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PubMed1989

Coexpression of intermediate filaments in squamous cell carcinomas of upper aerodigestive tract before and after radiation and chemotherapy.

Frozen sections of 48 squamous cell carcinomas and seven undifferentiated carcinomas of the upper aerodigestive tract were investigated immunohistochemically with monoclonal antibodies specific for keratin, vimentin, desmin, neurofilaments, and glial fibrillary acidic proteins. In nine squamous cell carcinomas (19%) and six undifferentiated carcinomas (86%) obtained before treatment coexpression of keratin and vimentin was detected in some tumor cells by double immunofluorescence studies. Nine squamous cell carcinomas expressed neurofilaments in scattered tumor cells. Coexpression of vimentin or neurofilaments was seen especially in the peripheral cell layer of the tumor nests and did not seem to correlate with the degree of differentiation. Three undifferentiated carcinomas additionally expressed desmin, and one tumor contained neurofilaments. Glial fibrillary acidic proteins were not detected. Increased coexpression of keratin with vimentin, desmin, or neurofilaments was seen in some tumors that were studied before and after radiation/chemotherapy, suggesting that the intermediate filament profile of tumor cells can be altered by external influences.

Carcinoma, Squamous Cell↗
PubMed1994

Intermediate filaments in rat pancreatic acinar tumors, human ductal carcinomas, and other gastrointestinal malignancies.

BACKGROUND/AIMS: Keratin is a member of the intermediate filament family in epithelial cells. Two-dimensional gel electrophoresis of different epithelial cells has shown 20 different keratin polypeptides. Therefore, mapping of the keratin polypeptides can be used to define a specific tissue. METHODS: Cytokeratin expression was investigated by using monoclonal antibodies in human surgical specimens and autopsy material of pancreatic, gastric, liver, and colon carcinomas and cholangiocarcinomas, and their metastasis to lymph nodes and liver was examined. In addition, rat acinar cell carcinomas were used to compare cytokeratin expression in ductal vs. acinar cell pancreatic carcinomas. RESULTS: Human pancreatic ductal carcinomas expressed keratins 7, 8, 18, and 19, whereas the majority of rat acinar carcinomas did not express keratins typical for ducts in rat pancreas. The keratin patterns of gastric and colon carcinomas were identical with keratins 8, 18, and 19. In contrast, hepatocellular carcinomas expressed the same keratin pattern as pancreatic acinar carcinomas with keratins 8 and 18, whereas cholangiocarcinomas expressed keratin 7, 8, 18, and 19, similar to pancreatic ductal carcinomas. Metastasis of pancreatic ductal and colon carcinomas retained their keratin patterns. CONCLUSIONS: Keratin polypeptide typing of unknown malignant cells can be a useful tool for cell identification.

Animals↗
PubMed1992

[Changes in the organization of the intermediate filament system of human fibroblasts in lysosomal storage diseases and their modeling].

The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.

Cells, Cultured↗
PubMed1989

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis.

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.

Animals↗
PubMed1992

A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human SW-13 cells.

Numerous studies have indicated that cytoplasmic intermediate filaments (cIFs) can associate with cellular lipids. To determine if these interactions might have functional consequences, we have studied the lipid metabolism of human SW-13 adrenal tumor cell lines that either contain vimentin-type cIFs (vim+) or lack any detectable cIF network (vim-). Although there were no significant differences in phospholipid or glyceride synthesis, vim- cell lines had elevated levels of cholesterol synthesis and decreased cholesterol esterification, compared with vim+ cells. These differences in cholesterol synthesis and esterification were found to be due to an impaired ability of vim- cells to utilize low density lipoprotein (LDL)-derived cholesterol, although receptor-mediated endocytosis of LDL and the capacity of these cells to esterify endogenously produced cholesterol were not affected. Expression of a mouse vimentin cDNA in stably transfected cell lines, derived from vim- cells, restored the capacity of these cells to utilize LDL cholesterol. The uptake and metabolism of [3H]cholesterol linoleate-loaded LDL showed that the impaired ability of vim- cells to esterify LDL cholesterol was not associated with an accumulation of cellular free cholesterol but rather an increase in the appearance of [3H]cholesterol in the culture medium. These studies indicate that in SW-13 cells, the intracellular movement of LDL-derived cholesterol from the lysosome to the site of esterification is a vimentin-dependent process.

Acetates↗
PubMed1998

Degradation of cytokeratin intermediate filaments by calcium-activated proteases (calpains) in vitro: implications for formation of Mallory bodies.

