Steroid hormone receptor expression and proliferation in rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Cunping Qiu, Liang Shan, Minshu Yu and Elizabeth G. Snyderwine*

Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

* To whom correspondence should be addressed at: Building 37, Room 4146, 37 Convent Drive MSC 4262, Bethesda, MD 20892-4262, USA. Tel: +1 301 496 5688; Fax: +1 301 496 0734; Email: elizabeth_snyderwine{at}nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet. Herein, the expression of estrogen receptor alpha (ER{alpha}), estrogen receptor beta (ERß) and progesterone receptor (PR) was examined in mammary gland carcinomas induced by PhIP in female Sprague–Dawley rats. Quantitative real-time polymerase chain reaction demonstrated that ER{alpha}, ERß and PR were statistically elevated by 3-, 4- and 8-fold in carcinomas compared with normal mammary glands. By immunohistochemistry, carcinomas showed statistically higher nuclear expression of all three steroid receptors with the majority of carcinomas showing at least 10% of epithelial cells stained for ER{alpha} (49/55, 89%), ERß (41/55, 75%) and PR (48/55, 87%). Furthermore, the level of expression of the three steroid hormone receptors was positively correlated with each other across the bank of carcinomas (Spearman analysis, P < 0.05). The expression of ER{alpha} in carcinomas was associated with tumor grade, extent of nuclear pleomorphism and cellular proliferation as measured by proliferating cell nuclear antigen (PCNA) and phospho-Rb immunostaining (Spearman analysis, P < 0.05). Confocal microscopy was used to measure the percentage of epithelial cells showing nuclear colocalization of receptors, PCNA, and cyclin D1. Colocalization of the receptors, and the colocalization of the receptors with PCNA and cyclin D1 was strikingly higher in carcinomas than in the normal mammary gland. In carcinoma cells, 37% of ER{alpha} positive epithelial cells were colocalized with PCNA in contrast to just 0.25% of cells in the normal mammary gland. The findings from this study indicate that ER{alpha}, ERß and PR were co-upregulated and nuclear localized in epithelial cells from rat mammary carcinomas compared with normal mammary glands, and that the co-upregulation was positively correlated with proliferation and cell cycle progression in carcinomas.

Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; ER{alpha}, estrogen receptor alpha; ERß, estrogen receptor beta; NMU, nitrosomethylurea; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 
The rat mammary gland is a widely used model for studying the pathogenesis, therapy and chemoprevention of breast cancer (13). Rat mammary gland carcinomas resemble human breast cancer in histopathogenesis, pathological features and hormone-dependence. Experimental rat mammary gland carcinogens 7,12-dimethylbenz[a]anthracene (DMBA) and N-nitrosomethylurea (NMU) induce carcinomas that are generally hormone-dependent (14) and estrogen and progesterone receptor (PR) positive (59). Ovarian steroids and their receptors are important in normal mammary gland development and carcinogenesis and estrogen is a major player in this regard (1013). It has been known for many years that ER{alpha} positive breast cancer cell lines proliferate in response to estradiol (14). In human breast cancer, the status of ER{alpha} expression provides a clinical biomarker of prognosis and response to therapies such as treatment with the antiestrogen tamoxifen (15).

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic amine, is prevalent in the western diet and implicated as an etiological factor in human breast cancer (16,17). In addition to epidemiological evidence linking PhIP exposure to an increased risk of breast cancer, PhIP is a known mammary gland carcinogen in rats (1820). Further supporting its possible involvement in human breast cancer, PhIP has recently been shown to have estrogenic activity in a human breast cancer cell line (21).

The molecular alterations in PhIP-induced rat mammary gland cancers have been examined in several studies (2227). Similar to a significant percentage of human breast cancers, PhIP-induced carcinomas show deregulation of the cyclin D1/cdk4 retinoblastoma pathway and enhanced cellular proliferation (27). Ovarian hormones induce the recruitment of quiescent mammary epithelial cells into the cell cycle, G1 progression and G1/S transition. One effect of estrogen on the cell cycle involves nuclear receptor ER{alpha}-mediated regulation of cyclin D1 transcription (28). Deregulation of estrogen-controlled pathways of proliferation has been implicated in breast carcinogenesis (1012). In the normal mammary gland a relatively small number of cells express receptors for estrogen and progesterone, and the expression of steroid receptors and proliferation-associated markers are almost mutually exclusive among the epithelial cells (10,11,13). However, breast cancer epithelial cells show a concomitant increase in nuclear expression of ER{alpha} and proliferation markers. These findings have supported the notion that while steroid hormones regulate proliferation in normal breast epithelial cells through a paracrine mechanism, presumably via steroid hormone-mediated release of growth factors from cells that subsequently stimulate proliferation in adjacent cells, deregulation in cancer epithelial cells involves a shift to an autocrine mechanism by which steroid hormones stimulate proliferation in the same cells harboring the receptors (12).

