Cyclin D1 expression during rat mammary tumor development and its potential role in the resistance of the Copenhagen rat

Abstract

Resistance to mammary tumorigenesis in Copenhagen rats is associated with loss of early preneoplastic lesions known as intraductal proliferations. The cause of this disappearance, however, is unknown. protein expression, bromodeoxyuridine labeling and apoptotic indices, or mast cell infiltration between Copenhagen and Wistar-Furth lesions at any time. expression in Copenhagen intraductal proliferations may play a role in the resistance of these rats to mammary tumorigenesis.

Background:

Resistance to mammary tumorigenesis in Copenhagen rats is associated with loss of early preneoplastic lesions known as intraductal proliferations. The cause of this disappearance, however, is unknown.

Results:

protein expression, bromodeoxyuridine labeling and apoptotic indices, or mast cell infiltration between Copenhagen and Wistar-Furth lesions at any time.

Conclusions:

expression in Copenhagen intraductal proliferations may play a role in the resistance of these rats to mammary tumorigenesis.

Introduction

].

(DCIS) and adenocarcinomas were detectable in the glands from Wistar-Furth rats. In contrast, the IDPs from Copenhagen rats failed to progress and instead declined in number, such that by 60 days after MNU treatment the glands were essentially free of lesions.

].

].

Animals and carcinogen treatment

Copenhagen and Wistar-Furth rats (6-7 weeks old) were purchased from Harlan Sprague Dawley (Indianapolis, Indiana, USA), maintained on a 12h light/dark cycle, fed Harlan Teklad rat chow (6% fat; Harlan Teklad, Madison, Wisconsin, USA), and were given free access to water. After 1 week of acclimatization the rats were given an intraperitoneal injection of 50 mg/kg MNU dissolved in acidified normal saline.

Bromodeoxyuridine treatment and mammary whole-mount

			preparation

].

Paraffin embedding, staining, and immunohistochemistry

-positive cells was determined as the number of positive cells divided by total cell number in a lesion.

The bromodeoxyuridine labeling index was determined by the number of bromodeoxyuridine-positive cells divided by total cells in a lesion. Small intestine from bromodeoxyuridine-treated rats or livers from partially hepatectomized rats were used as positive controls for staining.

For all immunohistochemistry, the specificity of the staining was ensured by replacing the primary antibody with 1% normal sheep serum. In all cases, no staining was observed.

]. Because this is a subjective method, samples were scored independently by two individuals, with similar results.

For staining of mast cells, samples were deparaffinized in xylenes, rehydrated through acetone and water, stained in 0.025% toluidine blue (Sigma) for 30s. Slides were washed in distilled water, dehydrated in acetone, cleared in xylenes, and mounted using Permount (Fisher). Mast cells were counted per high power (400×) field of view around the lesion.

Statistical analyses

-test was used.

Results

shows the same region of the inguinal mammary gland from typical whole-mounts from a Wistar-Furth and a Copenhagen rat, demonstrating the striking difference in development of lesions in the glands at 37 days.

].

expression levels. Suspected lesions must be microdissected from the whole-mounts, then embedded and sectioned to confirm their identity, leaving insufficient tissue for Western analysis.

). We observed similar levels of staining for this protein in all of the samples from both strains, including normal mammary tissue, IDPs, DCIS, and tumors.

expression levels in lesions from Wistar-Furth rats at this time.

. There were no significant differences between the Copenhagen and Wistar-Furth rats at 20, 30, or 37 days after MNU treatment.

Toluidine blue, which stains mast cells metachromatically, was used to visualize these cells within sections. Samples were scored for the number of mast cells per high power field of view around each lesion. There were 3.6 ± 0.5, 2.8 ± 0.3, and 6.4 ± 0.7 mast cells around Copenhagen IDPs, and 4.3 ± 0.5, 2.4 ± 0.5, and 6.6 ± 0.8 mast cells around Wistar-Furth IDPs at 20, 30, and 37 days after MNU, respectively (all values are means ± standard error of the mean). There were no significant differences in mast cell numbers between the two strains at any of the time points.

Discussion

].

]. There was no difference, however, in the apoptotic indices at 37 days between the strains.

].

overexpression or in the resistance of the Copenhagen rat.

protein and hence transformation. The β -catenin pathway, therefore, merits investigation in rat mammary tumorigenesis.

might play a fundamental role in the progression of IDPs to DCIS and adenocarcinomas during rat mammary tumorigenesis. Furthermore, this gene might also play a role in the resistance of Copenhagen rats to MNU-induced mammary tumorigenesis.

Acknowledgements

The authors to thank Dr Henry Thompson for discussions on bromodeoxyuridine staining of IDPs, Dr Alan Medline for help with identification and staining of mast cells, Dr Ross Cameron for help with photography, and Dr Laurie Stephen for her help with the animal work. This investigation was supported by a grant from the Canadian Breast Cancer Research Initiative. MCA is the recipient of a Natural Sciences and Engineering Research Council of Canada Industrial Research Chair and acknowledges support from the member companies of the Program in Food Safety (University of Toronto).

Figures and Tables

< 0.05, versus Wistar-Furth rats.

-nitrosourea treatment. Note the striking differences between the glands, with multiple preneoplastic lesions present in the Wistar-Furth gland but only one putative lesion present in the Copenhagen gland, as indicated by the arrows. Bar = 1 mm.

in Wistar-Furth lesions (b and c) but not in the Copenhagen IDP (a). All lesions are from mammary glands of rats 37 days after MNU treatment. (a-f) 1000× magnification and (g-I) 400× magnification.

Bromodeoxyuridine (BrdU) labeling indices in Copenhagen (Cop) and Wistar-Furth (WF) rats at 20, 30, and 37 days after MNU treatment.

Apoptotic indices in Copenhagen (Cop) and Wistar-Furth (WF) lesions at 20, 30, and 37 days after MNU.

Keywords

• Copenhagen rat
• cyclin D1
• intraductal proliferations
• mammary gland
• preneoplastic lesions