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Endocrinology Vol. 139, No. 6 2916-2925
Copyright © 1998 by The Endocrine Society

ARTICLES

Reconstitution of Estrogen-Dependent Transcriptional Activation of an Adenoviral Target Gene in Select Regions of the Rat Mammary Gland1

Meei-Huey Jeng, Chinghai Kao, Lakshmi Sivaraman, Susanne Krnacik, Leland W. K. Chung, Daniel Medina, Orla M. Conneely and Bert W. O’Malley

Department of Cell Biology (L.S., S.K., D.M., O.M.C., B.W.O.), Baylor College of Medicine, Houston, Texas 77030; Department of Internal Medicine (M.-H.J.), Division of Hematology/Oncology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908; and Department of Urology (C.K., L.W.K.C.), University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. Bert W. O’Malley, Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030. E-mail: berto{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen regulates proliferation and morphogenesis of mammary ductal epithelium by interacting with a specific intracellular estrogen receptor (ER) that acts as a hormone-dependent transcriptional regulator of gene expression. The mechanisms by which ER regulates transcription in response to estrogen have been analyzed extensively in tissue culture and in cell-free systems. These studies have demonstrated that the transcriptional activity of ER is strongly influenced by cellular context and highlight the need to address ER transcriptional activity in an appropriate cellular background. Thus, to gain insight into the mechanistic role of ER in mammary epithelial morphogenesis, we have used an adenoviral gene delivery strategy to introduce an estrogen-responsive reporter gene into the mammary epithelium and to monitor the activity of endogenous ERs in their natural environment where cellular context including stromal-epithelial interactions can be taken into account. Using this approach, we first demonstrated highly efficient adenoviral delivery throughout the mammary epithelium using a ß-galactosidase (ßgal) reporter gene under the control of the constitutively active cytomegalovirus (CMV) promoter. Next, we constructed an adenoviral vector by substituting the CMV promoter with an estrogen-dependent promoter fragment-linked ßgal (Ad-ERE-tk-ßgal). This adenoviral reporter system provides evidence that ER positive mammary epithelial cells display a differential sensitivity in a region-specific manner toward estrogen induction. Our data suggest that the availability of factor(s) other than ER is necessary for ER-mediated gene activation and may be important in modulating the differential responses of mammary epithelial cells to estrogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DEVELOPMENT of the mammary gland occurs primarily postnatally and is directed by a complex interplay between hormonal (polypeptide and steroid) and growth factor signals (1, 2). Progesterone and estrogen are the principle steroid hormones involved in normal breast development and tumorigenesis (3, 4, 5, 6, 7). During pregnancy, progesterone and estrogen promote growth and differentiation of normal mammary tissue by regulating ductal proliferation and branching, alveolar formation (8), and lobulo-alveolar development (9). In the case of mammary gland tumorigenesis, the effects of progesterone and estrogen can be either stimulatory or inhibitory or both, and such effects are dose and stage dependent (10, 11). The hormonal effects are known to be mediated by specific high affinity intracellular receptor proteins that are members of a superfamily of related transcription factors (12, 13, 14, 15, 16). Studies on the ontogeny of mouse mammary gland responsiveness to ovarian steroid hormones have indicated that receptors for estrogen and progesterone (ER and PR, respectively) are present in both stromal and epithelial cells. The estrogen and progesterone receptors in epithelial cells are responsive to their ligands at 4 and 7 weeks of age, respectively (17, 18). The essential role of these receptors in mediating mammary developmental responses to estrogen and progesterone has been confirmed recently by the generation of null mutant mice lacking functional receptors for both hormones (19, 20, 21). These mice display grossly impaired ductal epithelial proliferation and branching in the case of the estrogen receptor null mutants and significant ductal development but decreased arborization and an absence of alveolar differentiation in the case of the progesterone receptor null mutants.

