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Original Studies |
Barbara Ann Karmanos Cancer Institute (R.J.P., S.J.S., L.R.T., R.K.B.) and the Departments of Internal Medicine (R.J.P.), Pathology (L.R.T.), and Surgery (R.K.B.), Wayne State University School of Medicine, Detroit, Michigan 48201; and University of Virginia Health Sciences Center (R.J.S.), Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Robert J. Pauley Ph.D., Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201. E-mail: pauleyr{at}kci.wayne.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An emerging concept is that estrogen exerts a local effect near or at sites of synthesis via paracrine and autocrine mechanisms. This concept, based on evidence for human breast aromatase activity (7, 8, 9), Cyp19 messenger ribonucleic acid (mRNA) (10, 11), and estrogen production (12), has substantial experimental support (13, 14). Experiments in a xenograft model provided direct proof that locally produced estrogen can stimulate the growth of estrogen-dependent MCF-7 human breast cancer epithelial cell tumors to a greater extent than can estrogen delivered via an endocrine mechanism (15). The cell type(s) containing and their relative contribution to aromatase activity in human breast tissue remains a controversial issue (14), with evidence for aromatase in epithelial cells (16, 17), stromal cells (18, 19), and both cell types. Taken together, the data suggest that estrogen production and action in breast tumors predominately involve a paracrine mechanism with synthesis in fibroblasts and subsequent effects on tumor epithelial cells (13, 14, 20).
Transcriptional regulation of CYP19 is the major, although
not exclusive, mechanism controlling the amount of aromatase. The
complexity of CYP19 transcription is demonstrated by the
differential usage of at least nine alternative transcription
initiation sites among tissues (for review, see Ref. 14). Each
transcription initiation site determines unique first or 5' exons in
Cyp19 transcripts. These first exons include the multiple placental
exons; EI.1 is the major, EI.2 and EI.2a are minor, and both are
spliced 3' to EI.1 (1, 21, 22). PII is the major ovarian first exon
(23), which is 5', and in genomic DNA contiguous, to the common exon II
splice acceptor site. Adipose tissues and adipose cells treated
in vitro with modulators of CYP19
transcription contain Cyp19 mRNA first exons EI.3 and EI.4 in addition
to PII as well as a truncated EI.3tr and EI.2 spliced 3' to EI.4 (24).
Table 1
lists these CYP19
first exons and specifies the alternative nomenclature. Importantly,
Cyp19 transcripts with a given first exon indicate transcription
initiation through the function of distinct promoter and regulatory
sequences proximal to each CYP19 first exon (23, 24). Also,
transcripts initiated at each alternative promoter and containing
distinct first exons all have their first exon donor site spliced to a
common acceptor site in the noncoding region of exon II, 38 bp upstream
of the exon II universal translation initiation site (23).
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Materials and Methods |
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Tissues were acquired after protocol review and approval by the
Wayne State University human investigation committee. Tumors (G133,
WS-3, and WS-12), confirmed by histology, were digested, and
fibroblasts were cultured by methods similar to those previously used
(19, 33, 34). Briefly, tumor pieces were trimmed of fat, minced into
fine (1- to 2-mm) pieces and digested overnight with collagenase (150
U/mL) in DMEM/Ham’s F-12 (1:1) medium containing 20% calf serum.
Larger pieces, mostly parenchyma, were settled for 30 min. Cells in the
supernatant fraction, which were predominantly fibroblasts from the
stromal component, were recovered by centrifugation and seeded in
Waymouth’s MB752/1 medium with 10 mmol/L HEPES and 15% FCS (FIB
medium). Settled pieces, mostly from the epithelial cell-containing
parenchymal component, were transferred into fresh DMEM/F-12 containing
20% calf serum until attachment of the majority of the pieces (
1
week). The medium was changed to DMEM/F-12 with 10 mmol/L HEPES, 5%
horse serum, and 10 µg/mL insulin, from which fibroblasts were
removed by a 1- to 2-min trypsinization and cultured in FIB medium.
Fibroblasts pooled from supernatant, and settled fractions were
routinely cultured up to 15 passages in FIB medium.
