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Carcinogenesis Advance Access originally published online on November 25, 2005
Carcinogenesis 2006 27(3):491-498; doi:10.1093/carcin/bgi278
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Carcinogenesis vol.27 no.3 © Oxford University Press 2005; all rights reserved.

Overexpression of cyclins D1 and D3 during estrogen-induced breast oncogenesis in female ACI rats

S.John Weroha, Sara Antonia Li, Ossama Tawfik 1 and Jonathan J. Li *

Hormonal Carcinogenesis Laboratory, Department of Pharmacology, Toxicology, and Therapeutics and 1 Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA

* To whom correspondence should be addressed at: Department of Pharmacology, Mail Stop 1018, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA Email: jli1{at}kumc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
A common feature of human breast oncogenesis is cell cycle deregulation. The expression of cyclins D1 and D3 was examined during estradiol-17ß (E2)-induced mammary tumorigenesis in female August Copenhagen Irish (ACI) rats. Low serum E2 levels (~60–120 pg/ml) were sufficient to induce mammary gland tumors (MGTs) that remarkably resemble human ductal breast cancer (BC) at the histopathologic and molecular levels. Western blot analysis of the E2-induced MGTs revealed a marked rise in cyclins D1 (24-fold), D3 (9-fold) and cdk4 (3-fold) expression compared with age-matched untreated controls. Small focal dysplasias with large, pale staining nuclei were commonly seen at 3–3.6 months, large focal dysplasias, including atypical ductal hyperplasia at 3.6–4.3 months, ductal carcinoma in-situ (DCISs) at 4.3–5.0 months, and 100% incidence of invasive ductal BC/frank tumors at 5–6 months were detected after E2 treatment. Immunohistochemical analysis of serial sections of focal dysplasias, DCISs and invasive ductal carcinomas showed overexpression of cyclins D1, D3, estrogen receptor-{alpha} (ER{alpha}) and progesterone receptor (PR). However, cyclin D3 expression, unlike D1, was confined essentially to early pre-malignant lesions (focal dysplasias and DCISs) and primary MGTs with <1–5% of resting and normal hyperplastic breast cells staining positive. The kinase activity for cyclins D1 and D3, using retinoblastoma (Rb) as a substrate, in E2-induced MGTs and their binding to cdk4 was significantly elevated. Semi-quantitative reverse transcriptase PCR analysis of the E2-induced MGTs exhibited increased expression of cyclins D1 (2.9-fold) and D3 (1.4-fold) mRNA, indicating that their elevated protein expression was due in part to an increase in mRNA transcription. However, when analyzed by quantitative real-time Q-PCR, these genes were not amplified. These data indicate that in female ACI rat mammary glands, E2-induced pre-malignant lesions differentially and selectively express cyclins D1 and D3, thus contributing to a distinct growth advantage of these pre-neoplasias relative to E2-elicited normal hyperplasia.

Abbreviations: ACI, August Copenhagen Irish; ADH, atypical ductal hyperplasia; BC, breast cancer; Cdks, cyclin-dependent kinases; DCIS, ductal carcinoma in-situ; E2, estradiol-17ß; ER{alpha}, estrogen receptor alpha; H&E, hematoxylin and eosin; MGT, mammary gland tumor; PCR, polymerase chain reaction; PR, progesterone receptor; Q-PCR, quantitative real-time polymerase chain reaction; Rb, retinoblastoma; RT–PCR, reverse transcriptase PCR; SE, standard error


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Breast cancer (BC) is the most prevalent cancer afflicting women in developed countries with sporadic (non-familial) BC comprising >90% of all cases (1). One million new cases are reported worldwide each year (2). Based on epidemiologic and animal studies, it is evident that estrogen, both endogenous and exogenous, is the major risk factor for this disease (2–4). While estrogen clearly plays an important role in BC growth and progression, less is known about its involvement in BC causation.

