Identification of cytokeratin antigens in Novikoff ascites hepatoma.

Abstract

Further characterization of three polypeptide antigens present in Novikoff ascites hepatoma (molecular weights 39,000, 49,000, and 56,000-p39 p49, and p56, respectively), has established the identity of these proteins as cytokeratins. Two-dimensional gel electrophoresis revealed that each of the three proteins exists in several isoelectric forms, all characteristic of the keratins. The antigens p39 and p56 had amino acid compositions compatible with this family of proteins, and immunofluorescence microscopy localized the antigens on elaborate filament arrays indicative of the cytokeratins. Antibodies monospecific for the p39 antigen, purified by using affinity chromatography, were used to demonstrate that protein p39 was specific for Novikoff hepatoma cells. These results suggest that the cytokeratins contain cell-specific species that can be useful as markers for differentiation and neoplasia. (39,000,49,000, and 56,000-p39, p49, and p56, respectively) was originally purified from chromatin (3) and, in contrast to the higher molecular weight group, was also found in cytoplasmic fractions from Novikoff cells (4).The cytoskeletal fiber elements of most eukaryotic cells consist of the microtubules, microfilaments, and intermediatesized filaments. The latter system of filaments is collectively referred to as "intermediate filaments" because their size (6-11 nm) is between the thin microfilaments (5-6 nm) and the thick microtubules (20-25 nm). Examples ofthis category include the neurofilaments and the glial filaments, the 10-nm filaments of muscle, the keratin or prekeratin filaments of keratizing epithelia, and the desmosome-associated tonofilaments of most epithelial cells (5-15). Ofparticular relevance to this report are the keratins that are polypeptide components of the latter two groups. A number of reports have shown the specificity of the keratins not only to epidermis but to nonkeratizing epithelial cells as well (14,(16)(17)(18)(19)(20)(21)(22).The solubility properties ofthe keratins (23-26) are very similar to those we observed for the Novikoff hepatoma nuclear antigens during our purification attempts with these proteins. Moreover, the molecular weights ofthe denatured antigens are very similar to those reported for keratins or prekeratins from a number ofsources (14,15,(26)(27)(28)(29). In this report we have tentatively identified the Novikoff antigens p39, p49, and p56 as members ofthe family ofkeratins, on the basis oftheir preparative properties, isoelectric points, and amino acid compositions. We have used immunofluorescence microscopy to confirm their intracellular location on keratin-like intermediate filaments. Furthermore, the immunological specificity for at least thep39 antigen suggests that this polypeptide may offer distinct possibilities as a probe for neoplasia. MATERIALS AND METHODSNovikoff ascites hepatoma was maintained by weekly transplantations in male Sprague-Dawley rats. The hepatoma cells were washed three times with standard saline citrate as described (3) before us...

Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: cultured rat PC12 cells combine neuronal and epithelial features.

“…When preparations from PC12 cytoskeletons enriched in IFs were examined by one-(not shown) and two-dimensional gel electrophoresis (Fig. 6, a-c), two major polypeptides of Mr 55,000 and 49,000 were seen which were identical in positions to cytokeratins A and D as originally described in rat hepatocytes (Franke et al, 1981a) and intestinal cells (Franke et al, 1981d) as well as in various rat hepatoma cell cultures (Franke et al, 1981b,c;Schmidt et al, 1982;Venetianer et al, 1983). These two polypeptides, which occur in equimolar amounts in heterotypic tetrameric complexes (Quinlan et al, 1984), are the rat polypeptides equivalent to human cytokeratins Nos.…”

Intermediate filaments of the cytoskeleton in glandular cells of the rat fundic mucosa: Immunofluorescence and electron microscopy study

“…It has been clearly demonstrated that IF of epithelial cells grown in vivo or in culture are biochemically and immunologically related to prekeratin (Franke et al, 1978d;Sun et al, 1979). Based on this fact, antiprekeratin antiserum has been widely used to study the three-dimensional organization of this system of filaments in epithelial cells by immunocytochemical techniques (Franke et al, 1978a,b,c,d;Sun et al, 1979;Franke et al, 1980;Osborn et al, 1980;Asch et al, 1981;Dabike et al, 1981;Eckert and Daley, 1981;Franke et al, 1981a,b;Horwitz et al, 1981;Wu and Rheinwald, 1981;Banks-Schlegel, 1982;Schmidt et al, 1982;Staufenbiel and Deppert, 1982). The biological function of IF and the way they interact with other fibrillar systems is still unclear; however, they have been related to functions such as locomotion of cultured cells, organelle attachment and movement, and determination of the cell shape (Goldman et al, 1979).…”