Skip Navigation


Carcinogenesis Advance Access originally published online on October 4, 2007
Carcinogenesis 2008 29(1):177-185; doi:10.1093/carcin/bgm224
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/1/177    most recent
bgm224v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ren, X.
Right arrow Articles by Zarbl, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ren, X.
Right arrow Articles by Zarbl, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Comparative genomics of susceptibility to mammary carcinogenesis among inbred rat strains: role of reduced prolactin signaling in resistance of the Copenhagen strain

Xuefeng Ren1,2,{dagger}, Xun Zhang1,2,{dagger}, Andrea S. Kim1,{dagger}, Andrei M. Mikheev1, Mingzhu Fang4, Robert C. Sullivan1, Roger E. Bumgarner5 and Helmut Zarbl1,2,3,4,*

1 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
2 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105, USA
3 Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
4 Environmental and Occupational Health Sciences Institute, University of Medicine and Dentistry of New Jersey and Rutgers University, Piscataway, NJ 08854, USA
5 Department of Microbiology and Center for Expression Arrays, University of Washington, Seattle, WA 98195, USA

* To whom correspondence should be addressed. Tel: +1 732 445 0350; Fax: +1 732 445 0131; Email: zarbl{at}eohsi.rutgers.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
To elucidate the molecular basis for differential susceptibilities to mammary carcinogenesis, we compared the transcriptomes of normal mammary glands from pubescent female rats of the resistant Copenhagen (Cop) strain with those of the susceptible Fischer 344 (F344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N), Wistar–Furth (WF) strains and F1 (Cop x F344) progeny (F1). Gene expression profiles in mammary tissue within each rat strain were remarkably similar, indicating that gene expression was determined by genetic background. We next identified the subset of genes that were differentially expressed in all susceptible strains relative to the resistant Cop strain. Among these, the messenger RNAs encoding prolactin (Prl) and its cell surface receptor were significantly elevated in all susceptible strains. The expression levels of several Prl-regulated genes were also significantly elevated, indicating the presence of increased Prl signaling in mammary glands of all susceptible strains. Pathway analysis of gene expression profiles further identified the Prl-activated Jak/STAT-signaling pathway among the pathways that most distinguished sensitive rat strains from the resistant Cop rat. To test the hypothesis that reduced levels of the Prl signaling in mammary tissue partially contributed to the genetic resistance to mammary carcinogenesis, we used the neuroleptic drug, perphenazine, to transiently elevate serum Prl levels in the Cop strain. Whereas Cop rats are resistant to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis, ~5% of pubescent Cop females treated with perphenazine and NMU exposure developed mammary adenocarcinomas with latencies comparable with those of sensitive strains. Together, these finding indicated that in the rat, the molecular mechanisms underlying genetic susceptibility to mammary carcinogenesis include de-regulation of Prl signaling.

Abbreviations: ACI, August x Copenhagen Irish; Buf/N, Buffalo/N; Cop, Copenhagen; F344, Fischer 344; NMU, N-nitroso-N-methylurea; PCR, polymerase chain reaction; Prl, prolactin; PrlR, prolactin receptor; STAT, signal transducers and activators of transcription; TEB, terminal end bud; WF, Wistar–Furth


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The rat mammary tumor model has been widely used to study the biology, etiology, genetics and chemoprevention of human breast cancer. When compared with the mouse, mammary carcinogenesis in the rat more closely resembles human breast cancer with respect to histopathology, responsiveness to ovarian hormones, protective effects of full-term pregnancy and the absence of a viral etiology (1,2). Different laboratory rat strains vary widely with respect to their susceptibility to spontaneous and carcinogen-induced mammary carcinogenesis. Whereas feral rats and the Copenhagen (Cop) and Wistar–Kyoto laboratory strains are almost completely resistant to mammary carcinogenesis, most other inbred rat strains are highly susceptible to spontaneous and carcinogen-induced mammary tumors. Although there is considerable variability with respect to tumor incidence and latency among susceptible strains (3–6), the susceptibility to chemically induced carcinogenesis is largely concordant with their susceptibility to spontaneous mammary tumorigenesis. These differences provide a prime facie case for the importance of the genetic background of the host in determining susceptibility to mammary tumor. Collectively, genetic studies performed to date indicate that susceptibility in the rat is a polygenic trait, with >20 quantitative trait loci implicated in various genetic crosses (2,4,7). However, none of the tumor suppressor genes comprising these quantitative trait loci have been identified to date.

As an alternative approach to elucidate the molecular basis for differential genetic susceptibility to mammary carcinogenesis, we used DNA microarrays to compare expression profiles in normal, untreated mammary glands from pubescent female rats of the resistant Cop strain with those of the susceptible Fischer 344 (F344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N) and Wistar–Furth (WF) strains and the partially susceptible F1 (Cop x F344) hybrid progeny. We hypothesized that the genes that are differentially regulated between the resistant and susceptible strains will be a subset of genes comprising biochemical networks that contribute to susceptibility. Among the genes that were differentially expressed in mammary cells of all susceptible strains relative to the resistant Cop strain, we identified the genes encoding the peptide hormone, prolactin (Prl), the prolactin receptor (PrlR) and several genes shown previously to be regulated by Prl signaling.