The role of steroid hormone receptors in PhIP-induced rat mammary gland cancer and whether the expression of steroid hormone receptors is deregulated and associated with proliferation and cyclin D1 expression have not yet been examined. The current study examines the expression of steroid hormone receptors ER{alpha}, ERß and PR, as well as markers of cell-cycle proliferation and progression across a large bank of PhIP-induced rat mammary gland carcinomas.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 
Rat mammary gland samples and PhIP-induced carcinomas
PhIP-induced rat mammary gland carcinomas were archival samples that were generated in prior studies (18,20,23). Age-matched control rat mammary gland samples were also collected in these studies. Briefly, adolescent Sprague–Dawley rats had received 10 doses of PhIP (75 mg/kg), once a day for 10 days. Carcinomas and normal mammary gland samples were snap-frozen and stored at –80°C and stored in formalin-fixed paraffin embedded blocks at 4°C. Classification and grading of carcinomas were based on a scoring system adapted from the Scarff–Bloom–Richardson grading scheme as previously described (25) with the modification that proliferation was evaluated by proliferating cell nuclear antigen (PCNA) immunostaining instead of by mitotic figures. The highest grade, poorly differentiated carcinomas tended to show a solid growth pattern with marked nuclear pleomorphism and a higher percentage of PCNA positive cells.

Antibodies
Mouse monoclonal antibodies ER{alpha} (Ab-14) and PR (PR, Ab-5) were purchased from Lab Vision Corporation (Fremont, CA). Mouse monoclonal antibodies cyclin D1 (clone DOS-6) and PCNA (M0879) were obtained from DAKO Corporation (Carpinteria, CA). Polyclonal antibody ER{alpha} (H-184), ERß (H-150) and PR (C-19), PCNA (FL-261), phospho-Rb (sc-16671) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies for immunostaining were from Universal DAKO LSAB2 System (Carpinteria, CA). For confocal microscopy, secondary antibodies Alexa Fluor®488 goat anti-rabbit IgG (A-11008) and Alexa Fluor® 568 goat anti-mouse IgG (A-11031) were purchased from Molecular Probes (Eugene, Oregon).

RNA isolation and quantitative real-time polymerase chain reaction
Total RNA was isolated using TRIzol extraction reagent (Invitrogen Life Technology, Carlsbad, CA). The major stromal contaminant in the normal mammary gland is adipose tissue. Fat was effectively removed from normal mammary gland samples by avoiding the fatty layer after chilling the homogenate on ice prior to centrifugation at 4°C and by using phenol–chloroform extraction. The RNA was treated with DNA-free TM (Ambion, Inc., Austin, TX) prior to reverse transcription to avoid genomic DNA contamination. Reverse transcription was carried out with ThermoScriptTM RT–PCR System (Invitrogen Life Technology, Carlsbad, CA) using random primers according to manufacture instruction. An aliquot of 1 µg of RNA was used to synthesize cDNA and 1 µl of RT was used for the following experiment.

Relative quantitation of gene expression was determined by quantitative real-time polymerase chain reaction (PCR) analysis with the ABI Prism 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA) using the comparative method as described previously (26). Standard curves were generated with various amounts of cDNA from the NMU rat mammary carcinoma cell line (26). Reactions were also routinely run without template as a negative control. The sequence of primers and probes of ER{alpha}, ERß and PR for quantitative real-time PCR are given in Table I. TaqMan rodent GAPDH set (Applied Biosystems, Foster City, CA) was used for normalization of the target genes. GAPDH was chosen as the reference because it was consistently and reproducibly expressed in all samples. Relative gene expression was derived by the method outlined in ABI Prism Sequence Detection System User Bulletin #2.


View this table:
[in this window]
[in a new window]
 
Table I. ER{alpha}, ERß and PR primer sets used for quantitative real-time PCR analysis

 
Immunohistochemistry
Immunostaining was carried out using the Universal DAKO LSAB2 System. Briefly, 5 µm thick tissue sections were deparaffinized and rehydrated. Prior to being blocked by hydrogen peroxide, antigen retrieval was performed by microwaving. Slides were incubated sequentially with primary antibodies overnight at 4°C and then with HRP-labeled secondary antibodies for 30 min at room temperature. Antibody concentrations were as recommended by the manufacture. Negative controls were run without the primary antibody. Sections were developed using AEC substrate and counterstained with Mayer's hematoxylin. A total of 55 PhIP-induced rat mammary carcinomas and 5 study control rat mammary gland samples were stained. Cells with red-colored nuclei staining were scored as positive and the percentage of positively stained cells was calculated as previously described (26,27).

