Cytokeratin expression in simple epithelia

Differentiation

Differentiation (1986) 33: 69-85

0Springer-Verlag1986

Cytokeratin expression in simple epithelia 111. Detection of mRNAs encoding human cytokeratins nos. 8 and 18 in normal and tumor

cells by hybridization with cDNA sequences in vitro and in situ Rudolf E. Leube

’,Franx X. Bosch ’,Valentino Romano ’,Ralf Zimbelmann’,Heinz Hofler ’, and Werner W. Franke

’ Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG

* Institute of Pathology and Anatomy, University of Graz, Graz, Austria Abstract. We describe cDNA clones of mRNAs encoding human cytokeratins nos. 8 and 18, and the amino acid sequences deduced from their nucleotide sequences. Human cytokeratin no. 8 is a typical cytokeratin of the basic (type 11) subfamily, which is highly homologous to the corresponding bovine and amphibian (Xenopus lueuis) proteins; however, unlike the amphibian protein, it does not contain glycine-rich oligopeptide repeats in its carboxyterminal ‘tail‘ domain. Comparison with the reported amino acid sequences of two fragments of human ‘tissue polypeptide antigen’ (TPA), a widely used serodiagnostic carcinoma marker, revealed sequence identity, indicating that this serum component is derived from the intracellular cytokeratin no. 8 present in diverse kinds of epithelia and epitheliumderived tumors. Human cytokeratin no. 18 is very similar to the corresponding murine protein but contains two additional blocks of 4 and 5 amino acids in the ‘head’ portion. These cDNA clones and the RNA probes derived therefrom were used to detect specifically mRNAs by Northem-blot assays of RNAs from various carcinomas and cultured carcinoma cells. Using in situ hybridization on frozen sections of tumor-containing tissues, notably lymph nodes containing metastatic breast carcinoma, we were able to demonstrate the specificity and sensitivity of this procedure. The potential value for cell-biological research and pathology of being able to detect a mRNA encoding a given cytokeratin polypeptide in situ is discussed.

Introduction The identification and classification of a given cell according to certain types of differentiation (‘cell typing’) is an important criterion in histology and in work with cultured cells. Cell typing using differentiation markers has also become a valuable tool in pathology, notably in tumor diagnosis. In particular, the finding that different members of multigene families of proteins, including major proteins of the cytoskeleton, are selectivelyexpressed in different routes of cell differentiation has resulted in a long and still-growing list of differentiation markers. Among these, the cytoskeletal family of proteins forming intermediate-sized (7-12 nm) filaments (IFs) is of special interest, as the cell-type-specific expression of certain I F proteins is maintained in malignantly transformed cells and can thus be used to define the state of differentiation of normal as well as tumor cells

*

To whom offprint requests should be sent

[2429,35,871. In fact, antibodies to I F proteins are already in wide use in histology and pathology (e.g. [26, 35, 39, 43, 47, 55, 80, 87, 93, 94, 1121). Among the various I F proteins, the cytokeratins are epithelium-specific and therefore of special interest, both in developmental biology and in pathology [24-26, 108-1 lo]. These proteins represent a large multigene family comprising, in human tissues, at least 19 polypeptides which are differentially expressed in epithelia and epithelium-derived tumors [lo, 31, 35, 81-83, 1091. According to their immunological cross-reactivities, peptide maps, and genesequence relationships, the cytokeratins can be grouped into two subfamilies, i.e., acidic (type I) and basic (type 11). At least one representative of each subfamily is necessary to form the fundamental heterotypic cytokeratin subunit complex and the resulting IFs [lo, 12, 36, 42, 49-51, 63, 81, 91, 92, 102, 106, 109, 1151. As various epithelia and carcinomas express different sets of cytokeratin polypeptides, epithelium-derived tumors and cultured cells can be classified according to their specific cytokeratin patterns [31, 35, 81, 921. Typically, relatively simple combinations of cytokeratins are found in ‘simple’, i.e., one-layered epithelia and their tumors. For example, in several vertebrate species, hepatocytes, pancreatic epithelia, tubular epithelia of the kidney, ovarian granulosa cells, and several kinds of tumors and cultured cell lines derived from such simple epithelia, as well as cultured neuroendocrine cells of the line PC12, contain only two cytokeratin polypeptides, originally termed A, i.e., no. 8 of the human catalog [18], and D, i.e., human cytokeratin no. 18 [l, 13, 14, 18, 29-31, 34, 35, 38, 811. The same two cytokeratin polypeptides have also been found in early embryonal epithelia [9, 20, 33, 57, 58, 88, 89, 1031. A relatively simple cytokeratin pattern, comprising only three cytokeratins, i.e., nos. 8, 18, and 19 (‘the M,40,OOO cytokeratin’), is found in several other simple epithelia, such as the intestinal epithelia and their tumors, in certain hepatoma cells in culture, in various neuroendocrine cells, in some human carcinoids and small cell carcinomas, and in the epithelia of the rete testis and plexus choroideus [l, 7, 8, 30-32, 35, 53, 78, 79, 81, 82, 84, 92, 1141. Cytokeratins nos. 8 and 18 also occur, along with other cytokeratins, in the complex epithelia of lung, trachea, mammary gland, bladder urothelium, oviduct, uterine epithelium, and mesothelium and in tumors of these epithelia as well as in several cultured cell lines (1, 6, 10, 31, 39, 81-83, 92, 107, 1201. On the other hand, most stratified epithelia, squamous-cell carcinomas, and cells derived therefrom display a much greater complexity

70

of cytokeratin polypeptides, and several of these tissues and tumors apparently lack cytokeratins nos. 8 and 18 (cf. [l, 3, 10, 81--83, 85, 90, 92, 100, 110. 1181). Consequently, the presence of cytokeratins nos. 8 and/or 18 is an important diagnostic feature in two ways: first, the demonstration of their presence identifies epithelial or carcinomatous cells, which is especially valuable in analyses of metastatic tumors of equivocal morphology, and second, the identification of these cytokeratins provides, at least in certain combinations, an indication of the type of tumor present. The specific detection of cells expressing cytokeratin polypeptides nos. 8 and 18 has been possible so far only by applying gel electrophoresis [8@83] and immunocytochemical techniques using antibodies specific for cytokeratins nos. 8 and/or 18 (e.g. [16, 17. 39, 47, 65, 66, 84, 931). While the high sensitivity and topological resolution of immunocytochemical methods have been demonstrated in many instances. negative results based exclusively on the use of antibodies, notably those obtained using monoclonal antibodies, might be due to the selective masking of epitopes (e.g. [37, 52, 1191). Therefore, a valuable alternative approach for the determination of the presence of a given cytokeratin gene product would be to detect its mRNA either in vitro using isolated nucleic acids or, of more practical relevance, by in situ hybridization on sections. As a first step for this procedure, wc have recently isolated cDNA clones for several simple-epithelium-type cytokeratins such as human cytokeratin no. 18 [99] and bovine cytokeratins nos. 8 and 19 (human catalog equivalent numbers; component 19 is no. 22 of the bovine cytokeratin catalog given in [102]), which has allowed the specific detection of the corresponding mRNAs in Northern-blot analyses [2,73]. A short cDNA clone of mRNA encoding human cytokeratin no. 19 has been reported by Eckert and Green [21]. and a genomic clone of murine cytokeratin A (Endo A; equivalent to human cytokeratin no. 8) as well as a cDNA clone of murine component D (Endo B; equivalent to human cytokeratin no. 18) have been described by Duprey et al. [20] and Singer et al. [103]. In the present study, we describe a more extended cDNA clone for human cytokeratin no. 18 as well as a clone encoding human cytokeratin no. 8, and we show the sensitive detection of their mRNAs by Northern-blot analysis of extracted tumor RNA and by in situ hybridization on sections. Methods Enzymes, radiochemicals, sequencing, and in uitro trcinscriptinn system