The mechanism by which steroid hormone receptors mediate hormone-induced signal transduction has been studied extensively in tissue culture and cell-free systems. Binding of steroids to their cognate receptors results in the formation of activated receptor dimers that bind to specific enhancer DNA elements located in the promoter regions of hormone-responsive genes (22, 23). Ligand-dependent activation is accompanied by a removal of receptor-bound corepressor proteins (24) that inhibit transcriptional activation by steroid receptors and an induction of binding of coactivator proteins that facilitate functional interaction of steroid receptors with the general transcription machinery (15, 24, 25, 26). The activation or repression of specific genes by steroid receptors represents the manifestation of the hormonal response. Reconstitution of steroid receptor-dependent transcriptional responses in cultured cells has demonstrated that the receptors can be activated not only by their cognate ligand but also by intracellular signaling pathways initiated by growth factors and other extracellular signals in a ligand-independent manner (27). However, the impact of these ligand-independent pathways on ER or PR mediated regulation of transcription in situ in the mammary gland has not been established. Further, little information is available to date on the factors that influence localized ER and PR mediated regulation of transcription during mammary tissue development or tumorigenesis.

The objective of the present study was to develop a strategy that ultimately would allow us to localize and monitor changes in ER-dependent transcriptional responses in vivo in the mammary gland that occur as a function of developmental status in the presence or absence of hormonal or growth factor stimuli or in response to chemical or hormonal carcinogens. Our approach was to use an adenoviral gene delivery system (28, 29, 30) to introduce an exogenous estrogen-responsive reporter construct into the rat mammary gland. Using this system, we reconstituted estrogen-dependent reporter gene expression in situ and localized this response to a subpopulation of epithelial cells located in the branched small ducts. A significant portion of the ductal epithelium appears to be refractory to estrogen despite the presence of high concentrations of ERs. These data suggest that estrogen sensitivity of the ductal epithelium is regulated locally by the availability of additional factors other than ER that are necessary to impart a transcriptional regulation of ER target genes in mammary epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture cells and experimental animals
Hela, CV-1, 3T3, MCF-7, and 293 cells (American Type Culture Collection, Rockville, MD) were maintained in DMEM containing 10% FBS. Tryptose phosphate (0.26 g/liter) was added during homologous recombination and plaque assays. Medium components were obtained from Gibco BRL (Grand Island, NY).

Female Wistar Furth rats (28 days old) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). They were anesthetized and either sham-operated or ovariectomized before experimentation. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Recombinant adenovirus construction and large-scale production
Replication defective recombinant adenoviruses expressing ß-galactosidase (ß-gal) under the control of the cis-acting estrogen response element (ERE) and either the E1b or thymidine kinase (tk) minimal promoters were constructed. For pERE-tk-ßgal shuttle vector construction, the 191 bp XbaI-BglII ERE-tk fragment containing a single copy of the ERE sequence upstream of the tk promoter was isolated from plasmid pERE15 and ligated upstream of a 3.4-kb HindIII-DraI ß-galactosidase fragment from pCH110 (Pharmacia Biotech Inc., Piscataway, NJ), and the 153-bp poly A+ fragment from SV40 DNA in the pXCJL Ad vector (31). For the pERE4-Elb-ßgal shuttle vector construction, a synthetic oligonucleotide containing four copies of the ERE sequence located upstream of the E1b minimal promoter (32) was subcloned into the pqE1sp1 adenoviral shuttle vector (33). PXCJL, pqE1sp1, and pJM107 (containing the adenoviral genome) were obtained from Dr. Frank Graham (McMaster University, Hamilton, Ontario, Canada). Both adenoviral shuttle vectors were CsCl2-purified and were then cotransfected with pJM107 into 293 cells using N-(1-(2, 3-dioleoyloxyl)propyl)-N,N,N-trimethylammoniummethyl sulfate mediated transfection method according to the manufacturer’s instructions to allow homologous recombination to occur (Boehringer Mannheim Biochemicals, Indianapolis, IN). Individual plaques were isolated and amplified in 293 cells. Viral DNAs were prepared, and the recombinant adenovirus (Ad) was identified by PCR and Southern analyses according to the method of Graham and Prevec (34). Selected clones of Ad-ERE-tk-ßgal and Ad-ERE4-Elb-ßgal were obtained by plaque purification and propagated in 293 cells (34). Cells were harvested 36–48 h after infection. Cell pellets were then resuspended in PBS (50 mM Na phosphate, 100 mM NaCl, pH 7.4; PBS), lysed by three freeze/thaw cycles, centrifuged at 1000 x g for 5 min to remove cell debris, and the virus was purified by CsCl2 gradient centrifugation. Concentrated virus was immediately dialyzed, aliquoted, and stored at -80 C. Viral titers were determined by OD 260 nm measurement or plaque assay. The control virus Ad-CMV-ßgal where ßgal is under the control of the constitutive cytomegalovirus (CMV) promoter used in this study was constructed in a similar manner.

