Cytoskeletal Extracts Research Articles

Cytokeratins in the liver of the sea lamprey (Petromyzon marinus) before and after metamorphosis.

Cytokeratins of the liver were examined during the lamprey life cycle. This organ transforms, at metamorphosis, from a ductal larval structure to an aductal adult organ lacking the biliary passageways. Specifically, the relationship between liver morphology and cytokeratins 7, 18, and 19, and several unidentified cytokeratins was compared in larvae, transformers, and adults. Immunofluorescence staining showed that spatiotemporal cytokeratin distributions are cell-type-specific among hepatocytes, bile-duct cells, and gallbladder cells. In the larva, cytokeratins 7 and 19, and other unknown cytokeratins occur diffusely in the hepatocyte cytoplasm and prominently in the apices of hepatocytes near a bile canaliculus, so that collectively these cytokeratins form a pericanalicular ring. At mid-metamorphosis, the ring disappears, and cytokeratins are located predominantly in the periphery of bile-duct cells. In the adult, cytokeratin 19 has a sexually dimorphic distribution. Electrophoretic separations of cytoskeletal extracts of larval and adult livers have revealed larval cytokeratins 46, 49, 58, and 63.5, and adult male cytokeratins 46, 48, 49, 52.5, 58, 63.5, 64.5, 65, and 66. Immunoblotting has shown that certain cytokeratins are each related to at least two mammalian cytokeratins. We suggest that lamprey-specific liver cytokeratins exist, and that the metamorphosing liver can be used as a model for studying the relationship between cytokeratin organization and cell function.

Reconstituted human gingival epithelium: nonsubmerged in vitro model.

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

Glial cells of the lamprey nervous system contain keratin-like proteins.

Lamprey axons regenerate following spinal cord transection despite the formation of a glial scar. As we were unable to detect a lamprey homologue of glial fibrillary acidic protein (GFAP), a major constituent of astrocytes, we studied the composition of intermediate filament (IF) proteins of lamprey glia. Monoclonal antibodies (mAbs) were raised to lamprey spinal cord cytoskeletal extracts and these mAbs were characterized by using Western blotting and immunocytochemistry. On two-dimensional (2-D) Western blots, five of the mAbs detected three major IF polypeptides in the molecular weight (MW) range of 45-56 kD. Further studies were conducted to determine the relationship between the lamprey glial-specific antigen and other mammalian IF proteins. Antikeratin 8 antibody recognized two of the three polypeptides. Several of the glial-specific mAbs reacted with human keratins 8 and 18 on Western blots. Keratin-like immunoreactivity was found in all parts of the central and peripheral nervous systems in both larval and adult lampreys. The immunocytochemical staining patterns of glial-specific mAbs were indistinguishable on lamprey spinal cord sections. However, on brain sections, two distinct patterns were observed. A subset of mAbs stained only a few glial fibers in the brain, whereas others stained many more brain glia, particularly the ependymal cells. The former group of mAbs recognized only the two lower MW polypeptides on 2-D Western blots, but the latter group of mAbs recognized all three major IF polypeptides. This correlation is supported by the observation that the highest MW IF polypeptide has an increased level of expression in the brain relative to the spinal cord. Thus, in the lamprey, the glial cells of both spinal cord and brain express molecules similar to simple epithelial cytokeratins, but their IFs may contain these keratins in different stoichiometric proportions. The widespread presence in the lamprey of primitive glial cells containing keratin-like intermediate filaments may have significance for the extraordinary ability of lamprey spinal axons to regenerate.

Localized RNAs Are Enriched in Cytoskeletal Extracts of Drosophila Oocytes

Maternal mRNAs localized within the Drosophila oocyte encode positional information which specifies the pattern of the early embryo. With the goal of identifying molecules involved in RNA localization, we have developed a subcellular fractionation procedure which enriches for localized RNAs. Most RNAs and cellular proteins are solubilized by this method and are recovered in the supernatant fraction. However, five localized RNAs we examined are recovered in the detergent-insoluble pellet, despite temporal and spatial differences in their patterns of expression. These RNAs appear to be associated with a large, detergent-insoluble component of the oocyte. This association is specific for the oocyte proper, as localized RNAs in nurse cells and early embryos do not show this behavior. The fractionation behavior of these RNAs appears directly related to their localization in the oocyte, since fractionation and localization exhibit the same cytoskeletal and genetic requirements. The cortical cytoskeleton of the oocyte is a likely candidate for the localization substratum.