Confocal microscopy
Using antibodies for ER{alpha}, ERß, PR, cyclin D1 and PCNA, nuclear colocalization was determined by confocal microscopy. Sections were blocked with 5% bovine serum albumin in phosphate buffered saline, followed by an overnight incubation of the primary antibody at 4°C and fluorescent secondary antibody for 1 h at 37°C. The sections were air dried and mounted in Vectashield with DAPI (H-1200, Vector, Burlingame, CA). Tissue sections incubated with only secondary antibody served as the negative controls. Sections were analyzed for colocalization with a Zeiss LSM 510 NLO Confocal Microscope (Carl Zeiss Inc., Thornwood, NY). For each pair of the primary antibodies, 3–5 images were analyzed. The number of positively single stained (red or green only), double stained (both red and green, i.e. yellow) nuclei, and total number of epithelial cells within a field as indicated by DAPI single staining (blue) were counted using Image J software (NIH, Bethesda, MD).

Statistical analysis
Statistical analysis including Student's t-test (one- or two-tailed) and the Spearman Rank Order Correlation was carried out, where indicated, using SigmaStat Statistical Software, version 2.0 (Jandel, San Rafael CA). Statistical significance was assumed at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 
mRNA expression and immunostaining of ER{alpha}, ERß and PR
The mRNA expression of ER{alpha}, ERß and PR in normal mammary glands and mammary carcinomas were measured by quantitative real-time PCR. As shown in Table II, ER{alpha}, ERß and PR showed statistically significantly higher mRNA expression in mammary carcinomas than did normal mammary glands (Student's t-test, P < 0.05). The elevation of expression in carcinomas was highest for PR, followed by ERß and ER{alpha}.


View this table:
[in this window]
[in a new window]
 
Table II. Real-time PCR analysis of ER{alpha}, ERß and PR expression in PhIP-induced rat mammary carcinomas and normal mammary glands

 
The ER{alpha}, ERß and PR receptor proteins were detected in the nuclei of epithelial cells of both carcinoma and the normal mammary gland by immunohistochemistry (Figure 1). In carcinomas, ER{alpha} and PR positively stained epithelial cells were mainly distributed around the ductal elements, while ERß positive epithelial cells were located diffusely throughout the tumor. The percentage of epithelial cell nuclei stained for ER{alpha}, ERß, PR and PCNA was on average 2- to 4-fold higher in the carcinomas than in the normal mammary gland.



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 1. Immunohistochemical detection of ER{alpha}, ERß, PR and PCNA in normal rat mammary gland (upper images) and PhIP-induced rat mammary gland carcinomas (lower images). Magnification 400x.

 
By immunostaining a range in the level of steroid receptor expression was observed among the carcinomas, and the majority of carcinomas showed levels of epithelial cell expression >10% (Figure 2). A 10% level has been considered a standard cut off value for defining ER positive or negative carcinomas in human breast cancers (29). Rat mammary gland carcinomas have been considered ovarian hormone non-responsive at approximately <10% of positively stained cells (5). A few carcinomas (6/55, 11%) showed ER{alpha} nuclear staining levels comparable with normal mammary glands and <10% while the majority showed nuclear expression levels in the range of 11–38%. A similar range in the percentage of nuclear expression observed for ERß and PR and 14/55 (25%) and 7/55 (13%) carcinomas, respectively, showed <10% nuclear staining for both receptors. All ER{alpha} negative carcinomas were also ERß negative, whereas eight ERß negative carcinomas were considered ER{alpha} positive. By Spearman analysis, a statistically positive correlation in the level of expression was observed between ER{alpha} and ERß, and between each of the estrogen receptors and PR (Table III). ER{alpha} expression was statistically positively correlated with proliferation and cell cycle markers including PCNA, pRb and cdk4, as well as with the degree of nuclear pleomorphism and tumor grade. Neither ERß nor PR expression was statistically significantly correlated with PCNA or cdk4 expression but ERß was weakly correlated with pRb. PR but not ERß was statistically positively correlated with nuclear pleomorphism and tumor grade.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Quantitation of immunohistochemical staining for ER{alpha}, ERß, PR and PCNA in normal control rat mammary gland and PhIP-induced rat mammary gland carcinomas. For each antibody, the values represent the percentage of nuclear positive epithelial cells per 1000–1500 total epithelial cells counted. Each sample is represented individually by a dot. The overlaying of dots of similar values may have resulted in the appearance of fewer samples than indicated.