Mammary gland tumors (MGTs) induced in female August Copenhagen Irish (ACI) rats by estradiol-17ß (E2), at low serum E2 levels (5), have emerged as arguably the most germane animal model for studying human sporadic BC since it shares many salient histopathologic and molecular features, both in early pre-malignant lesions and in primary tumors (6,7). For example, both human and the E2-induced murine breast neoplasms and their ductal carcinoma in-situ (DCISs) exhibit high frequencies of c-myc/Myc and Aurora A (AurA) overexpression, centrosome amplification (6), chromosomal instability, c-myc amplification and aneuploidy (6,7) presumably via deregulation of specific cell cycle entities. Aneuploidy, a common feature of solid tumors, is the most defining characteristic of invasive ductal BC, both in human and E2-induced ACI rat breast (65–92%) (7–9).

In human BC, MYC is overexpressed, and subsequently activates downstream cell cycle regulatory targets, including cyclins and their respective cyclin-dependent kinases (cdks) (10,11). Three isoforms, D1, D2 and D3, comprise the cyclin D family, each of which binds and activates either cdk 4 or 6 (12–14). In human breast tumors, cyclin D2 also binds cdk2 (15). The D cyclins hyperphosphorylate retinoblastoma (Rb) protein, resulting in the release of E2F transcription factor, hence conferring a growth advantage to estrogen-susceptible breast cells when these proteins are overexpressed (16). One of the downstream targets of E2F-mediated transcription is cyclin E1 that, when bound to cdk2, maintains the phosphorylation of Rb. In addition, active cyclin D1-cdk4/6 complexes sequester p21 and p27, and therefore contribute to the sustained activation of the cyclin E1-cdk2 complexes (17). Aberrant expression of cyclin D1 has been well documented in human BC (18). Cyclin D1 mRNA and protein are frequently overexpressed in breast tumors but the gene is uncommonly amplified (19,20). Although less is known about cyclin D3 in BC, there is evidence that cyclins D1 and D3 have non-redundant functions (21,22) and are differentially expressed in breast and uterine cancers (23,24).

To assess whether cell cycle deregulation contributes to solely E2-induced ACI rat mammary oncogenesis, we have systematically analyzed D1 and D3 protein and mRNA expression levels in pre-malignant lesions during E2 oncogenesis and compared them with mammary glands from normal cycling rats, E2 elicited normal hyperplasia, and primary invasive ductal BCs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals and treatment
Intact female ACI rats were purchased from Harlan Sprague–Dawley Inc. (Indianapolis, IN) at 6–8 weeks of age. They were housed individually in an AAALAC-accredited facility under controlled temperature, humidity, 12 h light–dark cycles, and provided with Teklad Rodent Diet 8604, and tap water ad libitum. The animal studies were carried out in adherence to the guidelines established in the Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Resources, NIH, 1985). The rats were acclimated for at least 1 week prior to treatment, and then randomly distributed into four groups of E2-treated and corresponding equal number of age-matched untreated control groups with 6–10 animals per group. All rats in the treated groups received a single pellet containing either 20 mg cholesterol alone or 3 mg of E2 plus 17 mg of cholesterol (Hormone Pellet Press, Leawood, KS) implanted in the shoulder region for 3–6 months as described previously (6). The E2-treated groups were killed after 3, 4, 5 or 6 months (tumor bearing). Over the treatment period, the E2 serum levels were 123.5 ± 4.8 pg/ml (3 months) and 121.8 ± 3.0 pg/ml (6 months), similar to values reported previously (5). At the end of each individual treatment period, the rats were killed by decapitation, subjected to macroscopic examination, and the presence of MGTs—both number and location—was recorded. The abdominal-inguinal mammary glands and the MGTs, freed of connective and necrotic tissue, respectively, were quickly removed. Portions of these tissues were immediately frozen in liquid nitrogen and stored at –80°C for later analysis, while others were fixed in 10% buffered formalin, followed by a rapid paraffin-embedding process.