Prl is a peptide hormone synthesized by the anterior pituitary gland that plays an essential role in the proliferation and differentiation of normal mammary epithelium and in stimulating lactation (8), and increased Prl signaling has been associated with mammary carcinogenesis and tumor progression in both rodents and humans (9–13). However, the importance of Prl in the pathogenesis of mammary tumors remains controversial. For example, while a recent epidemiological study found a strong positive association between Prl levels and breast cancer risk in post-menopausal women (12), previous studies failed to detect a clear correlation between breast cancer risk and plasma Prl level (14,15). Moreover, recent observations indicate that both Prl and PrlR are synthesized and expressed in mammary epithelial cells of human (16,17), rat (18–20), goat and sheep (21). These findings suggest that Prl might act as an autocrine or a paracrine factor within the breast and could account for the inconsistent association of serum Prl levels with breast cancer risk (14). In rodents, numerous studies have implicated Prl in increased susceptibility to chemical carcinogens in the mammary glands (9,10). To test the hypothesis that reduced local expression of Prl and PrlR in mammary glands of rats relative to the susceptible strains can contribute to the decreased susceptibility of the resistant Cop strain, we treated pubescent female Cop rats with the neuroleptic drug, perphenazine, which induces the massive release of Prl from the pituitary gland by inhibiting dopamine receptors (22). As showing previously for susceptible strains, the resulting increase in Prl signaling induced rapid growth and differentiation of Cop mammary glands. More importantly, the resulting transient increase in Prl signaling within mammary cells also increased the susceptibility of the highly resistant Cop strain to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis. Taken together, these results support the hypothesis that the levels of Prl signaling in the mammary glands are not only regulated at the genetic level but also contribute at least in part to the mechanism of genetic susceptibility.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Animals and mammary gland samples
Before conducting any animal experiments, all protocols were reviewed and received the approval of the Institutional Animal Care and Use Committee. All experiments were performed in Fred Hutchinson Cancer Research Center vivarium, which is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care. Female ACI and F344 were purchased from Harlan Sprague Dawley (Indianapolis, IN); female Cop, Buf/N and WF were purchased from Charles River Laboratories (Wilmington, MA). Female F1 hybrids of Cop and F344 were generated by mating male Cop (Harlan Sprague Dawley) with female F344 between the ages of 20 and 24 weeks. All animals were provided a standard diet of rat chow and acidified water ad libitum and housed in an approved facility with climate control and a 12 h light/12 h dark cycle. Rats were euthanized individually in a CO2 chamber between 50 and 56 days of age. All mammary glands from the right side were then dissected and were immediately submerged in RNAlater RNA Stabilization Reagent (QIAGEN, Valencia, CA).

Mammary gland whole-mount analysis
The whole-mount procedure used was adapted from the method of Russo et al. (23). Briefly, rats were euthanized, and abdominal mammary glands were quickly excised and affixed to SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). The tissues on the slides were fixed in 10% buffered formalin, defatted in acetone for 2–4 days and hydrated through an ethanol series. Finally, the glands were stained in carmine alum or 0.025% toludine blue overnight. On the following day, the glands were dehydrated through an ethanol series and stored in xylenes overnight. Slides were then sealed in plastic bags containing methyl salicylate. Numbers of terminal end buds (TEBs) from untreated 55-day-old F344 and Cop rats were calculated.

RNA isolation, labeling and DNA microarray analysis
Total RNA was isolated from the mammary glands of each rat using RNeasy Maxi Kits (QIAGEN, Valencia, CA). A 20 ml volume of Buffer RLT and two columns were used per 1 g of wet tissue. Following ethanol precipitation, the RNA was re-suspended in deionized water, precipitated with 4 M LiCl overnight at 4°C and washed three times with 80% ethanol before re-suspension in water. Using 10 µg of each RNA sample as a template, labeled cRNAs was generated as described in the GeneChip Expression Analysis Manual (Affymetrix, Santa Clara, CA). Each cRNA sample representing a single animal was then hybridized to a single microarray and processed as described in the Affymetrix manual. Thirty GeneChip Rat Genome U34A Arrays (Affymetrix, Santa Clara, CA) were used to analyze the 30 RNA samples, including five samples per rat strain (five samples x five strains) and five for the F1 hybrids. Prior to the analyses, the quality of each of the labeled cRNA sample was assessed by hybridizing each to a Test 3 Array (Affymetrix). In all 30 samples, the ratios of 5'–3' ends were estimated to be >0.69 for glyceraldehyde-3-phosphate dehydrogenase and >0.64 for β-actin.

Image and data analysis
Using an Affymetix scanner, raw intensity data for each feature on the array were collected and scaled using a target intensity of 500 to account for differences in the global chip intensity using Affymetrix Microarray Analysis 5.0 (MAS 5.0) software. After the set of 59 probe sets comprising the Affymetrix Chip index used for quality control was removed, the remaining data were normalized using GC Robust Multi-Array Average statistical method and transformed using the logarithm base 2 using the Genetraffic statistical analysis software package 3.0 (Iobion Informatics LLC, La Jolla, CA). We next applied filtering and statistical analysis constraints to the expression data to exclude those genes that were not expressed above background levels. The log of the normalized expression data was analyzed using the BRB ArrayTools statistical analysis software package (Biometric Research Branch, National Cancer Institute, http://linus.nci.nih.gov/brb/). We identified genes that were differentially expressed among the two classes by using a multivariate permutation test (24,25). We used the multivariate permutation test to provide 90% confidence that the false discovery rate was <10%. The false discovery rate is the proportion of the list of genes claimed to be differentially expressed that are probably to be false positives. The test statistics used are random variance t-statistics for each gene (26). Although t-statistics were used, the multivariate permutation test is non-parametric and does not require the assumption of Gaussian distributions. To analyze further the reliability of finding from ArrayTool software, the same data set was re-analyzed using analysis of variance test from the TIGR Multi Experiment Viewer (The Institute for Genomic Research, Rockville, MD, http://www.tigr.org/software/). Genes were considered statistically significant if their P value was < 0.01 in both the analyses. In addition, the data were analyzed using unsupervised hierarchical clustering (Michael Eisen Stanford's Cluster and TreeView software package) with the centered correlation as the similarity metric and by average linkage clustering.

Pathway analysis was performed on gene expression profiles obtained from mammary glands of each strain using the Ingenuity Pathways Analysis (Ingenuity® Systems, http://www.ingenuity.com). The Functional and Canonical pathways analysis identified the biological functions or the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significantly altered among the data sets. The significance of the association between the data set and the canonical pathway was measured as a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to each canonical pathway. Fischer's exact test was used to calculate the probability (P value) that each biological function assigned to that data set, and the association between the genes in the data set and the canonical pathway was due to chance alone.