Benign tissues (WS-3 and WS-12) were obtained from grossly nonmalignant areas as distant as possible from the tumors. Benign tissues were processed similarly to tumors, except that fat was not trimmed before mincing, tissue digestion was for 48 h with the addition of hyaluronidase (100 U/mL), and epithelial cells were cultured as previously described (35) in DMEM/F-12 (1:1) with 10 mmol/L HEPES, 5% horse serum, 10 µg/mL insulin, 20 ng/mL epidermal growth factor, 0.5 µg/mL cortisol, and 100 ng/mL cholera toxin (EPI medium). Reduction mammoplasty tissues (WS-14 and WS-15) were processed similarly to benign breast tissues for culture of fibroblasts and epithelial cells. Portions of benign and reduction mammoplasty tissues used for culture were examined histologically, and all lacked carcinoma.
Fibroblast and epithelial cells were characterized by immunocytochemical methods we used to identify breast epithelial cells and breast fibroblasts (19, 35, 36) with minor modifications. Briefly, cells were cultured on glass chamber slides (Nunc, Naperville, IL), preserved in Methacarn fixative for 10 min, washed three times in phosphate-buffered saline, and blocked for 10 min in Super Block (ScyTek Laboratories, Logan, UT). Primary antibodies included the pan-cytokeratin monoclonal antibody AE1+AE3 cocktail (DAKO Corp., Carpenteria, CA), the cytokeratin 14 monoclonal antibody LL01 (Novacastra, Newcastle upon Tyne, UK), and the vimentin monoclonal antibody V9 (DAKO Corp.). Incubations with each antibody were overnight at 4 C, followed by reaction with biotinylated antimouse IgG and then peroxidase using the Elite ABC system (Vector Laboratories, Inc., Burlingame, CA), and visualized with 3',3'-diaminobenzidine (Sigma, St. Louis, MO). Cells were then counterstained with hematoxylin, and images were captured with a Sony DXC-970 MD 3cccd color video camera and processed with the MCID M5 Plus software (Imaging Research, Inc., St. Catherines, Canada).
Extended lifespan fibroblasts were developed by infection with the LXSN16E6E7 recombinant retrovirus encoding the human papilloma virus serotype 16 E6 and E7 transforming proteins (37). Actively proliferating fibroblasts were from one (WS-12) patient’s breast tumor and benign breast tissues. Infected cells were cultured in the same medium as uninfected cells, except that FIB medium contained 10% FCS. Surviving infected cells were defined as extended lifespan cells because proliferation continued beyond the approximately 15 passages when parallel uninfected cells senesced; proliferation, however, did diminish with further passage, and therefore, these are not immortalized cell lines.
Experimental protocol
The experimental protocol is identical to that of our prior study using cells cultured in serum-containing medium that demonstrated regulation of aromatase activity and Cyp19 mRNA levels in breast tumor and benign breast fibroblasts (19). The results demonstrated that Cyp19 transcripts and enzyme activity were at maximal levels with 54 h of treatment, and maximal mRNA accumulation and enzyme activity were at maximal levels in cells treated with the dexamethasone (Dex) along with phorbol diacetate (PDA) and dibutryl cAMP (db-cAMP). Experiments with fibroblasts were initiated by adding the test compounds in fresh serum-containing FIB medium for 54 h, with replacement at 24-h intervals and at 6 h before termination of treatment. Experiments with epithelial cells were performed similarly, except EPI medium was used. Test compounds were added as concentrated solutions to the specified final concentration. Compounds, either alone or in various combinations, were 100 nmol/L Dex, 1 mmol/L db-cAMP, and 100 nmol/L phorbol 12,13-diacetate (Sigma). The aromatase inhibitor CGS20267 or letrozole (1 µmol/L) was added in experiments in which one of the duplicate cultures was used for RNA isolation and one assayed for aromatase by tritiated water release (19).
RNA analyses
General molecular methods were described previously (19).
Oligonucleotide RT-PCR primers and hybridization probes were
synthesized by Genosys Biotech (The Woodlands, TX). At the end of
treatment, culture flasks were rapidly rinsed twice with sterile
phosphate-buffered saline, and RNA was extracted by the guanidium
isothiocyanate method using RNA isolator with minor modifications to
the manufacturer’s protocol (Genosys Biotech). RNA concentration was
determined spectrophotometrically. Assessment of RNA integrity and
verification of concentration involved either
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) Northern blotting or
G3PDH RT-PCR as we previously described (see Fig. 5C in Ref. 19).
