Abstract. In a rat mammary epithelial cell line, LA-7, cytokeratin bundles recognized in immunofluorescence by a monoclonal antibody (24B42) disappear after trypsinization of cultures and are gradually reformed after replating.We have followed the time course of cytokeratin filament reappearance by growing cells in low calcium medium (0.1 mM), which prevents desmosome formation, and then shifting to high calcium (1.8 mM) to start the process. By fixing the cells at various intervals and staining them in immunofluorescence for 24B42 cytokeratin and for desmosomal proteins, we found that cell to cell contact and desmosome formation are prerequisites for keratin filament formation in these cells. EGTA treatment, by disassembling desmosomes, causes the cytokeratin filaments to disappear and the 24B42 protein to pass into a soluble form in this cell line, as ascertained by 100,000 g fractionation and immunoenzymatic assay. Cycloheximide treatment also causes cytokeratin filaments to disappear, indicating that protein synthesis is needed for normal filament maintenance. In another related cell line (106A-10a) and in HeLa cells, trypsinization and EGTA exposure do not cause a complete loss of 24B42 immunofluorescence, although distinct filaments disappear, indicating the presence in these cells of different organizing centers, besides desmosomes, for cytokeratin bundle formation.LA7 cells therefore seem to have a cytokeratin system strictly dependent on the presence of desmosomes, which act as an organizing center for filament assembly. 106A-10a cells (also rich in desmosomes) and HeLa cells (showing instead a reduced number of desmosomes) have a cytokeratin system partially or totally independent from that of desmosomes, with different organizing centers.

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...

We have isolated two monoclonal antibodies to cytokeratins and determined their cell specificities. They display interesting localization within the rat mammary gland. One (lA10) shows specificity for myoepithelial cells; the other (24B42) is specific for lumenal cells at various stages of development. These two monoclonal antibodies and three others to cytokeratin previously isolated were used in conjunction with antibodies to myosin and collagen IV to confirm and extend our previous findings on epithelial cell types and development within the mammary gland.Antibodies, especially monoclonal antibodies, are very useful for identifying cell types using immunocytological methods. This approach has been used in our laboratory for studying the pathways of cell differentiation in the mammary gland (1). Antibodies to cytokeratins are especially useful in this context because there are many different kinds of cytokeratins, which display considerable cell specificity.The cytokeratins are expressed by a multigene family that is developmentally regulated (see refs. 2 and 3 for reviews). They are expressed mostly in epithelia but also in mesothelial cells (4). Every tissue has a complex pattern of these peptides, which can be distinguished by their isoelectric point and electrophoretic mobility in gels. Many tissues share the same peptides (5, 6). The distribution of the peptides, ascertained by either extraction or by immunohistochemical methods using polyclonal antisera, differs in different organs or in different parts of the same organ-for instance in different layers of the epidermis. Similar peptides are found in different mammalian species. Using monoclonal antibodies (7), the existence of both peptide-specific and common epitopes was observed (8). Monoclonal antibodies with considerable specificity (8-10), as well as some with broad crossreactivity (11), were identified by immunohistochemistry. Using polyclonal antisera to partially purified peptides it was shown by immunohistochemistry that cytokeratins are not uniformly distributed in the mammary gland (11-13). Quantitative differences were observed between myoepithelial and epithelial cells, and in the latter cells, between ductal and secretory alveolar cells (12)(13)(14).Cytokeratins show considerable crossreactivity among species. For instance, Lane (15) has shown that monoclonal antibodies to cytokeratins from PtK1 cells recognize cells in the rat mammary gland. We have found that one of these antibodies (Le6l) is highly selective for certain cell types (1). By taking advantage of this crossreactivity, we have prepared a set of monoclonal antibodies from mice immunized with total cow muzzle keratin. We report here on the properties of two antibodies that have considerable cell specificities. These antibodies are very useful for studying the cell types present in the rat mammary gland. They allow a further refinement of the pathway of cell differentiation in this organ. We also compare these monoclonal antibodies with three others from mice immunized w...

SUMMARYPristane-induced arthritis (PIA) is a murine disease resembling rheumatoid arthritis (RA) which is characterized by autoimmune responses to joint tissues. To identify the range of potential antigens targeted in PIA, proteins from arthritic or normal joint extracts were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and systematically screened for the ability to react with either serum IgG, or cultured splenic T cells, obtained from healthy or arthritic mice. Extracts from both normal and arthritic animals contained multiple proteins that were capable of reacting with murine serum IgG in immunoblotting experiments. In healthy controls, more bands were identified in extracts prepared from 30-week-old mice than from 8-week-old animals, but the widest range of proteins bound were derived from arthritic joints. Furthermore, the sera from PIA-positive mice reacted with more bands from each of the extracts than did normal sera. Fractionated extracts prepared from healthy joints failed to stimulate the in vitro proliferation of splenic T cells from either normal or arthritic animals. When arthritic joint components were screened, T cells from healthy mice responded weakly to some fractions, but multiple fractions elicited strong proliferation by T cells from mice with PIA. A band of apparent molecular mass 60 000 was the protein most commonly bound by serum IgG from arthritic mice, and the corresponding fraction stimulated the highest responses by T cells from PIA-positive animals. These results are consistent with the notion that the 60 000 MW mammalian heatshock protein is an important antigen in PIA, but that the autoimmune response diversifies with the development of arthritis to target multiple joint components.

To work toward an understanding of the mechanisms of cancer progression, we have followed the changes in expression of several genes in rat mammary carcinomas during the development of invasion. Tumors were induced by N-methyl-N-nitrosourea and serially transplanted to isogeneic rats. Gene expression in individual cells was followed by immunofluorescence microscopy. We show that invading cells do not express the same genes as the primary tumor or normal adult mammary gland but are similar to the primitive mammary duct cells present in late embryos. In the most advanced tumors, the most fundamental epithelial characteristic, polarity, is lost. Relative to the mechanism of invasion, we observe that the basal lamina, often thinner than normal, always coats the strands of invading cells and may be entirely absent from single invasive cells only. These observations suggest that the basal lamina ultimately disappears because of reduced expression of the genes or the failure to assemble the components. In these tumors it is the progressive alteration in gene expression rather than the enzymatic breakdown of the basal lamina that leads to invasion. These results show that the most important aspect of cancer progression is the progressive alteration of gene expression. They also raise questions about the mechanism that causes tumor cells to retrace the steps in their differentiation.