Quantitative real-time polymerase chain reaction analysis
Relative gene expression levels for selected genes were determined by quantitative real-time polymerase chain reaction (PCR) analysis. β-Casein messenger RNA levels were measured using the Taqman assay (Applied Biosystems, Foster City, CA). Other Prl signaling relative gene expression levels were measured using Sybr green assay (Invitrogen, Carlsbad, CA). PCR primers and the dual-labeled probes (6-carboxy-fluorescein and 6-carboxy-tetramethyl-rhodamine) (supplementary Table 1, available at Carcinogenesis Online for all genes were designed using ABI Primer Express v.1.5 software. Each sample was run in triplicate in separate wells for the target gene and for the reference β-actin gene. Amplification plots for β-actin derived from serial dilutions of an established reference sample (rat liver RNA) were then used to create a linear regression formula for calculation of target gene expression levels and β-actin gene expression levels were utilized as an internal control to normalize the data.

Immunoblotting
Mammary gland tissue and culture cell lines were homogenized in RIPA lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.25% sodium deoxycholate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF and other protease inhibitor). For western blot analysis of the PrlR, proteins (40 µg per lane) were separated in a precast 4–15% gradient polyacrylamide gel (Bio-Rad, Hercules, CA). The membranes were incubated with mouse anti-rat PrlR monoclonal antibody (1 µg/ml) (Affinity BioReagents, Golden, CO) at 4°C overnight, followed by incubation with IRDye 680 Conjugated Goat Anti-Mouse IgG (Li-Cor, Lincoln, NE) and the bands with florescence were determined by Odyssey Infrared Imaging System (Li-Cor). For analysis of signal transducers and activators of transcription 5 (STAT5) and phosphorylated STAT5, proteins (30 µg per sample) were separated on 8% polyacrylamide–sodium dodecyl sulfate gel and subjected to western blot analyses. The membranes were incubated overnight with primary antibodies and followed by horseradish peroxidase-conjugated secondary antibodies. Protein signals were detected using an enhanced chemiluminescent kit (SuperSignal WestDura Extended Duration Substrate; Pierce, Rockford, IL). β-Actin was used as an internal loading control. The following reagents were purchased from the respective companies: anti-STAT5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-STAT5a/b antibodies (Upstate, Lake Placid, NY) and anti-β-actin (Sigma–Aldrich Co., St Louis, MO).

Perphenazine treatment and tumorigenicity assay
Eighteen female Cop rats at the age of 50–55 days received three daily subcutaneous injections of perphenazine (3 p.p.m. in 0.01 N HCl), followed by a single intra-peritoneal injection of NMU (50 mg/kg) or saline 24 h after the last drug treatment. Another group of 31 female Cop rats of the same age were given NMU (50 mg/kg) treatment followed by three subcutaneous injections of perphenazine (3 p.p.m.) 24 h later. Mammary glands were palpated on a weekly basis. Eight months later, all experiments were terminated and rats were killed. At necropsy, all rats were necropsized for any abnormalities. All tumors and normal mammary glands were excised and fixed in normal buffered formalin for histological diagnosis.

Data analysis
Data were either the averages or representatives of at least three independent experiments. Statistical analyses of data were performed using one-way analysis of variance. In western blot results, the values presented are mean + SE (n = 3) and * or ** indicates statistical significance at P value <0.05 or <0.01 (Student's t-test).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Comparison of mammary gland development among rat strains
Rat mammary gland development is a continuous process that occurs between the ages of 35 and 60 days, which also corresponds to the period when the mammary glands of female virgin rats are most susceptible to chemical carcinogens. To assess strain differences in the timing of mammary development, we compared the histology of mammary gland whole mounts from 55-day-old F344 (Figure 1A) and Cop female rats (Figure 1B). Microscopic analysis failed to detect significant differences in the overall structure of the glands or in the number of TEBs (Figure 1A, B and C). Since TEBs are generally accepted to harbor the target cells for the carcinogen, these findings suggested that differential genetic susceptibility was not primarily the result of altered mammary development among strains, at least not at the gross anatomical level.


Figure 1
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Histological comparison of mammary gland development among rat strains. Whole-mount analysis of mammary tissue were prepared from 55-day-old, virgin, female rats that are genetically susceptible (F344) or resistant (Cop) to mammary carcinogenesis. Examples of TEBs in whole mounts of mammary glands from the F344 (panel A) and Cop rats (panel B) are indicated by bold arrows. TEB numbers per square millimeter are presented as a histogram which found no statistical significant differences between the resistant Cop and the susceptible F344 rat strains (panel C).

 
Gene expression profiles in mammary cells are defined by genetic background
To compare gene expression profiles among different strains, we isolated total RNA from normal mammary tissue of five individual animals from each strain. The RNA isolated from mammary glands of each animal was then used to generate labeled cRNA and hybridized to a single Affymetix Rat U34A Genome GeneChip expression array. Following data extraction, background subtraction and normalization, we excluded signals for ~1000 EST and any genes that were called as ‘absent’ in samples from all strains from further analyses. The remaining 2899 genes were then analyzed using unsupervised hierarchical clustering. Note that strain identifiers were not used for clustering, but were added to the resulting dendrogram after the analysis (Figure 2). The results indicated that gene expression profiles in mammary tissue were remarkably similar at both the qualitative and quantitative levels among individuals within a given strain. As a result, unsupervised hierarchical clustering of expression patterns grouped individual rats from each inbred strains into distinct clusters or clades of the dendrogram. Significantly, the resulting dendrogram recapitulated the genealogic tree of rat strains assembled on the basis of polymorphic marker segregation (27). Virtually, the same results were obtained if data from Expression Sequence Tags of unknown function were included in the analysis (data not shown). Taken together, these and the recently published data (28) indicated that gene expression profiles in mammary glands of different rat strains are genetically defined, and hence comparative analyses of expression profiles among strains may provide insights into the molecular bases for differential genetic susceptibility.


Figure 2
View larger version (64K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Unsupervised hierarchical clustering of gene expression data from mammary tissue of 30 female rats, representing six strains, individually analyzed on GeneChipTM arrays. Normalized gene expression data from individual animals were analyzed by unsupervised hierarchical clustering as described in Materials and Methods. Hierarchical clustering of mammary gland gene expression profiles from all 30 samples was performed with 2899 statistically significant genes using centered correlation linkage analysis. Clustering was performed solely using gene expression levels and was blind to strain identities. Data are presented in a heat map with individual animals represented by columns, whereas expression levels of individual genes relative to the mean of the same gene across all 30 arrays are represented by a red–green scale in rows. The red–green scale for the heat map indicates the level of expression of each gene. The results indicated that animals within a given strain clustered into clades of a dendrogram (indicated at the top of the heat map) based on their expression patterns. The positions of animals from the Cop, F344, ACI, F1 x hybrids, Buf/N and WF strains are bracketed above the dendrogram. The dendrogram to the left of the heat map indicates clustering of individual genes based on their expression levels.