Phosphorylation States of Actin Filament Adenine Nucleotides in Detergent-Extracted Neuronal Cytoskeletal Fractions

High performance liquid chromatography of nucleotides from Triton X-100 cytoskeletal extracts has permitted analysis of the ATP and ADP content of actin filaments isolated intact from PC12 pheochromocytoma cells. We observed that the adenine nucleotide content matched the actin content of these cytoskeletal extracts, a finding consistent with the unit stoichiometry of nucleotide binding. Efficient assembly-linked ATP hydrolysis occurs in vivo, and based on a boundary hydrolysis model for nucleotide-promoted assembly, the observed ADP/ATP ratio indicates that the average microfilament in nonmuscle cells has 24 ATP-actin molecules at its growing end. Studies with NB41A3 neuroblastoma cells indicate that the ATP content of assembled actin filaments is about 3-4 times lower.

A neurofilament polypeptide and the glial fibrillary acidic protein share common epitopes in the variable region

A polyclonal antibody against the high molecular weight neurofilament polypeptide (NF-H) obtained from cytoskeletal extracts of bovine spinal cord reacted with NF-H, with the middle molecular weight neurofilament polypeptide (NF-M) and with a M r 51,000 polypeptide, but not with the low molecular weight neurofilament polypeptide (NF-L). The M r 51,000 polypeptide corresponded to the glial fibrillary acidic protein (GFAP), which forms the intermediate filaments of astrocytes. The polyclonal antibody affinity-purified with GFAP, reacted with both purified GFAP and NF-H. Digestion of NF-H with α-chymotrypsin was used to determine the recognition site of the affinity-purified antibodies. Only fragments of the tail domain of NF-H reacted with the antibody. We propose that common epitopes exist between the variable C-terminal domains of NF-H and GFAP.

Monospecific monoclonal antibodies to keratin 1 carboxy terminal (synthetic peptide) and to keratin 10 as markers of epidermal differentiation.

Monospecific antibodies to individual keratin polypeptides can be used to examine the tissue and cellular coexpression of members of keratin pairs. Monospecific monoclonal and polyclonal antibodies have been raised to keratins 1 and 10 using both crude cytoskeletal extracts and synthetic peptides. The tissue distribution of these keratins has been determined against a panel of freshly frozen normal tissues from humans, rodents and pigs. Epidermal expression has been examined in psoriatic plaques, and healing wounds, as examples of epidermal hyperproliferation. Cultured keratinocytes in monolayer (low calcium), stratified (high calcium), and complex cultures, transformed keratinocytes, and tumour cell lines, have been examined for the in vitro expression of these keratins. The sensitivity and precise localization of reactivity with these monospecific antibodies gives a highly accurate picture of individual cell expression. There is confirmation of coexpression of keratins 1 and 10 in epidermal and mucosal sites, and with keratin 16 in hyperproliferative states. These monospecific antibodies provide an important means of examining keratin expression in epidermal tumours and keratinizing disorders, and of seeking keratin mutations in cell lines and in skin diseases.

A scanning electron-microscopic study of the effects of methylmercury on the neuronal cytoskeleton

Cultured mouse neuroblastoma cells, Neuro-2a (c1300), were exposed to 2.5 and 5.0 μM methylmercury (MeHg) with or without the concomitant administration of 10 mM glutathione for 24 h. Treated cells viewed by scanning electron microscopy (SEM) appeared sponge-like and were surrounded by fragments of cytoplasmic processes. SEM cytoskeletal preparations of treated cells showed a collapsed matrix containing globular bodies. Microtubules were not seen in treated cells, but intermediate and microfilaments were observed. SDS-PAGE analysis of cytoskeletal extracts revealed bands ranging in size from 90 to 27 kDa in all treatment groups except in the 5.0 μM-MeHg-treated group. This group showed a single band co-migrating with actin. Cells exposed to glutathione alone or concomitantly with MeHg appeared similar to control cells under all experimental conditions. These observations suggest that MeHg may predominantly affect microtubules to form a condensation product.

Characterization of a panel of neurofilament antibodies recognizing N‐terminal epitopes

Peptides corresponding to sequences from the amino-terminal "head" regions of the low, middle, and high molecular weight neurofilament proteins (NF-L, NF-M, and NF-H) were synthesized by a modification of the Merrifield solid-phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS-1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters.

Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.

