the absence of hormones. The experiment with the effects on CAT expression by hPRL alone was repeated 8 times and the data showed that hPRL (1.000 ngiml) alone always induced 2- to 3-fold CATactivity (p < 0.01 compared to medium alone control). Also, hPRL induced 2- to 3-fold CAT gene expression in T47D cells cultured in serumfree medium containing 25 pg/ml BSA (data not shown). The combination of hPRL and Dex resulted in a strong induction of CAT activity, i.e., a 21-fold increase. The synergistic action between hPRL and Dex was already seen 6 hr after addition of the hormones (data not shown). Additional experiments, also presented in Table I were then designed to study the effects of hPRL on MMTV LTR. In these experiments. T47D cells were stably transfected with a plasmid pMAMne,-CAT which is designed for high-level expression of CAT gene by linking the strong Rous sarcoma virus LTR enhancer to the LTR-CAT fusion gene. As shown in the Table (pMAM,,,-CAT), Dex and insulin caused a 11.67-fold induction of expression of CAT activity. However, CAT activity was increased 49-fold above the basal activity, when hPRL was added to the culture. Furthermore, hPRL acts synergistically with progesterone to induce the activation of LTR (Table I).
Prolactin dose-response c u n m Figure 2u shows the influence of various concentrations (10 to 5.000 ngiml) of hPRL on LTR activation. These concentrations of hPRL were used since our preliminary results showed that T47D cells under the conditions used here were less scnsitivc to hPRL at 10 p,g/ml (data not shown). As shown,
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hPRL induced CAT gene expression in a dose-dependent manner in T47D cells. By contrast, a significant induction of CAT activity was observed when the stable transfectants were stimulated with a combination of hPRL, Dex and insulin. The LTR in T47D cells responded to a concentration of hPRL as low as 10 ng/ml in the presence of Dex and insulin. The concentrations of hPRL used in the controls and the effects observed in Figure 2a are consistent with previous findings (Djiane et al., 1982; Muiioz and Bolander, 1989).
Effects of various concentrations of dexamethasone on prolactin induction of Mh47V L T R Since signals by Dex seem to be required to induce a strong reaction, we examined the effects of various concentrations M to M) of Dex on the prolactin regulation of LTR. As shown in Figure 2b, a significant induction of CAT activity, i.e., a strong synergism between hPRL and Dex, was observed when hPRL was added to the culture with M Dex. Furthermore, hPRL enhanced these responses. hPRL raised the levels of CAT activity in T47D cells treated with lo-* M Dex to the levels of CAT activity in T47D cells treated with M Dex (an influence attributable to a 100-fold difference in concentration of Dex). The concentrations of Dex used in the controls and the effects observed in Figure 26 are consistent with previous findings (Cato et al., 1986; Muiioz and Bolander, 1989). Evaluation of the effects of human prolactin on MMTV L T R expression using the TLC assay The TLC assay was performed for visual confirmation of the results described above. As shown in Figure 3, hPRL strongly enhanced the CAT activity induced by the combination of Dex and insulin or the combination of hydrocortisone and insulin. DISCUSSION
In this report we have applied the CAT reporter gene and gene transfection systems to investigate the effect of human prolactin on induction of MMTV LTR. W e provide direct
931
REGULATION OF MMTV LTR BY HUMAN PROLACTIN
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x
2
v
2. 5 ._ > ._ c
0
1
8
4 -
2 0
0
Y/ ' 0 10
3-
50 100
500 1000
Human Prolactin (ng/ml)
5000 2 1 -
'
0- Y
o
10-9
10.8
10'7
lo+
Dexamethasone (M)
FIGURE 2 - Dose-response curves for human prolactin and dexamethasone regulation of stably transfected MMTV LTR in T47D cells. Stable transfectants of T47D cells with plasmid MMTV,,,,-CAT were cultured in medium for 4 days and then treated with hormones in the fresh medium for an additional 2 days. (a) Human prolactin (hPRL) (as indicated), 1 pM dexamethasone (Dex) and 500 ng/ml insulin; 0,hPRL (as indicated) and 500 ngiml insulin; (b) 0, 1 pg/ml human prolactin (hPRL), dexamethasone, (,Dex),(as indicated) and 500 ngiml insulin; 0,Dex (as indicated) and 500 ngiml insulin. Crude cell extracts were tested for CAT activity using LSC assay. The data represent CAT activity in cellular extract containing 300 pg protein. Results are expressed as the mean of duplicate cultures. The variability of duplicate determinations in the CAT assay is less than 10%. Similar results were obtained in 2 additional experiments.