Restriction enzymes, the large (Klcnow-) fragment of DNA polymerase I. T4 ligase, DNA polymerase I (holoenzyme), alkaline phosphatase, and polynucleotide kinase were purchased from Boehringer (Mannheim, FRG). Radiochemicals and the kit for DNA sequencing according to Maxam and Gilbert [75] were obtained from Amersham-Buchler (Braunschweig, FRG) and New England Nuclear (Boston, Mass., USA). The T7 system for transcription in vitro was purchased from Pharmacia (Uppsala, Sweden).

kindly provided by Dr. Axel Ullrich (Genentech, South San Francisco, Calif.). Two-hundred-thousand phages were plated on three Petri dishes with C 600 hfl+ competent cells. DNA was transferred to nitrocellulose filters according to the method of Maniatis et al. [74]. The inserts of the human clone pKH18' [99] and the bovine clone pKB8' [73] - obtained by excision using the restriction endonucleases, Pst-I and Bam-H I/Sal-I, respectively - were separated by gel electrophoresis in low-melting-type agarose. Bands were excised, and the eluted DNA was nick-translated 1961. Hybridization with pKH18' was performed overnight at 42" C in 50% formamide, 5 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5 x Denhardt's solution [l x Denhardt's solution is 0.02% Ficoll, 0.02% bovine serum albumin (BSA), and 0.02% polyvinylpyrrolidon], 0.4% sodium dodecyl sulfate (SDS), and 100 pg/ml yeast tRNA. Hybridization with pKB8l was at somewhat lower stringency, i.e., overnight at 37" C. Final washes were also done at correspondingly high (65" C) and low (45" C) temperatures (in 0.1 x SSC and 0.4% SDS), respectively. Positive single plaques were obtained after three rounds of screening. Phage DNA was prepared essentially as described by Maniatis et al. [74] with the following modifications: 0.2 ml competent C 600 hfl+ cells (suspended in 1 0 m M MgS04) was inoculated with approximately lo7 plaque-forming units of phage suspension for 20 min at 37" C. Subsequently, 100 ml L-broth was added, and the cultures were further incubated at 37" C for 6-10 h with shaking. Lysis was completed by adding 0.5 ml chloroform and continuing shaking for 15 min at 37" C. The culture was cooled to 4" C and then digested with RNase and DNase (each at a final concentration of 10 pg/ml) at room temperature for 3@ 60 min. NaCl was added at a final concentration of 2%. Cell debris was pelleted at 4,000 rpm in a Sowall centrifuge (GSA rotor; Sorvall, Wilmington, Del) for 20 min at 4" C. Phage particles were then precipitated in 8% polyethylene glycol 6OOO overnight at 0°C. The phage pellet obtained after centrifugation (6,000rpm in a Sorvall GSA rotor for 20 min at 4" C) was suspended in 1 ml phage buffer (100 mM NaCI, 8 m M MgS04, and 50 mM Tris-HCI, pH 7.9, and 200 p1 lysis buffer (3% SDS, 250 mM EDTA, and 0.5 M Tris-HC1, pH 7.0) was added prior to incubation at 65" C for 15 min. This was followed by proteinase-K treatment (final concentration, 20 pg/ml) for 20 min at 37" C, and proteins were precipitated by adding 200 pl 5 M potassium acetate (pH 4.8) and keeping the solution on ice for 30 rnin. The supernatant obtained after centrifugation at 4,000 rpm in a laboratory centrifuge (Biofuge A; Heraeus Christ, Osterode, FRG) at 4" C was precipitated with 0.45 volumes of isopropanol for 2min at room temperature. The pellet was washed twice with 70% ethanol, and DNA was further purified by two cycles of chloroform/phenol extraction. DNA sequencing

The Eco-RI inserts from positive clones were excised and subcloned into the puC8 [113], pTZ19 R [15], and M13 vectors [77]. DNA sequencing was performed according to standard protocols [75. 1011.

Screening of' the cDNA library

Preparation of R N A

A cDNA library in AgtlO [56] constructed from poly(A)+RNA of the vulvar carcinoma cell line A-431 [ill] was

Total RNA and poly(A)' -RNA were extracted from cultured human cells of the vulvar epidermoid carcinoma-de-

71

rived line A-431 [22], the breast carcinoma cell line MCF-7 [104], and SV4O-transformed fibroblasts (cf. [28]) as described elsewhere [60,72, 731. RNA from small tumor tissue samples - mostly breast carcinomas and lymph nodes suspected of containing metastatic tumors - was isolated according to the following procedure. Approximately 0.5 g tissue was ground in a precooled porcelain mortar at -70' C (cf. (321). The finely ground cold tissue powder was directly transferred into 7.5 ml 4 M guanidinium isothiocyanate buffer and then vortexed extensively. This mixture was carefully loaded onto 4 ml 5.7 A4 cesium chloride and centrifuged at 32,000 rpm for 20 h at 20" C (SW40 rotor; Beckmann, Palo Alto, Calif.). The pelleted RNA was finally extracted with chloroform/phenol. Hybrid selection and translation

Positive clones from the plaque-hybridization screening assay were characterized by hybrid-selection release and subsequent translation in vitro as previously described [60,721 using 100 pg/ml poly(A)*-RNA obtained from A-431 cells. The 35S-methionine-labeledtranslational products from the selected mRNAs were separated by two-dimensional gel electrophoresis and were identified by co-electrophoresis with unlabeled cytoskeletal proteins from A-431 cells [81] using autoradiography. Northern-blot analysis

RNA was separated on agarose gels containing formaldehyde and was blotted on 'gene screen' filters (Gene Screen Plus TM, New England Nuclear, Boston, Mass) as described for nitrocellulose filters by Maniatis et al. [74]. The filters were hybridized overnight with RNA probes (see below) in 50% formamide, 5xSSC, 0.4% SDS, 5xDenhardt's solution, and 100 pg/ml yeast tRNA at 60' C. Two 20-min washes were carried out in 2 x SSC and 0.1% SDS at room temperature. RNase A (final concentration, 10 pg/ ml) was added to the next wash solution (2 x SSC and 0.1YO SDS), and digestion was allowed to proceed for 20min at room temperature. The filters were finally treated with 0.1 x SSC and 0.1% SDS for 1 h at 72" C and then processed for autoradiography. Preparation of radioactively labeled RNA

We used the in vitro transcription plasmids, pTZl8 R and pTZ19 R [15] obtained from Pharmacia (Uppsala, Sweden). The entire cDNA insert of AKH8' was excised from the RgtlO recombinants and was cloned into the Eco-RI site of the in vitro transcription plasmid, pTZ19 R (Pharmacia). In the case of pKH18', the unique Bam-HI site was used to subclone a 3'4erminal fragment into pTZ18 R. Plasmids containing the inserts in either orientation - yielding 'anti-sense' RNA when the poly(A) tract was proximal to the T7 promoter - were isolated and linearized with Hind111 (pTZl8 R-pKH18) and Bgl-I (pTZ19 R-pKH8), respectively. Transcription in vitro using 1 pg linearized DNA template and 10 units of T7 RNA polymerase was carried out according to the protocol provided by Pharmacia. For the preparation of 32P-labeledprobes, 100 pCi a-"P-UTP (specific activity, 600 Ci/mmol) was added to give a final concentration of 12.5 p M . For the synthesis of 'H-labeled probes, 50 pCi a-3H-UTP and 50 pCi a-'H-GTP (specific

Fig. la, b. Demonstration that cDNA clone pKH8' codes for human cytokeratin no. 8 by in vitro translation of hybrid-selectcd mRNA. a Coomassie-Blue-stained major cytokeratins of cultured human carcinoma cells of line A-431 (for entire complement, see (81, 921) separated by two-dimensional gel electrophoresis (NEPHGE, direction of nonequilibrium pH gradient electrophoresis; SDS, direction of polyacrylamide gel electrophoresis in the presence of SDS)together with the endogenous and in vitro translated products of the rabbit reticulocyte lysate system containing hybrid-selected poly(A)*-RNA. Cytokeratins nos. 5, 8, 13, and 18 that are expressed in A-431 cells are indicated. B and a indicate the positions of bovine serum albumin and muscle a-actin, respectively, that were used as markers in co-electrophoresis. The spot above human cytokeratin no. 5 is an endogenous component of the rabbit reticulocyte lysate. b Autoradiograph obtained after exposure of the gel shown in a, showing 3SS-methionine-labeledcytokeratin no. 8 to be the only detectable product of the in vitro translated mRNA selected by hybridization of A-431 poly(A)'RNA to pKH8'

activity, 3 M 5 Ci/mmol) were added at a final concentration of 50 pM.Transcription was terminated by digesting the DNA template with 20 pg/ml RNase-free DNase I (Worthington, Freehold, NJ) that had been treated with Macaloid (cf. [74]) for 15 min at 37" C. EDTA was then added at a final concentration of 10mA4, and the RNA was precipitated twice with ammonium acetate (final concentration, 2.5 M) and 0.6 volumes of isopropanol in the presence of Escherichia coli tRNA. The RNA was pelleted and dissolved in 100% formamide. The amount of RNA synthesized was estimated by trichloroacetic acid (TCA) precipitation and scintillation counting of the radioactivity.