We have isolated and characterized a full-length cDNA clone encoding human loricrin. Curiously, this protein displays major differences from the recently described mouse loricrin (Mehrel, T., Hohl, D., Nakazawa, H., Rothnagel, J.A., Longley, M.A., Bundman, D., Cheng, C.K., Lichti, U., Bisher, M.E., Steven, A. C., Steinert, P.M., Yuspa, S.H., and Roop, D.R. (1990) Cell 61, 1103-1112). Although both proteins are glycine-serine-cysteine-rich, the sequences have not been conserved. However, analysis of the sequences reveals a common motif of quasi-peptide repeats of an aliphatic or aromatic amino acid residue followed by several glycine and/or serine and cysteine residues. These sequences are interspersed and flanked by short glutamine- or glutamine/lysine-rich peptides. Thus loricrins consist of a family of cell envelope proteins of highly variable sequences that nevertheless retain common structural elements. We show that unlike all other putative protein components of the cell envelope, loricrins are highly insoluble, due at least in part to cross-linking by disulfide bonds. Furthermore, we have isolated four peptides from purified human cell envelopes that contain recognizable loricrin sequences and which are cross-linked by the N epsilon-(gamma-glutamyl)lysine isodipeptide bond. The presence of such bonds thus affords an explanation for the extraordinary insolubility of loricrin by cross-linking to the cell envelope and can also explain the low steady-state levels of monomeric loricrin in cytoskeletal extracts of epidermis. This study represents the first report of this isodipeptide cross-link in a protein component of the cornified cell envelope. We propose a model for the structure of loricrin in which (i) the unusual glycine-serine-rich sequences adopt a flexible loop conformation, indexed on the recurrent aliphatic residues; (ii) inter- or intramolecular isodipeptide and disulfide cross-links induce or stabilize folding of loricrin so as to form a more compact rosette-like structure; and (iii) the presence of the flexible glycine-rich loops necessarily will impact a flexible character to the cell envelope and entire epithelium.

Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I.

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.

Alpha-internexin, a novel neuronal intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain

alpha-Internexin is a 66 kDa protein that copurifies with intermediate filaments (IF) from rat spinal cord and optic nerve. This protein is axonally transported in rat optic nerve along with the neurofilament triplet proteins in slow component a. Polymerization in vitro and distribution in vivo confirm that alpha-internexin is a neuronal IF. We raised 2 highly specific monoclonal antibodies to alpha-internexin which were applied to frozen rat brain sections and Western blots of cytoskeletal extracts. These results indicate that alpha-internexin is primarily an axonal protein found in most, if not all, neurons of the CNS. Immunoreactive proteins of similar molecular weight were found in cytoskeletal extracts of CNS tissue from several additional species, including mouse and cow. While the distribution of alpha-internexin as given by immunocytochemical methods is similar to that of low molecular weight neurofilament protein (NF-L) in the adult, its distribution in the embryo is far more extensive. At embryonic day 16, when the expression of NF-L is still limited to a relatively small number of cells and levels of expression are low, alpha-internexin is already found at much higher levels and in cells not yet expressing NF-L in detectable quantities. Similar results are found at embryonic day 12. These data suggest that neuronal IF in the developing nervous system contain a higher proportion of alpha-internexin than their adult counterparts, and that expression of alpha-internexin precedes that of NF-L in many or most neurons of the developing brain.

Lipids noncovalently associated with keratins and other cytoskeletal proteins of mouse mammary epithelial cells in primary culture

Lipids noncovalently associated with cytoskeletal (CS) proteins of mouse mammary epithelial cells (MMEC) grown in primary culture were analyzed. A CS fraction, preapred by subjecting MMEC to 1.5 KCl and 1% Triton X-100 in phosphate buffered saline (pH 7.4), was extracted 4–6 times with chloroform/methanol. Thin-layer chromatography (TLC) indicated that in comparison to whole cell lipid extracts, CS lipids consisted mostly of neutral lipids, especially triacylglycerols and, possibly cholesteryl esters. TLC analysis of chloroform/methanol CS extracts prepared from MMEC that had been incubated 4 h in [ 3H]palmitate revealed similar results, with the majority of label appearing in triacylglycerols and other neutral lipids. By autoradiography of sodium dodecyl sulfate polyacrylamide gels, all of the major CS proteins appeared labelled. The major regions of autoradiographic density of the gel were excised, the protein solubilized, and the lipids extracted and subjected to TLC. Most of the radiolabel appeared at the origin and ion front and resolved as neutral lipids. In contrast, keratins of 54–55 kDa and 46 kDa appeared to be associated noncovalently with a higher ratio of polar lipids (possibly phospholipids) to nonpolar (neutral lipids). Very little radioactivity, mostly neutral lipid, was associated with actin. A previously unidentified CS component of 30 kDa had primarily noncovalently bound neutral lipid. The results are discussed in terms of the apparent interactions of keratin filaments with the plasma membrane, nuclear envelope and cytoplasmic organelles.