Immunohistochemistry
Sections (5–6 µm) were deparaffinized in xylene and rehydrated to water. Antigen retrieval was performed with Dako, Dako Corporation, Carpinteria, CA, target retrieval solution for 20–30 min. Non-specific staining was blocked with normal serum incubated overnight at 4°C with primary antibodies: cyclin D1 (SP4) and ER{alpha} (6F11) from Neomarkers (Fremont, CA), progesterone receptor (PR) (C19) from Santa Cruz Biotechnology (Santa Cruz, CA) and cyclin D3 (DCS22) from Biomeda (Foster City, CA). Appropriate secondary antibodies were incubated for 1 h at room temperature followed by 1 h with Vector Laboratories Elite ABC reagent (Burlingame, CA). Protein expression was evaluated by light microscopy after incubation with diamino-benzidine (DAB) for 10–15 min and counterstained with hematoxylin. Specificity of cyclins D1 and D3 expression was assessed by the absence of staining in the presence of their respective blocking peptide or in the absence of primary antibody, respectively. These studies and those related to the classification of the lesions were performed using the following groups: Group 1, untreated age-matched controls and Groups 2–5 were killed after 3, 4, 5 and 6 months of E2 treatment, respectively. To determine the frequency of ER{alpha}, PR, cyclins D1 and D3 positive cells in age-matched, untreated control mammary glands and E2-induced pre-malignant breast lesions, >200 cells were counted from one mammary gland of six individual rats at different E2-treatment periods. A minimum of 1200 cells were counted for each E2-treatment period.

Classification of lesions
For histopathological evaluation, mammary gland sections were prepared from paraffin-embedded blocks and stained with hematoxylin and eosin (H&E). The mammary gland lesions after E2 treatment were identified as follows: small focal dysplasia, commonly seen at 3–3.6 months, contained cells with large, light staining nuclei and prominent nucleoli with or without secondary lumens. Large dysplastic foci, observed between 3.6 and 4 months, were similar to the designation for human atypical ductal hyperplasia (ADH) (25) and were characterized by large, evenly spaced nuclei arranged in a regular pattern which occasionally also formed a rosette-like pattern around more evenly shaped secondary lumens. DCIS, also present after 4 months of E2 treatment, exhibited features described for ADH, but displayed more regular punched-out secondary lumens, with a cross sectional diameter of >2 mm. The majority of the DCISs seen were characterized as cribiform. Lower frequencies of comedo, papillary and solid-type DCISs were also observed. MGTs were recognized when atypical cells, similar to those observed in DCIS, were seen invading the surrounding stroma.

Western blot
Mammary glands were homogenized in lysis buffer containing 50 mM Tris–HCl, pH 7.4, 0.2 M NaCl, 2 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.5 mM Na3VO4, 20 mM tetra Na-pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM DTT. The supernatant fractions were collected and their protein content determined with BCA reagents (Pierce, Rockford, IL). Proteins were separated by gel electrophoresis on PAGEr Gold Precast Gels (Cambrex Bio Science Rockland, Rockland, ME) and transferred to a nitrocellulose membrane. Primary antibodies against ER{alpha} (6F11), cyclin D1, cdk2 (2B6) (Lab Vision Corporation, Fremont, CA), PR, cdk4 (C22) (Santa Cruz) or cyclin D3 were the same as those used for immunohistochemistry, and incubated overnight at 4°C. Appropriate secondary antibodies were incubated for 2 h and protein expression was visualized with ECL chemiluminescence (Amersham Biosciences, Piscataway, NJ). For these studies, age-matched untreated control rats and E2-treated for 4 and 6 months were used.

