Mammary epithelial cells of PR-A transgenic mice exhibit distinct alterations in gene expression and growth potential associated with transformation
Yu-Chien Chou,
Norihisa Uehara,
Jason R. Lowry and
G. Shyamala
Division of Life Sciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA
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Abstract
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Expression of the A and B forms of progesterone receptor (PR), in an appropriate ratio is critical for normal mammary development. As such, mammary glands of PR-A transgenic mice, carrying additional A form of PR as transgene, exhibit morphological and histological characteristics associated with transformation. Accordingly, in the present studies, we analyzed these mammary glands for the presence of transformed epithelial cells by examining for alterations in gene expressions and growth potential, known to be associated with different stages of transformation. These studies reveal that, in the aberrant mammary epithelial structures, there is a decrease in p21 expression, an increase in cyclin D1 expression accompanied by an increase in cell proliferation, and a decrease in estrogen receptor alpha (ER
). In mammary ducts with normal histology, there is a decrease in p21 expression without an elevation in cyclin D1 expression or cell proliferation or a decrease in ER
expression. Treatment of PR-A transgenics with anti-progestin, mifepristone, has no effect on cell proliferation, cyclin D1 or ER
expression in the aberrant epithelial structures. In contrast, mifepristone restored the loss of p21 expression in the epithelial cells of both the ducts with normal histology and aberrant structures. Parallel studies reveal no apparent differences between the mammary glands of wild-type and PR-B transgenic mice, which carry additional PR B form. Accordingly, we conclude that (i) mammary glands of PR-A transgenics contain at least two distinct populations of transformed epithelial cells, (ii) the epithelial cell population in the ducts with normal histology contain presumptive immortalized cells, indicative of early stages of transformation, (iii) the aberrant epithelial structures contain later stages of transformation associated with hyperplasias/pre-neoplasias and (iv) the transformation of mammary epithelial cells in PR-A transgenics might be due to a misregulation in progesterone action resulting from overexpression of PR A form.
Abbreviations: BrdU, 5-bromo-2-deoxyuridine; CDK, cyclin-dependent kinase; ER
, estrogen receptor alpha; ERKO, estrogen receptor null mutant; PCNA, polymorphic cell nuclear antigen; PR, progesterone receptor; RTPCR, reverse transcriptionpolymerase chain reaction.
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Introduction
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The female sex steroids, estradiol and progesterone, signaling through their respective receptors, ER
and PR are critical for normal mammary development, induction of mammary carcinogenesis and growth of some mammary tumors. A central role for PR in normal mammary development is established by the fact that in PR null mutant mice, which have ER
, there is a severe inhibition in lobulo-alveolar growth, normally accompanying pregnancy and occurring in response to estradiol and progesterone. At present the precise role of PR in mediating either normal mammary development or carcinogenesis is unknown (1,2).
PR exists in two molecular forms (the A and B forms) which actions can vary depending on cell and promoter context (3). Previous studies from our laboratory have shown that a regulated expression of the two isoforms of PR is critical for normal mammary development. As such, in transgenic mice carrying an imbalance in the native ratio of A to B forms by overexpressing either the A or B form (referred to as PR-A and PR-B transgenics, respectively) mammary development is abnormal (4,5). In particular, mammary glands of PR-A transgenics exhibit excessive ductal growth, contain aberrant epithelial structures with ducts composed of multiple layers of epithelial cells and a loss in basement membrane integrity and cellcell adhesion (4), characteristics frequently associated with transformed cells.
Extensive studies by Medina et al. have shown that normal, pre-neoplastic and neoplastic mouse mammary epithelial cells exhibit distinct patterns of gene expression and growth properties (68). In our previous studies, the aberrant features associated with mammary glands of PR-A transgenic mice were documented using morphological and histological criteria, which were not sufficient to determine if these glands contained epithelial cells with changes in gene expression correlated with transformed cells (4). Accordingly, in the present studies we have characterized the epithelial cells in the mammary glands of PR-A transgenics and report that they contain populations of epithelial cells with distinct alterations in gene expression and growth potential.
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Materials and methods
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Animal treatment and tissue collection
Nulliparous adult FVB mice (1014 weeks) were used in these studies. PR-A transgenic and PR-B transgenic mice have been described previously (4,5). Mammary glands of ER
null mutant (ERKO) mice were kindly provided by Dr Dennis B.Lubahn (9). The mice were housed and cared for in accordance with the NIH guide to humane use of animals in research.