 

View this table:
[in this window]
[in a new window]
 
Table III. Correlation among steroid receptors and histological parameters in PhIP-induced rat mammary carcinomas

 
Colocalization of ER{alpha}, ERß, PR, and cell cycle and proliferation markers
The colocalization of steroid hormone receptors, cyclin D1 and PCNA was examined in normal mammary glands and carcinomas by confocal microscopy. Representative images are shown in Figure 3 (and Supplementary Figures 4 and 5). In the normal mammary gland, relatively few normal epithelial cells showed colocalization of steroid receptors with the cell cycle markers (Table IV). The percentage of epithelial cells that showed colocalization between receptors ranged from 0.17 to 0.42% in the normal mammary gland. The percentage of steroid positive cells colocalizing with either cyclin D1 or PCNA ranged from 0.21 to 0.61. In carcinomas, the percentage of epithelial cells showing colocalization between receptors ranged from 15.6 to 29.9% in carcinomas. Epithelial cells showing colocalized staining for ER{alpha} and PR were located mainly around the ductal structures (Figure 3). The PR positive cells were nearly always positive for ER{alpha}. Cells showing colocalization of ER{alpha} (or PR) with ERß were more sparsely distributed throughout the carcinomas (Figure 3 and Supplementary Figure 4). The percentage of ER{alpha}, ERß, and PR expressing cells that colocalized with cyclin D1 and PCNA ranged from 16.3 to 37.0%.



View larger version (131K):
[in this window]
[in a new window]
 
Fig. 3. Representative confocal microscopy images showing nuclear colocalization of ER{alpha} with PR, ERß, cyclin D1 and PCNA in PhIP-induced rat mammary gland carcinomas. The single color of red or green stained nuclei represents one antibody. The overlay of the red and green color produces a yellow color that represents the colocalized staining of the ER{alpha} to another protein (third column). Magnification, 630x.

 

View this table:
[in this window]
[in a new window]
 
Table IV. Colocalization of steroid receptors and proliferation markers

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 
This study examined the expression of steroid hormone receptors ER{alpha}, ERß and PR in PhIP-induced rat mammary gland carcinomas. The mRNA and protein expressions of all three receptors were upregulated in carcinomas in comparison with the normal rat mammary gland. The majority of PhIP-induced rat mammary gland cancers were positive for ER{alpha} by immunostaining. Only 6 of 55 carcinomas showed levels of ER{alpha} expression at or below the level found in normal mammary gland and could be regarded as ER negative. The predominance of ER{alpha} positive over ER{alpha} negative carcinomas is consistent with the studies of other chemically induced rat mammary gland cancers (59). Across the bank of PhIP-induced carcinomas, the level of nuclear expression of ER{alpha} and PR were statistically associated; carcinomas with higher ER{alpha} tended to show higher PR. The expression of ER{alpha} and PR was also found to be colocalized in ~30% of epithelial cells in the carcinomas. The correlation between ER{alpha} and PR expression fits the widely accepted paradigm that PR is a downstream target of ER{alpha} activation (29). The results of the study indicate that the overwhelming majority of PhIP-induced rat mammary gland cancers possess an active ER{alpha}.

In rat mammary gland carcinomas, the level of expression of ER{alpha} was positively correlated by Spearman analysis with proliferation as assessed by PCNA, phosphorylated pRb, and cdk4 expression. Furthermore, there was a positive correlation between ER{alpha} expression and nuclear pleomorphism and between ER{alpha} and histologic grade. Our findings in rats are consistent with previous studies on the effect of soy diets on DMBA-induced rat mammary gland cancers that reported an association between ER{alpha} expression, PCNA and differentiation grade (6,7). In humans, ER{alpha} negative breast carcinomas are considered more proliferative and aggressive than ER{alpha} positive carcinomas (2932). Nevertheless, there are subclasses of human breast carcinomas that show an association between proliferation and ER{alpha} expression (3234). Rat mammary gland carcinomas in general are less aggressive than human breast cancers, tend to show a papillary or cribriform growth pattern, greater cellular differentiation and a low frequency of metastasis (2). The high percentage of ER{alpha} positive carcinomas in rats seems to be consistent with the lower grade appearance of these tumors in comparison with many human breast cancers.