Immunoprecipitation and kinase assay of primary E2-induced mammary tumors
Proteins were extracted as described for western blotting. Aliquots, 500 µg of total protein, were briefly precleared with appropriate normal IgG and A/G-agarose (Santa Cruz). Protein extracts were then incubated for 2 h with 1 µg of the same antibodies used for western blot for cyclins D1, D3 and cdk4, followed by 2 h incubation with 30 µl A/G agarose slurry. Normal IgG was used as a negative control. Immunoprecipitated complexes were washed several times with phosphate-buffered saline and immediately used for Rb-kinase assays (Upstate USA, Charlottesville, VA) following the manufacturer's standard protocol. Briefly, immunoprecipitates were incubated for 30 min, at 30°C with assay dilution buffer [20 mM MOPS, 25 mM ß-glycerol phosphate, pH 7.2, 1 mM EGTA, 1 mM sodium Na3VO4 and 1 mM DTT], 10 µCi [{gamma}-32P]ATP (Amersham) diluted in Upstate Mg/ATP cocktail. Rb protein was then subjected to SDS gel electrophoresis, dried in a gel dryer, exposed to phosphor plate and visualized in a Molecular Dynamics Phosphor Imager (Amersham). For these studies, age-matched untreated control rats and E2-treated for 6 months (tumor tissue only) were used.

Reverse transcriptase PCR (RT–PCR)
All reagents for RT–PCR were purchased from Invitrogen, Carlsbad, CA. First strand synthesis was carried out following the manufacturer's protocol for Moloney-murine leukemia virus (M-MLV) reverse transcriptase with 5 µg of total RNA and 250 ng of random primer. cDNA was then amplified by recombinant Taq DNA polymerase and gene-specific primers for cyclin D1 (FWD 5-CGCCTTCCGTTTCTTACTTCA-3'; REV 5-AACTTCTCGGCAGTCAGGGGA-3'; 251 bp amplicon), cyclin D3 (FWD 5-TCCAGTGCGTGCAAAAGGA-3'; REV 5-CCGTCCGGGCCTTACCT-3'; 80 bp amplicon) or ß-actin (FWD 5-ATGGTGGGTATGGGTCAGAA-3'; REV 5-TCCATATCGTCCCAGTTGGT-3'; 119 bp amplicon) for 30 cycles (95°C/45 s, 60°C/45 s and 72°C/45 s). PCR amplicons were separated by 1–2% agarose gel electrophoresis and visualized with ethidium bromide. Densitometry was performed using a Bio-Rad (Hercules, CA) Gel Doc EQ.

DNA isolation and quantitative real-time PCR (Q-PCR) for gene amplification
DNA from mammary glands from age-matched, untreated, controls and from E2-treated for 4 months, and tumor tissue from rats treated for 6 months was extracted by the LiCl of Gemmell and Akiyama (26). Genomic DNA was precipitated with 2 vol of ethanol, washed with 70% ethanol, briefly air dried and resuspended in Tris–EDTA buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0). DNA integrity was evaluated by agarose gel electrophoresis. Aliquots of 50 ng genomic DNA from control mammary glands and MGTs were amplified using Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) and gene-specific primers for cyclin D1 (FWD 5-GCGAGCCATGCTTAAGACTGA-3'; REV 5-CTCCCTCTGCACGCACTTG-3'; 70 bp amplicon), cyclin D3 (FWD 5-GCCGCGAGGCTCCTACTT-3'; REV 5-CATCCAGTACGCCAGCATCTT-3'; 76 bp amplicon) or ß-actin (same as for RT–PCR) in a 7300 Real Time PCR System (Applied Biosystems, Foster City, CA). Myc gene amplification was used as a positive control (FWD 5-CAAGAGGTGCCATGTCTCTACTCA-3'; REV 5-CAGCTGGATAGTCCTTCCTTGTG-3'; 74 bp amplicon). The baseline was set automatically and the threshold Ct value was measured during the exponential phase of the amplification. Each assay was performed in triplicate.

Statistical analyses
One way ANOVA with Dunn or Tukey post-hoc tests were used for statistical evaluation. Values were expressed as the mean ± SE. Statistical significance was assumed when P < 0.05 was obtained.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Western blot analysis of cyclins D1, D3 and cdk4 protein expression during E2-induced mammary oncogenesis
Cyclins D1 and D3 protein expression was assessed in mammary glands from 4-month E2-treated, 6-month E2-induced primary MGTs, and age-matched untreated controls. After 4 months of E2 treatment, the protein expression of cyclin D1 rose 3-fold, and it was statistically significant compared with age-matched untreated cycling mammary glands (Figure 1). A significant 24.8-fold increase, however, was observed in primary MGTs. Cyclin D3 expression was essentially undetected after 4 months of E2 treatment, but significantly rose 9-fold in MGTs when compared with age-matched untreated mammary glands. A significant 3.1-fold elevation in the expression of cdk4, an associated kinase for cyclins D1 and D3, was also detected in MGTs when compared with the evidently substantial levels already present in untreated age-matched cycling mammary glands (Figure 1A).


