Susceptibility of the mammary gland to carcinogenesis. III. The cell of origin of rat mammary carcinoma.

The rat mammary gland epithelium is composed of three cell types: dark cells (DCs), intermediate cells (ICs), and a layer of myoepithelial cells (MCs), which are evenly distributed along the mammary gland tree in rather constant proportions. The present study was carried out for a determination of the effect of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) on the distribution and proliferative activity of these cell populations. The proportion and the DNA labeling index (DNA-LI) of each cell type were determined in terminal end buds (TEBs), terminal ducts (TDs), alveolar buds (ABs), and alveoli of the normal Sprague-Dawley rat mammary gland and in intraductal proliferations (IDPs) and carcinomas removed at selected intervals after DMBA administration. DMBA-induced changes in cell distribution were limited to TEBs and TDs, whereas ABs and alveoli were unaffected. The alterations consisted in an increment in ICs from 11% in TEBs and TDs to 90% in tumors and a decrease in DCs from 77% in TEBs and TDs to 7% in tumors. MCs were relatively unaffected. The DNA-LI of DCs, which in the normal gland TEB was 14%, was depressed by DMBA to 6%, whereas the DNA-LI of ICs remained unchanged from the basal level of 40% during the process of carcinogenesis. The progressive increment in number of ICs with a steady DNA-LI suggested that the IC is the target cell of the carcinogen and the cell of origin of mammary carcinomas.     

Effect of exercise on the rat mammary gland: implications for carcinogenesis

Physical activity has been associated with decreased risk for developing breast cancer yet to date, the mechanism remains unknown. The purpose of this investigation was to evaluate the effects of moderate exercise training on the normal mammary gland in an attempt to identify alterations or differences that might be associated with tumour inhibition. A total of 170 female Sprague-Dawley rats were randomized to baseline (n=10), exercise (EX; n=80), or sham-exercise groups (SHAM; n=80). Treadmill training (20-25 m min-1, 15% grade, 30 min day-1, 5 days week-1) was started at 28 days of age (DOA). Animals were killed at 28, 42, 56, 70 and 84 DOA. Mammary glands were evaluated by histology and immunohistochemistry. Terminal end buds (TEB), structures susceptible to carcinogenesis, were counted. Sexual maturation, estradiol and progesterone, and organ and muscle weights were also evaluated. No differences in growth, sexual maturation, or steroid hormones were observed in response to training. No difference in the number of TEBs was observed at any timepoint between EX and SHAM. Proliferation was significantly increased at 56 DOA and tended to be increased at 42 and 70 DOA in the EX animals whereas cell death was significantly increased at 70 DOA and tended to be increased at 84 DOA in the EX animals. These data suggest no difference in the number of carcinogen-susceptible structures as a result of moderate exercise. The changes in cell proliferation and apoptosis with exercise training suggest altered cell turnover that will necessitate future study particularly with relevance to carcinogenesis.     

Susceptibility of the mammary gland to carcinogenesis: I Differentiation of the mammary gland as determinant of tumor incidence and type of lesion.

The influence of age and mammary gland differentiation on the incidence of tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) was studied by correlating the development of the mammary glands of 20-180-day-old, virgin Sprague-Dawley rats with the number and type of tumors induced by DMBA administered at those various ages. The number of terminal end buds (TEBs), terminal ducts (TDs), and alveolar buds (ABs)/sq mm and their DNA-labeling indices (DNA-LI) were determined. Highest density of TEB occurred when the rats were 20 days old, decreasing thereafter. DNA-LI ranged between 25.2 and 29 in TEB of rats aged 30-55 days, which was coincident with the highest incidence of carcinomas. With aging, the number of TEBs and their DNA-LI decreased and the number of TDs and ABs increased, although with a low DNA-LI, which correlated with a lower incidence of carcinomas and higher incidence of benign lesions.     

Gene expression in fibroadenomas of the rat mammary gland in contrast to spontaneous adenocarcinomas and normal mammary gland.

Fibroadenomas are considered a benign lesion in rodent carcinogenicity studies. However, the entity adenocarcinoma arising in fibroadenoma does exist and in humans there is evidence of certain forms of fibroadenomas to confer greater risk of subsequent breast cancer. In this study, we aim to elucidate the molecular features of both spontaneous fibroadenomas and adenocarcinomas. The gene expression of the two tumour types is examined and compared to mammary gland in the same developmental state and examined for similarities which might indicate common molecular pathways. In the present study no similarities were discovered. We conclude that in the tumours examined here, no progression to adenocarcinoma is likely. Further studies are needed, examining a greater number of tumours and including cases of adenocarcinoma arising in fibroadenoma.     