The degradation of rat hepatic intermediate filament (IF) proteins cytokeratin A (CK-A, 55-kDa) and cytokeratin D (CK-D, 48-kDa) by purified rat liver calcium-activated proteases (calpains I and II) was evaluated in vitro. Calpain-mediated IF proteolysis was monitored by SDS-PAGE and Western blotting with antibodies to CK-A and CK-D and compared to microtubule protein actin. Both cytokeratins underwent rapid yet limited proteolysis by calpain I and II. Despite the conserved nature of cytokeratins and limited substrate specificity for calpains, distinct fragmentation patterns were obtained for calpain I) CK-A, 46- and 43-kDa/CK-D, 41-, and 39-kDa; and calpain II) CK-A, 46- and 43-kDa/CK-D 41-kDa. The 46-kDa CK-A fragment was the predominant fragment for both calpains. Two-dimensional electrophoresis (IEF/SDS-PAGE) of CK fragments revealed the presence of classic "staircase" patterns consistent with endogenous proteases. Furthermore, proteolytic fragments showed a 2-D electrophoretic shift to lower pI suggesting that the limited hydrolysis occurred within the N-terminal arginine-rich region of CK, a region believed essential for IF interactions in vivo. Thus, calpains may represent an initial step in the turnover of these stable and long-lived proteins and as such, may be relevant to diseases characterized by abnormal disruption and bundling of IF such as formation of Mallory bodies in alcoholic hepatitis.

Actins↗
PubMed1988

Distribution of a novel 57 kDa intermediate filament (IF) protein in the nervous system.

A 57 kDa protein, that is not vimentin, is the major component of intermediate filaments (IF) obtained after 2 cycles of in vitro assembly from PC12 cells (Parysek and Goldman, 1987). By use of an antiserum to the 57 kDa protein, a cross-reacting antigen (of identical molecular weight) was detected on immunoblots of IF preparations and by immunofluorescence of various rat tissues. Immunolocalization studies on 3-4 micron frozen sections of tongue, small intestine, and adrenal gland showed bright labeling of nerve bundles and fine-caliber nerve fibers. The chromaffin cells and ganglion cells of the adrenal medulla also were labeled. In the nervous system, intense labeling was seen in small-caliber nerve fibers in sciatic nerve and spinal cord dorsal roots, as well as in the dorsal white columns and dorsal root ganglia. Of the ganglion cells, preferential labeling was seen in small-sized ganglion cells, whereas a monoclonal antibody to the 150 and 200 kDa neurofilament triplet (NFT) components labeled the large-sized ganglion cells. In the areas of the brain thus far examined with 57 kDa antiserum, there was labeling of components of cranial nerves and labeling of thin fibers in several areas, including the granular layer of the cerebellum and the corticospinal tract in the brain stem. For each tissue, adjacent sections treated with vimentin or glial fibrillary acidic protein antibody revealed labeling patterns distinct from that seen with either the 57 kDa or NFT antibodies. These results indicate that the 57 kDa IF protein is a neural IF component. Furthermore, this protein is distributed in only certain neuronal elements; these elements may be unified by an as yet unrecognized pattern of function in the nervous system.

Animals↗
PubMed1991

Tris-tricine and Tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system.

Human mesothelial cells contain a number of well defined intermediate filament proteins (IFs) that have been completely sequenced including vimentin and the cytokeratins (K7, K8, K18, and K19). The electrophoretic migration of these IFs was monitored as a function of second dimension gel buffer composition using various systems including Tris-glycine (pH 8.3 or 9.2), Tris-glycine with 20% methanol, Tris-borate, Tris-tricine, and sodium phosphate. All of the second dimension buffer chemistries yielded patterns of sufficient resolution to identify the major cytoskeletal proteins but differed in the relative mobilities of the IFs. Using gene sequence calculated molecular weight data, the major cytoskeletal polypeptides of human mesothelial cells were ranked from highest molecular weight to lowest molecular weight. This rank order of sequence calculated molecular weights was then compared to the rank order determined form the actual migration of the polypeptides in the different gel systems. With the Tris-tricine and the Tris-borate gel systems as well as gene sequence data, KS = vimentin greater than beta-tubulin = K7 greater than K18 greater than K19 greater than actin. With the pH 8.3 and 9.2 Tris-glycine systems, as well as the sodium phosphate gel system, the rank order of the polypeptides did not correspond to gene sequence data. Adding 20% methanol to the Tris-glycine system resulted in IF migration that more closely corresponded to the gene sequence derived data. Migration position of the IFs depended upon the temperature of the second dimension separation as well. In mesothelial cells, the migration of a total of 15-25% of the polypeptides was influenced by differing buffer systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Boric Acids↗
PubMed1988

Lack of lamins A and C in mammalian hemopoietic cell lines devoid of intermediate filament proteins.