Figure 1
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  		Fig. 1. Cyclins D1, D3 and cdk4 expression in mammary glands and MGTs. (A) Representative western blots of individual, age-matched untreated control mammary glands (C1, C2), 4-month E2-treated mammary glands (E4-1, E4-2) and bona fide MGTs (T1, T2) from 6-month E2-treated rats (n = 6). (B) Protein expression was measured using a Molecular Dynamics PDSI with ImageQuant software and graphed as fold increase. After 4 months of E2 treatment and in E2-induced MGTs, cyclin D1 expression increased 3- and 24.8-fold, respectively; cyclin D3 expression did not change at this time period, but increased 9-fold in MGTs; cdk4 expression increased 1.8- and 3-fold, respectively. ß-actin was used as a loading control. Data represent the mean ± SE. Statistical significance was determined by one way ANOVA with a Tukey or Dunn post hoc test, *, P < 0.05 versus control.

 
Cyclin D1/D3 expression in age-matched untreated mammary glands and primary breast tumors
Untreated age-matched mammary glands from virgin cycling rats exhibited a limited number of lobular units containing small terminal ducts surrounded by occasional ductules or acini. Cyclin D1 protein expression was detected along the cells in the inner luminal epithelial layer (Figure 2A), but cyclin D3 expression was uncommonly seen in these cells (Figure 2B). E2-induced MGTs were characterized by cells uniformly showing nuclear enlargement, hyperchromasia, pleomorphism and prominent nucleoli. Both cyclin D1 and cyclin D3 were consistently overexpressed in E2-induced primary MGTs (Figure 2C and D) relative to age-matched untreated mammary glands.


Figure 2
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  		Fig. 2. IHC staining of cyclins D1 and D3 protein in age-matched control untreated mammary glands and MGTs. Six to nine untreated mammary glands and MGTs from 6-month E2-treated female ACI rats were analyzed by IHC. (A) Strong nuclear cyclin D1 staining was detected in epithelial cells of untreated control mammary glands and (C) MGTs. The surrounding stroma was largely negative. (B) Epithelial cells in untreated control mammary glands exhibited a low frequency of cyclin D3 staining while (D) epithelial cells of MGTs exhibited marked positive nuclear staining.

 
ER{alpha}, PR and cyclin D1/D3 expression in pre-malignant mammary lesions
Female ACI rats, treated with E2 for 3–6 months showed marked lobular–alveolar hyperplasia, consisting of acini lined by epithelial cells with small nuclei and some lactational changes (Figure 3A). Focal dysplasias consisted of cells with large pale nuclei compared with those in adjacent hyperplasias (Figure 3F). The most common histopathologic type of DCIS seen was cribiform (Figure 3K). A few of the cell nuclei were giant in size and consistently found in focal dysplasias, DCISs and primary MGTs during different periods of E2 treatment (Figure 3O, arrows).


Figure 3
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  		Fig. 3. Representative H&E and IHC of E2-induced rat mammary pre-malignant lesions. Mammary gland serial sections were prepared from age-matched untreated controls and 4-month E2-treated rats. Each serial section from normal, hyperplasia, focal dysplasia, and DCIS were stained sequentially for H&E (A, F, K), ER{alpha} (B, G, L), PR (C, H, M), cyclin D1 (D, I, N) and cyclin D3 (E, J, O) (20x, 40x inset). Giant-size nuclei were often seen in DCIS (arrows) and dysplasia (not shown). Distinct ER{alpha}, PR, cyclins D1 and D3 positive stained cells were detected in dysplastic foci and DCIS. Cyclin D3 positive cells were only present in dysplastic foci and DCIS. Some weak cyclin D3 stained cells were observed in untreated age-matched control mammary glands (data not shown) undergoing normal hyperplasia.