6,
experimental evidence that human prolactin can enhance the expression of LTR in a human breast-cancer cell line. A potential mechanism in tumorigenesis in mice attributable to MMTV appears to be transcriptional activation of cellular proto-oncogenes by enhancer and/or promoter sequences in the viral LTR (Nusse, 1986). To evaluate whether human prolactin and other hormones regulate enhancer andlor promoter sequences in LTR directly, we constructed a recombinant plasmid consisting of LTR of MMTV upstream of CAT gene. The effects of hormones on the activation of LTR were monitored by the enzyme activity of the bacterial CAT. The LTR-CAT fusion gene was stably o r transiently transfected into human breast-cancer cell line T47D cells. T47D cells contain a high concentration of prolactin receptors and are biologically responsive to human prolactin (Shiu, 1979). This cell line appears to be particularly useful for studying the effects of prolactin in breast cancer at a molecular level. Our results show that treatment with human prolactin plus dexamethasone induces significant levels of CAT activity in T47D cells transfected with a LTR-CAT fusion gene, but not in cells transfected with LTR or CATgene alone. Also, human prolactin plus dexamethasone exerts no effect on SV40 promoter and enhancer activity. These results indicate that the hormones used induce CAT activity through activation of the LTR. Furthermore, they clearly show that human prolactin can act synergistically with glucocorticoid hormone to induce LTR-directed gene expression in transfected T47D human breast-cancer cells. Strong synergism between hPRL and Dex was observed when hPRL was added to the culture with M Dex. T47D cells are also known to have high levels of
progesterone receptors. Also, glucocorticoid receptors and progesterone receptors are known to interact with the same sites (hormone-responsive element) in LTR (von der Ahe et al., 1985). The synergistic effects at higher concentrations of Dex observed in this study may be mediated through interaction with the progesterone receptors, as suggested by other studies (Cato et a/., 1986; Muiioz and Bolander, 1989). In this context, we have shown that hPRL also acts synergistically with progesterone to induce the activation of LTR, suggesting that the synergism could occur through the progesterone pathway. In either case, the results indicate that the positive influence of prolactin in the present studies operates through the induction of promoter and/or enhancer sequences in the viral LTR. Synergistic relationships between prolactin and glucocorticoid have previously been reported to operate on alteration of cell shape, adhesion and lipid accumulation in T47D cells (Shiu and Paterson, 1984) and to induce expression of p-casein gene promoter in a mouse mammary epithelial cell line (Doppler ct al., 1989). These latter investigators, using ovine prolactin and p-casein gene promoter constructs, demonstrated that only stably but not transiently transfected constructs were induced with both PRL and dexamethasone. Our results differ in that we were able to produce strong synergetic effects using either stable or transient constructs. The reason for this difference could be that Doppler et al. used rat p-casein gene promoter constructs and a mouse mammary epithelial cell line, whereas we used MMTV LTR promoter constructs and a human cell line derived from a patient with mammary adenocarcinoma. The exact mechanisms by which prolactin controls the regulatory elements in LTR are still not understood. The
932
HARAGUCHI E T A L .
FIGURE3 - Induction of CAT activity by human prolactin in T47D cells stably transfected with pMMTV,,,-CAT. Culture conditions are the same as in Figure 2. Crude cell extracts were tested for their ability to convert ['4CJchloramphenicol (Chl) to the acetylated form (Ac-Chl) using TLC assay. hPRL. 1 kglml human prolactin; Dex, 1 pM dexarnethasone; HC, 50 ngiml hydrocortisone; insulin, 500 ngiml insulin. The experiment was repeated twice, and one of the typical results is shown in the Figure. protein kinase C (PKC) activator 12-0-tetradecanoylphorbol 13-acetate (TPA) has been found to have prolactin-like activity on mouse mammary gland explants (Rillema and Waters, 1986). It has been postulated that the mechanism by which prolactin stimulates proliferation involves the generation of second messengers coupled to PKC activation (Waters and
Rillema, 1989). Also. it has been found that TPA induces the nuclear transcription factor AP-1, composed of heterodimeric combinations of proto-oncogene products, a Jun family protein with a Fos family protein formed through the leucine zippers in these proteins (Kouzarides and Ziff. 1989). In this context, it is interesting to note that AP-1 consensus binding sites are present in MMTV LTR. Alternatively. transcription factors, whose consensus binding sites are present in MMTV LTR and which can interact synergistically with steroid receptors, have been reported (Schule et al., 1988; Bruggemeier et al., 1991). Also, a novel mammary cell-line-specific enhancer element in the MMTV LTR, which interacts with the hormoneresponsive clement in MMTV LTR, has been described (Yanagawa e l al., 1991). These investigators have identified nuclear factors that specifically interacted with this enhancer element in the nuclear extract from T47D cells (Yanagawa et al., 1991). Taking these previous reports into consideration, an attractive interpretation of the observed results in the present study is that the mammotropic polypeptide hormone prolactin might induce transcription factors, which can relay in the rcgulatory circuit for the induction of LTR in mammary cells. The sequences that confer steroid hormone inducibility on the MMTV promoter have been extensively characterized. The characterization of functional elements in the LTR involved in thc synergistic action between prolactin and steroid hormones can be pursued in future analyses. The identification of prolactin-responsive elements in LTR that is responsible for prolactin-induced gene transcription represents a subject of great interest. Additional experiments aimed at understanding the molecular mechanisms responsible for induction of MMTV LTR by prolactin are already in progress, and may better determine the significance of the role of prolactin in the development of breast cancer. ACKNOWLEDGEMENTS This work was supported by grants from the Eleanor Naylor Dana Charitable Trust, National Institutes of Health AGO5633 and CA41061, Ronald McDonald Charities and the Newland Foundation. We thank Dr. William G. Bradley for reviewing the manuscript. We also appreciate the assistance of Ms. C. Gotlieb and Ms. S. Littlc in the preparation of the manuscript.