 
Identification of differential expressed candidate genes and biological pathways associated with genetic susceptibility to chemical carcinogenesis
Using the resistant Cop strain as the reference, we next performed a pairwise comparison of mammary gland gene expression profiles with each of five susceptible rat strains. Beginning with the set of genes whose differences in expression levels were reproducible and highly significant (P < 0.01), we arbitrarily selected the subset of genes whose expression was increased or decreased >2-fold (measured as a log 2 ratio of Cop versus susceptible strains). We identified 31, 100, 32, 111 and 50 genes that, respectively, distinguished the susceptible F344, F1 hybrid, ACI, Buf/N and WF strains from the resistant Cop strain (supplementary Table 2, available at Carcinogenesis Online). Included among these genes, identified were several previously implicated in mammary carcinogenesis, including the PrlR, the Jun D proto-oncogene, insulin-like growth factor 1 (Igf1), as well as its binding factors, Igfb2 and Igfb5.

Our data indicated that expression of the PrlR gene was ~2- to 3-fold higher in the all the sensitive rat strains as compared with resistant Cop strain. These findings were confirmed by real-time PCR analysis (Table I). Prl, the ligand for the PrlR, and its expression was very low in mammary tissue and was not detectable by DNA microarray analysis. However, quantitative real-time PCR was capable to detect Prl expression with a relative high-cycle number 36. More importantly, the results demonstrated that Prl gene expression in the mammary glands of almost all susceptible strains was consistently elevated relative to the Cop strain (Table I). Further analysis of the microarray data revealed that the expression of several known targets of Prl signaling, including whey acidic protein and casein alpha, beta and kappa, were also significantly elevated in susceptible strains relative to the resistant Cop strain, and all increases were confirmed by real-time PCR analysis (Table I). Notably, expression of the β-casein gene, an established marker of mammary gland differentiation, was elevated at least 10-fold in mammary cells of almost all the susceptible strains relative to the resistant Cop strain.


View this table:
[in this window]
[in a new window]

  		Table I. Average fold change of Prl-signaling gene expression in susceptible rat strains versus Cop rat confirmed by real-time data

 
The same data sets, we next used for functional categorization of genes and pathway mapping. Given that ~10% of genes examined were differentially expressed between any pair of rat strains, it was not surprising that a large number of biochemical and physiological functions were altered among strains. The biological functions of the genes whose differential expression levels were highly significant in all sensitive strains relative to the resistant Cop rat are listed in Figure 3A. The cellular processes that were altered between the resistant and all susceptible strains included cell death, cancer, lipid metabolisms, etc. Detailed pathway analysis showed that pathways involved in fatty acid biosynthesis, bioenergetics and membrane transportation (supplementary Table 2, available at Carcinogenesis Online) were the most significantly affected metabolic pathways (Figure 3B). Among the signaling pathways that were most significantly altered between the resistant and susceptible strains was the Janus kinase/STAT signal transduction pathway (Figure 3B), which plays a key role in Prl signaling. To test if Prl signaling was constitutively elevated in susceptible rat strains, we compared PrlR protein levels and the steady-state levels of phosphorylation status of STAT5a/b protein in mammary tissues of all six rat strains by immunoblotting. Using anti-PrlR antibody, we detected two different forms of PrlR protein with molecular weights of~90 and 45 kDa in rat mammary gland (Figure 4A). The results showed that the 90 kDa isoform of the PrlR protein was expressed at a relatively low level in mammary gland, and its expression level was significantly reduced in Cop rat when compared with F344 and F1 hybrid rat strains (Figure 4A and B). In contrast, protein levels of the 45 kDa PrlR was expressed in relatively higher levels compared with 90 kDa isoform in each rat strains, and its expression is significantly higher in F344 rat strain than in other strains (Figure 4A and B). Western blot analysis further showed that the levels of total STAT5a/b proteins expressed in mammary tissues varied among rat strains (Figure 4C). However, the steady-state levels of phosphorylated STAT5a/b protein were significantly elevated in the F344 and ACI strains (Figure 4C and D). Together, these results support the hypothesis that increased Prl signaling contributes to the genetic susceptibility to mammary carcinogenesis in some susceptible rat strains.


Figure 3
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Comparative analysis of biological functions and pathways among all inbred strains. The Ingenuity® Systems Pathway Analysis program was used to identify the most reproducible and significant biological functions (panel A) or pathways (panel B) that differentiated each of the susceptible rat strains to resistant Cop strain. Data are plotted as the negative logarithm of statistical significance (P < 0.01) relative to individual function group (A) or biochemical pathway (panel B) defined by the algorithm for each pairwise strain comparison. The Functional and Canonical Pathways Analysis was used to identify the biological functions or the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significantly altered among the data sets. The significance of the association between the data set and the canonical pathway was measured as a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to each canonical pathway. Fischer's exact test was used to calculate the probability (P value) that each biological function assigned to that data set, and the association between the genes in the data set and the canonical pathway was due to chance alone.

 


Figure 4
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Prl signaling in mammary gland is reduced in the resistant Cop strain when compared with susceptible rat strains. Whole mammary gland lysate prepared from the six rat strains [Cop, F344, F1 Hybrid, ACI, Buf/N and WF] were analyzed by western blotting using antibodies against PrlR, in which 90 kDa long form of PrlR was shown in upper band and 45 kDa short form of PrlR was shown in lower band in panel (A). Total STAT5a/b and phospho-STAT5a/b protein levels were shown in panel (C). In all analyses, β-actin was used as an internal loading control. Western blot analysis was performed in triplicate, and representative results are presented in panels (A) and (C). The levels of each relevant protein from each analysis were then measured and normalized to β-actin levels in the same lane. The mean of the relative levels of expression were then semi-quantified for the PrlR (panel B) and P-STAT5a/b protein levels (Panel D). The values presented are mean + SE (n = 3), and * or ** indicates statistical significance compared with the Cop strain at P value <0.05 or <0.01 (Student's t-test).