The ERß is a second estrogen receptor with different ligand binding specificity and distinct functions from ER{alpha} (35,36). The ERß has been detected in human and rat mammary gland carcinomas as well as in normal mammary gland (5,8,3741). As observed previously with rat mammary gland carcinomas induced by hormones or DMBA (5,8), the immunohistochemical pattern of ERß expression in PhIP-induced carcinomas is different from ER{alpha}. The epithelial cells showing ERß expression are found more diffusely throughout the carcinoma while ER{alpha} positive cells are located primarily around the lumen in putative luminal epithelial cells. We found that ~21% of epithelial cells in PhIP-induced carcinomas showed nuclear colocalized expression of both ER{alpha} and ERß using confocal microscopy. This finding indicates that the expression of these receptors colocalizes in a minority of epithelial cells and supports the notion that the function of each receptor is largely unique.

The role that ERß might play in mammary gland carcinogenesis is not yet known with certainty. Several studies have suggested potentially opposing actions of ER{alpha} and ERß on the expression of genes involved in cell-cycle regulation and proliferation (4244). ERß may also possibly influence the expression of proliferation markers in human breast cancers (45). In our study, however, ERß did not appear to independently influence proliferation in carcinomas. The level of expression of ERß tended to parallel the expression of ER{alpha}. Furthermore, all ER{alpha} negative tumors were also ERß negative. Unlike ER{alpha}, ERß was not statistically associated with PCNA or cdk4 expression, and although ERß was positively associated with the levels of pRb by Spearman analysis, the association was weaker than the association between ER{alpha} and pRb. Furthermore, there was no statistical difference in PCNA or pRb expression between ER{alpha} positive/ERß positive and ER{alpha} positive/ERß negative carcinomas (Student's t-test, P > 0.05). The results suggest that ER{alpha} rather than ERß is the main estrogen receptor driving proliferation in PhIP-induced rat mammary gland carcinomas.

The majority of human breast cancers have been shown to be positive for both ER{alpha} and ERß and studies have suggested that ERß may be a favorable prognostic factor in human breast cancer (35,38,40,46,47). PhIP-induced rat mammary gland carcinomas appear to be similar to a percentage of human breast cancers in the coexpression of both estrogen receptors, and rat mammary gland carcinomas would be akin to human breast cancers with good prognosis. Further studies are clearly required to evaluate the impact of ERß expression on rat mammary gland carcinogenesis.

As observed in human breast cancer (11,12), we observed differences in nuclear colocalization of steroid receptors in normal rat mammary gland and carcinomas. While the percentage of epithelial cells showing nuclear colocalization of any three of the steroid hormone receptors and PCNA was low in normal mammary gland, the percentage of epithelial cells showing colocalization increased well over 50-fold in mammary gland carcinomas. Consistent with studies in human breast cancer, there appeared to be a paracrine to autocrine shift in the regulation of proliferation in PhIP-induced rat mammary gland carcinomas such that a higher percentage of epithelial cells now showed coexpression of steroid hormone receptors with PCNA. Our studies also showed an increased nuclear localization of the steroid hormone receptors with cyclin D1 during carcinogenesis. Estrogens and progesterone have been shown to promote persistent cyclin D1 activation during the G1 phase of the cell cycle (28,44). Cyclin D1 appears to be a focal point for the cell-cycle deregulation occurring in rat mammary gland carcinomas and there is mounting evidence for the central role of cyclin D1 specifically in PhIP-induced rat mammary gland cancers (23,27). For example, PhIP-induced rat mammary gland cancers show a high frequency of mutations in H-ras, a gene linked to the cell cycle via cyclin D1, and overexpression of Stat5a, a gene that transactivates the cyclin D1 promoter, as well as overexpression and amplification of cyclin D1 itself (22,25,27). Steroid hormone receptors may also partly mediate the aberrant proliferation in PhIP-induced rat mammary gland cancer via cyclin D1. Continued effort to clarify the molecular mechanisms of rat mammary gland cancer especially with regard to steroid hormone receptors expression is expected to provide further insight into factors involved in breast cancer development.


    Supplementary Material
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 
Supplementary material is available online at: http://www.carcin.oupjournals.org


    Acknowledgments
 
The authors thank Ms Susan Garfield and Mr Stephen Wincovitch, Confocal Microscopy Core Facility, Laboratory of Experimental Carcinogenesis, NCI, for assistance with confocal microscopy; and Dr Insa Schroeder, Cellular and Molecular Biology Section, Laboratory of Experimental Carcinogenesis, NCI, for help with quantitative real-time PCR. We further acknowledge NCI-CCR Fellows Editorial Board for editorial assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary Material
 References
 