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Publication of the internalional Union Against Cancer Publication de I'Union Internationale Contre le Cancer
HUMAN PROLACTIN REGULATES TRANSFECTED MMTV LTR-DIRECTED GENE EXPRESSION IN A HUMAN BREAST-CARCINOMA CELL LINE THROUGH SYNERGISTIC INTERACTION WITH STEROID HORMONES Soichi HARAGUCHI, Robcrt A. GOOD,Robcrt W. ENGELMAN and Noorbibi K. DAY AN Children's Hospital, University of South Florida, College of Medicine, 801 Sixth Street South, St. Petersburg, FL 33701, USA. Prolactin plays a key role in the regulation and growth of mammary cells, and influences tumor promotion. W e have shown that chronic energy restriction intake depresses prolactin levels, inhibits production of MMTV proviral D N A and proto-oncogene expression in mammary glands and prevents development of mammary tumors. Since the expression and proto-oncogene activation of MMTV are regulated by promoter/ enhancer elements within its long terminal repeat (LTR), in the present study we used a chloramphenicol acetyl transferase (CAT) reporter gene system and gene transfection methods to study the effect of prolactin on MMTV LTR using a human ductal carcinoma cell line T47D stably or transiently transfected with a plasmid consisting of the LTR upstream of CAT gene. Human prolactin or dexamethasone induced, respectively, a 2-fold or 6-fold increase in CAT activity compared with background CAT activity in the absence of hormones. However, the combination of human prolactin and dexamethasone strongly enhanced (20-fold) induction of the LTR compared with the control. Human prolactin also showed a synergistic effect with progesterone on LTR induction. Both LTR and CAT genes needed to be linked for induction of CAT activity by prolactin and dexamethasone. Our results indicate that human prolactin can act synergistically with steroid hormones to regulate MMTV LTR-directed gene expression in transfected T47D cells.
o 1992 Wilqv-Liss,Inc. Prolactin, a polypeptide hormone secreted by lactotropic cells of the pituitary gland, plays an important role in the regulation of growth and differentiation of mammary cells (Buckley et al., 1988) including tumor growth. Mouse mammary tumor virus (MMTV), a major etiologic agent in the development of mammary adenocarcinoma in mice, persists in infected mammary epithelial cells as a proviral copy and induces mammary adenocarcinoma through activation of ccllular proto-oncogenes, the int genes. Activation of the protooncogenes is then directed by the enhancer and promoter elements in the long terminal repeat (LTR) of MMTV (Nusse, 1986). We have shown that dietary chronic encrgy restriction intake (CEIR) suppresses proliferation of mammary alveolar cells, prevcnts tumor development, reduces MMTV and inr gene expression and also depresses prolactin levels (Sarkar et nl., 1982; Chen et al., 1990; Hamada et nl., 1990; Engelman et al., 1991). A crucial role for prolactin in thc early expression of MMTV mRNA in vivo using C3H mice was also recently reported from our laboratory (Hamada et al., 1990). The studies were designed to alter prolactin and energy levels in order to evaluatc their effects on MMTV mRNA expression as follows. Mice on restricted diets were grafted with adenohypophyses in order to increase their prolactin levels. Alternatively, mice fed ad libitum were treated with the dopaminomimetic agent octahydrobenzo[g]quinoline to lower their prolactin levels. The results showed that adenohypophyseal grafting significantly increased prolactin levels in CEIR mice and this effect was associated with an increase in MMTV mRNA expression within thc mammary gland. A linear correlation between prolactin levels and MMTV mRNA expression was found. Conversely, elimination of the nocturnal peak of circulating prolactin by i.p. injection of the dopamine analog delayed by 8 weeks and reduced (even for as long as 25 weeks) mammary-