Assessment of in vitro estrogen-induced transactivation of ERE-reporter activity in cultured mammary epithelial cells via infection with an adenoviral vector
To evaluate the effects of estrogen and antiestrogen on the expression of recombinant adenovirus reporter constructs in tissue culture cells, 4 x 105 MCF-7 cells were plated in six-well plates and deprived of estrogen for 1–3 days before transfection in phenol red-free DMEM containing 5% dextran-coated charcoal-stripped FBS (DCC-FBS) (35, 36). Transfection was done by exposing the culture cells to recombinant adenovirus for 2 h in serum-free and phenol red-free DMEM. Medium was then removed and replaced with fresh phenol red-free DMEM containing 5% DCC-FBS. 17-ß estradiol (E2, 10-8–10-12 M) and/or ICI 164, 384 (10-7 M) were dissolved in ethanol and added to the medium for 24 h to demonstrate steroid specificity in transactivation of ER target genes. Cells were then fixed for X-gal staining or harvested for liquid ß-galactosidase assay. Data are presented as the average of duplicate values. The experiments were repeated at least three times. E2 was purchased from Sigma Chemical Co. (St. Louis, MO). ICI 164, 384 was obtained from Zeneca Pharmaceuticals (Macclesfield, UK).

Assessment of in vivo estrogen-induced transactivation of ERE-reporter activity in rat mammary gland via adenoviral vector infection
Twenty-eight-day-old female Wistar Furth rats were anesthetized and ovariectomized to reduce the circulating estrogen and progesterone. Ten days later, rats receiving the adenovirus were first anesthetized and infused with 10 µl adenovirus in conjunction with a vital tracking dye (indigo carmine, 50 µg/10 µl) through intraductal injection with a blunt-ended 20–26 gauge needle (37). At the same time, rats receiving hormonal treatment were given estrogen benzoate (EB) suspension in sesame oil (100 µg/0.2 ml) sc. Twenty-four hours later, the animals were then anesthetized, and the mammary fat pad was removed for X-gal staining. Rats were then euthanized with CO2.

ß-galactosidase assay and X-gal staining in cultured cells and in tissues
For ß-galactosidase liquid assay in tissue culture cells, cell monolayers were rinsed once with PBS followed by the addition of 1 ml of 40 mM Tris HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl) and incubated on ice for 5 min. Cells were harvested, pelleted, and suspended in 100 µl ice-cold 0.25 M Tris HCl, pH 7.5. Cell extracts were prepared by three freeze/thaw cycles. Cytosols containing equal amount of protein were used for ß-galactosidase activity assay (38).

For X-gal staining in situ in tissue culture cells, cells were rinsed twice with PBS and fixed with 0.5% glutaraldehyde in PBS for 5 min at room temperature. Cells were then rinsed twice with PBS and stained with X-gal staining solution (1.3 mM MgCl2, 15 mM NaCl, 44 mM HEPES, pH 7.4, 3 mM K3Fe(CN6) 3 mM K4Fe(CN6), and 0.5 mg/ml X-gal).

For X-gal staining in the mammary gland, rats were anesthetized and fat pads containing the mammary gland were removed. The staining procedure was performed according to the method described previously (39) with modification. The fat pads were fixed in fresh cold 2% paraformaldehyde solution containing 0.1 M PIPES, pH 6.9, 2 mM MgCl2, 1.25 mM EGTA for 1–2 h, washed with PBS containing 2 mM MgCl2 three times, and permeabilized with 0.02% NP40, 0.01% Na deoxycholate, and 2 mM MgCl2 in PBS for 1 h. The fat pads were then stained immediately with staining solution containing 25 mM K3Fe(CN6), 25 mM K4Fe(CN6), 2 mM MgCl2, 0.02% NP40, 0.01% Na deoxycholate, 0.5 mg/ml X-gal in PBS, pH 8.1 at 37 C for 12–16 h. After staining and photography, the glands were subsequently dehydrated, embedded in paraffin, and sectioned serially for microscope examination and photography.