72 1 ATG AAC AAG GTA GAG CTG GAG TCT CGC CTG GAA GGG CTG ACC GAC GAG ATC AAC TTC CTC AGG CAG CTG TAT GAA

M e t A s n L y s V a l G l u L e u G l u S e r A r g L e u G l u G l y L e u T h r A s p G;.J

Ile A s n P h e L e u A r g G l n L e u T y r G l u

7 6 GAG GAG ATC CGG GAG CTG CAG TCC CAG ATC TCG GAC ACA TCT GTG GTG CTG TCC ATG GAC AAC AGC CGC TCC CTG G l u G I u Ile A r g G l u L e u G l n S e r G l n Ile S e r A s p T h r S e r V a l V a l L e u S e r Met A s p A s n S e r A r g S e r L e u 1 5 1 GAC ATG GAC AGC ATC ATT GCT GAG GTC AAG GCA CAG TAC GAG GAT ATT GCC AAC CGC AGC CGG GCT GAG GCT GAG A s p Met A s p S e r Ile I l e A l a G l u V a l L y s A l a G l n T y r G I u A s p IIe A l a A s n A r g S e r A r g A l a G l u A l a G l u 2 2 0 AGC ATG TAC CAG ATC AAG TAT GAG GAG CTG CAG AGC CTG GCT GGG AAG CAC GGG GAT GAC CTG CGG CGC ACA AAG S e r Met T y r G l n Ile L y s T y r G l u G l u L e u G l n S e r L e u A l a G l y L y s His G l y A s p A s p L e u A r g A r g T h r L y s 3 0 1 ACT GAG ATC TCA GAG ATG AAC CGG AAC ATC AGC CGG CTC CAG GCT GAG ATT GAG GGC CTC AAA GGC CAG AGG GCT T h r G l u Ile S e r G l u M e t A s n A r g A s n I l e S e r A r g L e u G l n A l a G l u Ile G l u G l y L e u L y s G l y G l n A r g A l a

3 7 6 TCC CTG GAG GCC GCC ATT GCA GAT GCC GAG CAG CGT GGA GAG CTG GCC ATT AAG GAT GCC AAC GCC AAG TTG TCC S e r L e u G I u A l a A l a Ile A l a A s p A l a G I u G l n A r g G l y G l u L e u A l a Ile L y e A s p A l a A s n A l a L y s L e u S e r 4 5 1 GAG CTG GAG GCC GCC CTG CAG CGG GCC AAG CAG GAC ATG GCG CGG CAG CTG CGT GAG TAC CAG GAG CTG ATG AAC G l u L e u G l u A l a A l a L e u G l n A r g A l a L y s G l n A s p Met A l a A r g G l n L e u A r g G l u T y r G l n G l u L e u Met A s n 5 2 6 GTC AAG CTG GCC CTG GAC ATC GAG ATC GCC ACC TAC AGG AAG CTG CTG GAG GGC GAG GAG AGC CGG CTG GAG TCT V a l L y s L e u A l a L e u A s p Ile G l u Ile A l a T h r T y r A r g L y s L e u L e u G l u G l y G l u G l u S e r A r g L e u G l u S e r

t

6 0 1 GGG ATG CAG AAC ATG AGT ATT CAT ACG AAG ACC ACC GGC GGC TAT GCG GGT GGT TTG AGC TCG GCC TAT GGG GAC G l y M e t G l n A s n M e t S e r I I e His T h r L y s T h r T h r G l y G l y T y r A l a G l y G l y L e u S e r S c r A l a T y r G l y A s p 6 7 0 CTC ACA GAC CCC GGC CTC AGC TAC AGC CTG GGC TCC AGC TTT GGC TCT GGC GCG GGC TCC AGC TCC TTC AGC CGC L e u T h r Asp P r o G l y L e u Ser T y r Ser L e u G l y Ser Ser Phe G l y Ser G l y A l a G l y Ser Ser Ser Phe Ser A r g

7 5 1 ACC AGC TCC TCC AGG GCC GTG GTT GTG AAG AAG ATC GAG ACA CGT GAT GGG AAG CTG GTG TCT GAG TCC TCT GAC T h r Ser Ser Ser A r q A l e V a l V a l V a l L y s L y s IIe G l u T h r A r g A s p G l y L y s L e u V a l Ser G l u S e r Ser Asp 8 2 6 GTC CTG CCC AAG TGA A CAGCTGCGGC AGCCCCTCCC AGCCTACCCC TCCTGCGCTG CCCCAGAGCC TGGGAAGGAG GCCGCTATGC V a l L e u P r o L y s *+*

9 1 2 AGGGTAGCAC TGGGAACAGG AGACCCACCT GAGGCTCAGC CCTAGCCCTC AGCCCACCTG GGGAGTTTAC TACCTGGGGA CCCCCCTTGC

1 0 0 2 CCATGCCTCC AGCTACCAAAA CAATTCAATT GCTTTTTTTT TTTGGTCCAA AATAAAACCT CAGCTAGCAA AAAAAAAAAA AA

Fig. 2. Nucleotide sequence of clone pKH8' and the deduced partial amino acid sequence of human cytokeratin no. 8. The cDNA extends from a position within the a-helical rod domain l b of the protein to the poly(A) tail region of the mRNA. The arrowhead indicates the end of the a-helical rod after the TYR(X)LLEGE consensus sequence. The three asterisks denote the termination codon. The polyadenylation signal is underlined. The overlined sequence corresponds to the Bgl-I site at which the plasmid was cleaved, prior to T7 transcription, in order to provide the anti-sense RNA probe

The specific activity of the 3H-labeled RNA probes was approximately 8-9 x lo7 cpm per microgram of transcript. In the case of 32P-labeledprobes, the specific activity had a range of 5-8 x lo* cpm per microgram of transcript. The probe length was adjusted to approximately 200 nucleotides by limited alkaline hydrolysis [l 11 : RNA transcripts exceeding 200 nucleotides in length were incubated in 0.1 M NaOH for 20 rnin at 0" C and were then neutralized by the addition of 0.1N HCl and 0.2 M sodium acetate (pH 5.5). Under the conditions used for in situ hybridization, this alkaline treatment increased the hybridization signal by a factor of at least 2 for RNA transcripts longer than 400 nucleotides. Hybridization in sifu