gland MMTV mRNA expression. Furthermore Engelman et al. (data not shown) observed that > 9 0 % of grafted mice restricted in energy intake with elevated prolactin levels developed high incidence of tumor. Similarly, when prolactin levels were suppressed, tumor incidence was markedly reduced in fully-fed mice. In the present study using CAT reporter gene and gene transfection systems, we present evidence that human prolactin regulates MMTV LTR-directed gene expression in a human breast-cancer cell line. MATERIAL AND METHODS
Cell culture T47D is a human breast-cancer cell line derived from the pleural effusion of a patient with disseminated carcinoma of the breast (Keydar et al., 1979). T47D cells contain a high concentration of prolactin receptors (Shiu, 1979). T47D cells were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO), 10 pgiml bovine insulin (Sigma, St. Louis, MO), 100 Uiml penicillin, 100 pgiml streptomycin and 2 mM L-glutamine in a humidified atmosphere of 5% COz in air.
Hormones Human pituitary prolactin (hPRL) was kindly provided by Dr. S. Raiti from the National Hormone and Pituitary Program, NIDDK (Baltimore, MD). Bovine pancreas insulin, dexamethasone (Dex), progesterone (Prog) and hydrocortisone (HC) were obtained from Sigma. Construction of plasmid.7 The constructs used in the present study are shown in Figure 1. Plasmid pMMTV,,,-CAT was constructed by ligating the BamHI fragment containing the neo gene, a dominant selection gene encoding resistance to the antibiotic G418 from pMAM,,, (Clontec, Palo Alto, CA) to the BamHI fragment containing the MMTV LTR-CAT fusion gene from pMSGCAT (Pharmacia LKB, Piscataway, NJ) by using T4 DNA ligase (GIBCO BRL, Gaithersburg, MD). Plasmid pMMTV,,, was constructed by ligating the BamHI fragment containing neo gene from pMAM,,,, to the BamHI fragment containing MMTV LTR gcne from pMSG (Pharmacia LKB). Plasmid pMAM,,,-CAT consisting of Rous sarcoma virus LTR enhancer upstream of MMTV LTR-CAT fusion gene was obtained from Clontech. Plasmid pCAT-control which contains SV40 promoter and enhancer sequences and a CAT gene was obtained from Promega (Madison, WI). Plasmid pCAT-basic which contains a CAT gene lacking eukaryotic promoter and cnhancer sequences was obtained from Promega. Trarisfection and hormone induction experiments Transient transfection of T47D cells was performed by the DEAE-dextranidimethyl sulfoxide (DMSO) shock procedure (Golub et nl., 1989) with slight modifications as follows. The cell suspension of T47D cells (2 x lo7) dissociated by incubaReceived: May 20. 1992 and in revised form July 28. 1992.
REGULATION OF MMTV LTR BY HUMAN PROLACTIN I
1
0
MMTV-LTR
2
3
5
4
CAT
pMMTVneo-CAT MMTV-LTR
CAT
neo
CAT
pCAT-Basic
47-
~ C A ; Control
-ClCl\S$E-
CAT
.,
6kb
929
activity were monitored by 2 alternative methods as follows: ( I ) Liquid scintillation counting (LSC) assay (a phaseextraction assay) (Seed arid Sheen, 1988). The reaction products were extracted with xylene. After 2 brief back-extractions of the xylene phase with 0.25 M Tris-HCI (pH 8.0) to reduce the background, a portion of the xylene phase was mixed with scintillant (ICN, Irvine, CA) and counted in a scintillation counter (LS 3801, Beckman, Irvine, CA). (2) Thin-layer chromatography (TLC) assay. The reaction mixture described above was extracted with ethyl acetate, and then the butyryl chloramphenicol products were separated from unmodified chloramphenicol substrate by ascending TLC on silica plates (J.T. Baker, Phillipsburg. NJ) using a chloroform:methanol (97:3) solvent system. The resolved reaction products were detected by autoradiography.