 
Serial sections of E2-induced hyperplasia, focal dysplasia and DCIS were examined for the expression of ER{alpha}, PR, cyclins D1 and D3 (Figure 3). A moderate percent of cells positively stained for ER{alpha} and PR were detected in normal hyperplasia adjacent to MGTs, 21.4 ± 2.5 and 17.4 ± 0.8%, respectively (Figure 3B and C and Figure 4). These levels of expression, however, did not significantly differ from those detected in age-matched untreated cycling mammary glands. Strong nuclear ER{alpha} and PR positive stained cells were prevalent in focal dysplasias, 76.0 ± 5 and 96.0 ± 0.7%, and in DCISs, 74.6 ± 8 and 79.8 ± 7%, respectively (Figure 3G, H, L and M and Figure 4).


Figure 4
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  		Fig. 4. Frequency of ER{alpha}, PR, cyclins D1 and D3 positive cells in age-matched, untreated control mammary glands and E2-induced pre-malignant breast lesions. The frequency of positive cells was analyzed in formalin-fixed, paraffin-embedded mammary gland sections. A minimum of 200 cells from each mammary gland of six individual rats (1200 total) were counted from six hyperplastic regions and pre-neoplastic lesions. The frequency of positive cells for ER{alpha}, PR, cyclins D1 (D1) and D3 (D3) in untreated age-matched controls were 33 ± 2, 28 ± 2, 13 ± 1 and 0.9 ± 0.2%, respectively; normal hyperplasia was 21.4 ± 2.5, 17.4 ± 0.8, 55 ± 4 and 5 ± 1%, respectively; in large dysplasia, 76.0 ± 5, 96.0 ± 0.7, 83.0 ± 2 and 48.0 ± 5%, respectively; and in DCIS, 74.6 ± 8, 79.8 ± 7, 63.0 ± 5 and 57.0 ± 5%, respectively. Data represent the mean ± SE.

 
In subsequent serial sections of focal dysplasias and DCISs, the proportion of positive stained nuclear cyclins D1 and D3 cells was 83.0 ± 2 and 48.0 ± 5%, and 63.0 ± 5 and 57.0 ± 6%, respectively (Figure 3I, J, N and O and Figure 4). While E2-induced hyperplasias also showed high levels of cyclin D1 expression, 55.0 ± 4 (Figures 3D and 4), remarkably few cells expressed cyclin D3, 5.0 ± 1% (Figures 3E and 4).

Cyclins D1 and D3 immunoprecipation and kinase assay
To determine whether the elevated protein expression of cyclins D1 and D3 was functionally active in MGTs, co-immunoprecipitation and kinase assays were performed using Rb as a substrate. Since a prerequisite to the formation of a functional cyclin/cdk holoenzyme is the association of a cyclin with its cdk binding partner, specific antibodies to cyclins D1, D3 and cdk4 were used to immunoprecipitate their respective proteins, and later subjected to western blot analysis with antibodies to cyclin D1 or D3. A direct association between cdk4 and cyclins D1 or D3 was observed (Figure 5A). As expected, neither cyclin D1 nor D3 formed complexes with cdk2 (Figure 5A). To further assess the functionality of cyclin/cdk complexes, immunoprecipitated cyclins D1 and D3 proteins were subjected to kinase assays utilizing [{gamma}-32P]ATP and Rb substrate (Figure 5B). Both cyclins D1– and D3–cdk4 complexes showed markedly elevated kinase activity in E2-induced MGTs relative to their corresponding age-matched untreated mammary tissue.