For cell proliferation studies, mice were administered 160 µg/g body wt of 5-bromo-2-deoxyuridine (BrdU, Sigma, St Louis, MO) 2 h before death. For studies with mifepristone (RU486, Sigma), mice were treated with mifepristone 16 µg/g body wt daily for 4 days. For immunohistochemical analyses on paraffin sections, mammary tissues were collected, fixed in 4.7% formalin (same as 10% buffered formalin phosphate, Fisher Scientific, Pittsburgh, PA), dehydrated, embedded in paraffin and cut into 5 µm thick sections. For immunohistochemical analyses on frozen sections, mammary glands were mounted in OCT and quick-frozen in a mixture of dry ice and ethanol. Cryostat sections (510 µm thick) were cut and mounted onto glass slides and fixed for 2 min in methanol/acetone (1:1). For immunoblot analyses tissues were frozen in liquid nitrogen and stored at 70°C until use.
Antibodies
The antibodies used were: anti-BrdU, rat monoclonal antibody (Harlan Sera-Lab Ltd, Loughborough, UK); anti-polymorphic cell nuclear antigen (PCNA), clone PC10 (DAKO, Carpinteria, CA); anti-cyclin D1, mouse monoclonal antibody (Biocare Medical, Walnut Creek, CA); anti-ER
, mouse monoclonal antibody 6F11 (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK); anti-p21WAF1, mouse monoclonal antibodies Ab-5 and Ab-11 (Lab Vision, Fremont, CA).
Immunohistochemistry
BrdU-, cyclin D1-, p21- and ER
-positive cells were analyzed in paraffin embedded sections as described previously (10,11). All the mouse monoclonal antibodies used in these studies corresponded to IgG1. Accordingly, in experiments using mouse monoclonal antibodies, for negative controls, the primary antibodies were substituted at equivalent concentrations with an irrelevant mouse IgG1 (DAKO). The antigenantibody complexes were identified using Universal DAKO LSAB2 labeled streptavidinbiotin peroxidase kit (DAKO). The sections were counterstained with Mayers hematoxylin solution (DAKO). After counterstaining, nuclei negative for the antigen appeared purple-blue and positive nuclei appeared brown. Analyses for PCNA-positive cells were performed on frozen sections using an indirect immunofluorescence assay as described previously (12). In each experiment, mammary glands from a minimum of three mice were examined and mammary glands for each mouse were analyzed in triplicate. In each experiment, the percentage of immuno-positive cells was obtained by counting a minimum of 500 cells per gland. The differences between the various experimental groups were analyzed by means of a two-sided Students t-test and were considered significant when P < 0.05 was obtained.
Western blot analyses
Protein extracts were prepared from mammary tissues of wild-type and PR-A transgenic mice by homogenization in lysis buffer [50 mM TrisHCl (pH 8.0), 125 mM NaCl, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate and 1 mM PMSF] containing the following protease inhibitors: leupeptin, pepstatin, aprotinin, each at a final concentration of 1 µg/ml. The homogenates were sonicated, centrifuged at 110 g and the pellets were discarded. Protein concentrations in the supernatants (lysates) were determined by DC protein assay (Bio-Rad, Hercules, CA). Aliquots of lysates equivalent to 20 µg of protein were subjected to electrophoresis through 816% SDSPAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 10% non-fat powdered milk prior to treatment with the primary antibodies. Subsequently, the blots were washed and treated with appropriate secondary antibodies. The resulting antigenantibody complexes were detected by ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK), the films were scanned and subjected to densitometric analyses using the PC version of NIH image (Scion Corporation).
cDNA synthesis and quantitative RTPCR analysis for ER
Total cellular RNA was extracted using Totally RNA isolation kit (Ambion, Austin, TX) according to the protocol provided by the manufacturer. For cDNA synthesis, 6 µg of total RNA, prepared as described above was treated with DNase I, to remove any contaminating genomic DNA, and then used for Reverse Transcriptase (RT) coupled cDNA synthesis using oligo-(dT)15 primers and Superscript II (Life Technologies, Bethesda, MD). The RT reaction was performed at 42°C for 50 min, followed by heating at 70°C for 10 min. The resultant cDNA was either used immediately for quantitative RTPCR or stored at 20°C for later use.
