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(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...

We describe reversible changes of intermediate filaments of fibroblastic cells associated with changes in the functional state of the cells. The changes are revealed by comparing the immunofluorescence patterns given by a monoclonal antibody and a polyclonal serum, both recognizing vimentin. The state of the filaments depends on culture density; this effect cannot be attributed to the nutritional state of the cells, their growth rate, or substances released into the medium. It seems to depend mainly on the aggregation of filaments during strong cell movements. The possible significance of these findings for the functional role of intermediate filaments is discussed.Intermediate filaments, 8-10 mm in diameter, are present in most cells. They are made up of different proteins that share some common epitope (1). In fibroblastic cells, the main constituent of the filaments is vimentin (2, 3), with a molecular weight of 58,000; smaller related peptides (molecular weight, >40,000) are also observed (4, 5) and are thought to be cleavage products of vimentin by a specific protease (6). Vimentin molecules associate to form protofilaments: various models have been proposed for this association (7,8). Protofilaments are associated in bundles; in muscle cells and nucleated erythrocytes, they are crosslinked by another protein, synemin (9, 10); protofilaments are aggregated by cationic proteins (11). Vimentin undergoes phosphorylation, which may have a regulatory significance (12,13). Under the action of colchicine, intermediate filaments collapse into a coiled mass near the nucleus (14); collapse is also caused by intracellular injection of a monoclonal antibody to a common antigen of the intermediate filaments (15) and, in lymphocytes, by capping of surface molecules (16). This and other evidence shows that intermediate filaments are connected to the plasma membrane (10, 17). They also form a cage around the nucleus (18) and appear to be connected to the nuclear membrane (19 for 2 hr; they were then fixed in neutral formalin for 20 min, washed in phosphate-buffered saline, and dried. They were overlaid with photographic emulsion, developed after 1 wk, and counterstained with hematoxylin.Characterization of Material Stained by Hybridoma TllA9e. The material stained by Tl1A9e was characterized by the immunological blotting technique. Cytoplasmic extracts of sparse or confluent NIH 3T3 cultures were prepared (23) by lysing monolayers in 1% (wt/vol) deoxycholate/1% (vol/vol) Nonidet P-40/1 mM phenylmethylsulfonyl fluoride/7 mM NaCI/1 mM MgCl2/100 mM NaF/7 mM Tris-HCI, pH 7.4. The lysates were centrifuged at 4°C for 10 min at 3,000 X g to sediment nuclei. The supernatants were run on 10% NaDodSO4 gels and blotted to nitrocellulose paper (Schleicher & Schuell; BA85, 0.45 ,tm) (24). The nitrocellulose paper was incubated in undiluted culture supernatant of T1lA9e hybridoma or a 1:30 dilution of Tl1A9e mouse serum. Bound TllA9e was located by using either l"I-labeled goat anti-mouse Ig or rabbit anti-mouse IgM followed...

Cells of a mammary cell line isolated from a DMBA-induced rat mammary carcinoma undergo differentiation in vitro . A reversible differentiation leads to the formation of two types of microstructure (domes and ridges); this paper is concerned with the mechanism of dome formation. This differentiation is initiated by inducers, some of which are generated in the cultures and act locally; their effect is strongly dependent on cell concentration and requires hydrocortisone. There are, in addition, exogenous inducers as well as inhibitors. In the pathway to dome formation important roles are played by cAMP (probably both intracellular and extracellular), the organization of the cytoskeleton, and the Thy-1 antigen. The pathway and the significance of the phenomenon for mammary gland development are discussed.

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