  1. Nandi,S., Guzman,R.C. and Yang,J. (1995) Hormones and mammary carcinogenesis in mice, rats, and humans: a unifying hypothesis. Proc. Natl Acad. Sci. USA, 92, 3650–3657.[Abstract/Free Full Text]
  2. Russo,J., Gusterson,B.A., Rogers,A.E., Russo,I.H., Wellings,S.R. and van Zwieten,M.J. (1990) Comparative study of human and rat mammary tumorigenesis. Lab. Invest., 62, 244–278.[ISI][Medline]
  3. Welsch,C.W. (1985) Host factors affecting the growth of carcinogen-induced rat mammary carcinomas: a review and tribute to Charles Brenton Huggins. Cancer Res., 45, 3415–3443.[Abstract]
  4. Thompson,H.J., McGinley,J., Rothhammer,K. and Singh,M. (1998) Ovarian hormone dependence of pre-malignant and malignant mammary gland lesions induced in pre-pubertal rats by 1-methyl-1-nitrosourea. Carcinogenesis, 19, 383–386.[Abstract]
  5. Knott,K.K., McGinley,J.N., Lubet,R.A., Steele,V.E. and Thompson,H.J. (2001) Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea. Breast Cancer Res. Treat., 70, 171–183.[CrossRef][ISI][Medline]
  6. Gallo,D., Giacomelli,S., Cantelmo,F., Zannoni,G.F., Ferrandina,G., Fruscella,E., Riva,A., Morazzoni,P., Bombardelli,E., Mancuso,S. and Scambia,G. (2001) Chemoprevention of DMBA-induced mammary cancer in rats by dietary soy. Breast Cancer Res. Treat., 69, 153–164.[CrossRef][ISI][Medline]
  7. Gallo,D., Ferrandina,G., Giacomelli,S., Fruscella,E., Zannoni,G., Morazzoni,P., Riva,A., Bombardelli,E., Mancuso,S. and Scambia,G. (2002) Dietary soy modulation of biochemical parameters in DMBA-induced mammary tumors. Cancer Lett., 186, 43–48.[CrossRef][ISI][Medline]
  8. Cheung,S.Y., Yuen,M.T., Choi,H.L., Cheng,H.K., Huang,Y., Chen,S. and Chan,F.L. (2003) An expression study of hormone receptors in spontaneously developed, carcinogen-induced and hormone-induced mammary tumors in female Noble rats. Int. J. Oncol., 22, 1383–1395.[ISI][Medline]
  9. Allred,C.D., Allred,K.F., Ju,Y.H., Clausen,L.M., Doerge,D.R., Schantz,S.L., Korol,D.L., Wallig,M.A. and Helferich,W.G. (2004) Dietary genistein results in larger MNU-induced, estrogen-dependent mammary tumors following ovariectomy of Sprague–Dawley rats. Carcinogenesis, 25, 211–218.[Abstract/Free Full Text]
  10. Anderson,E. (2002) The role of oestrogen and progesterone receptors in human mammary development and tumorigenesis. Breast Cancer Res., 4, 197–201.[CrossRef][ISI][Medline]
  11. Clarke,R.B., Howell,A., Potten,C.S. and Anderson,E. (1997) Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res., 57, 4987–4991.[Abstract]
  12. Clarke,R.B. (2003) Steroid receptors and proliferation in the human breast. Steroids, 68, 789–794.[CrossRef][ISI][Medline]
  13. Russo,J., Ao,X., Grill,C. and Russo,I.H. (1999) Pattern of distribution of cells positive for estrogen receptor alpha and progesterone receptor in relation to proliferating cells in the mammary gland. Breast Cancer Res. Treat., 53, 217–227.[CrossRef][ISI][Medline]
  14. Thompson,E.W., Reich,R., Shima,T.B., Albini,A., Graf,J., Martin,G.R., Dickson,R.B. and Lippman,M.E. (1988) Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens. Cancer Res., 48, 6764–6768.[Abstract]
  15. Katzenellenbogen,B.S. and Frasor,J. (2004) Therapeutic targeting in the estrogen receptor hormonal pathway. Semin. Oncol., 31, 28–38.
  16. Layton,D.W., Bogen,K.T., Knize,M.G., Hatch,F.T., Johnson,V.M. and Felton,J.S. (1995) Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis, 16, 39–52.[Abstract]
  17. Sinha,R., Gustafson,D.R., Kulldorff,M., Wen,W.Q., Cerhan,J.R. and Zheng,W. (2000) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen in high-temperature-cooked meat, and breast cancer risk. J. Natl Cancer Inst., 92, 1352–1354.[Free Full Text]