Controls with no input RNA were included in G3PDH RT-PCR experiments;
in none of the reported experiments were PCR products detectable in the
no input RNA control. Within the experiments reported here, the
variation in G3PDH RT-PCR products among the RNA samples in a single
experiment was less than 0.1-fold, and that between two experiments
analyzed simultaneously was less than 0.2-fold (data not shown); hence,
Cyp19 transcripts were not standardized to G3PDH transcript levels.
RT-PCR for Cyp19 mRNA used amplimer pairs in the coding regions of
exons III–V and exons IX and X, as we previously described (19).
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. The 5'-sense primers included those specific for the
noncoding regions in exons EI.1, EI.3, EI.4, and PII. The AEII control
5'-sense primer primes Cyp19 mRNA regardless of transcription
initiation site because it is complementary to sequences in the
untranslated 5'-portion of exon II that are present in all Cyp19 mRNAs
(23). All Cyp19 mRNA RT-PCR products were identified and verified by
molecular hybridization with an oligonucleotide probe for the coding
region of exon II. Therefore, Cyp19 mRNA first exon-specific and AEII
control RT-PCR products, which all contain 266 bp of identical
3'-sequences, were distinguished by both primer specificity and product
size (Table 1). Preliminary RT-PCR experiments demonstrating specific
Cyp19 mRNA 5' exons (in parentheses) using human placenta
(EI.1, EI.2a, EI.2, and PII), lung (EI.1, EI.2a, EI.2, EI.4, and PII),
liver (negative), and breast tissue (EI.3, EI.4, and PII) RNAs
(CLONTECH, Palo Alto, CA) verified this method to detect alternative
first exons in Cyp19 transcripts (data not shown). RT reactions contained 100 ng/µl total cellular RNA and 1 µmol/L exon III antisense 3'-primer as well as a no input RNA control. Additional components were 1.25 U/µL each Moloney murine leukemia virus reverse transcriptase and ribonuclease inhibitor; 1 mmol/L each of deoxy (d)-ATP, dCTP, dGTP, and dTTP; 3 mmol/L MgCl2; 10 mmol/L Tris-HCl (pH 8.3); and 50 mol/L KCl from the RNA PCR kit (Perkin-Elmer Corp., Foster City, CA). Reactions were performed for 60 min at 42 C. PCR used equivalent aliquots of the RT product, containing the equivalent of 20 ng/µL input total cellular RNA with 0.2 µmol/L exon III antisense 3'-primer that were added to parallel reactions containing 0.2 µmol/L of each 5'-primer. Additional components were 0.2 U/µL AmpliTaq DNA polymerase, 2.5 mol/L MgCl2, 10 mol/L Tris-HCl (pH 8.3), and 50 mol/L KCl from the RNA PCR kit. The initial step was at 94 C for 60 s, followed by 25 cycles of 94 C for 30 s, 55 C for 60 s, and 72 C for 90 s, and then 72 C for 300 s in a Delta Cycler II thermocycler (Ericomp, San Diego, CA). Higher primer concentrations and additional cycles (38) increased nonspecific products (data not shown).
Aliquots containing the equivalent of 100 ng input RNA were electrophoresed through 8% PAGE gels with 1 x Tris borate ethylenediamine tetraacetate buffer (Novex, San Diego, CA) and included 100-bp DNA ladder molecular size standards (GenSura Laboratories, Del Mar, CA). Gels were stained with Vistra Green (Amersham Pharmacia Biotech, Arlington Heights, IL) and imaged by fluorescence (Storm 860, Molecular Dynamics, Inc., Sunnyvale, CA). DNA was electroblotted from each gel to a charged nylon membrane according to the supplier’s recommendations (Novex). The CYP19 exon II coding region oligonucleotide probe was end labeled with T4 polynucleotide kinase, hybridized to membranes, and washed to a maximum stringency of 56 C in 5 x SSPE and 0.1% SDS for 10 min (19). Autoradiography was performed for the specified time. Membranes were exposed to a phosphorscreen, imaged with the Storm 860, and analyzed by ImageQuant (Molecular Dynamics, Inc.). Intensity is in relative units with background subtracted. The fold increase in PCR product is expressed relative to the untreated control value, a minimum control value for this calculation was set at 10 because this is the lower limit of reliable detection over the background. PCR controls included the no RNA input RT product and the 5' EII control primer; in none of the reported experiments were PCR products detectable in the no input RNA control. Additional controls commonly included RNA from the MCF-7 aromatase-transfected cell line (39, 40) with or without reverse transcriptase or pooled human breast tissue (data not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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with reduction mammoplasty cells.