A cytokeratin-immunohistochemical study of hepatoblastoma

Six cases of hepatoblastoma (five epithelial, one mixed epithelial-mesenchymal) were studied on serially cut cryostat sections, using a panel of monoclonal antibodies directed against individual cytokeratins, vimentin, and desmin, in an indirect immunoperoxidase procedure. Embryonic and fetal-type tumor cells expressed the “hepatocellular” cytokeratins no. 8 and 18 but, surprisingly, also expressed the “bile duct type” cytokeratin no. 19. In addition, two cases had a number of tumor cells which were also positive for the “bile duct type” cytokeratin no. 7. Cells embedded in osteoid-like material were immunoreactive for vimentin but also for cytokeratins no. 7, 18, and 19. Gel electrophoresis, and Western blotting of cytoskeletal extracts, confirmed the immunohistochemical data. The implications of these findings for the histogenesis of hepatoblastoma are discussed in this report.

Phosphorylation of the peripherin 58-kDa neuronal intermediate filament protein: Regulation by nerve growth factor and other agents.

Abstract Peripherin, a recently described member of the intermediate filament multigene family, is present in peripheral and certain central nervous system neurons as well as in cultured neuron-like cell lines, including PC12 pheochromocytoma cells. In PC12 cells, peripherin appears to be the major intermediate filament protein and its relative levels and synthesis are specifically increased during nerve growth factor (NGF)-promoted neuronal differentiation. The present study examines the phosphorylation of peripherin and the regulation thereof by nerve growth factor and other agents in cultured PC12 cells. Immunoblotting experiments using a peripherin-specific antiserum show five distinct isoforms of this protein in whole cell and cytoskeletal extracts resolved by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of these isoforms incorporate detectable quantities of [32P]phosphate during metabolic radiolabeling. The small proportion (approximately 6%) of total cellular peripherin that is extractable with 1% Triton X-100, does not appear to incorporate phosphate. NGF increases peripherin phosphorylation by 2-3-fold within 1-2 h of treatment. Epidermal growth factor and insulin have no effect. The relative levels of phosphorylated peripherin are markedly elevated (17-fold) by long term NGF exposure, and peripherin becomes a major cytoskeletal phosphoprotein. Activators of protein kinases A and C and treatment with depolarizing levels of K+ also enhance peripherin phosphorylation by 2-3-fold, in cultures both with and without prior long term NGF treatment. Evidence is presented that NGF regulates peripherin phosphorylation by a mechanism independent of protein kinases A and C and of depolarization. The large increase in phosphorylated peripherin brought about by NGF treatment suggests that this neuronal filament protein may play a role in the elaboration and maintenance of neurites. The presence of multiple independent pathways that acutely enhance peripherin phosphorylation indicates that this role is subject to modulation by extrinsic signals.

Anti‐200 kDa neurofilament antibody cross‐reacts with microtubule‐associated protein‐2 (MAP‐2)

An antibody prepared against purified 200-kDa neurofilament (NF) subunit was found to cross-react with a high molecular weight polypeptide present in rat brain cytoskeletal extracts starting from early postnatal development. This polypeptide was found to be highly concentrated in microtubule preparations and was specifically recognized as the 280-kDa microtubule-associated protein-2 (MAP-2). Our results, besides suggesting the existence of common epitopes between the heavy NF subunit and MAP-2, suggest the possibility of an interaction between two of the most important neuronal cytoskeletal organelles: NFs and microtubules.

Modulation of Keratin Expression in Type II Pneumocytes by the Extracellular Matrix

The expression of specific keratin intermediate filaments during differentiation of rat type II pneumocytes in primary culture on various matrices was investigated. Changes in keratin expression were assessed using a monoclonal antikeratin antibody, 24A3, known to react strongly with alveolar epithelial cells in injured lung. Type II cell differentiation was modulated by culture on extracellular matrices known to either accelerate or retard loss of differentiated morphology and metabolic function. During culture on a plastic or fibronectin-rich surface, loss of cell differentiation correlates with increased staining with 24A3 antikeratin antibody by indirect immunofluorescence and with increased abundance of a family of acidic 46,000-dalton keratin isoforms detected in two-dimensional polyacrylamide gels of type II cell cytoskeletal extracts. Loss of type II cell differentiation is retarded or prevented by culture on substrata of purified laminin or of EHS tumor-derived basement membrane (matrigel). 24A3-linked fluorescence and expression of the 46 kDa keratins are reduced in parallel, although at 7 days in culture on matrigel or laminin, keratin expression increases. The results show that changes in type II cell differentiation effected in primary culture by the extracellular matrix correlates with changes in expression of the 24A3-reactive keratins. Loss of differentiated shape and function favors expression of these cytoskeletal antigens, which may provide quantifiable markers of the type II to type I cell transition that occurs during alveolar remodeling.