  18. Snyderwine,E.G., Thorgeirsson,U.P., Venugopal,M. and Roberts-Thomson,S.J. (1998) Mammary gland carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Sprague–Dawley rats on high- and low-fat diets. Nutr. Cancer, 31, 160–167.[ISI][Medline]
  19. Ito,N., Hasegawa,R., Sano,M., Tamano,S., Esumi,H., Takayama,S. and Sugimura,T. (1991) A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Carcinogenesis, 12, 1503–1506.[Abstract]
  20. Ghoshal,A., Preisegger,K.H., Takayama,S., Thorgeirsson,S.S. and Snyderwine,E.G. (1994) Induction of mammary tumors in female Sprague–Dawley rats by the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and effect of dietary fat. Carcinogenesis, 15, 2429–2433.[Abstract]
  21. Lauber,S.N., Ali,S. and Gooderham,N.J. (2004) The cooked food derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is potently oestrogenic: a mechanistic basis for its tissue specific carcinogenicity. Carcinogenesis, 25, 2509–2517.[Abstract/Free Full Text]
  22. Yu,M. and Snyderwine,E.G. (2002) H-ras oncogene mutations during development of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland cancer. Carcinogenesis, 23, 2123–2128.[Abstract/Free Full Text]
  23. Shan,L., He,M., Yu,M., Qiu,C., Lee,N.H., Liu,E.T. and Snyderwine,E.G. (2002) cDNA microarray profiling of rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 23, 1561–1568.[Abstract/Free Full Text]
  24. Shan,L., Yu,M., Qiu,C. and Snyderwine,E.G. (2003) Id4 regulates mammary epithelial cell growth and differentiation and is overexpressed in rat mammary gland carcinomas. Am. J. Pathol., 163, 2495–2502.[Abstract/Free Full Text]
  25. Shan,L., Yu,M., Clarke,B. and Snyderwine,E.G. (2004) Possible role of Stat5a in rat mammary gland carcinogensis Breast Cancer Res. Treat., 88, 263–272.[CrossRef][ISI][Medline]
  26. Qiu,C., Yu,M., Shan,L. and Snyderwine,E.G. (2003) Allelic imbalance and altered expression of genes in chromosome 2q11-2q16 from rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Oncogene, 22, 1253–1260.[CrossRef][ISI][Medline]
  27. Qiu,C., Shan,L., Yu,M. and Snyderwine,E.G. (2003) Deregulation of the cyclin D1/Cdk4 retinoblastoma pathway in rat mammary gland carcinomas induced by the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Res., 63, 5674–5678.[Abstract/Free Full Text]
  28. Cicatiello,L., Addeo,R., Sasso,A., Altucci,L., Petrizzi,V.B., Borgo,R., Cancemi,M., Caporali,S., Caristi,S., Scafoglio,C., Teti,D., Bresciani,F., Perillo,B. and Weisz,A. (2004) Estrogens and progesterone promote persistent CCND1 gene activation during G1 by inducing transcriptional derepression via c-Jun/c-Fos/estrogen receptor (progesterone receptor) complex assembly to a distal regulatory element and recruitment of cyclin D1 to its own gene promoter. Mol. Cell Biol., 24, 7260–7274.[Abstract/Free Full Text]
  29. Saji,S., Omoto,Y., Shimizu,C., Warner,M., Hayashi,Y., Horiguchi,S., Watanabe,T., Hayashi,S., Gustafsson,J.A. and Toi,M. (2002) Expression of estrogen receptor (ER) (beta)cx protein in ER(alpha)-positive breast cancer: specific correlation with progesterone receptor. Cancer Res., 62, 4849–4853.[Abstract/Free Full Text]
  30. Silvestrini,R., Daidone,M.G., Bertuzzi,A. and Di Fronzo,G. (1984) Relationship between estrogen receptors and cellular proliferation. Recent Results Cancer Res., 91, 163–168.[ISI][Medline]
  31. Meyer,J.S., Rao,B.R., Stevens,S.C. and White,W.L. (1977) Low incidence of estrogen receptor in breast carcinomas with rapid rates of cellular replication. Cancer, 40, 2290–2298.[ISI][Medline]
  32. Bertuzzi,A., Daidone,M.G., Di Fronzo,G. and Silvestrini,R. (1981) Relationship among estrogen receptors, proliferative activity and menopausal status in breast cancer. Breast Cancer Res. Treat., 1, 253–262.[Medline]
  33. Paradiso,A., Lorusso,V., Tommasi,S., Schittulli,F., Maiello,E. and De Lena,M. (1988) Relevance of cell kinetics to hormonal response of receptor-positive advanced breast cancer. Breast Cancer Res. Treat., 11, 31–36.[ISI][Medline]