 
Perphenazine-induced hyperprolactinemia increases the susceptibility of the Cop mammary gland to chemical carcinogenesis
To further test the latter hypothesis, we used the dopamine receptor antagonist, perphenazine, to induce transient hyperprolactinemia in the highly resistant Cop strain. Perphenazine is a neuroleptic drug, which induces a massive release of Prl from the pituitary gland by inhibiting dopamine receptors, leading to 5- to 10-fold elevation of serum Prl levels (22,29). Twelve 50-day-old, female Cop rats were treated with daily injection of perphenazine (3 p.p.m.) for three consecutive days. An equal number of control animals were injected with the vehicle alone. As had been previously observed in several susceptible strains (30), perphenazine treatment induced rapid growth and differentiation of mammary tissue in the Cop rat. Thus, by 3 days after treatment the mammary tissue of the Cop rat resembled that of a lactating female rat (Figure 5A–D). On the fourth day after exposure to perphenazine, animals were treated with a single dose of NMU at 50 mg/kg, which is sufficient to induce tumors in 70–90% of females from the various susceptible rat strains. We also performed the experiment by first exposing rats to a carcinogenic dose of NMU followed by 3 days of treatment with perphenazine. As expected, the Cop females treated with perphenazine alone failed to develop any mammary tumors for up to 1 year after injection (Table II). In contrast, ~5% (2/37) of pubescent Cop female rats exposed to a combination of NMU and perphenazine developed mammary adenocarcinomas between 3 and 9 months after exposure (Table II), including a highly vascularized mammary adenocarcinoma that attained a diameter of 2 cm within 3 months of exposure (Figure 5E–G).


View this table:
[in this window]
[in a new window]

  		Table II. Number of mammary adenocarcinomas induced by exposure of pubescent Cop female rats to a combination of NMU and perphenazine

 


Figure 5
View larger version (138K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Effects of perphenazine treatment in resistant Cop rats and induction of mammary adenocarcinomas by NMU in resistant Cop rats treated with perphenazine. Whole-mount analyses of mammary gland morphology with and without exposure to the drug perphenazine were performed as described in the text. Mammary gland whole mounts were prepared from 55-day-old, virgin, female Cop rats after three consecutive daily injection with either the vehicle alone (A, whole mount and B, x4 magnification) or after treatment with perphenazine (C, whole mount and D, x4 magnification). (E) Anatomical position of a mammary carcinoma that developed 3 months after treating a virgin female rat with three daily doses of perphenazine followed by a single 50 mg/kg dose of NMU. Histopathology of the hematoxilin and eosin-stained mammary adenocarcinoma at (F) low (x4) and (G) high magnification (x20) under light microscopy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Genetic linkage studies performed using crosses between resistant rat strains and various susceptible strains indicated that in the rat, susceptibility to mammary carcinogenesis is a polygenic trait, and >20 distinct quantitative trait loci have been linked to the incidence of mammary carcinogenesis following exposure to various carcinogens (1,2,31). These studies did not, however, exclude the possibility that different susceptibility loci mutated in different strains could affect the same biological processes. The possibility that common biochemical mechanisms are involved in the susceptibility of different strains is supported by the observation that mammary tumors induced in all susceptible rat strains by chemical carcinogens are predominantly papillary adenocarcinomas that do not metastasize (32). In addition, all susceptible strains show comparable frequencies of H-ras oncogene mutations when exposed to different mammary carcinogens (33–36). We therefore hypothesized that genetic susceptibility in rats would result in the presence of a characteristic gene expression signature, and that comparisons of these genetically determined signatures could provide insights into the biochemical basis for differential susceptibilities. We reasoned that if susceptible strains harbor mutations in different susceptibility loci that affect the same biochemical pathway, we should be able to identify a specific set of genes and biochemical pathways that were similarly altered in all susceptible strains relative to the resistant Cop strain. Moreover, since ~30% of (Cop x F344) F1 progeny develop mammary tumors after carcinogen exposure, we also expected that in the F1 progeny, the pattern of expression for any genes implicated in genetic susceptibility should approach or recapitulate the levels seen in the susceptible F344 parent.

Among the genes that showed reproducible and statistically significant differences in steady-state levels between the resistant Cop strain and in all susceptible inbred strains, including the F1 hybrids, we identified numerous gene implicated in the Prl-signaling pathway, which plays a crucial role in the normal growth and development of mammary glands. For example, mammary glands of genetically engineered Prl knockout mice fail to develop beyond the early ductal stage. In contrast, both the PrlR and Prl have been implicated in the growth of spontaneous and carcinogen-induced rodent. The elevated Prl signaling was observed in rat mammary tumor induced by NMU treatment when compared with normal mammary tissue collected from the same generated tumor rats, in which PrlR and STAT5 was elevated 4.9 and 2.9 in rat mammary tumor relative to the normal mammary tissue, respectively (37). Transgenic mice expressing human Prl have increased rates of mammary cancer (38), and antagonists of Prl signaling have been shown to inhibit the growth of chemically induced mammary tumors in mice (39). Elevated expression of biologically active Prl and the PrlR are also frequently detected in human breast cancers, and epidemiological data also suggest a relatively strong positive association between circulating Prl levels and breast cancer risk in post-menopausal women (40). Likewise, studies in genetically susceptible WF and Sprague–Dawley rats found an association between the serum Prl levels and the sensitivity (tumor frequency and latency) to carcinogen induction of mammary tumors (41,42). Together, these studies suggest that elevated Prl signaling can contribute to both normal mammary growth and development, but can also contribute tumor promotion and/or progression. Our analysis indicated that all susceptible strains expressed elevated levels of the transcripts encoding the peptide hormone, Prl, its cognate receptor, and several downstream targets of Prl signaling.

Western blot analyses using antibodies specific for PrlR indicated that two different isoforms of PrlR protein are present in the rat mammary gland. The expression levels of 90 kDa isoform of PrlR protein are significantly reduced in Cop rats when compared with susceptible F344 and F1 hybrid rat strains, in contrast, the 45 kDa isoform was only significantly elevated in F344 rat compared with all other rat. Previous studies showed that although both forms of PrlR can be activated by Prl binding, only 90 kDa form activates signaling pathways associated with mammary cell differentiation (43,44). In contrast, activation of either PrlR isoform can promote cell growth by recruiting and activating the Janus kinase 2 and mitogen-activated protein kinase (43,45,46). These finding suggested that Cop mammary cells would receive reduced mitogenic signals in response to Prl binding.