Figure 5
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  		Fig. 5. In vivo complex formation between cyclin D1 or D3 with cdk4 and in vitro kinase activity. (A) Cyclins D1, D3, cdk2 and cdk4 were immunoprecipitated from bona fide E2-induced MGTs from 6-month E2-treated rats and subjected to western blot analysis for the expression of either cyclin D1 or D3. Cyclins D1 and D3 co-immunoprecipitated with cdk4, but did not with cdk2 used as negative control. (B) Cyclins D1 and D3 immunoprecipitates were subjected to in-vitro kinase assays. The immunoprecipitates were incubated with [{gamma}-32P]ATP and Rb substrate. The [32P]-labeled Rb was analyzed by 15% SDS–PAGE electrophoresis and autoradiography. Kinase activities were higher in mammary tumors (T) than in age-matched untreated controls (C).

 
Cyclins D1 and D3 mRNA expression
Cyclins D1 and D3 mRNA expression was determined by semi-quantitative RT–PCR analysis of mammary gland tissues from female ACI rats treated with E2 for 4 months and primary MGTs (Figure 6). Compared with age-matched untreated mammary glands, a significant rise in cyclin D1 mRNA was detected after 4 months and a further rise, 2.9-fold, in MGTs, compared with age-matched untreated cycling mammary glands. In primary MGTs, cyclin D3 mRNA levels also exhibited a significant, but more modest, 1.4-fold, increase when compared with age-matched untreated mammary glands.


Figure 6
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  		Fig. 6. Expression of cyclins D1 and D3 mRNA by semi-quantitative RT–PCR. Top, A representative gel showing amplified cDNA of cyclins D1 (251 bp), D3 (80 bp) and ß-actin (119 bp) transcripts subjected to 1–2% agarose gel electrophoresis in untreated age-matched control mammary glands (C1-2), 4-month E2-treated mammary glands (E4,1–2), and bona fide E2-induced MGTs from 6-month E2-treated rats (T1-2). Bottom, Densitometric values for cyclins D1 and D3 were normalized against ß-actin. Fold increases are represented as the mean ± SE (n = 6). Statistical significance was determined by one way ANOVA with a Tukey post-hoc-test, *, P < 0.05 or **, P < 0.001 versus control.

 
Q-PCR analysis of cyclins D1 and D3 genes
To assess gene copy number, Q-PCR analysis of cyclins D1 and D3 genomic DNA was performed on 10 age-matched untreated mammary glands and an equal number of primary MGTs. The threshold for amplification was set at a 2-fold increase relative to the gene copy number detected in the age-matched untreated group. The MGTs examined did not exhibit an amplification of either cyclin D1 (0.80 ± 0.5) or D3 (0.73 ± 0.05) gene. The amplification of c-myc was used as a positive control as previously reported (7).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Cell cycle deregulation, particularly overexpressed cyclin D, has been implicated in various human cancers, including invasive ductal BC (27). Cyclin D1 protein is overexpressed in >50% of human BCs (28) and has been frequently detected early in breast pre-neoplasias (29). The 11q13 amplicon, in which cyclin D1 resides, has been shown to be amplified in a modest number of primary human BCs (30) and in several human BC cell lines (31–33). Much less is known about cyclin D3 in human BC; however, its overexpression in high grade human breast tumors has been reported (34). MGTs develop in transgenic mice overexpressing cyclin D1 (35), while cyclin D3 overexpression leads to squamous cell carcinomas in mouse mammary glands (36). Taken together, these findings support an oncogenic role for cyclins D1 and D3, but their effects may involve different pathways.