Epithelial cells (left column) had the following
characteristics: a highly uniform cuboidal morphology, intense staining
of all cells with the epithelial cell specific intermediate filament
pan-cytokeratin antibody cocktail (row A), intense staining of a
significant portion of these cells with the breast myoepithelial and
progenitor cell type (41) cytokeratin 14 antibody (row B), and staining
of less than half of these cells with the alternative intermediate
filament vimentin antibody (row C), which is expressed in breast
parenchymal myoepithelial cells in vivo and in
vitro (42). On the other hand, as demonstrated in the right
column, fibroblasts had a highly uniform spindle morphology (row
D), minimal or no detectable staining above background with the
pan-cytokeratin and cytokeratin 14 antibodies (rows A and B,
respectively), and intense staining of all cells with the vimentin
antibody (row C). Fibroblasts from benign and tumor tissues had the
same staining pattern with these cytokeratin and vimentin antibodies
(data not shown). These results demonstrate that epithelial cells are
not a detectable component of the fibroblasts and that fibroblasts are
not a significant component of the epithelial cells examined in this
study.
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, lanes 1–5) and
phosphorscreen image analysis (Fig. 2C), an approximately 12-fold
increase in Cyp19 mRNA accumulation in cells exposed to Dex alone or in
combination with PDA and cAMP; the aromatase enzyme inhibitor
letrozole had no effect on this increase. These results
are consistent with our previous report in terms of specificity and
fold induction of Cyp19 mRNA in breast tumor-derived fibroblasts
(19).
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, B, lanes 1–5, and
C). First exon PII transcripts were increased by PDA and cAMP in
combination with Dex, but not by Dex alone (Fig. 2, B, lanes 6–10, and
C). First exon EI.3 initiated transcripts were only marginally
detectable in the Dex, PDA, and cAMP treatment group (Fig. 2, A, lanes
6–10, and C). Benign breast tissue-derived fibroblasts from the same
patient demonstrated similar up-regulation of Cyp19 mRNA using EI.4 and
PII (data not shown). We recognized several confounding problems in the interpretation of these results. Basal levels of Cyp19 mRNA and first exons were exceedingly low compared to levels of RNA prepared from whole breast tissue and without the isolation of specific cells. The ability to stimulate transcription in vitro could be due in part to adaptation of fibroblasts during long term culture. Also, we detected only marginal use of first exon EI.3 compared to whole breast tumor RNA. Accordingly, additional experiments, including the use of first passage cells, were designed to obviate these problems.
Fibroblasts from breast tumor and benign breast tissues were examined
from primary cultures rather than multiply passaged cells (Fig. 3). Fewer treatments could be tested
because of the limited number of cells. The basal level of Cyp19 mRNA
also was very low in these breast tumor-derived fibroblasts (Fig. 3A, lane 1). No expression or up-regulation of EI.1 and associated EI.2 and
E2a was observed in this experiment (Fig. 3A, lanes 5–8) or in other
experiments. Dex alone caused an approximately 12-fold increase in
Cyp19 mRNA that was associated solely with increased EI.4 transcripts
(Fig. 3, A and B, lanes 2, 6, and 11). PDA and cAMP to a lesser extent
increased Cyp19 mRNA that was associated solely with PII transcription
(Fig. 3, A and B, lanes 4, 8, and 12). The increase in Cyp19 mRNA
produced by Dex in combination with PDA and cAMP was associated with 1)
an increase in EI.3 transcripts, not observed with other treatments
(Fig. 3A, lanes 11–12); 2) an increase in EII transcripts, not
observed with Dex alone and greater than that produced by PDA and cAMP
(Fig. 3B, lanes 7–8); and 3) an increase in EI.4 transcripts, not
observed with PDA and cAMP alone and greater than that produced by Dex
alone (Fig. 3B, lanes 2–4). These results substantiate expression of
Cyp19 mRNA in short term cultures of breast tumor fibroblasts and
indicate complex and differential regulation of transcription
initiation from first exons EI.3, EI.4, and PII that are detected in
RNA isolated from breast tissues.