FIGURE 1 - Diagrams of constructs. The basic features of these Long terminal repeat of constructs are described in the text. mouse mammary tumor virus (MMTV LTR); 0, chloramphenicol acetyl transferase reporter gene (CAT); 0, neo gene (neo); s LTR enhancer; m, SV40 early promoter; RESULTS , SV40 on, early promoter and enhancer; 19, Experimental strategy SV40 early splicing region plus SV40 polyadenylation. To define the influence of human prolactin on LTR, we used both transient and stable gene transfection assays. In these tioii with 0.05% trypsin/0.53 mM EDTA (GIBCO) was washed experiments, hormone-activated LTR of MMTV provides thc with suspension Tris-buffered saline (STBS; 25 mM Tris CI, regulatory elements such as promoter and enhancer for the pH 7.4, 137 mM NaCI, 5 mM KCI, 0.6 mM Na2HP04,0.7 mM expression of the bacterial chloramphenicol acetyltransferase CaCI2, 0.5 mM MgCI2) and subsequently incubated with a (CAT) reporter gene not normally expressed in T47D cells. mixture containing 10 pg of plasmid DNA and 500 yg The effects of hormones on the activation of LTR were D E E - d e x t r a n (Pharmacia) in 1 ml of STBS for 30 min at measured by the enzyme activity of the bacterial CAT. 37°C. After incubation, DMSO was added to the tube to a final concentration of 10%. After DMSO treatment for 2 min at Hiimun prolactin and daumethasone can regulate trunsietitly room temperature. the cells were washed twice with STBS and transfected MMTV LTR-CATfiision gene in T47D cells T47D cells were transiently transfected with various plasonce with RPMI 1640 medium without serum. Stable transfection of T47D cells was carried out using the Polybrene method mids and treated with hormones, i.e., they were transfected (Kawai and Nishizawa, 1984) with slight modifications as with pMMTV,,,-CAT consisting of the complete LTR upfollows. T47D cells (1.5 x lo5) were seeded in 60-mm dishes stream of the bacterial CAT gene. T47D cells were also (Costar, Cambridge, MA) and incubated for 20 hr. At the end transfected with pMMTV,,, which contains the complete LTR of the incubation period, the culture fluid was removed and 1.5 alone or pCAT-basic which contains the CAT gene alone. In ml of RPMI 1640 containing 30 yg of polybrene (Aldrich, preliminary experiments, we had observed that hPRL and Dex Milwaukee, WI) were added to the dishes. After 1 hr incuba- require confluent cell culture to induce high LTR activation. tion, 1 yg of plasmid DNA and 10 yg salmon-sperm DNA The transiently transfected T47D cells were therefore cultured (Sigma) were added to the dishes, and the cultures were in medium for 4 days and treated with hPRL, Dex and insulin incubated for 6 hr with occasional shaking. The culture fluid in the fresh medium for another 2 days, and then the CAT was then removed, and the culture was treated with 2.5 ml of activity was determined. Significant CAT activity was induced medium containing 30% DMSO at room temperature for 4 in T47D cells transfected with pMMTV,,,-CAT by the stimulamin. The culture fluid was removed, and the cells were rinsed tion of hPRL, Dex plus insulin. No detectable level of CAT with medium and then incubated with fresh medium. The activity was observed with treatment using hPRL, Dex and transfected T47D clones were selected in culture medium insulin in T47D cells transfected with LTR alone (pMMTV,,,) supplemented with 600 pg/ml G418 (GIBCO). After 2 to 4 or CAT gene alone (pCAT-Basic) (data not shown). These weeks. clones with stable transfectants appeared. The clones results indicate that both CAT and LTR genes need to be were pooled and expanded in medium containing G418. linked for induction of CAT activity by prolactin and dexaTransiently transfected T47D cells (1.2 x lo6) or T47D stable methasone. transfectant cells (6 X lo5) were cultured in media in a 60-mm To examine the specificity of the hormonal response, T47D cell-culture dish (Costar) for 4 days. The appropriate hor- cells were transiently transfected with pCAT-control consistmones were then added after medium change and cultured for ing of SV40 promoter and enhancer sequences upstream of an additional 2 days. CAT activity was assayed as described CAT gene. CAT activity was comparable with that of hPRL, below. Dex and insulin or insulin alone (data not shown). The above results clearly indicate that hPRL and Dex can act to regulate CA T assay the promoter and/or enhancer elements in the LTR in human CAT assay was performed according to the method dc- breast-cancer cells, whereas hPRL and Dex have no effect on scribed by Gorman et al. (1982) with a slight modification. The SV40 promoter and/or enhancer. These control experiments cells were disrupted 3 times by freezing in a dry-iceiethanol were performed in transiently transfected cells, because pCATbath and thawing at 37°C. The crude extracts were taken for basic and pCAT-control plasmids do not contain neo gene to protein determinations using a protein assay kit (Bio-Rad, select stable transfectants. Richmond, CA) and assayed for CAT activity to convert [I4C] chloramphenicol to the acetylated form. Typically, CAT en- Human prolactin can replate stably transfected MMTV zyme activities were determined in a reaction containing 300 LTR-CATfusion gene 111 T47D cells pg of proteins with 0.1 pCi [ ' T I chloramphenicol (55.3 Following the above transient transfection experiments, a mCi/mM, NEN, Boston, MA), 25 yg n-butyryl Coenzyme A stable transfection assay was conducted to further analyze the (Sigma) in 125 pl of 0.25 M Tris-HCI (pH 8.0). Reactions were influence of hPRL on the activation of LTR. The recombinant allowed to proceed for 120 min at 37°C. Quantitations of CAT plasmid pMMTV,,,-CAT was transfected into T47D cells.