  34. Fanelli,M.A., Vargas-Roig,L.M., Gago,F.E., Tello,O., Lucero,D.A. and Ciocca,D.R. (1996) Estrogen receptors, progesterone receptors, and cell proliferation in human breast cancer. Breast Cancer Res. Treat., 37, 217–228.[ISI][Medline]
  35. Murphy,L., Cherlet,T., Lewis,A., Banu,Y. and Watson,P. (2003) New insights into estrogen receptor function in human breast cancer. Ann. Med., 35, 614–631.[CrossRef][ISI][Medline]
  36. Palmieri,C., Cheng,G.J., Saji,S., Zelada-Hedman,M., Warri,A., Weihua,Z., Van Noorden,S., Wahlstrom,T., Coombes,R.C., Warner,M. and Gustafsson,J.A. (2002) Estrogen receptor beta in breast cancer. Endocr. Relat. Cancer, 9, 1–13.[Abstract/Free Full Text]
  37. Dotzlaw,H., Leygue,E., Watson,P.H. and Murphy,L.C. (1997) Expression of estrogen receptor-beta in human breast tumors. J. Clin. Endocrinol. Metab., 82, 2371–2374.[Abstract/Free Full Text]
  38. Jarvinen,T.A., Pelto-Huikko,M., Holli,K. and Isola,J. (2000) Estrogen receptor beta is coexpressed with ERalpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am. J. Pathol., 156, 29–35.[Abstract/Free Full Text]
  39. Saji,S., Jensen,E.V., Nilsson,S., Rylander,T., Warner,M. and Gustafsson,J.A. (2000) Estrogen receptors alpha and beta in the rodent mammary gland. Proc. Natl Acad. Sci. USA, 97, 337–342.[Abstract/Free Full Text]
  40. Skliris,G.P., Carder,P.J., Lansdown,M.R. and Speirs,V. (2001) Immunohistochemical detection of ERbeta in breast cancer: towards more detailed receptor profiling? Br. J. Cancer, 84, 1095–1098.[CrossRef][ISI][Medline]
  41. Speirs,V., Parkes,A.T., Kerin,M.J., Walton,D.S., Carleton,P.J., Fox,J.N. and Atkin,S.L. (1999) Coexpression of estrogen receptor alpha and beta: poor prognostic factors in human breast cancer? Cancer Res., 59, 525–528.[Abstract/Free Full Text]
  42. Kass,L., Durando,M., Ramos,J.G., Varayoud,J., Powell,C.E., Luque,E.H. and Munoz-de-Toro,M. (2004) Association of increased estrogen receptor beta2 expression with parity-induced alterations in the rat mammary gland. J. Steroid Biochem. Mol. Biol., 91, 29–39.[CrossRef][ISI][Medline]
  43. Liu,M.M., Albanese,C., Anderson,C.M., Hilty,K., Webb,P., Uht,R.M., Price,R.H.,Jr, Pestell,R.G. and Kushner,P.J. (2002) Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression. J. Biol. Chem., 277, 24353–24360.[Abstract/Free Full Text]
  44. Strom,A., Hartman,J., Foster,J.S., Kietz,S., Wimalasena,J. and Gustafsson,J.A. (2004) Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc. Natl Acad. Sci. USA, 101, 1566–1571.[Abstract/Free Full Text]
  45. Jensen,E.V., Cheng,G., Palmieri,C., Saji,S., Makela,S., Van Noorden,S., Wahlstrom,T., Warner,M., Coombes,R.C. and Gustafsson,J.A. (2001) Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc. Natl Acad. Sci. USA, 98, 15197–15202.[Abstract/Free Full Text]
  46. Nakopoulou,L., Lazaris,A.C., Panayotopoulou,E.G., Giannopoulou,I., Givalos,N., Markaki,S. and Keramopoulos,A. (2004) The favourable prognostic value of oestrogen receptor beta immunohistochemical expression in breast cancer. J. Clin. Pathol., 57, 523–528.[Abstract/Free Full Text]
  47. Fuqua,S.A., Schiff,R., Parra,I., Moore,J.T., Mohsin,S.K., Osborne,C.K., Clark,G.M. and Allred,D.C. (2003) Estrogen receptor beta protein in human breast cancer: correlation with clinical tumor parameters. Cancer Res., 63, 2434–2439.[Abstract/Free Full Text]
Received October 25, 2004; revised December 13, 2004; accepted December 18, 2004.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
26/4/763    most recent
bgi013v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Qiu, C.
Articles by Snyderwine, E. G.
PubMed
PubMed Citation
Articles by Qiu, C.
Articles by Snyderwine, E. G.