It is evident that the elevated levels of cyclin D1 observed in mammary glands during E2-induced oncogenesis in the ACI rat may provide a distinct growth advantage to cells undergoing focal dysplasia, DCIS and MGT development. A number of studies have reported that normal resting human breast, ductal hyperplasia and ADH exhibit very weak levels of cyclin D1 expression (37,38). Similarly, in the ACI rat, the normal cycling mammary gland expresses low levels of cyclin D1, but in E2-elicited hyperplasia higher levels of this cyclin were detected. This is likely due to the sustained E2 serum levels in these treated rats. In relation to cyclin D1 expression, moderate to strong positive nuclear staining has been reported in 39.4% of the cells contained in human ADH/dysplasia (29), and in 59% of the cells within human DCISs and invasive ductal BCs (33,37). Cyclin D3 expression has been detected in 10% of human BCs (39); however, its expression in early pre-malignant stages has not been assessed. The overexpression of both cyclins D1 and D3 in early stages of E2-induced breast oncogenesis in ACI rats, reported herein, suggests their involvement during incipient stages of MGT development. These data are consistent with a report demonstrating that of the total number of specimens analyzed, 75% showed elevated cyclin D1 mRNA in low grade DCISs and 87% in comedo-type DCISs (37). Unlike ER{alpha}, PR and cyclin D1, the nuclear expression of cyclin D3 in normal hyperplasia was weak and detected in only few hyperplastic cells. With continued E2 treatment, cyclin D3 overexpression was confined largely to focal dysplasias, DCISs and primary MGTs, indicative of its selective expression, likely driven by the sustained E2 exposure. Consequently, the early overexpression of cyclin D3 may also have a distinctive role in E2-induced rat mammary oncogenesis. Interestingly, hepatocytes and human breast epithelial cells, designed to overexpress cyclin D1, resulted in centrosome amplification (40). However, it is unclear whether this is a direct or indirect effect mediated by a downstream alteration occurring as a result of continued cell cycle progression.

In the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat BC model, the resulting MGTs did not overexpress cyclin D3 (41). This illustrates differences between chemical carcinogen- versus estrogen-induced murine mammary oncogenesis. Indeed, differences have also been reported in relation to aneuploid frequencies in MGTs induced by synthetic and environmental chemical carcinogens (<15%) and solely estrogen-induced MGTs (>84%) (7); the MGTs generated by chemical carcinogens were highly diploid, whereas E2-induced MGTs were found to be highly aneuploid, a feature that is characteristic of both human DCISs and invasive ductal BCs (40).

Compared with a moderate rise in cyclin D1 mRNA, only a modest increase in mRNA cyclin D3 expression was detected. This finding suggests that the elevated levels of cyclin D3 in particular may be in part due to post-translational stabilization of the protein. Similar results have been reported for cyclin D3 in a human uterine sarcoma cell line (42), as well as in primary human BCs (34). While the rise in cdk4 protein does not coincide with the much greater elevation in cyclin D1/D3 protein in E2-induced MGTs, there is growing evidence that the D-type cyclins have cdk-independent functions (43,44). For instance, cyclin D1 forms physical associations with >30 transcription factors/co-regulators, such as DMP1 (cyclin D1-interacting myb-like protein), C/EBPß, and STAT3 (signal transducers and activation of transcription), as well as several nuclear receptors, including the ER{alpha} (45,46). In this instance, cyclin D1 binds with p300/CBP-associated factor and potentiates activation of ER{alpha} (47).

Our observation that cyclins D1 and D3 genes were not amplified in E2-induced ACI rat MGTs is consistent with either a relatively low or undetected amplification levels of these genes (0–16%) in human BC, frequently associated with an elevated level of ER{alpha} expression (20). However, cyclin D1 gene amplification may not be required to drive the cyclin D1 mRNA and protein overexpression in female ACI rat mammary glands, since persistent low levels of estrogen exposure may be a sufficient stimulus. The studies detailed herein show, for the first time, that the deregulation of cyclins D1 and D3 expression at the mRNA and protein levels are clearly incipient events during solely E2-induced ACI rat mammary oncogenesis, thus suggesting they have an important role in the growth of pre-malignant breast cells leading to tumor development.


   Acknowledgments
 
Our special thanks to Ms Tandria Price, for her excellent secretarial assistance in the preparation of this manuscript. This investigation was supported by Grant CA 58030 (to S.A.L.) from the National Cancer Institute.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 6, 2005; revised November 14, 2005; accepted November 20, 2005.


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E. W. Blank, P.-Y. Wong, R. Lakshmanaswamy, R. Guzman, and S. Nandi
Both ovarian hormones estrogen and progesterone are necessary for hormonal mammary carcinogenesis in ovariectomized ACI rats
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