The STAT5 transcription factor is the major downstream effect molecule of PrlR. After Prl binds to the PrlR, the recruited Janus kinase 2 kinase activity becomes activated and phosphorylates the STAT5a/b protein, activating its signaling capacity (47). We therefore used western blot analysis to compare steady-state levels of total and phosphorylated STAT5a/b protein among strains. Western blot results for whole mammary tissues demonstrated that the steady-state levels of phosphorylated STAT5a/b were significantly lower in the mammary glands of the Cop rats compared with that of F344 and ACI strains. Although the observed steady-state increases in STAT5a/b phosphoylation were not very large, it must be remembered that the assays were performed on whole mammary tissue, and hence the actual increase would be diluted if only a fraction of the mammary cells were affected. Consistent with the latter contention, studies with Prl knockout mice suggested that the effect of Prl signaling is mediated primarily in cells of the TEBs. Taken together, these observations suggest that the F344 and ACI strains share overlapping mechanisms of genetic susceptibility that includes constitutively activated Prl signaling. Consistent with the latter contention, both the F344 and ACI strains are highly susceptible to estrogen-induced mammary carcinogenesis relative to WF and Buf/N strains (48,49).

Previous studies comparing the response of mammary tissue from differentially susceptible rat strains to carcinogen exposure indicated that resistance in the Cop strain is mediated at the level of tumor promotion or progression. These studies demonstrated comparable kinetics of DNA adduct formation, repair and mutagenesis in the mammary cells of susceptible and the resistant Cop strains (33,50). Moreover, histopathological analyses demonstrated that pre-malignant lesions, which are reminiscent of human ductal carcinoma in situ, are also formed at similar frequencies in the sensitive and resistant strains (51). However, pre-malignant lesions induced by carcinogen exposure regress and fail to progress in the resistant Cop strain, suggesting that the resistance of the Cop strain is at least in part mediated by gene that suppress the outgrowth or progression of pre-malignant lesion. Given Prl’s demonstrated role in promoting mammary tumor growth, it was reasonable to posit that the reduced level of Prl signaling was insufficient to promote the outgrowth of these pre-malignant lesions present in mammary glands of the resistant Cop strain. The latter hypothesis predicts that increasing Prl signaling in the mammary glands of Cop should partially overcome their resistance to chemically induced mammary carcinogenesis. To explore the contribution of increased Prl signaling to genetic susceptibility, we tested the effect of perphenazine-induced hyperprolactinemia on the susceptibility of pubescent female Cop rats to NMU-induced mammary carcinogenesis (52). The results demonstrated that among 37 pubescent female Cop rats treated with perphenazine before or after exposure to NMU, ~5% developed mammary tumors in 3–9 months, suggesting that even transient elevation of serum Prl levels sensitizes Cop female rats to chemically induced carcinogenesis. Several previous studies have demonstrated that mammary tumors can be induced in Cop rat by in utero and perinatal exposures (51) or following implantation of NMU crystal directly into mammary glands (53). In addition, two recent studies showed that about 30–40% pubescent Cop females exposed to NMU with or without exposure to estrogen will develop mammary tumors after a prolonged latency of >1 year (28,54). However, the present study, which combined NMU exposure with perphenazine, is the first to demonstrate the induction of mammary carcinomas in the Cop rat with kinetics comparable with those seen among susceptible strains following systemic exposure to NMU.

Our results are also consistent with the findings of two previous studies which suggested a link between perphenazine-induced elevation of serum Prl and genetic sensitivity to mammary carcinogenesis. One of these studies showed that the relative sensitivities of different mouse strains to chemically induced carcinogenesis is correlated with the level of serum Prl induced by perphenazine (55). Another study in three genetically susceptible rat strains found a correlation between susceptibility to mammary carcinogenesis and serum Prl levels in female rats chronically treated with 17β-estradiol (41). Another study showed that perphenazine enhanced the ability of retrovirally transduced oncogenes to induce mammary carcinomas in the Cop strain (56). Taken together with previous studies, the results of the present study strongly suggest that the reduced levels of Prl signaling present in the mammary tissue of Cop rat are insufficient to promote the efficient outgrowth of initiated cells. When Prl signaling is enhanced by even a transient induction of prolactinemia by perphenazine, a fraction of the NMU-initiated cells present in the mammary glands of Cop females progress toward malignancy with latency comparable with that of the susceptible strains.

Although perphenazine and many other anti-psychotic drugs are potent inducers of Prl release from the pituitary gland in humans (22,57–59), the implications of our finding in the rat for individuals on anti-psychotic therapy are unclear. However, there exist preliminary epidemiological data to suggest an association between the use of dopamine receptor antagonists and an increased risk of developing breast cancer. Although many of these studies have been challenged on methodological grounds or lacked adequate statistical power, recent studies have suggested that exposure to anti-psychotic drugs is associated with a significantly elevated risk of breast cancer (40,60). Among these is a large cohort study that compared the incidence of breast cancer in 52 819 women exposed to dopamine antagonists and 55 289 controls. This study found a statistically significant 16% increase in breast cancer risk among women treated with anti-psychotic drugs, and risk increased with dose of the drugs (60). Among the neuroleptic drugs that antagonize dopamine, risperiodone is commonly used to treat attention deficit hyperactivity disorder, and concerns have been raised regarding it proclivity to induce hyperprolactinemia in male and female children and adults taking the drug (59). Our observation indicating that dopamine agonists sensitize pubescent, virgin female rats of the normally resistant Cop rat strain to mammary carcinogenesis reinforces the need to revisit these concerns.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Tables 1 and 2 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Public Health Services (U19ES011387, P30ES007033 and P30ES005022).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Jeff Delrow and the staff of the Genomics Shared Resource of the Fred Hutchinson Cancer Research Center for performing DNA microarray analyses. Some of the data analyses were performed using BRB ArrayTools version 3.2.3 developed by Dr Richard Simon and Amy Peng Lam. All gene expression profiling data discussed in this manuscript were submitted to National Center for Biotechnology Information Gene Expression Omnibus database that is public available and the accession number is GSE2581 [NCBI GEO] .

