Cell types present in the mammary gland and their evolution were studied by labeling female rats with radioactive thymidine at various phases of the estrus cycle. The results suggest that the stem cells for mammary development are present in the terminal end buds and that they generate a lineage for lumenal cells and possibly a distinct one for myoepithelial cells. Growth and differentiation are controlled by both hormones and local factors.The mammary. gland is the site of very frequent cancers in humans as. well as in animals. The formation of these cancers appears to be influenced by the developmental changes occurring in the gland after birth: in female rats the inducibility of cancer by chemicals.is maximal at 50-60 days ofage, when the changes are at their peak; pregnancy, lactation, and hormone treatments affect the incidence of mammary cancer in women.and in animals. Therefore, a knowledge of the cell types present in the mammary gland and their evolution may be useful for understanding carcinogenesis.In the young rat the mammary gland consists of a system of branching ducts, which terminate with actively growing structures, the terminal end buds (TEB); the ducts.show branching (sometimes extensive) into ductules, also-called alveolar buds. Essentially only two cell types are clearly recognized in these structures: the epithelial cells that line the lumen of ducts and ductules and the myoepithelial cells that surround them. However, three types are recognized from nuclear morphology (1). In lactation the alveoli (which derive from ducts and ductules) are lined by functionally different cells, those producing milk, which also are surrounded by myoepithelial cells. The differentiation ofthe mammary gland is under the control ofmultiple hormones. Many observations show that estrogens. promote end-bud. development and duct elongation and that progesterone promotes duct enlargement and ductule formation and growth (2-7).We have taken advantage ofthe hormonal changes occurring during the normal estrus cycle of the mature virgin female rats to identify cell types with differential growth responses. The basic approach was to label the cells by an injection of [3H]dThd into the animal at defined phases of the estrus cycle and to detect the. DNA-synthesizing cells in histological slices by autoradiography (4-10). By allowing time periods ofvarious lengths between injection and removal of the gland for examination, pulse-chase experiments also were performed. These approaches identify several kinds of epithelial mammary cells and suggest a possible developmental pathway. The experiments were complemented by studies with various cell markers, which will be reported separately. MATERIALS AND METHODSForty-two female Sprague-Dawley rats were used, most as cycling adults (50-60 days of age) and some as immature animals (ca. 25 days). The estrus phases were determined by vaginal smears. Groups ofanimals were injected intraperitoneally with[3H]dThd (20 Ci/mmol; 1 Ci = 3.7 x 10W°becquerels), by using 0.5 mCi for a...
(1); they also, however, generate cells offusiform shape (fibroblast like) that do not form domes. It has been suggested that RAMA 25 cells are derived from a mammarv stem cell and that the fusiform cells may be the equivalent of the myoepithelial cells of the normal mammary gland (1). If so, the formation of fusiform cells in the cultures might be the in vitro equivalent ofa normal differentiation step and thus represent an interesting model system for studying differentiation in vitro.The present work was carried out to determine whether the cuboidal-to-fusiform transition observed in RAMA cultures indeed reproduces a differentiation step occurring in the animal. In this context, we asked two questions: (i) Is this transition a general, or at least a frequent, property of rat mammary cell lines and (ii) how does the distribution of available markers in the cuboidal and the fusiform cells in vitro compare with those of mammary epithelial and myoepithelial cells in vivo?The results show that the cuboidal-to-fusiform transition can be regarded as a general property of mammary cells. The transition, however, does not correspond to that of a stem cell to a myoepithelial cell. Rather, the data suggest that the fusiform cells are a new cell type. MATERIALS AND METHODSCell Lines. The RAMA 25 (cuboidal) and RAMA 4 and 29 (fusiform) lines were obtained from D. Bennett; the LA7 line (cuboidal) was a derivative of RAMA 25 (2). The F2408 line (rat fibroblast) was obtained from W. Eckhart.Tumor Induction. Tumors were induced in 50-dav-old virgin female Wistar-Furth rats by single intravenous injection of Nnitrosomethylurea (K & K) at 5 mg/100 g of body weight (3). The day after injection, the animals were put on a high fat diet (20% lard/36.5% cornstarch/7.5% dextrose/25% casein/5% alphacel/4% salt mixture/2% vitamin fortification). Seven ofthe 18 rats injected developed mammary tumors within a year.Isolation of RANI Cell Lines. Cells were isolated from a slowgrowing adenocarcinoma of the fifth right inguinal mammary gland, which appeared 7 months after injection. Histologically, this carcinoma conformed to a papillary adenocarcinoma. A fairly soft part of the tumor was cut into small fragments and digested with collagenase and hyaluronidase (4). The cell suspension was plated on a feeder layer of RAMA 29 (1) cells previously exposed overnight to mitomycin C (0.5 jig/ml). The medium was Ham's F-12 that had been conditioned for 24 hr by RAMA 29 cells and then centrifuged and filtered through a Nalgene filter unit (0.45-pL pore size) to remove cells. It was supplemented with 20% fresh Dulbecco's modified Eagle's medium/5% fetal calf serum/hydrocortisone (50 ng/ml)/insulin (50 ng/ml). In subsequent transfers, medium conditioned by LA7 cells (2) was sometimes used, without discernible difference in the results.Cloning. When the cultures became confluent, they were transferred bv the use of trypsin (250 mg/liter)/EDTA (90 mg/ liter) in Tris-buffered saline (pH 7.5). After three such transfers, the cells were cloned. A culture w...
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