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, lanes 1, 9, and 5,
respectively). Because of the limited number of cells, only a DEX, PDA,
plus cAMP treatment group was examined. Increased transcripts with PII
predominated over those of EI.3 or EI.4 (Fig. 3C, lanes 10, 6, and 8,
respectively), but all were increased compared to basal levels. These
results demonstrate in short term cultures of benign breast fibroblasts
complex and differential regulation of transcription initiation from
first exons EI.3, EI.4, and PII, similar to that in fibroblasts from
the same patient’s malignant breast tissue. Normal breast tissues in vivo also express Cyp19 transcripts (10, 11). Therefore, both fibroblasts and epithelial cells derived from the same reduction mammoplasty tissues were examined for Cyp19 mRNA and first exon usage. Reduction mammoplasty tissue was examined because it is from patients without breast cancer and therefore permits examination of cells that have not been altered by proximity to a breast tumor. Also, epithelial cells can be readily cultured from reduction mammoplasty specimens, but rarely from breast tumors, making it possible to examine whether fibroblasts and epithelial cells from the same tissue are similar or different for Cyp19 mRNA expression and regulation.
Patterns of regulation paralleled those observed in cells derived from
malignant and benign tissues. Cyp19 mRNA was up-regulated by Dex, PDA,
and cAMP in fibroblasts from both tissues, although in WS-14 cells Dex
appeared to be the primary regulator whereas in WS-15 cells the primary
regulator was the Dex, PDA, and cAMP combination (Fig. 4, B and A, respectively). These
responses were associated with EI.4 usage in WS-14 fibroblasts, but in
WS-15 the Dex, PDA, and cAMP combination predominantly up-regulated
PII. Usage of EI.3 was stimulated by the DEX, PDA, and cAMP combination
in both fibroblast preparations. In contrast, Cyp19 mRNA and first exon
usage by epithelial cells was at best very marginally above the limit
of detection, indicating that in vitro regulators of
fibroblast Cyp19 mRNA transcription lack the ability to up-regulate
transcription in epithelial cells. These results are consistent with
our prior conclusion (19), using mostly immortalized cell lines, that
aromatase in epithelial cells in vitro is not responsive to
regulators of aromatase in fibroblasts.
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). For both benign breast and breast
tumor fibroblasts, the expression and regulation of Cyp19 mRNA in short
term cultures and extended life cells were qualitatively similar.
Therefore, the results are described only for the extended life
fibroblasts.
Breast tumor-derived extended life fibroblasts (Fig. 5C) had EI.4 usage
regulated by Dex alone or in combination with other additives, with an
approximately 14-fold induction over the control treatment. In
contrast, PII usage, although slightly increased by cAMP alone,
demonstrated a substantial increase in combination with Dex and/or PDA.
EI.3 usage was also up-regulated, but principally by the combination of
cAMP plus DEX and/or PDA. Benign breast extended life fibroblasts
exhibited a similar pattern of Cyp19 mRNA and first exon regulation
(Fig. 5D). Therefore, extended life fibroblasts from both benign breast
and breast tumor tissues exhibited regulated expression of
CYP19 gene first exon usage comparable to breast tissues
in vivo.
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Discussion |
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The present study addressed two fundamental issues pertaining to the concept of local aromatase-mediated estrogen synthesis. The first issue concerns the cellular origin of Cyp19 mRNA and aromatase in breast tissues, which prior in vivo and in vitro observations, including ours (18, 19) and others (10), indicated predominantly involved stromal fibroblasts. The current results provide the first demonstration that fibroblasts from breast reduction mammoplasty specimens, from benign breast tissues peripheral to tumors, and from tumor tissues all exhibit regulated CYP19 transcription from first exons EI.3, EI.4, and PII. As noted, these are the same first exons detected in RNA directly isolated from the same types of breast tissues (25, 26, 28, 44). These results provide additional support for the concept that fibroblasts are the cells in breast tissues that accumulate Cyp19 transcripts originating from these same first exons.