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Shepel LA, et al. The genetic components of susceptibility to breast cancer in the rat. Prog. Exp. Tumor Res. (1999) 35:158–169.[Medline]
  2. Isaacs JT. Genetic control of resistance to chemically induced mammary adenocarcinogenesis in the rat. Cancer Res. (1986) 46:3958–3963.[Abstract/Free Full Text]
  3. Maita K, et al. Spontaneous tumors in F344/DuCrj rats from 12 control groups of chronic and oncogenicity studies. J. Toxicol. Sci. (1987) 12:111–126.[Medline]
  4. Gould MN, et al. Genetic regulation of mammary carcinogenesis in the rat by susceptibility and suppressor genes. Environ. Health Perspect. (1991) 93:161–167.[Web of Science][Medline]
  5. Segaloff A. The role of the ovary in estrogen production of mammary cancer in the rat. Cancer Res. (1974) 34:2708–2710.[Abstract/Free Full Text]
  6. Wood GA, et al. Tissue-specific resistance to cancer development in the rat: phenotypes of tumor-modifier genes. Carcinogenesis (2002) 23:1–9.[Abstract/Free Full Text]
  7. Shepel LA, et al. Genetic identification of multiple loci that control breast cancer susceptibility in the rat. Genetics (1998) 149:289–299. [erratum appears in Genetics, 1998, 149, 1627].[Abstract/Free Full Text]
  8. Freeman ME, et al. Prolactin: structure, function, and regulation of secretion. Physiol. Rev. (2000) 80:1523–1631.[Abstract/Free Full Text]
  9. Welsch CW. Prolactin and the development and progression of early neoplastic mammary gland lesions. Cancer Res. (1978) 38:4054–4058.[Abstract/Free Full Text]
  10. Welsch CW, et al. Inhibition of mammary tumorigenesis in carcinogen-treated Lewis rats by suppression of prolactin secretion. J. Natl Cancer Inst. (1979) 63:1121–1124.[Web of Science][Medline]
  11. Russo J, et al. Experimentally induced mammary tumors in rats. Breast Cancer Res. Treat. (1996) 39:7–20.[CrossRef][Web of Science][Medline]
  12. Hankinson SE, et al. Plasma prolactin levels and subsequent risk of breast cancer in postmenopausal women. J. Natl Cancer Inst. (1999) 91:629–634.[Abstract/Free Full Text]
  13. Clevenger CV, et al. The role of prolactin in mammary carcinoma. Endocr. Rev. (2003) 24:1–27.[Abstract/Free Full Text]
  14. Clevenger C, et al. Prolactin as an autocrine/paracrine factor in breast tissue. J. Mammary Gland Biol. Neoplasia (1997) 2:59–68.[CrossRef][Medline]
  15. Tworoger S, et al. Prolactin and breast cancer risk. Cancer Lett. (2006) 243:160–169.[CrossRef][Web of Science][Medline]
  16. Clevenger C, et al. Expression of prolactin and prolactin receptor in human breast carcinoma. Evidence for an autocrine/paracrine loop. Am. J. Pathol. (1995) 146:695–705.[Abstract]
  17. Ginsburg E, et al. Prolactin synthesis and secretion by human breast cancer cells. Cancer Res. (1995) 55:2591–2595.[Abstract/Free Full Text]
  18. Steinmetz R, et al. Transcription of prolactin gene in milk secretory cells of the rat mammary gland. J. Endocrinol. (1993) 136:271–276.[Abstract/Free Full Text]
  19. Mershon J, et al. Prolactin is a local growth factor in rat mammary tumors. Endocrinology (1995) 136:3619–3623.[Abstract]
  20. Kurtz A, et al. Mammary epithelial cells of lactating rats express prolactin messenger ribonucleic acid. Biol. Reprod. (1993) 48:1095–1103.[Abstract]
  21. Le Provost F, et al. Prolactin gene expression in ovine and caprine mammary gland. Neuroendocrinology (1994) 60:305–313.[Web of Science][Medline]
  22. Ben-David M. Mechanism of induction of mammary differentiation is Sprague-Dawley female rats by perphenazine. Endocrinology (1968) 83:1217–1223.[Abstract/Free Full Text]
  23. Russo IH, et al. Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12-dimethylbenz[a]anthracene. J. Natl Cancer Inst. (1978) 61:1439–1449.[Web of Science][Medline]
  24. Korn E, et al. Controlling the number of false discoveries: application to high-dimensional genomic data. J. Stat. Plan. Inference (2004) 124:379–398.[CrossRef]
  25. Simon R, et al. Design and Analysis of DNA Microarray Investigations (2003) New York: Springer-Verlag.
  26. Wright GW, et al. A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics (2003) 19:2448–2455.[Abstract/Free Full Text]
  27. Canzian F. Phylogenetics of the laboratory rat Rattus norvegicus. Genome Res. (1997) 7:262–267.[Abstract/Free Full Text]
  28. Blakely CM, et al. Hormone-induced protection against mammary tumorigenesis is conserved in multiple rat strains and identifies a core gene expression signature induced by pregnancy. Cancer Res. (2006) 66:6421–6431.[Abstract/Free Full Text]
  29. Stringer B, et al. Effect of raised serum prolactin on breast development. J. Anat. (1989) 162:249–261.[Web of Science][Medline]
  30. Gould MN, et al. Effects of endocrinologic conditions associated with acute versus chronic lactation on the incidence of mammary carcinomas in irradiated rats: brief communication. J. Natl Cancer Inst. (1978) 60:469–471.[Web of Science][Medline]
  31. Gould MN. Inheritance and site of expression of genes controlling susceptibility to mammary cancer in an inbred rat model. Cancer Res. (1986) 46:1199–1202.[Abstract/Free Full Text]
  32. Briand P. Hormone-dependent mammary tumors in mice and rats as a model for human breast cancer (review). Anticancer Res. (1983) 3:273–281.[Web of Science][Medline]
  33. Lu SJ, et al. Formation and repair of O6-methylguanine and methylation of the Ha-ras proto-oncogene by N-methyl-N-nitrosourea are not associated with mammary tumor resistance in the Copenhagen rat. Carcinogenesis (1992) 13:857–861.[Abstract/Free Full Text]