Hybridizations were performed on cryostat sections (approximately 5 l m ) of human tissues and tumors that had been obtained during surgery or as autopsy material and had been shock frozen [l, &8,26, 35, 82, 841. The sections were mounted on gelatine-coated glass slides [44]and fixed for 20 min at 20" C in 4% formaldehyde freshly prepared from paraformaldehyde in phosphate-buffered saline (PBS) and 5 mM MgC12. The sections were then rinsed in PBS, dehydrated through an ascending ethanol series, and dried at 42OC for 12-24 h. Prior to hybridization, the sections were rehydrated in 2 x SSC containing 0.1% Triton X-100 and were treated with 0.5 pg/ml predigested proteinase K

for 30 min at 37" C. The reaction was stopped by rinsing the sections with 2 x SSC containing 0.1 M glycine. At this point, some sections were treated with 50 pg/ml RNase A in 2 x SSC for 30 min at 37" C, as a negative hybridization control. The sections were then postfixed for 5 rnin at 20" C in 4% formaldehyde. The acid- and heat-pretreatment steps of the protocol of Hafen et al. [48] were omitted from our protocols since they did not improve the hybridization signal. After the second fixation, the sections were rinsed in 50% formamide and 2 x SSC, and were then incubated for approximately 30 min in hybridization solution (60% formamide, 2 x SSC, 10% dextran sulfate, 1 x Denhardt's solution, 10 mM Tris-HCI, 0.25% nuclease-free BSA, and 500 pg/ml E. coli tRNA, pH 7.5). In some experiments, the hybridization solution contained 0.5% SDS. The RNA probes were denatured in 100% formamide for 10min a 80" C and were added directly to the hybridization solution at a temperature of 40"-50" C. Usually, 2 ng (1 p1) probe was applied to each section (final concentration, 0.08 pg/ ml). The hybridization reaction was carried out by placing the slides (without coverslips) into tightly sealed moist chambers at 42O-45"C for 5-7 h. After hybridization, the sections were washed in 50% formamide and 2xSSC for 1 h at 42" C with at least two changes of wash buffer. They were then equilibrated with 2 x SSC for 15 min and treated for 30 rnin at 37" C with 50 pg/ml RNase A (DNase free). The sections were then washed again for 30min in 50% formamide and 2xSSC at 37°C and finally for 30min

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b Fig. 3a, b. Comparison of the amino acid sequence of human cytokeratin no. 8 (H 8) with: a that of its bovine equivalent 'A' (designated B A ( 8 ) ; taken from [73]; and b that of the BrCN fragments C and El of the serodiagnostic tumor marker TPA, as reported by Redelius et al. [95]. The asterisks show identical residues; numbers denote conservative exchanges, such as 1 (for S and T), 2 for acidic amino acids (D and E), 3 for basic residues (H, R, K), and 5 for hydrophobic residues (M, I, L, V, A), whereas 4 denotes exchanges between charged amino acids irrespective of the kind of charge. a The dotted line under the sequence indicates a possible heptad repeat pattern typical of cx-helical coiled coil formation. Three possible skip positions (denoted by semicircles) are discussed. The conserved sequence, DGKLVSE, in the tail domain is ooerlined. Note the additional amino acids in the tail region of human cytokeratin no. 8. b Comparison of the BrCN fragments E l and C of TPA with human cytokeratin no. 8. The aligned sequences, where comparable (asterisks),are identical. The arrowheads denote methionine residues defining cleavage sites

in 0.1 x SSC at 50" C, dehydrated in graded ethanol solutions containing 0.3 M ammonium acetate, and air-dired. Autoradiographic exposure using Kodak NTB2 emulsion (Kodak, Rochester, NY) lasted for 1-3 weeks. The sections were stained with hematoxylin and eosin. Autoradiographs of adjacent areas of the same size were selected, and the grain densities over cells within these areas were determined by counting. Results Isolation and identification of a cDNA clone encoding human cytokeratin no. 8

A AgtlO cDNA library of the human carcinoma cell line, A-431 [l 111, was chosen to isolate a clone encoding cytokeratin no. 8, because this cytokeratin is a major constituent of the IF cytoskeleton of this cell type (cf. [81, 921). The phages were screened with the isolated and nick-translated Bam-HI/Sal-I insert of a plasmid (pKB8') encoding the bovine equivalent cytokeratin no. 8 [73] at relatively low stringency. The Eco-RI insert of one of the clones selected (IZKH8') was subcloned into the plasmid vector, pTZ19 R, and one of these plasmid clones (pKH8l) was subjected to the hybridization-selection-translation assay using poly(A)+-RNA from A-431 cells. The "S-methionine-labeled products obtained by in vitro translation of the released mRNA (together with unlabeled cytoskeletal pro-

teins of A-431 cells; Fig. 1 a) was examined by two-dimensional polyacrylamide gel electrophoresis and was identified as being human cytokeratin no. 8 (Fig. 1 b). Sequence characteristics of human cytokeratin no. 8 and comparison with the corresponding bovine and amphibian sequences

The clone pKH8' contains 1,083 nucleotides (Fig. 2), starting in the 'coil-lb' region of the a-helical rod portion that is typical of all I F proteins (cf. [45, 49, 50, 106, 1151) and extending into the 3'-noncoding region. The 3'-end presents a canonic polyadenylation signal located 12 nucleotides upstream from the poly(A) stretch of which the clone contains only 14 residues. As expected, the predicted sequence of 279 amino acids encoded by this clone differs only slightly from the sequence of bovine cytokeratin no. 8 and displays the same structural characteristics (Fig. 3a). The extent of homology (93%; 90% identity) between the two species is almost equally high in the rod domain (94% homology; 91% identity) and the carboxyterminal 'tail' domain (90% homology; 88% identity). The heptad repeat pattern typical of the ahelical coiled coil rod domain and its interruptions in coil 2 are also indicated in Fig. 3a. A non-a-helical spacer region of 24 amino acids appears to separate coil 1 b from coil 2. Only a short region in the center of the carboxyterminal tail exhibits significant interspecies divergence, this being

