Most cell types start expressing vimentin when brought into tissue culture. Using both vimentinexpressing (HeLa) and vimcntin-negative (MCF-7) epithelial cell lines, we have identified the cisregulatory DNA elements involved in this process. Sequenccs located 1 . I -0.6 kb upstream of the vimentin transcription-initiation site strongly enhance expression in HeLa cells, but are silenced in MCF-7 cells. Other regulatory elements in the vimentin promoter (an enhancer 3.2. -2.6 kb upstream and a minimal promoter region including the CAAT-box) are potentially active in both cell types, but are silenced by the 0.5-kb fragment in MCF-7 cells. Deletion of this fragment restores transcriptional activity of a transfected vimentin promoter. Our data indicate that a double AP lljun-binding site present in the 0.5-kb fragment mediates the induction of vimentin expression in cultured epithelial cells, while silencing sequences located within the same fragment are responsible for the absence of vimentin expression in MCF-7 cells.In contrast to MCF-7 cells, a transfected vimentin promoter and gene arc transcriptionally active in the vimentin-negative epithelial cell line T24. Transfection studies show that type-111-intermediatefilament expression is not impaired at any level in these cells.Upon traasfection and expression of a desmin construct in T24 cells not only desmin, but also vimentin was detected. Both proteins assembled into intermediate filaments. This induction of vimentin expression appeared to be regulated at the post-transcriptional level.Intermediate filaments (IF) represent a group of cytoskelctal structures of approximately 10 nm in diameter which, along with microtubules and microfilamcnts, occur in the cytoplasm of virtually all mammalian cells. The IF protcins are encoded by a large multigene family and can be divided into six different types on the basis of gene structure and homology [l-41. Apart from the lamins, the different types of IF subunits are expressed in a developmentally regulated and tissuespecific fashion. This highly conserved specificity of expression suggests that each type or combination of subunits plays an important role in ccllular differentiation [l -61.The vimentin-expression pattern is more complex than that of the other IF subunits. In adult tissues, vimentin is mainly expressed in cells of mcsenchymal origin. Moreover, during differentiation of a variety of cell types vimentin expression is both positively and negatively regulated [l -3, 61. Depending on cell type, vimentin expression can precede the expression of the IF subunit specific for a particular tissue, it can be transiently coexpressed during development, or it can

Abstract. The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non‐epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates.The DNA content of the GO/Gl fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage I1 and higher all had a higher DNA content than that of normal endometrium.The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and postmenopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage I1 and higher had intermediate values compared to those of carcinomas below stage 11.The nuclear protein/l)NA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for postmenopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage I1 and higher.