The second issue concerns the differences reported in CYP19 transcription initiation site usage among breast reduction mammoplasty specimens, benign breast tissues peripheral to tumors, and tumor tissues. In reduction mammoplasty tissue from cancer-free women, transcripts with first exon EI.4 predominate, whereas first exons EI.3 and PII predominate in benign and tumor breast tissues from women with cancer (25, 26, 28, 44). The present study questioned whether this phenomenon, termed exon switching, occurs as a result of intrinsic differences in fibroblasts between normal tissue and benign or neoplastic breast tissues (31), or because of differences in transcription regulatory molecules in these tissues. Our data provide the first evidence for qualitatively similar responses in CYP19 first exon usage by various regulatory molecules with fibroblasts derived from reduction mammoplasty specimens, from benign areas surrounding breast tumors, and from the tumors themselves. Therefore, these data indicate that differences in transcription regulatory molecules, not intrinsic differences among fibroblasts, account for the variation in CYP19 first exon usage among cancer-free, benign and malignant breast tissues.
Estrogen influences breast cancer clinical outcome in patients by stimulating the proliferation of estrogen receptor-positive tumor epithelial cells. This could occur by an endocrine mechanism by which estradiol is taken up from plasma, by a stromal to epithelial paracrine mechanism, and/or by an autocrine mechanism with estrogen synthesis in tumor epithelial cells. The relative contribution of any one of these mechanisms is likely to vary with the physiological status of the female and possibly also with the local and systemic changes occurring during breast tumorigenesis and progression. Experimental evidence supports the potential of each mechanism to contribute to estrogen synthesis and influence breast tumorigenesis. Discerning the relative contribution of each mechanism in women at a given stage of normal development, aging, breast tumorigenesis, and cancer progression, however, is a complex task. Our long term goal is to gain insight into this complex and dynamic problem.
The concept that local aromatase-mediated estrogen synthesis occurs in stromal fibroblasts with action by a paracrine mechanism in breast epithelial cells develops from characterization of and evidence for estrogen synthesis in breast fibroblasts. Normal breast tissues contain fibroblasts as a component of the intralobular and extralobular stroma. Proliferation of fibroblasts contributes to breast cancer desmoplasia, the excessive collagen deposition and hard tumor consistency that commonly characterizes breast tumors. Therefore, stromal fibroblast-like cells, although not targets of tumorigenic transformation, are a significant cellular feature of normal and neoplastic breast tissues (30). Our prior studies demonstrated that two thirds of human breast tumors can aromatize androgens to estrogens, as detected by radiochemical methodology (43). Further, by immunohistochemistry we reported that stromal cells are the predominant type of cell in breast tumors that contain aromatase, and that enzymatic activity correlated with immunohistochemical staining in stromal fibroblast cells (18). Finally, aromatase activity and mRNA were increased significantly by regulatory molecules in isolated benign breast and breast tumor fibroblasts grown in culture (19). The current results extend these observations and provide substantive additional evidence for the idea that stromal cells contribute to the increased aromatase and levels of estrogens present in breast tissue of women with breast cancer.
Experimental evidence supports the potential significance of estrogen synthesis in tumor epithelial cells with action in these estrogen-dependent cells by an autocrine mechanism. Aromatase activity, immunohistochemically reactive aromatase, and Cyp19 mRNA have been demonstrated in breast tumor epithelial cells (14, 16, 17, 45, 46). Additional evidence is the ability of androstenedione, after aromatization to estrogen, to stimulate growth of aromatase-transfected MCF-7 breast cancer cells (40). Importantly, estrogen produced locally in tumors arising from these xenografted cells may exceed the amount taken up from plasma (15). Deregulated aromatase expression in two mouse models is associated with increased mammary cell proliferation and neoplastic development (47, 48). Thus, autocrine mechanisms of estrogen action can produce physiologically significant effects on the mouse mammary gland, including tumorigenesis. Notwithstanding these results regarding autocrine effects, the current and our prior data (19) in human breast epithelial cells do not support a major role for estrogen synthesis in this cell type. Basal Cyp19 mRNA and aromatase activity were low, and the same regulatory molecules produced significantly smaller effects than in fibroblasts. These data emphasize that inherent differences between breast epithelial and fibroblast cells probably determine responsiveness to regulators of CYP19 transcription. Nevertheless, the possibility remains that the necessary and optimal conditions for CYP19 expression and regulation in epithelial cells have not been identified.