PCR reactions were performed using the ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, CA). The primers used for detection of ER
were the same as described previously (13). In preliminary studies, optimal experimental conditions were established and a standard curve was generated using serially diluted samples. The amount of transcripts in each sample was calculated from the standard curve and normalized to ß-actin gene, run as an internal control.
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Results
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Rate of epithelial cell proliferation is augmented in mammary glands of PR-A transgenic mice and is restricted to aberrant epithelial structures
The proliferative status of the mammary epithelial cells in PR-A transgenic mice was examined by immunocytochemistry using two independent parameters, i.e. PCNA and BrdU. As expected, few BrdU- or PCNA-positive cells were detected in mammary ducts of wild-type mice (Figure 1A, C and E
). Similarly, in mammary glands of PR-A transgenics, few BrdU- (3.3 ± 0.7%) or PCNA-positive cells (5.0 ± 2.2%) were detected in ducts with normal histology and were comparable with those observed in ducts of wild-type mice (BrdU: 2.9 ± 1.3%; PCNA: 5.2 ± 0.3%; Figure 1
). In contrast, there was a significant increase in both BrdU- (24.3 ± 4.1%) and PCNA-positive cells (14 ± 3.9%) in aberrant mammary epithelial structures of PR-A transgenic mice (Figure 1B, D and E
).

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Fig. 1. Analyses for cell proliferation in mammary glands of wild-type and PR-A transgenic mice. Mammary glands from wild-type (A and C) and PR-A transgenic mice (B and D) were analyzed for immunoreactive BrdU (A and B) and immunoreactive PCNA (C and D), as described in text. Scale bar represents 20 µm. (E) The number of BrdU- and PCNA-positive cells in the different morphological structures were analyzed as described in text; ND: ducts with normal histology; AD: aberrant duct. ***BrdU- and PCNA-positive cells in aberrant structures are significantly higher than that in ducts with normal histology (P < 0.001).
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Cyclin D1 expression is elevated in the aberrant epithelium of PR-A transgenics
In mouse mammary glands, cyclin D1 is essential for mammary epithelial cell proliferation (14,15) and overexpression of cyclin D1 can lead to ductal hyperplasia (16). Therefore, we examined if the increase of mammary epithelial cell proliferation in PR-A transgenic mice was also accompanied by changes in cyclin D1 expression. Immunoreactive cyclin D1 was detected in the epithelial cell nuclei of all genotype (Figure 2AC
). The number of cyclin D1-positive cells was similar between mammary ducts in wild-type mice (5.5 ± 0.6%) and ducts with normal histology in PR-A transgenic mice (4.6 ± 0.6%; Figure 2D
). In contrast, there was an apparent increase in the intensity of immunostaining in the aberrant structures of PR-A transgenics (Figure 2
, compare C with A and B); there was also an increase in the number of cyclin D1-positive cells (7.2 ± 0.8%; Figure 2D
).

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Fig. 2. Analyses for cyclin D1 expression in mammary glands of wild-type and PR-A transgenic mice. Mammary glands from wild-type (A) and PR-A transgenic mice (B and C) were analyzed for immunoreactive cyclin D1 as described in text. Scale bar represents 20 µm. (D) The number of cyclin D1-positive cells among the various morphological structures were analyzed as described in text; ND: ducts with normal histology; AD: aberrant duct. *Cyclin D1-positive cells in aberrant structures are significantly higher than that in ducts with normal histology (P < 0.05).
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Expression of p21 is decreased in mammary epithelial cells of PR-A transgenics
Our foregoing observations on cyclin D1 expression, taken together with the patterns of immunostaining for BrdU and PCNA, strongly implied the involvement of cyclin D1 in the aberrant mammary epithelial growth. It is well known that the growth promoting effects of cyclins are achieved through their assembly with their catalytic partners, cyclin-dependent kinases, CDK4 and CDK6 and whose activities, in turn, can be constrained by CDK inhibitors (17). One of the CDK inhibitors, p21, can either act as a growth promoting or growth inhibitory factor, depending on its cellular concentration; at low concentration p21 acts as an assembly factor and thus promotes the formation of active CDK complexes while at high concentrations it inhibits CDK kinase activity (18). Consistent with this, p21 concentration has been shown to be elevated during growth suppression in human mammary tumor cells (19).