930
HARAGUCHI E T A L . TABLE I -SYNERGISTIC EFFECTS OF HUMAN PROLACTIN WITH STEROID HORMONES ON MMTV LTR INDUCTION Hormone
Construct
pMMTV,,,-CAT'
CAT' activity
hPRL
Dex
+ + + + -
+
Prog
Insulin
-
+ + + ++
(cpm)
Induction ratio. -fold
8,188 23.46 7,114 20.56 855 2.45 799 2.29 + 2,573 7.37 +2,317 6.64 335 0.96 349 1.oo pMAM,,,-CATI ++ 147,000 49.00 ++ 35,000 11.67 3,000 1.00 pMMTV,,,,-CAT2 + + 25,810 77.28 +885 2.65 + 9.908 29.66 334 1.oo 'T47D cells stably transfected with pMMTV,,,-CAT or pMAM,,,-CAT construct were cultured in medium for 4 days and then incubated with 1 pgiml human prolactin (hPRL), 1 pM dexamethasone (Dex). and/or 500 ng/ml insulin in the fresh medium for another 2 days.-*T47D cells were transiently transfected with PMMTV,,,-CAT, cultured in medium for 4 days, and treated with 1 pgiml human prolactin (hPRL) and/or 10 nM progesterone (Prog) in the fresh medium for another 2 day~.-~Crudecell extracts were tested for CAT activity using LSC assay. The data represent CAT activity in cellular extract containing 300 pg protein. Results are expressed as the mean of duplicate cultures. The variability of duplicate determinations in the CAT assay is less than 10%. Similar results were obtained in 2 additional experiments.
+-
Stably transfected T47D cells were selected on the basis of their resistance to the antibiotic G418. The stably transfected T47D cells were cultured in medium for 4 days and treated with hormones in the fresh medium for another 2 days, and then CAT activity was determined. Table I (representative of 3 separate experiments) shows the effects of various combinations of hPRL, Dex and insulin on the induction of LTR. Addition of hPRL or Dex alone caused approx. 3-fold or approx. 7-fold induction above the basal CAT activity detected in T47D cells cultured in the absence of hormones. The experiment with the effects on CAT expression by hPRL alone was repeated 8 times and the data showed that hPRL (1.000 ngiml) alone always induced 2- to 3-fold CATactivity (p < 0.01 compared to medium alone control). Also, hPRL induced 2- to 3-fold CAT gene expression in T47D cells cultured in serumfree medium containing 25 pg/ml BSA (data not shown). The combination of hPRL and Dex resulted in a strong induction of CAT activity, i.e., a 21-fold increase. The synergistic action between hPRL and Dex was already seen 6 hr after addition of the hormones (data not shown). Additional experiments, also presented in Table I were then designed to study the effects of hPRL on MMTV LTR. In these experiments. T47D cells were stably transfected with a plasmid pMAMne,-CAT which is designed for high-level expression of CAT gene by linking the strong Rous sarcoma virus LTR enhancer to the LTR-CAT fusion gene. As shown in the Table (pMAM,,,-CAT), Dex and insulin caused a 11.67-fold induction of expression of CAT activity. However, CAT activity was increased 49-fold above the basal activity, when hPRL was added to the culture. Furthermore, hPRL acts synergistically with progesterone to induce the activation of LTR (Table I).