A potential criticism regarding this and our previous studies is that isolated cells in culture do not completely reflect in vivo conditions. Although fibroblasts appear to lose CYP19 expression in vitro, regulatory molecules permitted fibroblasts to recapitulate at least some features of in vivo CYP19 expression. Our current use of cells during the first passage diminishes this criticism somewhat. The similarity of effects observed during early and late passages reported here and previously (19) and even with extended lifespan cells suggests that in fibroblasts there are no fundamental changes in aromatase regulation taking place. Consequently, it appears that the results provide valid information, particularly regarding the ability to stimulate aromatase in stromal compared to epithelial cells.
Our prior studies demonstrated that aromatase enzyme activity reached about 10 pmol/mg protein·h after 54 h of treatment with the combination of Dex, cAMP, and PDA in benign breast and breast tumor fibroblasts cultured in serum-containing medium (19), an activity comparable to primary reduction abdomenoplasty stromal cells cultured in serum-containing medium with Dex (49) or in serum-free medium with dB-cAMP (50) for a similar time. These latter in vitro studies generally resulted in examination for Dex and dB-cAMP effects under serum-containing and serum-free conditions, respectively, and the demonstration that Dex and db-cAMP regulation of CYP19 transcription primarily involved the EI.4 and the EI.3 plus PII promoter regions, respectively (24, 51, 52, 53). However, in breast tissues from cancer-free and breast cancer patients, transcripts from all three promoters accumulate (25, 26, 27), implying that in the same tissue, multiple regulatory pathways control transcription initiation from more than one promoter. The results presented here, using our prior experimental plan of breast fibroblasts in serum-containing medium, demonstrate that Dex acted alone to regulate promoter EI.4 transcription, but also and importantly that cAMP acted alone and in combination with Dex to regulate transcription from promoters PII and EI.3. Therefore, these in vitro results resemble the observed pattern of transcription from EI.3, EI.4, and PII in breast tissues. On the other hand, cAMP alone or in combination with Dex and/or PDA increased PII transcripts primarily and EI.3 transcripts secondarily, whereas there were similar levels of PII and EI.3 transcripts in breast tissues (26). Adipose tissue fibroblasts cultured under serum-free conditions also had PII transcripts increased primarily and EI.3 transcripts secondarily by cAMP and a phorbol ester (53), implying that our observations are not necessarily due to the presence of serum. This apparent discordance between in vivo and in vitro studies may reflect the functional status of positive (54) as well as negative (54, 55) regulators of EI.3 transcription initiation other than cAMP.
In summary, the results presented provide new and substantial evidence consistent with the concept that local aromatase-mediated estrogen synthesis in breast tissues predominantly involves fibroblast cells in the stromal component. All three first exons expressed in breast tissues could be expressed in cultured fibroblasts. If EI.3 and PII had not been expressed or regulated, for example, the implication could be that epithelial, not fibroblast, cells transcribe these first exons in vivo or that serum-free conditions (50, 56) may be required to demonstrate cAMP effects. Our results also are consistent with the concept that breast stromal fibroblasts contribute to a paracrine effect of estrogen. The relative importance of breast fibroblast aromatase and estrogen production may vary with a woman’s physiological status, as well as during breast tumorigenesis and progression, and could be concordant or discordant with estrogen production from other tissues. Taken together, these observations indicate that breast stromal fibroblasts mimic many aspects of the complex regulation of aromatase activity in vivo. The evidence presented here suggests that stromal fibroblasts derived from breast tissues can be used to identify additional primary transcription factors and local mediators that control differential CYP19 transcription, which include PGE2, an effector of the protein kinase A and protein kinase C pathways (52), and insulin-like growth factors I and II (33, 34).
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Acknowledgments |
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Footnotes |
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Received June 23, 1999.
Revised October 18, 1999.
Accepted October 22, 1999.
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