Immunohistochemical analysis
The right and left no. 4 abdominal mammary glands from EB treated rats were sectioned into proximal and distal regions relative to the nipple and fixed in 4% paraformaldehyde. Tissues were embedded in paraffin and sectioned into 5-µm thick sections. The sections were then deparaffinized, rehydrated through graded alcohols followed by incubation in PBS. They were then incubated for 30 min each in 0.2% glycine and 0.3% hydrogen peroxide in methanol. Sections were rehydrated in PBS and blocked with 10% goat serum in PBS for 30 min followed by incubation overnight with rabbit anti-ER IgG, MC-20 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:200 dilution. Sections were incubated without primary antibody or with rabbit serum as control. Sections were rinsed several times in PBS (5 min each) and then incubated with biotinylated goat antirabbit secondary antibody at a 1:500 dilution for 15 min at 40 C, washed several times in PBS and then developed using the Vectastain ABC kit (Vector Labs., Burlingame, CA). All sections were counterstained with hematoxylin. Brown staining diaminobenzidine positive cells were visualized using a Zeiss Axioskop microscope (Carl Zeiss Inc., Thornwood, NY) at 40x magnification coupled to a Hamamatsu C5810 CCD camera (Hamamatsu Corp., Bridgewater, NJ) and were processed using Adobe Photoshop 4.0 (Adobe Systems, Inc., San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen-dependent activation of adenoviral reporter genes in MCF-7 breast cancer cells
To examine the feasibility of reconstituting estrogen-dependent reporter gene expression in vivo in the rat mammary gland, we constructed two recombinant adenovirus vectors bearing ERE-driven reporter constructs. The reporter constructs (Ad-ERE-tk-ßgal and Ad-ERE4-E1b-ßgal) contained one copy of the ERE located upstream of the thymidine kinase (tk) or four copies of the ERE located upstream of the adenoviral E1b minimal promoters (32) and the ß-galactosidase reporter gene, respectively. To test hormone responsiveness of these reporter constructs in cultured cells, MCF-7 cells were infected first by these adenoviral expression vectors before exposure to estrogen. Twenty-four hours later, cell cytosols were prepared and analyzed for ß-galactosidase activity. Figure 1Go (A and B) shows that ß-galactosidase expression is induced at comparable levels in MCF-7 cells infected with either Ad-ERE-tk-ßgal or Ad-ERE4-E1b-ßgal. Estrogen induces the expression of reporter activity in a dose-dependent manner, and the induction by estrogen is blocked in both cases by the presence of an estrogen receptor antagonist, ICI 162, 384.



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  		Figure 1. Induction of ß-galactosidase activity by E2 treatment after Ad-ERE-tk-ßgal or Ad-ERE4-E1b-ßgal infection in MCF-7 cells. MCF-7 cells were deprived of estrogen and infected with Ad-ERE-tk-ßgal (A) or Ad-ERE4-E1b-ßgal (B) for 2 h in serum free DMEM. Media were removed and fresh media containing 5% DCC-FBS, and the indicated amount of E2 were added to cells for an additional 24 h. ICI 164, 384 at a concentration of 10-6 M was used to block the effect of E2 at the concentration of 10-9 M. Cell pellets were then collected and cytosols prepared. An equal amount of protein was used for ß-galactosidase assay. Each bar represents the mean value from duplicates, the experiments were repeated at least three times.

 
The degree of cell infection obtained using the Ad-ERE4-E1b-ßgal target virus is shown in Fig. 2Go. Panel A indicates that, in the absence of estrogen, approximately 30% of cultured cells express basal levels of ß-galactosidase. However, incubation with estrogen resulted in robust expression of ß-galactosidase activity in approximately 70% of the cells (panel B). Further, both the basal and estrogen induced activity were blocked by incubation with ICI 162, 384 (panels C and D). Thus, the adenoviral strategy for introducing synthetic target genes into cultured cells results in a high degree of infection, and the target gene retained responsiveness to estrogen.



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  		Figure 2. X-gal staining of MCF-7 cells after E2 and ICI 164, 384 treatments. MCF-7 cells were deprived of estrogen and infected with Ad-ERE4-E1b-ßgal for 2 h in serum-free DMEM. Media were removed and fresh media containing 0.2% ETOH vehicle (A), 10-10 M E2 (B), 10-7 M ICI 164, 384 (C), and 10-10 M E2 and 10-7 M ICI 164, 384 (D) was added for an additional 24 h. Cells were then fixed with glutaraldehyde and stained with X-gal solution to visualize the blue cells in which the ß-galactosidase was expressed.