Analyses for the status of p21 expression, by immunoblot assays, revealed that it was decreased in mammary glands of PR-A transgenics (Figure 3A
), which was also apparent in immunohistochemical analyses (Figure 3B
). As such, while in both wild-type and PR-A transgenics, immunoreactive p21 was detected in the nuclei of epithelial cells; the overall intensity of p21 immunostaining was reduced in the ducts of PR-A transgenics as compared with the ducts of wild-type mice (Figure 3B
, compare a and b). A decrease in the level of p21 immunoreactivity was also apparent in the aberrant epithelial structures (Figure 3B, c
).

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Fig. 3. Analyses for p21 expression in mammary glands of wild-type and PR-A transgenic mice. (A) Immunoblot analyses: the bar graphs show quantitative analyses of immunoblots by densitometry. PR-A (a) represents the data for a single sample from three separate experiments to demonstrate the low intra-sample variability; PR-A (b) shows the data corresponding to three different samples to demonstrate inter-sample variability. ***p21 expression in the mammary glands of PR-A transgenic mice are significantly lower than that in wild-type mice (P < 0.001). There is no significant difference between PR-A (a) and PR-A (b). The inset shows a representative immunoblot corresponding to mammary gland lysates from wild-type (lane 1) and PR-A transgenics (lanes 24) with (+) and without () treatment with the primary antibody. The position of the molecular weight standards is indicated on the left. (B) Immunolocalization of p21: (a) shows mammary glands of wild-type mice; (b and c) show respectively a duct with normal histology and an aberrant duct of PR-A transgenic mice; (d) shows the absence of immunoreactivity with irrelevant mouse IgG (top) and deletion of primary antibody (bottom). Scale bar represents 20 µm.
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ER
expression is decreased in aberrant epithelial structures of PR-A transgenics
In a series of comprehensive studies, Medina et al. have identified certain molecular markers unique to mammary epithelial cells in various stages of transformation. In particular, they have shown that in mouse mammary epithelial cells, a decrease in p21 expression, without an increase in cyclin D1 expression is indicative of immortalization and precedes the onset of hyperplasias/pre-neoplasias (7). Thus, from the patterns of cyclin D1 and p21 expression (shown in Figures 2 and 3
), it appeared that while the ducts in PR-A transgenics had retained a normal histology they might, nevertheless, contain immortalized epithelial cells. If this were so, it would also imply that the epithelial cells in the aberrant structures contained cells in later stages of progression with increased growth potential, a characteristic of hyperplasias. Another feature that distinguishes the hyperplasias from the presumptive immortalized cells is a decrease in the expression levels of ER
(7,8). Therefore, to further verify the separate identity of the epithelial cells in the aberrant structures from those in the ducts with normal histology, we examined the status of ER
. As shown in Figure 4A
, the level of ER
transcripts, analyzed by quantitative real-time RTPCR, was significantly decreased in the mammary glands of PR-A transgenics. In immunochemical analyses both the intensity of staining and the number of ER
-positive cells in the ducts with normal histology of PR-A transgenics (24.9 ± 1.8%) were comparable with that seen in the ducts of wild-type mice (26.9 ± 1.4%) (Figure 4
, compare B and C and D). In contrast, there was a decrease in both the intensity and number of ER
-positive cells (12.7 ± 0.8%) in the aberrant epithelial structures (Figure 4C and D
). Figure 4
also shows the absence of ER
immunostaining in mammary glands of ERKO mice.
Mifepristone (RU486) restores the loss of p21 in mammary epithelial cells of PR-A transgenic mice but has no effect on the expression of cyclin D1, ER
or cell proliferation
Next, we examined whether progesterone/PR signaling is involved in misregulation in cell proliferation and changes in expression levels of p21, cyclin D1 and ER
in the mammary glands of PR-A transgenics. To this end, we tested the effects of the anti-progestin, mifepristone on these various molecular parameters. Administration of mifepristone to intact PR-A transgenic mice had no effect on the morphology of mammary glands such that excessive ductal growth was still present (data not shown). Furthermore, in these glands, BrdU-positive cells were still detected in the aberrant epithelial structures (Figure 5A
) and the number of these cells (26.3 ± 3.6%) was equivalent to those seen with untreated mice (Figure 1E
, Table I
). In contrast, mifepristone abolished BrdU immunostaining in the ducts with normal histology (Figure 5A
) and also in the ducts of wild-type mice (Table I
). Mifepristone also did not have any effect in the aberrant epithelial structures of PR-A transgenics with regard to cyclin D1 expression (Figure 5B
) such that the number of cyclin D1-positive cells (6.6 ± 1.0%) was comparable with that observed in untreated mice (Figure 2D
, Table I
). Similarly, in the aberrant epithelial structures, a decrease in ER
expression, both with regard to intensity (Figure 5C
) and the number of ER
-positive cells (14.3 ± 1.0%, Table I
) was unaffected in mifepristone treated mice. In contrast to BrdU and cyclin D1 expression, mifepristone abolished the differences between mammary glands of wild-type and PR-A transgenics by restoring the loss of p21 as shown by immunoblot analyses and immunostaining for p21 (Figure 5DF
). There were no apparent differences between mammary glands of wild-type and mifepristone treated wild-type mice with regard to the expression patterns of p21 (data not shown).