Prolactin dose-response c u n m Figure 2u shows the influence of various concentrations (10 to 5.000 ngiml) of hPRL on LTR activation. These concentrations of hPRL were used since our preliminary results showed that T47D cells under the conditions used here were less scnsitivc to hPRL at 10 p,g/ml (data not shown). As shown,
-
hPRL induced CAT gene expression in a dose-dependent manner in T47D cells. By contrast, a significant induction of CAT activity was observed when the stable transfectants were stimulated with a combination of hPRL, Dex and insulin. The LTR in T47D cells responded to a concentration of hPRL as low as 10 ng/ml in the presence of Dex and insulin. The concentrations of hPRL used in the controls and the effects observed in Figure 2a are consistent with previous findings (Djiane et al., 1982; Muiioz and Bolander, 1989).
Effects of various concentrations of dexamethasone on prolactin induction of Mh47V L T R Since signals by Dex seem to be required to induce a strong reaction, we examined the effects of various concentrations M to M) of Dex on the prolactin regulation of LTR. As shown in Figure 2b, a significant induction of CAT activity, i.e., a strong synergism between hPRL and Dex, was observed when hPRL was added to the culture with M Dex. Furthermore, hPRL enhanced these responses. hPRL raised the levels of CAT activity in T47D cells treated with lo-* M Dex to the levels of CAT activity in T47D cells treated with M Dex (an influence attributable to a 100-fold difference in concentration of Dex). The concentrations of Dex used in the controls and the effects observed in Figure 26 are consistent with previous findings (Cato et al., 1986; Muiioz and Bolander, 1989). Evaluation of the effects of human prolactin on MMTV L T R expression using the TLC assay The TLC assay was performed for visual confirmation of the results described above. As shown in Figure 3, hPRL strongly enhanced the CAT activity induced by the combination of Dex and insulin or the combination of hydrocortisone and insulin. DISCUSSION
In this report we have applied the CAT reporter gene and gene transfection systems to investigate the effect of human prolactin on induction of MMTV LTR. W e provide direct
931
REGULATION OF MMTV LTR BY HUMAN PROLACTIN
B
A 4
z 0
9 8 -
5
0
z
7 -
x
v
?L
2.
xz
c
'5 .- 2
u a
6 -
x
2
v
2. 5 ._ > ._ c
0
1
8
4 -
2 0
0
Y/ ' 0 10
3-
50 100
500 1000
Human Prolactin (ng/ml)
5000 2 1 -
'
0- Y
o
10-9
10.8
10'7
lo+
Dexamethasone (M)
FIGURE 2 - Dose-response curves for human prolactin and dexamethasone regulation of stably transfected MMTV LTR in T47D cells. Stable transfectants of T47D cells with plasmid MMTV,,,,-CAT were cultured in medium for 4 days and then treated with hormones in the fresh medium for an additional 2 days. (a) Human prolactin (hPRL) (as indicated), 1 pM dexamethasone (Dex) and 500 ng/ml insulin; 0,hPRL (as indicated) and 500 ngiml insulin; (b) 0, 1 pg/ml human prolactin (hPRL), dexamethasone, (,Dex),(as indicated) and 500 ngiml insulin; 0,Dex (as indicated) and 500 ngiml insulin. Crude cell extracts were tested for CAT activity using LSC assay. The data represent CAT activity in cellular extract containing 300 pg protein. Results are expressed as the mean of duplicate cultures. The variability of duplicate determinations in the CAT assay is less than 10%. Similar results were obtained in 2 additional experiments.