  34. Sukumar S, et al. Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature (1983) 306:658–661.[CrossRef][Medline]
  35. Zarbl H, et al. Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature (1985) 315:382–385.[CrossRef][Medline]
  36. Zarbl H, et al. Activation of H-ras-1 oncogenes by chemical carcinogens. Basic Life Sci. (1986) 38:385–397.[Medline]
  37. Shan L, et al. Global gene expression profiling of chemically induced rat mammary gland carcinomas and adenomas. Toxicol. Pathol. (2005) 33:768–775.[CrossRef][Web of Science][Medline]
  38. Rose-Hellekant TA, et al. Prolactin induces ERalpha-positive and ERalpha-negative mammary cancer in transgenic mice. Oncogene (2003) 22:4664–4674.[CrossRef][Web of Science][Medline]
  39. Tomblyn S, et al. The role of human prolactin and its antagonist, G129R, in mammary gland development and DMBA-initiated tumorigenesis in transgenic mice. Int. J. Oncol. (2005) 27:1381–1389.[Web of Science][Medline]
  40. Moorman PG, et al. Antidepressant medications and their association with invasive breast cancer and carcinoma in situ of the breast. Epidemiology (2003) 14:307–314.[CrossRef][Web of Science][Medline]
  41. Blankenstein MA, et al. Prolactin concentration in plasma and susceptibility to mammary tumors in female rats from different strains treated chronically with estradiol-17 beta. Breast Cancer Res. Treat. (1984) 4:137–141.[CrossRef][Web of Science][Medline]
  42. Boyns AR, et al. Basal prolactin blood levels in three strains of rat with differing incidence of 7,12-dimethylbenz(a)anthracene induced mammary tumours. Eur. J. Cancer (1973) 9:169–171.[Medline]
  43. Das R, et al. Transduction of prolactin's (PRL) growth signal through both long and short forms of the PRL receptor. Mol. Endocrinol. (1995) 9:1750–1759.[Abstract/Free Full Text]
  44. Hennighausen L, et al. Prolactin signaling in mammary gland development. J. Biol. Chem. (1997) 272:7567–7569.[Free Full Text]
  45. Piccoletti R, et al. Rapid stimulation of mitogen-activated protein kinase of rat liver by prolactin. Biochem. J. (1994) 303:429–433.[Web of Science][Medline]
  46. Buckley A, et al. Prolactin-induced phosphorylation and nuclear translocation of MAP kinase in Nb2 lymphoma cells. Biochem. Biophys. Res. Commun. (1994) 204:1158–1164.[CrossRef][Web of Science][Medline]
  47. Hennighausen L, et al. Developing a mammary gland is a stat affair. J. Mammary Gland Biol. Neoplasia (1997) 2:365–372.[CrossRef][Medline]
  48. Dunning W, et al. Strain differences in response to estrone and the induction of mammary gland, adrenal, and bladder cancer in rats. Cancer Res. (1953) 13:147–152.[Abstract/Free Full Text]
  49. Shellabarger C, et al. Rat differences in mammary tumor induction with estrogen and neutron radiation. J. Natl Cancer Inst. (1978) 61:1505–1508.[Web of Science][Medline]
  50. Cha RS, et al. Ha-ras-1 oncogene mutations in mammary epithelial cells do not contribute to initiation of spontaneous mammary tumorigenesis in rats. Carcinogenesis (1996) 17:2519–2524.[Abstract/Free Full Text]
  51. Lu SJ, et al. Mammary tumor induction by N-methyl-N-nitrosourea in genetically resistant Copenhagen rats. Cancer Res. (1992) 52:5037–5041.[Abstract/Free Full Text]
  52. Erb RJ, et al. Serum prolactin level increase in normal subjects following administration of perphenazine oral dosage forms: possible application to bioavailability testing. J. Pharm. Sci. (1982) 71:883–888.[CrossRef][Web of Science][Medline]
  53. Takahashi H, et al. Induction of mammary carcinomas by the direct application of crystalline N-methyl-N-nitrosourea onto rat mammary gland. Cancer Lett. (1995) 92:105–111.[CrossRef][Web of Science][Medline]
  54. Shull JD, et al. Susceptibility to estrogen-induced mammary cancer segregates as an incompletely dominant phenotype in reciprocal crosses between the ACI and Copenhagen rat strains. Endocrinology (2001) 142:5124–5130.[Abstract/Free Full Text]
  55. Sinha YN, et al. Serum, pituitary and urine concentrations of prolactin and growth hormone in eight strains of mice with varying incidence of mammary tumors. Int. J. Cancer (1979) 24:430–437.[CrossRef][Web of Science][Medline]
  56. Wang B, et al. Overcoming the activity of mammary carcinoma suppressor gene in Copenhagen rats by v-H-ras oncogene transfer into mammary epithelial cells in situ. Cancer Res. (1991) 51:5298–5303.[Abstract/Free Full Text]
  57. Ben-David M, et al. Acute changes in blood and pituitary prolactin after a single injection of perphenazine. Neuroendocrinology (1970) 6:336–342.[Web of Science][Medline]
  58. Guzman RC, et al. Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy. Proc. Natl Acad. Sci. USA (1999) 96:2520–2525.[Abstract/Free Full Text]
  59. Dickson RA, et al. Neuroleptic-induced hyperprolactinemia. Schizophr. Res. (1999) 35(suppl.):S75–S86.[CrossRef][Web of Science][Medline]
  60. Wang PS, et al. Dopamine antagonists and the development of breast cancer. Arch. Gen. Psychiatry (2002) 59:1147–1154.[Abstract/Free Full Text]
Received February 7, 2007; revised September 20, 2007; accepted September 21, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Vet PatholHome page
L. Rush

Vet. Pathol., May 1, 2008; 45(3): 434 - 435.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/1/177    most recent
bgm224v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ren, X.
Right arrow Articles by Zarbl, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ren, X.
Right arrow Articles by Zarbl, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?