74 1 TCC ACC TTC TCC ACC AAC TAC CGG TCC CTG GGC TCT GTC CAG GCG CCC AGC TAC GGC GCC CGG CCG GTC AGC AGC S e r T h r Phe S e r T h r A s n T y r A r g S e r L e u G l y S e r V a l G l n A l a P r o S e r T y r G l y A l a A r g P r o V a l S e r S e r 7 6 GCG GCC AGC GTC TAT GCA GGC GCT GGG GGC TCT GGT TCC CGG ATC TCC GTG TCC CGC TCC ACC AGC TTC AGG GGC A l a A l a S e r V a l T y r A l e G l y A l a G l y G l y S e r G l y S e r A r g Ile S e r V a l S e r A r g S e r T h r S c r P h e A r g G l y 1 5 1 GGC ATG GGG TCC GGG GGC CTG GCC ACC GGG ATA GCC GGG GGT CTG GCA GGA ATG GGA GGC ATC CAG AAC GAG AAG G l y Met G l y Ser G l y G l y L e u A l a T h r G l y I l e A l a G l y G l y L e u A l a G l y Met G l y G l y I I e G i n Asn G l u L y s 2 2 6 GAG ACC ATG CAA AGC CTG AAC GAC CGC CTG GCC TCT TAC CTG GAC AGA GTG AGG AGC CTG GAG ACC GAG AAC CGG G l u Thr Met G l n Ser L e u Asn Asp A r g L e u A l a Ser T y r L e u Asp A r g V a l A r g Ser L e u G l u Thr G l u Asn A r g 3 0 1 AGG CTG GAG AGC AAA ATC CGG GAG CAC TTG GAG AAG AAG GGA CCC CAG GTC AGA GAC TGG AGC CAT TAC TTC AAG A r g L e u G l u S e r L y s I l e A r g G l u H i s L e u G l u L y s L y s G l y P r o G l n V a l A r g A s p T r p S c r His T y r Phe L y s 3 7 6 ATC ATC GAG GAC CTG AGG GCT CAG ATC TTC GCA AAT ACT GTG GAC AAT GCC CGC ATC GTT CTG CAG ATT GAC AAT I l e I l e G l u Asp L e u A r g A l a G l n I L e Phe A l a A s n T h r V a l A s p A s n A l a A r g I l e V a l L e u G l n I l e A s p Asn A 5 1 GCC CGT CTT ‘ X T GCT GAT GAC TTT AGA GTC AAG TAT GAG ACA GAG CTG GCC ATG CGC CAG TCT GTG GAG AAC GAC A l a A r g L e u A l e A l a A s p A s p Phe A r g V a l L y s T y r G l u T h r G l u L e u A l a M e t A r g G l n Ser V a l G l u Asn Asp 5 2 6 ATC CAT GGG CTC CGC AAG GTC ATT GAT GAC ACC AAT ATC ACA CGA CTG CAG CTG GAG ACA GAG ATC GAG GCT CTC I l e H I S G l y L e u A r g L y s V a l I l e A s p A s p T h r A s n I l e T h r A r g L e u G l n L e u G l u T h r G l u I l e G l u ALa L e u 6 0 1 AAG GAG GAG CTG CTC TTC ATG AAG AAG AAC CAC GAA GAG GAA GTA AAA GGC CTA CAA GCC CAG ATT GCC AGC TCT L y s G l u G l u L e u L e u Phe M e t L y s L y s A s n H i s G l u G l u G l u V a l L y s G l y L e u G l n A l a G l n I l e A l a Ser Ser 6 7 6 GGG TTG ACC GTG GAG GTA GAT GCC CCC AAA TCT CAG GAC CTC GCC AAG ATC ATG GCA GAC ATC CGG GCC CAA TAT G l y L e u Thr V a l G l u V a l Asp A I a P r o L y s Ser G l n Asp L e u A l a L y s I l e M e t A l a Asp I l e A r g A l e G l n T y r 7 5 1 GAC GAG CTG GCT CGG AAG AAC CGA GAG GAG CTA GAC AAG TAC TGG TCT CAG CAG ATT GAG GAG AGC ACC ACA GTG Asp G l u L e u A l a A r g L y s Asn A r g G l u G l u L e u Asp L y s T y r T r p Ser G l n G l n l i e G l u G l u Ser T h r Thr V a l 8 2 6 GTC ACC ACA CAG TCT GCT GAG GTT GGA GCT GCT GAG ACG ACG CTC ACA GAG CTG AGA CGT ACA GTC CAG TCC TTG V a l T h r T h r G l n Ser A l a G l u V a l G l y A l a A l a G l u T h r T h r L e u T h r G l u L e u A r g A r g T h r V a l G l n Ser L e u 9 0 1 GAG ATC GAG CTG GAC TCC ATG AGA AAT CTG AAG GCC AGC TTG GAG AAC AGC CTG AGG GAG GTG GAG GCC CGC TAC G l u I l e Asp L e u Asp Ser Met A r g A s n L e u L y s A l a Ser L e u G l u Asn Ser L e u A r g G l u V a l G l u A l a A r g T y r 9 7 6 GCC CTA CAG ATG GAG CAG CTC AAC GGG ATC CTG CTG CAC CTT GAG TCA GAG CTG GCA CAG ACC CGG GCA GAG GGA A l a L e u G l n M e t G l u G l n L e u A s n G l y I l e L e u L e u His L e u G l u S e r G l u L e u A l a G l n T h r A r g A l a G l u G l y 1 0 5 1 CAG CGC CAG GCC CAG GAG TAT GAG GCC CTG CTG AAC ATC AAG GTC AAG CTG GAG GCT GAG ATC GCC ACC TAC CGC G l n A r g G l n A l a G l n G l u Tyr G l u A l a Leu L e u Asn I l e L y s V a l L y s L e u G l u A l a G l u I l e A l a Thr Tyr A r g 1 1 2 6 CGC CTG CTG GAA GAT GGC GAG GAC TTT AAT CTT GGT GAT GCC TTG GAC AGC AGC AAC TCC ATG CAA ACC ATC CAA A r g L e u L e u G l u A s p G l y G I u A s p Phe A s n L e u G l y A s p A l a L e u A s p Ser Ser A s n Ser M e t G l n Thr I l e G l n

t

1 2 0 1 AAG ACC ACC ACC CGC CGG ATA GTG GAT GGC AAA GTG GTG TCT GAG ACC AAT GAC ACC AAA GTT CTG AGG CAT TAA L y s T h r T h r T h r A r g A r g I l e V a l A s p G l y L y s V a l V a l S e r G l u T h r A s n A s p T h r L y s V a l L e u A r g H I S *I* 1 2 7 6 GCCAGCAGAA GCAGGGTACC CTTTGGGGAG CAGGAGGCCA ATAAAAAGTT CAGAGTTCAA AAAAAAAAAA AA

Fig. 4. Nucleotidc sequence of clone pKH18’ and the amino acid sequence of human cytokcratin no. 18 deduced therefrom. The mine in position 1 of the deduced amino acid sequence probably corresponds to the sixth amino acid (see Fig. 5). The overlined nucleotide sequence, GGATCC, is the Bam-HI restriction site used for subcloning into the transcription vector, pTZl8 R. The arrowhead indicates the end of the or-helical rod domain. Thc canonical polyadenylation signal is underlined

due to the insertion of 12 amino acids in a somewhat scattered fashion in the human sequence (Fig. 3 a). We have recently published the complete amino acid sequence of the corresponding cytokeratin of the frog, Xenopus laevis, termed cytokeratin 1 [8], which exhibits a remarkably high homology with bovine cytokeratin no. 8 [40]. The sequence of human cytokeratin no. 8 is also highly homologous to that of the amphibian protein: where comparable, the rod exhibits 90% homology (78% idcntity), whereas in the tail region only 62% homology (46% identity) is found (data not shown). The heptapeptide sequence, DGKLVSE, which is located shortly before the carboxyterminus (Fig. 3 a), has been relatively well conserved during evolution from amphibia to man. This is true not only for the type-I1 cytokeratin no. 8 but also for several type-I cylokeratins and, in a modified version, for desmin and vimentin (e.g. [40,59, 60, 62, 92,991). With the exception of a single GGYAGG hexapeptide, the tail of human cytokeratin no. 8, like that of bovine cytokeratin no. 8, has a relatively low glycine content and does not display glycine-rich oligopeptide repeats, a notice-

able difference as compared to the amphibian protein (cf. [40]; for other type-I1 cytokeratins containing tails with glycine-rich repeats, see [46, 49, 59, 105, 1061). Comparison of the amino acid sequences of human cytokeratin no. 8 and ojthe ‘tissue polypeptide antigen ’ A complex protein fraction containing polypeptides ranging in M, from 20,000 to 45,000, termed ‘tissue polypeptide antigen ’ (TPA), has been described in diverse carcinoma cells and in placenta, and is widely used in clinical diagnosis as a serum marker for the presence of carcinomas (5, 71, 1 171. Figure 3 b shows a comparison of the amino acid sequence of the TPA fragments C and El obtained by cyano- , gen bromide cleavage [95] and that of human cytokeratin no. 8. The sequences are identical. Fragment El of TPA is clearly derived from the start of coil 2 of the rod domain, and the three missing amino acids in the sequence published by Redelius et al. [95] can now be identified as serine, isoleucine, and asparagine. The larger fragment C can also

H 18

-

_ _ _ _ _ ST-FSTNYRSLGSVQAPSYGRPVSSAASVYAGGGSGSRISVSRSTSFRGGMGSGGLATGIAGG ** * * * * * * * * i t * ** 5 * * 5 * * * * * * * * * * * * * * * * * * * * * * ** * * *

M D ( 1 8 ) SFTTRSTTFSTNYRSLGSVRTPSQRVRPASSAASVYA~GGSGSRISVSRSV----WGGSVGSA-----G

, coilla

I

H 18

.

I

LAGMGGIQNEKETMOSLNDRLASYLDRVRSLETENRRLESKIREHLEKKGPQ-VRDWSHYFKIIEDLRAQ *I**** .................................... I*** M D ( 1 8 ) LAGMGGIQTEKETMDLNDRLASYLDKVKSLETENRRLESKIREHLEKKGPQGVRDWGHrFKI~ED~RAQ

********

************

* == =

coillbH 18

*

MD(18)

ILANSVDNARIVLQIDNARLAADDFRVKYETELAMRQSVESDIHGLRKWDDTNITRLQLETEIEALKEE * z : z z z : : = ?