 
Introduction of foreign genes into the mammary gland by adenoviral delivery
To determine the feasibility of introducing foreign genes into the mammary gland, we first injected indigo carmine as a vital tracking dye to examine the physical penetration of the dye throughout the mammary epithelial compartments (37, 40). Rats were anesthetized, and the main ducts were cannulated with a blunted 21–26 gauge needle. A single gland can accommodate injections of 2–40 µl of dye suspension depending on the age of the animal. Using this procedure, we were able to monitor the success of each injection and visualize the complete glandular structure within seconds after the injection (Fig. 3Go, A and B). The tracking dye diffused out of the mammary ducts completely within 20 h and became invisible. This dye was therefore used in combination with the adenovirus preparations to monitor the success of injection. In the first experiments, an adenoviral construct that contains the ß-galactosidase gene under the control of the constitutive CMV promoter (Ad-CMV-ßgal) in combination with the tracking dye was injected into the mammary gland to examine its degree of infectivity in the mammary epithelium. To determine the conditions under which infection by the adenovirus is optimal, studies were performed to determine the appropriate titer of adenovirus to deliver to the gland and the appropriate length of time between administration of the virus and measurement of gene expression. Mammary glands were infused with varying amounts of the virus in conjunction with tracking dye and the mammary fat pads were dissected, fixed, and stained with X-gal at various time points after the injection. A multiplicity of infection of 10:1 was found to be sufficient to infect the epithelial cells with the assumption that the number of mammary epithelial cells per gland is approximately 3 x 107. The results in Fig. 4Go demonstrate that while the tracking dye had completely disappeared at 48 h after injection of the dye alone (panel A), strong ß-galactosidase expression was observed and persisted throughout the gland (panels B and C). Interestingly, analysis of this expression at high magnification revealed that the ß-galactosidase staining was localized to the luminal epithelial compartment of the small ducts but not in the stromal compartments, indicating that the luminal epithelial cells preferentially take up the virus. The expression of ß-galactosidase persisted but diminished dramatically 4 days after infusion of the virus (panel E) and was undetectable 9 days after infusion (panel F).



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  		Figure 3. Intraductal injection of vital tracking dye into rat mammary gland. Four-week-old female Wistar-Furth rats were anesthetized and cannulated with a 22-gauge blunt-ended needle into the mammary main duct and 10 µl of the indigo carmine blue tracking dye (5 mg/ml in PBS) was infused within seconds into the mammary gland (A). The dye outlined the arborized structures consisting of primary, secondary, and tertiary branches and reached the end buds (B) (used with permission of Plenum Press; Ref. 40).

 


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  		Figure 4. Ad-CMV-ßgal infection of rat mammary gland. Rats were anesthetized and mammary gland infused with 10 µl of tracking dye alone (A) or in combination with 4 x 107 pfu Ad-CMV-ßgal (B–F). The dye diffused out in less than 24 h. Two days post intraductal injection, the mammary fat pads were dissected, fixed, and stained with X-gal solution. The blue cells represent the induction of ß-galactosidase reporter activity. Panels A–C and E–F are whole mount staining, and panel D is the tissue section obtained from the gland stained in C. Panels E and F show that ß-galactosidase expression (blue color) is diminished after 4 days (E) and is undetectable at 9 days after infusion (F). S, Stromal cells; LE, luminal epithelium; ME, myoepithelium.

 
In situ localization of estrogen receptor-dependent gene expression
To reconstitute estrogen receptor-dependent reporter gene expression in situ in the mammary gland, we next tested the estrogen responsiveness of the two adenoviral reporter gene constructs we had generated. We injected glands with either the Ad-ERE-tk-ßgal or Ad-ERE4-E1b-ßgal reporter viruses together with the tracking dye. Rats were then injected with estradiol benzoate or control vehicle and the fat pads were removed 24 h later, fixed, and stained for ß-galactosidase activity. Surprisingly, despite the comparable levels of induction of both reporter genes by estrogen when tested in MCF-7 cells, only the Ad-ERE-tk-ßgal reporter gene responded to estrogen by expression of ß-galactosidase when tested in the mammary gland, indicating that reporter gene expression was dependent on a strong promoter in the tissue. The estrogen responses obtained with the Ad-ERE-tk-ßgal reporter virus are shown in Figs. 5Go and 6Go. In the absence of estrogen (Fig. 5AGo and Fig. 6Go, A and B) very low basal expression of ß-galactosidase was observed in the gland. However, in the mammary glands of rats treated with estradiol benzoate for 1 day, strong localized expression of ß-galactosidase was detected (Fig. 5BGo and Fig. 6Go, C and D). The expression pattern was strikingly different from that observed using the constitutive ß-galactosidase expression construct, Ad-CMV-ßgal (Fig. 5CGo). Despite the ability of the virus to penetrate epithelial cells located throughout the mammary ductal system as demonstrated using the CMV-ßgal adenovirus (Fig. 5Go, C, F, and I), estrogen-dependent reporter gene expression was concentrated in the small ducts (Figs. 5HGo and 6DGo) as compared with regions close to nipples (Figs. 5EGo and 6CGo). These data indicate that the estrogen receptor activity is minimal in the large ductal epithelia at this time and is localized to the small ducts of the gland.