Role of vitamin a in chemical carcinogenesis of the mammary gland

The effect of feeding rats with large doses of vitamin A on the concentration of the polycyclic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) and its metabolites in various organs and in the blood and also on the rate of metabolism in the liver of rats after intravenous injection of the carcinogen were studied. In hypervitaminosis A the quantity of DMBA and its metabolites was found to be considerably reduced in all the organs tested and in the blood. The rate of DMBA metabolism in the liver of the animals increased with an increase in the dose of vitamin A.Deceased.Department of Biophysics, Biological Faculty, Moscow University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 273–275, March, 1979.     

Susceptibility of the mammary gland to carcinogenesis. II. Pregnancy interruption as a risk factor in tumor incidence.

In the rat, pregnancy and lactation prior to carcinogen administration protect the mammary gland from developing carcinomas and benign lesions. In this study, the influence of pregnancy interruption versus full pregnancy and pregnancy plus lactation on the incidence of carcinomas and benign lesions was studied in the mammary glands of rats treated with 7,12-dimethylbenz(a)anthracene (DMBA). Fifty-nine Sprague-Dawley rats were separated into 5 groups: I) rats that had had one pregnancy and one lactation; II) rats that had had one pregnancy without lactation; III) rats that had had pregnancy interrupted at the 12th day of gestation; IV) age-matched virgin rats as a control Group I; and V) age-matched virgin rats as a control for groups II and III. The 5 groups received a single intragastric dose of DMBA (10 mg/100 g body weight), with the exception of 2 animals per group, which were killed 1 hour after an intraperitoneal injection of 2.5 mu Ci 3H-thymidine/g body weight. The number of labeled nuclei per 100 cells (DNA labeling index, LI) was counted in terminal end buds (TEBs), terminal ducts (TDs), and alveolar buds (ABs) of the glands. The number of structures and the DNA-LI were correlated with the incidence of tumors at 22 weeks after DMBA. Pregnancy, with or without lactation, resulted in elimination of TEBs and reduction in the DNA-LI of TDs and ABs. These groups did not develop carcinomas. After the interruption of pregnancy the mammary gland contained numerous TEBs, with a high DNA-LI; 77% of these animals developed carcinomas, and all of them developed benign lesions. Therefore, while pregnancy and lactation protected the mammary gland from developing carcinomas and benign lesions by induction of full differentiation, pregnancy interruption did not elicit sufficient differentiation in the gland to be protective, and these animals were at the same risk as virgin animals treated with the carcinogen.     

Molecular analysis of rat mammary carcinogenesis: an approach from carcinogenesis research to cancer prevention

A rat strain carrying the human c-Ha-ras proto-oncogene is highly susceptible to chemically induced mammary carcinogenesis. All the transgenic rats develop preneoplastic mammary lesions within 20 days of an injection of N-methyl-N-nitrosourea, and mammary carcinomas appear within 8 weeks of treatment with a variety of chemical carcinogens. In this review, we summarize molecular aspects of mammary carcinogenesis in transgenic rats and the potential application of this model for studies of breast cancer prevention.     

Genetically engineered rodent models of mammary gland carcinogenesis: an overview

Breast cancer is a multifactorial disease that develops as a result of interactions among genetic, environmental, and hormonal factors. Multiple genetic derangements are believed to be involved in the pathogenesis of breast cancer, including the inactivation of tumor suppressor genes and/or the disregulation of proto-oncogenes. Age, hormones, and environmental factors further influence these genetic derangements. Spontaneous and chemically induced animal models of breast cancer have been limited in their usefulness. The advent of targeted gene mutations has allowed for a more specific exploration of the pathogenesis of breast cancer by creating mouse models that mimic single or multiple gene alterations found in human mammary tumors. The genes targeted in these models include mouse mammary tumor integration sites and genes that encode for growth regulators, signal transduction proteins, cell cycle proteins, and cell matrix proteinases. In this review, I summarize tumor morphology and the relevance of each model to the pathogenesis and progression of human breast cancer. These models have great potential for elucidating the multistep process of mammary gland carcinogenesis and for contributing to the identification of novel therapeutic targets.     