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Table I. Number of BrdU, PCNA, cyclin D1 and ER -positive cells in mammary glands of wild-type and PR-A transgenic micea
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Mammary epithelial cells of PR-B transgenic mice do not exhibit alterations in cell proliferation or in p21, cyclin D1 or ER
expression
The distinguishing characteristic of PR-A transgenic mice is that they carry an imbalance in the native ratio of A:B isoforms of PR and hence, an alteration in signaling through PR. Therefore, it was important to identify if the changes observed in the mammary epithelial cells of PR-A transgenics were indeed related to its mammary phenotype or simply resulted from abnormal signaling through PR, due to an overall imbalance in the ratio of the two isoforms. An imbalance in the native ratio of A:B isoforms of PR also exists in PR-B transgenic mice but the mammary phenotypes of these mice are distinct from PR-A transgenics (5). Accordingly, we also examined the mammary glands of PR-B transgenics. As shown in Figure 6
, as compared with mammary ducts of wild-type mice, there was no detectable increase in the number of BrdU-positive cells or cyclin D1 expression in ducts of PR-B transgenics (Figure 6B and D
). The expression of p21 was also not diminished in the ducts and, in fact, appeared to be somewhat elevated (Figure 6F
).

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Fig. 6. Analyses for cell proliferation, cyclin D1, p21 and ER in mammary glands of PR-B transgenic mice. Mammary glands from wild-type (A, C, E and G) and PR-B transgenic mice (B, D, F and H) were analyzed for immunoreactive BrdU (A and B), cyclin D1 (C and D), p21 (E and F) and ER (G and H). Note that there are no apparent differences between wild-type and PR-B transgenic mice in the patterns of immunostaining for BrdU, cyclin D1, p21 and ER . Scale bar represents 20 µm.
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Similarly, ER
expression was also unaffected in the mammary glands of PR-B transgenic mice (Figure 6H
).
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Discussion
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Using morphological and histological criteria, we had documented previously that mammary development in PR-A transgenic mice was abnormal. In particular, we had demonstrated that these glands exhibited extensive ductal growth, loss in basement membrane integrity and cellcell adhesion (4), characteristics commonly associated with transformed cells. In this report, we demonstrate that the expression patterns of p21, ER
and cyclin D1 are altered in the mammary epithelial cells of PR-A transgenics and are accompanied by a higher rate of proliferation as revealed by immunostaining for BrdU and PCNA (summarized in Table I
). Mouse mammary epithelial cells exhibiting a decrease in p21 without an elevation in cyclin D1 are believed to correspond to immortalized cells with limited growth rate (7). In PR-A transgenics, ductal epithelial cells with normal histology have a decrease in p21 expression without an increase in cyclin D1 expression or cell proliferation. Mouse mammary epithelial cells are also presumed to be immortal if they can be propagated in vivo, through serial transplantation, beyond five to seven generations (20). We have serially transplanted tissue fragments from mammary glands of PR-A transgenics up to eight generations (data not shown). Thus, we conclude that these ducts contain the presumptive immortalized epithelial cells, indicative of early stages of transformation. Among the characteristics that distinguish the presumptive immortalized epithelial cells from those in hyperplasias/pre-neoplasias are increases in cyclin D1 and cell proliferation and a decrease in ER
expression (8), features associated with the aberrant epithelial structures of PR-A transgenics. Accordingly, we also conclude that these structures contain epithelial cells in later stages of transformation and correspond to hyperplasias. In this context, it is relevant to note that when PR-A transgenics are crossbred with transgenic mice overexpressing the unactivated form of C-Neu, the hybrid mice develop mammary tumors with a shorter latency (21). These observations, taken together with our previous histological and morphological studies, therefore, establish that mammary glands of PR-A transgenics contain transformed epithelial cells. In turn, they reinforce our earlier proposal that an imbalance in the relative expression levels of A:B isoforms of PR can lead to abnormal mammary development and transformation of epithelial cells.