Association of reovirus proteins with the structural matrix of infected cells

The interaction of reovirus with the cytoskeleton was investigated. The soluble components of infected cells were extracted with the nonionic detergent NP-40 in a physiological buffer, and a cytoskeletal extract was prepared from the detergent-insoluble fraction. We observed a selective association of viral-specified products with the cytoskeleton that was temporally controlled. Viral dsRNA appeared first on the framework but after several hours was found also in the soluble phase, encapsidated in mature virions. The initial viral translation products were associated exclusively with the soluble fraction, but concomitant with the appearance of dsRNA, viral proteins μ NS and σ 3 were detected on the cytoskeleton. Several hours later, all viral proteins were detected on the framework. Viral polypeptide μ NS exhibited unique spatial distribution patterns that correlated with viral assembly: Before dsRNA replication, it appeared as diffusely distributed protein; a few hours later, it was detected in punctate foci interconnected by tiny filaments; several hours later, it appeared as an extensive fiber network that traversed the foci. The other viral proteins were detected only within viral foci. μ NS remained bound to the matrix fraction after treatment with DNase, Mg 2+, and high salt, treatments that released other viral proteins. This distribution pattern was virus-directed because passage of virus at high multiplicity of infection induced mutations that prevented assembly of the μ NS-coated filament organization. A small fraction of the viral-specified products that included polypeptide μ NS, but not viral dsRNA, was coprecipitated from cytoskeletal extracts with proteins of mol wt ∼55K by monoclonal antibodies that recognized tubulin and vimentin. Disruption of this interaction by long exposure to colchicine did not prevent association of viral proteins or RNA with the matrix, indicating that viral products were not transported through these interactions. The results indicate that reovirus morphogenesis includes temporal and spatial controls not described previously.

Cytokeratin‐specific monoclonal antibodies are reactive with tumours of smooth muscle derivation. An immunocytochemical and biochemical study using antibodies to intermediate filament cytoskeletal proteins

Formalin fixed and paraffin wax embedded tissue from 13 leiomyosarcomas, three leiomyoblastomas, five leiomyomas and fresh tissue from one leiomyosarcoma and one leiomyoma was studied. Tumours were stained by the avidin-biotin-peroxidase technique with antibodies to desmin, vimentin, cytokeratins and S-100 protein. Neoplastic cells in all the cases were positive for desmin and vimentin but were negative for S-100; antibody CAM 5.2 gave strong reactivity with tumour cells of 12 leiomyosarcomas and all leiomyomas, but failed to stain the leiomyoblastomas; LP34 weakly stained two leiomyosarcomas and one leiomyoblastoma. Immunoblot studies using whole cell extracts of one leiomyosarcoma and one leiomyoma respectively, showed 55-58 kd and 35-38 kd bands reactive with monoclonal antibody CAM 5.2. However, no cytokeratin components were demonstrated when cytoskeletal extracts of the same tissue samples were immunoblotted with CAM 5.2. The implications of these findings for the diagnostic histopathologist are discussed.

Keratin protein identification in dimethylbenzanthracene‐induced hamster cheek‐pouch squamous cell carcinomas

Individual keratin proteins were identified in 10 DMBA induced squamous cell carcinomas (SCC) of the hamster cheek pouch. SDS-polyacrylamide gel electrophoresis of water-insoluble cytoskeletal extracts from the tumor tissue demonstrated alterations in the protein distribution normal for the site. Immunoblot analysis with a broad spectrum polyclonal antikeratin antiserum identified the keratins in the preparations and confirmed changes in their distribution in the tumor preparations. The major keratin species for all the tumor tissues ranged in molecular weight from 45 to 57kd. The normal tissues had keratins with molecular weights from 45 to 73kd. The absence of high molecular weight keratins was a prominent feature in all the cancers. The histologic appearance of the tumors was varied but the distribution of the keratins was not correlated with the various histologies. The results demonstrate that changes in keratin gene expression occur in DMBA-induced cheek-pouch carcinomas but the precise alterations in the keratin proteins from those seen normally are not predictable.