IFANTVDNARIVLQIDNARLAADDFRVKYETELAQSVENDIHGLRKVIDDTNITRLQLETEIEALKEE **1*********************************** ********5********************

: =

=

I

H 18

=

= :

: =

= =

I

LLFMKKNHEEEVKGLQAQIASSGLTVEVDAPKSQDLAKIMADIRAQYDELARKNREELDKYWSaQIEEST **********2 M D ( 1 8 ) LLFMKKNHEEEVQGLEAQIASSGLTVEVDAPKSQDLSKIMADIRAQYEALAQKNREELDKYYS~IEEST b z 2 - -

************ ** . . . . . . . . . . . . . . . . . . . .

** ******************

-

- -

-

-

-

H 18 TWTTQSAEVGAAETTLTELRRTVQSLEIDLDSMRNLKASLENSLREVEARYALQMEQLNGILLHLESEL *it*** ***5 ***********5+1********3* 5 2***** 5*******5******** M D ( 1 8 ) TWTTKSAEIRDAETTLTELRRTLQTLEIDLDSMKNQNINLENSLGDVEARYKA~EQLNGVLLHLESEL - - - -

- -

-

-

- -

- -

- -

***** -- -

--

I

H 18 MD(18)

H 18

15

-

-

- -

- -

A

AQTRAE~RQAQEYEALLNIKVKLEAEIATYRRLLEDGEDFNLGDALDSSNSMQTIQKTTTRRIVDGKW

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * ***********5******3****3** AQTRAEGQRQAQEYEALLNIKVKLEAEIATYRRLLEDGEDFSLNDALDSSNSMTVQKTTTRKIVDGRW - - - _ - _ - - -- _- -- -- -GTNDTKVLAH

******3**** M D ( 1 8 ) SETNDTRVLRH

be assigned to coil 2 of the rod domain corresponding to a position located 36 amino acids away from fragment E l . This comparison also shows that the differences noted previously between bovine cytokeratin no. 8 and human TPA fragment C [73] are due to species differences. Identification and sequence analysis of clones encoding human cytokeratin no. 18 We have previously described a relatively short (756 nucleotides, excluding the poly(A) stretch) cDNA clone encoding human cytokeratin no. 18 (pKH18’) isolated from a liver expression library [99]. Using this clone as a probe, we selected a nearly full-length clone from an A-431 cDNA library (AKH18’) and subcloned it into the plasmid vector, pUC8. The presence in the sequence of this clone (pKH18’) of two terminal Eco-RI and two internal Pst-I sites, together with the unique Bam-HI and Sma-I sites, was used to generate fragments which were sequenced either after subcloning into M13 vectors [loll or after end-labeling [75]. Clone pKH18’ contains 1,347 nucleotides, including a poly(A) stretch of 14 nucleotides (Fig. 4). It encodes 424 amino acids that represent the entire polypeptide, except for the first 5 amino acids between the initial methionine and the first codon of P K H (Ser), ~ ~ as~ indicated by comparison with the complete sequence of human cytokeratin no. 18 kindly provided by R. Oshima (Cancer Research Foundation, La Jolla, Calif.) and the corresponding murine cytokeratin (Fig. 5). The sequence determined from clone pKH1S2 also reveals four different amino acid positions in comparison with the previously published sequence of clone pKH18’, these being located at positions 196 (from Gln to Glu), 240 (from Ser to Ala), 303 (from Arg to Asp), and 306 (from Arg to Ser). Re-examination of the actual sequencing gels of clone pKH18l [99] and comparison with the sequence of the cytokeratin-no.-18 cDNA clone isolated by R. Oshima and colleagues (see above) convinced us that

Fig. 5. Comparison of the amino acid sequence of human cytokeratin no. 18 ( H 18) with that of the corresponding murine protein termed D or Endo B ( M D18) reported by Singer et al. [103]. The asterisks denote identical amino acids; numbers denote homologies (for other symbols, see the legend to Fig. 3). The upper lines demarcate the three a-helical coiled coil rod domains, la, 1 b, and 2. The lower dotted lines show a possiblc heptad repeat pattern typical of coiled coil formation which is interrupted in three positions in coil 2 (small semicircles around heptad dots). The insertion of 9 amino acids in the amino terminal head region of human cytokeratin no. 18 is noteworthy. The conserved motiv, DGKWSE, in the carboxyterminal tail region is overlined

the above-mentioned differences were due to reading mistakes in the pKH18l sequence. The murine and human sequences are highly homologous (88% identical residues), and this homology is not restricted to the rod portion but extends into both the head and the tail. The sequence representing the a-helical coiled coil rod (see Fig. 5) is interrupted twice, i.e., by a spacer of 13 amino acids between coil l a and coil Ib, and by a 21-amino-acid-long spacer between coil l b and coil 2. The two sequences also exhibit the same pattern of heptad repeats, the typical feature of a-helices that form coiledcoils. This heptad pattern appears to be disturbed in some positions in coil 2 (as indicated in Fig. 5), including a distinct ‘stutter’ in the second half of coil 2, which is characteristically found in all I F proteins examined so far (for reviews, see [105, 106, 1151). In order to maximize the alignment of the two sequences, certain ‘gaps’ have to be introduced, which indicate that the human cytokeratin contains two additional clusters of 4 and 5 amino acid residues in the head portion (Fig. 5). This suggests that an insertional event occurred in the head domain of cytokeratin no. 18 during the evolution of the two species. The total apparent molecular mass of human cytokeratin no. 18, i.e., 48, 167, as calculated from the amino acid sequence (including the initial methionine, which is probably not retained in the mature protein), slightly exceeds the M , values of 45,000-46,000 estimated from SDS-polyacrylamide gel electrophoresis (cf. 118, 31, 35, 811). This deviation, however, is not without precedent in other cytokeratin polypeptides. For example, the bovine counterpart of human cytokeratin no. 19, which runs in some (although not all) systems of SDS-polyacrylamide gel electrophoresis in a position corresponding to an M,of -40,000, has an apparent molecular mass of 43,893 [2]. A molecular mass of about 47,400 has been calculated for the equivalent murine cytokeratin no. 18 (component D, Endo B) by Singer et al. [103], although this cytokeratin runs significantly

76

to an estimated size of -1.95 kb, was detected in several epithelia and carcinomas as well as in cultured cells of epithelial origin such as lines MCF-7 and A431 (Fig. 6, lanes 2 and 3) and in two different kinds of breast carcinomas (Fig. 6, lanes 4 and 5), whereas no hybridization was obtained with RNA from various non-epithelial cells such as SV40-transformed fibroblasts (Fig. 6, lane 1). The reported lengths of the mRNA of human cytokeratin no. 8 differ somewhat from the values reported for bovine cytokeratin no. 8 (1.85 kb [73]) and murine cytokeratin no. 8 (approximately 1.64 kb [20]), but results obtained from co-electrophoresis of RNA from all three species would be needed before significance could be ascribed to these apparent differences. When clone pKH18' or its subclones were used in such Northern-blot experiments, a band of approximately 1.4 kb was detected in samples from cells and tissues that were also positive for cytokeratin no. 8 (for an example, see Fig. 6, lane 6), thus confirming our previous results obtained with clone pKH18' [99]. In situ hybridization experiments