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  		Figure 5. Estrogen-dependent transcriptional activation of Ad-ERE-tk-ßgal reporter gene in the mammary gland. Ovariectomized rats were anesthetized and the mammary gland infused with Ad-ERE-tk-ßgal (1.6 x 108 pfu) (A and B) or Ad-CMV-ßgal (4 x 107 pfu) (C) in conjunction with a tracking dye in a final volume of 10 µl. Rats receiving Ad-ERE-tk-ßgal were injected with control vehicle sesame oil (A) or 100 µg estrogen benzoate (B) in a volume of 0.2 ml. The next day, mammary fat pads were dissected, fixed, and stained with X-gal. Arrows indicate the location of nipples that connect to the mammary main ducts and subsequently the lobulo-alveolar structures. High power magnification was also obtained to further illustrate the location of ß-galactosidase at regions close to nipples (D–F) and at distal regions of the glands (G–I). D and G, High power magnification from panel A. E and H, Derived from panel B. F and I, Derived from panel C. Blue cells are the cells expressing ß-galactosidase.

 


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  		Figure 6. Localization of ER-dependent ß-galactosidase activity. Analysis of ß-galactosidase stained sections (40x magnification) from control (panels A and B) and EB treated (panels C and D) rats showing large (A and C) and small (B and D) ducts. Sections were counterstained with nuclear fast red. Blue cells are the cells expressing ß-galactosidase. Animals were treated the same way as mentioned in Fig. 5Go.

 
Localization of ER expression in mammary epithelium
The selective responsiveness of the small duct epithelium to estrogen suggested that either estrogen receptors are selectively expressed in the hormone responsive cells or that receptors in these cells are selectively more active in response to estrogen than those residing in the proximal ductal epithelium. To distinguish between these possibilities, we used an immunohistochemical approach to examine the expression of ER throughout the mammary gland. The results in Fig. 7Go demonstrate that ER is expressed in both the epithelial and stromal cells with the most intense expression observed in the nuclei of the epithelium. These findings are similar to those previously obtained in adult mice using anti-ER antibodies and radioactive ligand binding assays (18, 41). Within the epithelial compartment, strong expression of ER was observed in numerous cell nuclei of the proximal large ducts (panel A) and more distal small ducts (panels B and C) with rare ER expression in the terminal end buds (panel D). In both large and small ductal epithelia, over 50% of the cells were positive for ER. These results clearly demonstrate that the lack of estrogen-dependent reporter gene expression in the proximal ductal epithelium is not due to a lack of estrogen receptor expression in this region but rather to a decreased sensitivity of these receptors to the hormonal stimulus.



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  		Figure 7. Immunohistochemical localization of ERs in rat mammary epithelial and stromal cells. Five-micrometer sections through large (A) and small (B and C) ducts and end buds (D) probed with rabbit anti-ER IgG followed by peroxidase labeled secondary antibody and counterstained with hematoxylin (magnification, 40x). Animals were treated the same way as mentioned in Fig. 5Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated the feasibility of introducing heterologous genes into the rat mammary gland in vivo using an adenoviral gene delivery approach. The adenovirus efficiently infected rat mammary epithelial cells throughout the mammary ductal system but did not reach the stromal compartment of the gland. Thus, it is most likely that the adenovirus cannot penetrate through multiple layers of the cells, especially the basal membrane that underlies the base of the mammary epithelial cells. Our results indicate that the adenoviral delivery strategy serves as a powerful tool to deliver specific genes to mammary epithelium, but its utility may be restricted to epithelial cell targets. Nevertheless, the approach has several attractive features for monitoring and manipulating gene expression in the mammary gland. First, the virus can be purified at high titers allowing injection of minimal volume of material into the gland. Second, viral gene expression persists up to 4 days in the tissue, allowing ample time for experimental and hormonal manipulation of the animals and monitoring of transcriptional responses. Third, the ability of the virus to infect both proliferating and resting cells makes it ideally suited to localizing changes in hormone responsiveness that occur in proliferating and differentiated epithelial cells as a consequence of developmental stage, hormonal manipulation, or in response to carcinogen challenge.