Using immunofluorescence microscopy and immunoblot analysis, we have examined the composition of the nuclear lamina in several murine and human cell lines. Whereas it was shown that intermediate filament-positive Ehrlich ascites tumor and HeLa-S3 cells contain the three major mammalian lamin subspecies, only lamin B could be detected in several myeloid- and lymphoid-derived cell lines representative of distinct stages in hemopoietic differentiation but all devoid of cytoplasmic intermediate filament proteins. These included the murine plasmacytoma cell types MPC-11 and MOPC-31C, murine myeloma cells X63-Ag8.6.5.3 and human promyelocytic leukemia cells HL-60. Our results provide the first evidence that mammalian somatic cells capable of normal proliferation may lack both cytoplasmic intermediate filament proteins and a normal complement of nuclear lamins.

Animals↗
PubMed1997

Complexity of expression of the intermediate filaments of six new human ovarian carcinoma cell lines: new expression of cytokeratin 20.

Six permanent human ovarian carcinoma cell lines (OVISE, OVTOKO, OVMANA and OVSAYO from clear cell adenocarcinoma, and OVSAHO and OVKATE from serous papillary adenocarcinoma) were established from solid tumours. The cell lines have been in culture for 5-8 years, the passage number varying from 62 to 246. Immunohistochemical analysis has shown that five of the six cell lines express at least six cytokeratin (CK) polypeptides. OVISE and OVSAYO expressed CKs 6, 7, 8, 18, 19 and 15 and/or 16. OVTOKO was positive for CKs 7, 8, 18, 19 and 15 and/or 16. OVSAHO expressed CKs 6, 7, 8, 14, 18, 19 and 15 and/or 16. OVMANA expressed CKs 6, 7, 8, 18, 19, 20 and 15 and/or 16. OVKATE expressed CKs 6, 7, 8, 13, 17, 18, 19, 20 and 15 and/or 16. The expression of CK7, additional expression of vimentin, and clinical and histopathological findings enabled us to confirm that six cell lines had been established from primary ovarian cancers. Two of the six cell lines were positive for CK20, although CK20 was not expressed in the original tumours. The heterotransplanted tumours produced by CK20-positive cells also expressed CK20. This is the first report of ovarian carcinoma cell lines that express CK20 irrespective of their histological type. CK20 has been found in all colon carcinoma cell lines, but only in the mucinous type of ovarian tumours. These new ovarian carcinoma cell lines will therefore provide a relevant experimental system for elucidating the regulatory control mechanisms of intermediate filament expression.

Adenocarcinoma, Clear Cell↗
PubMed1985

A study of intermediate filaments (cytokeratin, vimentin, neurofilament) in two cases of Merkel cell tumor.

In two cases of Merkel cell tumor, the study of intermediate filaments, using monoclonal antibodies (vimentin, cytokeratin, neurofilaments), confirmed the double differentiation (neuroendocrine and epithelial) of this tumor as previously observed in histological, electron microscopical and histochemical analyses. Labelling of the tumor cells was positive with monoclonal antibodies against neurofilament proteins and cytokeratin.

Adenocarcinoma↗
PubMed1991

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.

Amino Acids↗
PubMed1983

Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments.

We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers averaging 33 and 35 kdalton/nm, respectively, which probably represent mature IF. Taken together with earlier results on IF of other subclasses, these results indicate that the average linear density of IF scales according to the average mass of their constituent subunits, both for "minimal form" and for mature IF. As underlying mechanism for this homology, we propose that the fundamental building-blocks of all these IF contain a common structural element whose packing within the various IF is likewise conserved and which specifies the overall structure. The variable amounts of mass in the nonconserved moieties account for the observed proportionality. This scheme fits with amino acid sequence data for several IF subunits that have revealed, as a likely candidate for the common element, an essentially conserved alpha-helical domain, contrasting with the highly variable sequences of their non-alpha-helical terminal domains.

Animals↗
PubMed1987

Characterization of intermediate filaments expressed by Ewing tumor cell lines.

The histogenesis of Ewing sarcoma is still controversial; we therefore studied the expression of intermediate filaments (IF) in cell lines derived from Ewing tumors since identification of IF in tumor cells is considered a reliable marker of tissue origin and differentiation. All nine lines studied expressed vimentin IF; in addition, a small number of Ewing cells from three lines expressed keratin filaments. After treatment with phorbol esters, a high percentage of cells from these three lines synthesize keratin IF identified by immunoblotting as keratin 8 and 18 polypeptides, which are expressed by single epithelia and epithelial cells in early embryonic development. Furthermore cells from a fourth line synthesize keratins after transplantation in nude mice. These data indicate that, under certain conditions, undifferentiated Ewing cells may acquire an IF phenotype related to that of epithelial cells.

