An attempt has been made to put forward a unifying hypothesis explaining the role hormones play in the genesis of mammary cancers of different phenotypes and genotypes in mice, rats, and humans. Most mammary cancers in these species originate in luminal mammary epithelial cells lining the mammary ducts and alveoli. These cancers are histopathologically diverse and are classified on the basis of growth requirements as hormone-dependent or hormone-independent tumors. In most strains of mice, mammary cancers at the time of detection are largely of the hormone-independent type; in rats, almost all mammary cancers are hormone-dependent, while humans have both phenotypes. In spite of these differences, in vivo studies show that hormones (ovarian and pituitary) are essential for luminal mammary epithelial cell proliferation and also for the development of mammary cancers of both hormone-independent and hormone-dependent types. This article, based on our extensive in vivo and in vitro studies and on current literature, proposes a model to explain the central role of hormones in the genesis of all types of mammary cancers. The model attempts to address the following questions: (i) how hormones regulate luminal mammary epithelial cell proliferation, (ii) why hormones are required for the genesis of mammary cancers of all phenotypes and genotypes, including those which are always classified as hormone-independent tumors, and (iii) why the three species (mouse, rat, and human) have consistently different ratios of hormone-dependent to hormoneindependent tumors.Breast cancer is the most common cancer in American women and is second only to lung cancer as a cause of cancer deaths. Despite advances in the early detection and treatment of breast cancer, the morThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tality rate from breast cancer has not changed appreciably in the last few decades. Unfortunately, in contrast to the known increased risk for lung cancer associated with cigarette smoking, no single major etiological agent is known for breast cancer. Epidemiological studies have identified various risk factors, including hormone-related factors, but taken together, these risk factors seem to account for only a third of breast cancer cases (1, 2). A search for appropriate preventive measures will require a thorough understanding of the etiology of this disease and of the mechanisms involved in the genesis of breast cancers of different phenotypes and genotypes.Animal and human studies indicate that an appropriate hormonal milieu is required for proliferation of mammary epithelial cells and is also prerequisite for breast carcinogenesis. This article, based on our extensive in vivo and in vitro studies and on an analysis of the literature, will put forward a unifying hypothesis that offers an explanation of the role that hormones may play in the genesis of ma...

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Full term pregnancy early in life is the most effective natural protection against breast cancer in women. Rats treated with chemical carcinogen are similarly protected by a previous pregnancy from mammary carcinogenesis. Proliferation and differentiation of the mammary gland does not explain this phenomenon, as shown by the relative ineffectiveness of perphenazine, a potent mitogenic and differentiating agent. Here, we show that short term treatment of nulliparous rats with pregnancy levels of estradiol 17␤ and progesterone has high efficacy in protecting them from chemical carcinogen induced mammary cancers. Because the mammary gland is exposed to the highest physiological concentrations of estradiol and progesterone during full term pregnancy, it is these elevated levels of hormones that likely induce protection from mammary cancer. Thus, it appears possible to mimic the protective effects of pregnancy against breast cancer in nulliparous rats by short term specific hormonal intervention.

Administration of a single i.v. injection of 50 mg N-methyl-N-nitrosourea (MNU)/kg body wt to 50- to 60-day old virgin rats, 120-day-old virgin rats, and 120-day-old parous rats (Sprague-Dawley; n = 18-37) resulted in a high incidence of mammary carcinomas in the virgin animals (97.3% in 50- to 60-day-old virgin rats; 75.0% in 120-day-old virgin rats), but mammary carcinomas did not develop in the parous rats. The concentrations in serum of various mammotropic hormones were measured in identical groups of rats at the time of MNU treatment. Growth hormone (GH) concentration was significantly reduced in parous rats, as compared with young or age-matched virgin rats. The concentrations of prolactin, 17 beta-estradiol, progesterone, corticosterone and thyroxine were not significantly altered in the parous rats compared to the two groups of virgin animals. Histological examination of the mammary glands from the three groups of rats showed that the epithelia of the parous animals were in a stage of regression, whereas the mammae of the young virgin rats showed the highest degree of lobulo-alveolar development. The levels of estrogen receptor (ER), epidermal growth factor (EGF) receptor (EGF-R) and GH receptor (GHR) in the mammary glands of the animals were also measured. We found a reduction in the receptor levels for both estrogen and EGF in mammary tissues from parous animals. Receptors for GH were present in normal mammary tissues from both virgin and parous rats. We hypothesize that the reduction in the circulating concentration of GH caused the reduced susceptibility of parous rats to mammary carcinogenesis possibly by decreasing the levels of ER and/or EGF-R in the mammary gland.

We have develo ed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H] The limited in vitro growth capacity of mammary epithelial cells in conventional primary culture in tissue culture dishes is a well-known phenomenon. The cells generally undergo a few rounds of division, but proliferation cannot be sustained nor can these cells be passaged. Mouse mammary epithelial cells cultured at low density become flattened and multinucleated, and rarely do they attain confluence (1, 2). Cells plated at high density are maintained well but little growth is ever achieved.Most studies to date dealing with proliferation of mammary epithelial cells in vitro have used established cell lines adapted to grow in conventional monolayer culture (3-6). Considerable effort is being devoted in several laboratories to analysis of hormone and drug sensitivity in these selected cell lines. An inherent limitation of this approach is that such lines may not be representative of the original cell population. Therefore, it is of considerable importance to improve the conditions for culture of primary cells. Our demonstration of differentiated function in mouse mammary cells cultured on floating collagen gels, as indicated by levels of casein (7), mammary tumor virus (8), and prolactin receptor (9), has prompted us to examine collagen as an appropriate substrate for growth. The most encouraging results were obtained when mammary cells were embedded within the collagen gel. The present study describes (10), and normal mammary gland from C3H/Crgl and BALB/cNIV/Crgl mice were dissociated by a modification of a previously described method (11,12). Briefly, finely minced mammary tissues were placed in 125-or 250-ml erlenmeyer flasks containing 0.1% collagenase (CLS III; 120-150 units/mg; Worthington) in Hanks' balanced salt solution (10 ml/g of tissue) and swirled on a gyratory water bath shaker (model G76, New Brunswick) at 120-150 rpm at 370C for approximately 90 min or until the suspensions were uniform without macroscopic lumps. The suspension was passed through Nitex cloth (mesh size, 150 ,um); the cells were collected by centrifugation at 80 X g for 5 min, and washed twice with Hanks' solution. The resulting preparation consisted mainly of small clumps of cells. Cell number was estimated by mixing 1 vol of cell suspension with 9 vol of 0.02% crystal violet in 0.1 M citric acid and counting stained nuclei in a hemocytometer. Dissociated human mammary epithelial cells were obtained from excised tissue (reduction mammoplasties and mastectomies) by modification of the above procedure and subsequent gradient centrifugation (details will be reported elsewhere).Culture Procedure. Collagen solution and ge...

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Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level. Preparation of Floating Collagen Membranes. The collagen solution and collagen gels were prepared according to the method of Michalopoulos and Pitot (11) as modified by us (7).Eighteen-twenty hours after the cells were seeded onto collagen-gel-coated dishes, the gels were removed from the plastic substrate by rimming the gels with a scalpel blade and gently

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