Recombinant adenoviral vectors have been targeted to several cell lines in vitro and several organs in vivo. Adv-RSV-ßgal has been used to infect several tissue culture cell lines like RKO, MDA-435, T47D, MCF-7, HT29, SHSY-5, SK-N-SH, IMR-32, K-562, and primary breast carcinoma cells (42) with greater than 95% cell efficiency. The adenovirus containing ß-galactosidase gene alone demonstrated various degrees of toxicity in different cancer cell lines. The same group studied the functional role of bcl-xs adenovirus in the same cell lines and demonstrated the induction of apoptosis in over 90% of the cells. Recently, adenovirus-mediated overexpression of transcription factor E2F-1 has been shown to induce apoptosis in several human breast and ovarian carcinoma cell lines (43). Also, HC11 cells have been shown to be infected with high efficiency by an inactive LacZ adenoviral reporter and expression of LacZ achieved using Cre recombinase (44). The same expression has been reconstituted in vivo in transgenic mice expressing Cre-recombinase spatially and temporally under the control of the WAP or MMTV promoter. The inactive LacZ adenoviral vector was injected directly into the mammary gland, although it was not obvious if the delivery was by the intraductal route or into the adipose stroma. Reporter genes using adenoviral vectors have been delivered to several organs in vivo including salivary gland, lung, liver, gut, blood vessels, brain, CNS, chondrocytes, T cells, etc. The experiments reported herein extend previous studies by demonstrating the feasibility of intraductal injections in the normal mammary gland and examining the functional role of an estrogen-dependent target gene.

Using this strategy, we have reconstituted localized estrogen-dependent activation of an exogenously introduced reporter gene in the mammary epithelium of the small ducts. In contrast to the constitutive reporter gene, the estrogen-dependent target gene response was concentrated at the level of the small ducts with very low activity detected in the large ductal epithelium. Thus, while the adenoviral vector can efficiently infect the large primary and secondary ductal epithelium, these cells appear to be refractory to the estrogen stimulus. Analysis of the expression of estrogen receptors throughout the gland demonstrated that lack of estrogen response is not due to a lack of estrogen receptor expression in large ductal epithelia. In contrast, this region of the gland is densely populated with estrogen receptors that appear to be relatively insensitive to the hormonal stimulus. These observations indicate that factors other than the ER that are necessary for the estrogen-dependent transactivation response are differentially expressed in estrogen sensitive epithelial cells.

Estrogen regulation of gene expression is known to be mediated by a hormone-dependent removal of corepressor proteins from the estrogen receptor and a stimulation of binding of coactivator proteins to the ER (25, 26). Binding of receptor coactivators enhances ER interaction with the general transcription apparatus and results in strong enhancement of the estrogen-dependent transactivation response. The central role of these coactivators in estrogen receptor activation suggests that the lack of ER activity in ductal epithelia may be due to a differential expression of estrogen receptor coactivators in subpopulations of mammary epithelial cells. In this regard, it will be of interest to determine which coactivators are coexpressed with estrogen receptors in the mammary epithelia and to evaluate how these proteins may contribute to the differential transcription regulatory responses of ER positive epithelial cells to the estrogen stimulus.


   Acknowledgments
 
We would like to thank Elizabeth A. Hopkins for tissue embedding and sectioning, Debbie Townley for photographic work, and Lin-Ching Ho for the construction of recombinant adenoviruses. We also would like to thank Dr. Frank Graham for providing adenoviral vectors, and Drs. John Cidlowski and Victoria E. Allgood for providing pERE-E1b-CAT plasmid.


   Footnotes
 
1 This work was supported by Grants PO-1-CA64255 and DAMD17–96-1–6233. Back

Received December 12, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1106[Free Full Text]
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  3. Dickson RB, Thompson EW, Lippman ME 1990 Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids. J Steroid Biochem Mol Biol 37:305–316
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