Fig. 6. Autoradiograph showing a Northern-blot hybridization of total RNA from SV40-transformcd fibroblasts (fane I), poly(A)+RNA from the brcast carcinoma cell line MCF-7 (fune2). poly(A)* RNA from thc vulvar carcinoma cell line A-431 (fane3). and total RNA from two different brcast carcinomas (fanes4 and 6, an invasive lobular carcinoma of thc signet ring type; lane 5, an invasive ductal mammary carcinoma with microadenoid elements) aftcr rcaction with 32P-UTP-labeled RNA transcripts of the truncated clone pKH8' (lanes 1-5) and the Bam-HI-site-terminated subclone of pKHl8' (lane 6). The horizonral bars on the left indicate the positions of the molecular weight markers (28s and 18s eukaryotic rRNA, and 23s and 16s prokaryotic rRNA). Lanes 1-3 are from the same blot, as are fanes 4 and 5

more slowly in SDS-polyacrylamide gel electrophoresis than human cytokeratin no. 18 ( M I estimates of 48,00050,000 have been given in [18, 29-31, 88, 891; note, however, the value of M , 46,000 determined from the gel system used in [57]). Apparently, these differences in the relative gel electrophoretic mobility of the SDS complexes of human and the murine polypeptides are due to relatively minor changes of amino acid composition. Deiection oJ'mRNA encoding cytokeratins nos. 8 and 18 by Northern-blo t analysis

RNA from several cultured cells and tissues, including tumors, was examined on filters by Northern-blot analysis using a T7 transcript of the Bgl-I-truncated clone, pKH8l, as a probe for hybridization. A single band, corresponding

The availability of the cDNA clones for an 'expression pair' of human simple-epithelium-type cytokeratins, i.e., nos. 8 and 18, enabled us to initiate detailed studies of the expression of these cytokeratins in normal and transformed simple epithelial cells, including metastatic tumors, by hybridization in situ. In initial experiments to establish in situ hybridization as a technique for detecting and localizing simple-epithelium-type cytokeratin mRNAs in tissue sections, we used nick-translated cDNA of human cytokeratin no. 18 excised from pKH18' or pKH18' as probes. However, the signals obtained were relatively weak. We then subcloned pKH8' and pKH18l into transcription vectors in order to prepare labeled RNA probes ('riboprobes'), which are generally assumed to increase the sensitivity of in situ hybridization (e.g. [ l l , 541). PTZ18 R plasmids, containing a unique BamHI fragment of pKH18', and pTZ19 R plasmids containing the pKH8l insert, were isolated, and T7 RNA polymerase was used to synthesize "P-UTP-labeled transcripts complementary to the corresponding mRNAs. The anti-sense orientation and the specificity of these RNA probes were confirmed by Northern-blot analysis (Fig. 6). These probes were then applied to frozen sections of mammary-gland tissue, and the hybridization signals obtained were significantly enhanced when the tissue sections were pretreated with proteinase K. Further enhancement of the hybridization signal was obtained when the size of the RNA transcripts was shortened to about 200 nucleotides, as recommended by Cox et al. [ll]. Finally, the inclusion of detergent such as 0.5% SDS in the hybridization solution, although somewhat detrimental to the morphological preservation of the tissue, further increased the hybridization efficiency (data not shown).

Fig. 7a, b. Detection of mRNA coding for human cytokeratin no. 8 in a lymph-node metastasis of a ductal-lobular breast carcinoma by in situ hybridization using the BgI-I linearized subclone pKH8'. In this experiment, the hybridization solution did not contain SDS. The a-32P-UTP-labeled RNA probe was subjected to limited alkaline hydrolysis to reducc the average length to about 200 nucleotides. The autoradiographic cxposure time was 3 weeks. Both microphotographs show specific localization of mRNA for human cytokeratin no. 8 in the tumor cells, whereas the surrounding stromal and lymph-node cells (dark-stained nuclei) arc not significantly labeled. a Kodak Ektachromc daylight color film; b Agfapan film. Bars, 25 pm

I1

78

79

Cytokeratins nos. 8 and 18 have been detected by in situ hybridization in various normal tissues and tumors known to contain these proteins from the results of immunocytochemical studies. A detailed comparison of various methods and tissues will be presented elsewhere (F.X. Bosch, R. Leube, V. Romano, and W.W. Franke, manuscript in preparation). In the present study, we restrict ourselves to a representative situation that is probably also the diagnostically most important one, i.e., the detection of cytokeratins in metastatic tumors. Figures 7-9 show that the RNA probes described above can be used to localize cytokeratin mRNAs in metastatic carcinoma cells in lymphnode biopsies. The tissue sections shown were obtained from the axillary lymph nodes of a patient suffering from an invasive breast carcinoma with both ductal and lobular elements. Figure7 shows the results of experiments in which the 32P-UTP-labeled anti-sense RNA probe for cytokeratin no. 8 mRNA was used; both color and black-andwhite photomicrographs are presented in order to illustrate the specific silver grain distribution, the resolution of grains, and their correlation with the cytoplasm of only one cell type, i.e., tumor cells. Significant labeling, as compared to background grain densities over extracellular regions and in regions outside of the section, was restricted to the nests of metastatic tumor cells (Fig. 7a, b). Figure 7 also shows considerable variations in grain density even in the same tumor region, differing by a factor of 2 to 3. This may reflect local variations in hybridization efficiency, tumorcell heterogeneity with respect to the concentrations of cytokeratin mRNAs, or the inclusion of scattered lymphoid cells in the tumor (see also Figs. 8 and 9). More detailed analyses will be required to show whether such differences in silver grain density relate to cell-type heterogeneities in regions containing admixtures of morphologically different tumor cells. The corresponding in situ hybridization of an adjacent section of the same lymph node with the 32P-UTP-labeled anti-sense probe for cytokeratin no. 18 is presented in Fig. 8a, which shows enrichment of the silver grain density over tumor cells. In the area shown, the signal-to-background ratio is approximately 7.5: 1; the average value for the entire preparation being 6.2: 1. As a control, the reaction with the similarly labeled ‘sense strand’ RNA probe is shown in Fig. 8 b. Such controls exhibited almost background-level silver grain densities over tumor cells, thus demonstrating the specificity of the reaction in Fig. 8a. Detailed quantitative evaluation, however, revealed a slight but significant reaction of the sense-strand probe over tumor cells (signal-to-background noise ratio, 1.3:l), a finding that we cannot explain at present. Another routinely used control was to pretreat the sections with RNase A prior to hybridization, which resulted in diminished labeling (data not shown). Figure 9 shows, for comparison, ’H-labeled anti-sense RNA probes for human cytokeratins no. 8 (Fig. 9a) and

no. 18 (Fig. 9b) in an experiment in which the hybridization solution contained 0.5% SDS, resulting in somewhat poorer preservation of the tissue structure. Again, the specific reaction over the tumor cells is evident. After a similar autoradiographic exposure time (3 weeks), the silver grain densities obtained with the 3H-labeled probes were comparable with those obtained using the 32P-labeled probes, even though the specific radioactivity of the former was sixfold lower at the time of application to the section. Figure9a and b also shows tumor regions in which remarkable differences with respect to silver grain density are visible over adjacent cells. As already mentioned, the reasons for these cell-tocell differences are not yet clear. In the various kinds of tissues examined, we did not observe consistent accumulations of silver grains over specific cell regions such as the perinuclear cytoplasm. This indicates that a juxtanuclear distribution, as recently reported for the mRNA encoding vimentin in certain cultured cells [68], does not exist for cytokeratin mRNAs in tissues. Discussion The simple-epithelium-type cytokeratins, nos. 8 and 18 also termed A and D or Endo A and Endo B in rodents - are a frequently occurring ‘expression pair’ [lo, 29-36, 81, 83, 1091. These two cytokeratins are found not only in one-layered epithelia but also in some complex epithelia comprising several different cell types (for references, see [38, 81, 92]), in basal cells of certain stratified epithelia ([39]; W.W. Franke, unpublished data), and in a variety of epitheliumderived tumors, notably carcinomas [81, 92, 1021. The present study describes the first cDNA probes for this expression pair that will be valuable tools in studies of cytokeratin expression at the mRNA level. Our sequence data obtained from the cDNA clone, pKH18’, encoding human cytokeratin no. 18 confirm and extend earlier results reported for a shorter human cDNA clone (pKH18l [99]) and for a complete cDNA clone of the murine equivalent of cytokeratin no. 18 [103]. Comparison of the amino acid sequence of cytokeratin no. 18 with the sequences of other cytokeratins identifies this protein as being a typical member of the acidic (type I) cytokeratin subfamily, although several differences from other type-I cytokeratins are evident (for a detailed discussion, see [99, 1031). Remarkably, human cytokeratin no. 18 has 9 additional amino acids in the head domain of the molecule, as compared to the murine protein, which is indicative of insertion events during mammalian evolution. Our analysis of the partial amino acid sequence of human cytokeratin no. 8 shows that it is a typical cytokeratin of the basic (type 11) subfamily, displaying a high sequence homology to the corresponding bovine and Xenopus proteins (for detailed discussions of sequence features, see [40, 721). An obvious difference between human and bovine cytokeratin no. 8, on the one hand, and the amphibian pro-