Animals↗
PubMed1990

Cell differentiation in benign cutaneous fibrous histiocytomas. An immunohistochemical study with antibodies to histiomonocytic cells and intermediate filament proteins.

Seven benign cutaneous fibrous histiocytomas (BFHs) were investigated immunohistochemically by using a panel of antibodies to histiomonocytic cells and intermediate filament proteins. Immunoreactivity with antimacrophage antibodies was observed in all tumors in a significant but varying proportion of the tumor cells. All tumors were also positive for vimentin. In addition, two of the BFHs were positive for cytokeratin and one for desmin. The results suggest that BFHs either display a true histiomonocytic trait or, alternatively, contain a prominent histiomonocytic infiltration. Some BFHs may be related to smooth muscle cells as indicated by desmin positivity. The occasional occurrence of cytokeratin in BFHs could also be explained by smooth muscle cell differentiation.

Adult↗
PubMedDate not supplied

Characterization of disulfide crosslink formation of human vimentin at the dimer, tetramer, and intermediate filament levels.

We have investigated the structural interactions of individual molecules of human vimentin in the soluble state and in filaments. Oxidative crosslinking experiments were conducted with wild-type vimentin aimed at the single cysteine in the helical domain coil 2b, mutated cysteine-free vimentin, and derivatives engineered to carry cysteines in presumed d positions of the heptad amino acid repeats in coils 1a and 2b. We provide conclusive evidence that crosslinking of the cysteine 328 in wild-type vimentin, when in the filamentous or tetrameric forms, occurs outside of the coiled-coil dimer, i.e., between staggered dimer molecules. This occurs despite the close axial register of the dimers and contradicts previous deductions. The extent of crosslinking increases with temperature as well as with the concentration of the crosslinking reagent. We conclude therefore that the cysteines are not in an ideal position for crosslinking but that molecular motion is needed to enhance the reaction. The occurrence of collision complexes, which has been speculated in the literature, does not occur and cannot explain these results. Furthermore, using tailless vimentin with the corresponding mutations, we provide compelling evidence that in type III intermediate filament proteins exchange of individual chains between dimers occurs only if the proteins are incubated in urea at concentrations above 3 M. In 5 M urea, however, the exchange is completed within seconds. The same reaction occurs between human vimentin and mouse desmin at a comparable speed, indicating that both type III intermediate filament proteins have a high affinity for one another at the coiled-coil level.

Amino Acid Sequence↗
PubMed1986

Production of a monoclonal antibody reacting with vimentin, a structural protein of intermediate filaments.

This paper reports on the production of a monoclonal antibody (i-18) reacting with vimentin, the major structural component of intermediate filaments in cells of mesenchymal origin. The antibody was obtained following immunization with hamster fibroblasts and was selected for its ability to bind to the cytoskeleton fraction of the aforementioned cells. It decorated a perinuclear filamentous network characteristic of vimentin filaments in cells of mesenchymal origin of avian through human species. The specificity of the reagent was further ascertained on the basis of the sensitivity of the decorated filaments to colcemide. The strict antibody specificity for cells of mesenchymal versus epithelial origin was confirmed also in vivo on histological specimens from solid tissue. The i-18 monoclonal antibody precipitated a molecule of about 57 Kd from metabolically labelled cellular extracts. The broad cross-reactivity of this monoclonal antibody among different animal species, as well as its strict in vivo mesenchymal tissue specificity makes this antibody a useful reagent for both experimental and diagnostic purposes.

Animals↗
PubMed1990

The role of the vimentin intermediate filaments in rat 3Y1 cells elucidated by immunoelectron microscopy and computer-graphic reconstruction.

The three-dimensional arrangement of vimentin intermediate filaments (IF) was studied in 3Y1, rat fibroblastic cell line, to elucidate its biological role in the cell. While actin filaments were observed exclusively in the superficial part of the cell, vimentin IF were found to be abundantly present in the inside of the cell where microtubules were occasionally discovered. By whole-mount immunoelectron microscopy and computer-graphic reconstruction of serial thin sections, it was observed in more detail that vimentin IF are located very close to the nucleus, endoplasmic reticulum, and mitochondria. Vimentin IF were observed to be attached to these organelles laterally or terminally. Thus, we can reasonably assume that vimentin IF are major cytoskeletal structures deep inside the cell and that they play an important role in supporting the location of the organelles. This is the first report which has visualized the three-dimensional relationship between vimentin IF and the organelles of the cell.

Animals↗