6,
experimental evidence that human prolactin can enhance the expression of LTR in a human breast-cancer cell line. A potential mechanism in tumorigenesis in mice attributable to MMTV appears to be transcriptional activation of cellular proto-oncogenes by enhancer and/or promoter sequences in the viral LTR (Nusse, 1986). To evaluate whether human prolactin and other hormones regulate enhancer andlor promoter sequences in LTR directly, we constructed a recombinant plasmid consisting of LTR of MMTV upstream of CAT gene. The effects of hormones on the activation of LTR were monitored by the enzyme activity of the bacterial CAT. The LTR-CAT fusion gene was stably o r transiently transfected into human breast-cancer cell line T47D cells. T47D cells contain a high concentration of prolactin receptors and are biologically responsive to human prolactin (Shiu, 1979). This cell line appears to be particularly useful for studying the effects of prolactin in breast cancer at a molecular level. Our results show that treatment with human prolactin plus dexamethasone induces significant levels of CAT activity in T47D cells transfected with a LTR-CAT fusion gene, but not in cells transfected with LTR or CATgene alone. Also, human prolactin plus dexamethasone exerts no effect on SV40 promoter and enhancer activity. These results indicate that the hormones used induce CAT activity through activation of the LTR. Furthermore, they clearly show that human prolactin can act synergistically with glucocorticoid hormone to induce LTR-directed gene expression in transfected T47D human breast-cancer cells. Strong synergism between hPRL and Dex was observed when hPRL was added to the culture with M Dex. T47D cells are also known to have high levels of
progesterone receptors. Also, glucocorticoid receptors and progesterone receptors are known to interact with the same sites (hormone-responsive element) in LTR (von der Ahe et al., 1985). The synergistic effects at higher concentrations of Dex observed in this study may be mediated through interaction with the progesterone receptors, as suggested by other studies (Cato et a/., 1986; Muiioz and Bolander, 1989). In this context, we have shown that hPRL also acts synergistically with progesterone to induce the activation of LTR, suggesting that the synergism could occur through the progesterone pathway. In either case, the results indicate that the positive influence of prolactin in the present studies operates through the induction of promoter and/or enhancer sequences in the viral LTR. Synergistic relationships between prolactin and glucocorticoid have previously been reported to operate on alteration of cell shape, adhesion and lipid accumulation in T47D cells (Shiu and Paterson, 1984) and to induce expression of p-casein gene promoter in a mouse mammary epithelial cell line (Doppler ct al., 1989). These latter investigators, using ovine prolactin and p-casein gene promoter constructs, demonstrated that only stably but not transiently transfected constructs were induced with both PRL and dexamethasone. Our results differ in that we were able to produce strong synergetic effects using either stable or transient constructs. The reason for this difference could be that Doppler et al. used rat p-casein gene promoter constructs and a mouse mammary epithelial cell line, whereas we used MMTV LTR promoter constructs and a human cell line derived from a patient with mammary adenocarcinoma. The exact mechanisms by which prolactin controls the regulatory elements in LTR are still not understood. The
932
HARAGUCHI E T A L .
FIGURE3 - Induction of CAT activity by human prolactin in T47D cells stably transfected with pMMTV,,,-CAT. Culture conditions are the same as in Figure 2. Crude cell extracts were tested for their ability to convert ['4CJchloramphenicol (Chl) to the acetylated form (Ac-Chl) using TLC assay. hPRL. 1 kglml human prolactin; Dex, 1 pM dexarnethasone; HC, 50 ngiml hydrocortisone; insulin, 500 ngiml insulin. The experiment was repeated twice, and one of the typical results is shown in the Figure. protein kinase C (PKC) activator 12-0-tetradecanoylphorbol 13-acetate (TPA) has been found to have prolactin-like activity on mouse mammary gland explants (Rillema and Waters, 1986). It has been postulated that the mechanism by which prolactin stimulates proliferation involves the generation of second messengers coupled to PKC activation (Waters and
Rillema, 1989). Also. it has been found that TPA induces the nuclear transcription factor AP-1, composed of heterodimeric combinations of proto-oncogene products, a Jun family protein with a Fos family protein formed through the leucine zippers in these proteins (Kouzarides and Ziff. 1989). In this context, it is interesting to note that AP-1 consensus binding sites are present in MMTV LTR. Alternatively. transcription factors, whose consensus binding sites are present in MMTV LTR and which can interact synergistically with steroid receptors, have been reported (Schule et al., 1988; Bruggemeier et al., 1991). Also, a novel mammary cell-line-specific enhancer element in the MMTV LTR, which interacts with the hormoneresponsive clement in MMTV LTR, has been described (Yanagawa e l al., 1991). These investigators have identified nuclear factors that specifically interacted with this enhancer element in the nuclear extract from T47D cells (Yanagawa et al., 1991). Taking these previous reports into consideration, an attractive interpretation of the observed results in the present study is that the mammotropic polypeptide hormone prolactin might induce transcription factors, which can relay in the rcgulatory circuit for the induction of LTR in mammary cells. The sequences that confer steroid hormone inducibility on the MMTV promoter have been extensively characterized. The characterization of functional elements in the LTR involved in thc synergistic action between prolactin and steroid hormones can be pursued in future analyses. The identification of prolactin-responsive elements in LTR that is responsible for prolactin-induced gene transcription represents a subject of great interest. Additional experiments aimed at understanding the molecular mechanisms responsible for induction of MMTV LTR by prolactin are already in progress, and may better determine the significance of the role of prolactin in the development of breast cancer. ACKNOWLEDGEMENTS This work was supported by grants from the Eleanor Naylor Dana Charitable Trust, National Institutes of Health AGO5633 and CA41061, Ronald McDonald Charities and the Newland Foundation. We thank Dr. William G. Bradley for reviewing the manuscript. We also appreciate the assistance of Ms. C. Gotlieb and Ms. S. Littlc in the preparation of the manuscript.
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