PR-B transgenic mice also carry an imbalance in the native ratio of A:B isoforms. We have shown previously that the mammary phenotype of PR-B transgenics is somewhat opposite to that of PR-A transgenics and, in particular that they do not exhibit excessive ductal growth (5). Our present studies show that mammary epithelial cells of PR-B transgenics do not exhibit significant changes in the expression levels of the various defined molecular markers, used for the identification of transformed cells in PR-A transgenics. Thus, our present studies also reveal that the transformation of mammary epithelial cells in PR-A transgenics is simply not the result of an imbalance in the ratio of A:B isoforms but specifically due to overexpression of PR A form. In this context, it is noteworthy that an imbalance in the relative ratio of A:B isoforms of PR has also been observed in certain human mammary tumors, and this is often associated with overexpression of PR A form (2224).
Progesterone/PR signaling has been shown to regulate p21 expression indirectly through SP-1 sites on p21 promoter (25). A distinguishing feature of both the presumptive immortalized epithelial cells and those in hyperplasias is the loss in p21 expression. In PR-A transgenics, the loss in p21 expression is restored in both these cell populations with the antiprogestin, mifepristone, suggesting the involvement of progesterone/PR signaling. However, it is important to note that, in mammary epithelial cells of PR-A transgenics, changes in p21 expression levels do not appear to be tied to degree of cell proliferation. As such, the decrease in p21 expression in the presumptive immortalized epithelial cells is not accompanied by an increase in cell proliferation and conversely, when the loss in p21 is restored with mifepristone in the hyperplasias, they continue to proliferate. p21 is a multifunctional protein and as such, has been implicated in a vast array of regulatory networks (26). To this end, the significance of decreased p21 expression accompanying the immortalization of mouse mammary epithelial cells must await future studies.
In mammary glands of wild-type mice, cell proliferation is initiated at the onset of pregnancy in response to progesterone/PR signaling, which requires cyclin D1 (14,15). Similarly, long-term (21 days) administration of estradiol and progesterone to ovariectomized wild-type mice increases cell proliferation analogous to that occurring during pregnancy (27) and this is accompanied by an increase in cyclin D1 expression (28). In the hyperplasias of PR-A transgenics, similar to mammary epithelial cells of wild-type mice, the increase in cell proliferation is accompanied by elevated expression of cyclin D1 except that it occurs in the absence of pregnancy. Furthermore, in these cells both the increases in cell proliferation and cyclin D1 expression are insensitive to mifepristone. In contrast, mifepristone abolishes both the basal epithelial cell proliferation observed in the immortalized cells in the ducts of PR-A transgenics (Figure 5
) and in mammary ducts of wild-type mice (Table I
). This suggests that a principal trigger for the progression of immortalized cells to hyperplasias may be a derangement in progesterone/PR-dependent regulation of cyclin D1 expression resulting, in turn, in progesterone independent proliferation. In fact, it may even be that the mammary epithelial cells of hyperplasias have acquired a resistance to progesterone/PR signaling due to overexpression of cyclin D1, as found with T47-D human mammary tumor cells (29).
In summary, our studies show that mammary glands of PR-A transgenics contain distinct populations of epithelial cells in different stages of transformations, and hence, with different growth potential. In addition, they show that the altered growth potential of these epithelial cells is at least, in part, due to misregulation in progesterone action at the level of cell cycle. Thus, our present studies establish that an imbalance in the expression of the two isoforms of PR, resulting from overexpression of PR A form, can lead to transformation of mammary epithelial cells. These studies also highlight that PR-A transgenic mice can serve as an important experimental model for dissecting the mechanisms underlying ovarian steroid dependent mammary epithelial cell transformation and progression in vivo.
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Notes
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1 To whom correspondence should be addressed Email: shyamala_harris{at}lbl.gov 
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Acknowledgments
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These studies were supported by a grant from the National Institutes of Health Grant CA 66541. N.U. was supported by institution training grant DAMD17-00-1-0224. We thank A.Asaithambi for assistance with the real-time RTPCR analyses and Derek Wong for assistance with the immunohistochemistry experiments.
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Received August 7, 2002;
revised October 24, 2002;
accepted November 25, 2002.