Fat droplet formation in rat lactating mammary gland and mammary carcinomas viewed by freeze-fracture.

The freeze-fracture morphology of the endoplasmic reticulum and Golgi apparatus membranes was analyzed in lactating rat mammary glands and in mammary carcinomas induced by 7,12-dimethylbenzanthracene or N-nitrosomethylurea. Membranes in close proximity with fat droplets present a spectrum of transformations, from the normal, wavy, particle-rich appearance of cytoplasmic membranes to rigid, particle-free bilayers. The following sequence of events is proposed for the biogenesis of fat droplets: (1) formation of particle-free areas in the endoplasmic reticulum and Golgi membranes; (2) apposition of the bilayered lipid membranes to the growing fat droplet; and (3) progressive conversion of the membrane lipids (or amphypathic lipid precursors) into triglycerides at the periphery of the fat droplet. Our results suggest that membranes are not only involved in the synthesis and secretion processes, but that some of their components are incorporated in the fat droplets and contribute to the secretory product itself.     

Site of lodination in rat mammary gland

The ability of the mammary gland to take up and organically bind radioiodide was studied in non-pregnant, pregnant, and lactating rats. Autoradiography was used to determine whether duct cells or alveolar cells are responsible for iodination in the rat mammary gland. Iodination was not detected in mammary glands from non-pregnant rats, but occurred late in the twelfth day of gestation and continued throughout pregnancy and lactation. Protein-containing vacuoles in alveolar cells and casein-like proteins in milk were the major sites where iodination occurred within the gland. Milk proteins in the lumens of ductules adjacent to alveoli were also iodinated. In contrast, ducts, myoepithelial cells, fat cells, blood vessels and other histological components of the gland did not show iodinating capability. Cytochemistry was also used to identify endogenous mammary peroxidase activity in the same glands, and it was found that the presence and location of this enzyme was correlated with the ability to iodinate.     

Nonrandom frequency distribution of mitoses in rat lobuloaveolar mammary gland epithelium.

A quantitative microscopic technique was employed to examine the distribution of mitotic activity in the rat mammary gland. The frequency distribution of mitoses per unit volume of lobuloalveolar mammary gland epithelium in virgin Lewis/Mai rats at each phase of the estrous cycle were determined and compared to the expected Poisson frequency distributions, assuming random mitotic activity. Both pooled data and data from individual rats were compared to expected Poisson distributions. At each phase of the estrous cycle, the pooled observed distributions deviated significantly from Poisson distributions. Sixty-seven percent (72/108) of the observed frequency distributions obtained from individual rats also deviated significantly from expected Poisson distributions. These data indicate a nonrandom distribution of mitoses in rat lobuloalveolar mammary gland epithelium. This observation suggests that local cell products and/or a variation in the extent of replicative synchrony of lobuloalveolar cell populations may determine in part the pattern of mitotic activity in this tissue. A nonrandom distribution of mitoses in mammary epithelium may have significance in relation to the genesis of hyperplastic and neoplastic lesions of the mammary gland.     

Cellular proliferation in the rat mammary gland during pregnancy and lactation

Cellular proliferation during different stages of pregnancy and lactation in the rat mammary gland was studied by use of autoradiography at the ultrastructural level or in combination with immunocytochemistry. The study consisted of two groups of animals, young 45-day-old rats undergoing their first pregnancy and old multiparous rats undergoing their third pregnancy. Both groups of animals were injected with [3H]thymidine, 2 hours before sacrifice. The timing of proliferative activity through pregnancy and lactation was similar in the two groups. A peak level of proliferation was observed on the 5th day of gestation and the rate of proliferation was at its lowest at the end of gestation. A second small peak was observed on the 3rd day of lactation. In virgin animals, both epithelial and myoepithelial cells showed a high level of proliferative activity. The epithelial cells continued to divide during all stages of their differentiation, throughout pregnancy and lactation. Myoepithelial cells showed active division in early pregnancy but their rate of proliferation declined as the cells acquired abundant cytoplasmic filaments for their contractile function. Intra-alveolar dendritic cells, which have been recently identified in the lactating rat breast, showed a high proliferative rate at the end of gestation.