Fig. 8a, b, In situ hybridization of the Same metastatic lymph-node tumor as that shown in Fig. 7 using ”P-UTP-labeled RNA probes derived from cDNA clones encoding human cytokeratin no. 18. The hybridization solution did not contain SDS, and the autoradiographic exposure time was 3 weeks. a Hybridization with anti-sense RNA, i.e., RNA complementary to mRNA for human cytokeratin no. 18. Note the specific labeling of the tumor cells (center and feft ham and the absence of significant labeling over the lymph-node cells. The signal-to-noise ratio in this specimen was 7.5:l. b Hybridization with the corresponding sense RNA, i.e., RNA sequences identical with the sequence of cytokeratin-n0.-18 mRNA. The signal-to-noise ratio in this specimen was 1.3 :1. Kodak Ektachrome artificial light film. Burs, 25 pm

80

81

tein, on the other, is the absence of glycine-rich oligopeptide repeats in the tail domain of the two mammalian species. The absence of glycine-rich oligopeptide repeats in the tail of human cytokeratin no. 8 is also remarkable in view of the existence of such repeats in human cytokeratin no. 7 [&I, a protein with a similar molecular mass and isoelectric point which is co-expressed with cytokeratin no. 8 in several cell types (cf. [31, 35, 39, 63, 72, 80-83, 92, 1201). The presence of such glycine-rich regions in the head and tail domains has been discussed in relation to the increased hydrophobicity, the relatively higher insolubility [105], and the higher ‘melting points’ of the different chains [36] of these cytokeratins [73], but experimental evidence for functional differences between cytokeratins with and without glycine-rich tails is lacking. It is worth mentioning in this context that similar glycine-rich repeats can be identified in various other proteins such as the precursor of the prion protein that forms ‘scrapie-associated fibrils’ ([98] ;this protein contains four complete and two incomplete repeats of the motif, WGQPHGGG), the ‘core protein A1 ’ associated with heterogenous nuclear ribonucleoprotein particles [97] and a protein called ‘nucleolin’ which is assumed to interact with pre-rRNA ([67l; in both nuclear proteins, similar repeats of the basic motif gGG!RGG and modifications thereof are remarkable). Fisher et al. [23] have recently drawn attention to the existence of a GGGS and a GGSGGG motif in nuclear lamins A and C, which share considerable sequence homology with IF proteins in the a-helical rod domain [76]. The identity of the amino acid sequence of human cytokeratin no. 8 with that of two fragments of the serum component TPA proves that this protein is a cytokeratin, and that the protein fraction referred to as TPA contains fragments of cytokeratin no. 8 which are probably derived by proteolysis from the cytokeratin IFs of carcinomas and possibly also other epithelial cells - upon cell lysis in necrotic tissue (cf. [73, 1161). These data as well as the relationship of at least one other TPA-fragment (B1 subfragments A and B) to type-I cytokeratins (the preparation called B1 in [95] appears to be heterogeneous) add further support to the concept that TPA is a degradation product of IF proteins [70], specifically cytokeratins [I 161. This also explains most of the immunohistochemical results obtained using TPA antibodies [4, 86, 1161. As the identity of TPA and cytokeratin no. 8 has now been demonstrated, it should be possible to design improved serodiagnostic assays for specific cytokeratins. These could be based on the use of established monoclonal cytokeratin antibodies recognizing epitopes in the rod portions that are relatively more protected from proteolysis, or by producing antibodies directed against selected cytokeratin sequences located in these protected regions. As we have demonstrated by Northern-blot analysis in the present and a previous study [99], cDNA clones for human cytokeratin nos. 8 and 18 can be used to detect

selectively their mRNAs and to distinguish them from mRNAs encoding other IF proteins, including cytokeratins of the same subfamily. In the present study, we showed that such cDNA clones can be used to prepare DNA or RNA probes for the detection of mRNAs for cytokeratins nos. 8 and 18 in cells and tissues by in situ hybridization. The demonstration of these mRNAs in cell clusters of lymph nodes from a patient with breast carcinoma unequivocally identifies these cells as a metastatic carcinoma. This is in agreement with gel electrophoretic demonstrations of the presence of cytokeratins nos. 8 and 18 in all forms of ductal and lobular carcinomas ([80-821; most of these tumors contain, in addition, cytokeratins nos. 7 and 19). The in situ hybridization technique complements the immunocytochemical staining techniques that are already widely used in pathology for the detection of carcinoma metastases (cf. [43, 80, 81, 871). The sensitivity and resolution of the in situ hybridization procedures described in our study (both with ”P- and 3H-labeledprobes) are clearly sufficient to detect ‘micrometastases’, i.e., small groups of carcinoma cells or even scattered individual carcinoma cells. It will be interesting to apply these cDNA-derived probes for simple-epithelium-type cytokeratin mRNAs in combination with probes of mRNAs for cytokeratins characteristic of certain stratified epithelia, in order to characterize certain types of carcinomas in greater detail and to examine cell-type heterogeneity of cytokeratin expression in the same tumor. The occurrence of cell-type heterogeneity of cytokeratin expression in certain tumors has already been shown by immunocytochemical observations using specific monoclonal cytokeratin antibodies (e.g. [17, 39, 80, 1121). In situ hybridization of mRNAs encoding other IF proteins has recently been demonstrated, although at lower magnifications, for nick-translated 35S-labeledcDNAs encoding glial filament protein [69] or certain murine cytokeratins specific for some stratified epithelia [a]. Although it is clear that, in histology and diagnostic pathology, immunocytochemistry will continue to be preferred for routine tests, in situ hybridization is necessary to resolve certain questions such as the possible existence of mRNAs in the absence of the protein encoded, be it due to mRNA inactivation, low steady-state concentrations of the protein, or selective degradation of the protein(s) in a particular tissue sample. In situ hybridization of mRNAs of IF proteins will also be an important control for negative immunocytochemical results that may be due to inaccessibility (masking) of the specific epitope or epitopes (for a discussion, see also [54]). Acknowledgements. We thank Thomas M. Magin for help, cooperation, and valuable discussions. We are also grateful to Dr. Frank Longo for reading the manuscript and Irmgard Purkert for its careful typing. This work was supported by the Deutsche Forschungsgemeinschaftand the Research Council ‘ Rauchen und Gesundheit’.

Fig. 9a, b. Detection of mRNAs encoding human cytokeratins no. 8 (a) and no. 18 @) in a lymph-node metastasis of a breast carcinoma by in situ hybridization using RNA probes (same tissue as that shown in Figs. 7 and 8). In this experiment, both probes were labeled with 3H-UTP and ”-GTP, and were subjected to limited alkaline hydrolysis. The hybridization solution contained 0.5% SDS. The autoradiographic exposure time was 3 weeks. In a, note the marked heterogeneity of the silver grain density over the tumor cells. Also note that lymphoid cells included in the tumor areas are devoid of significant labeling. The signal-to-noise ratio with the cytokeratinn o . 4 probe was 7.8:1 (a), while that with the human cytokeratin-n0.-18 probe was 3.4:l 0).Kodak Ektachrome daylight film.Burs, 25 pm

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References 1. Achtstaettcr T, Moll R, Moore B, Franke WW (1985) Cytokeratin polypeptide patterns of different epithelia of human male urogenital tract : lmmunofluorescence and gel electrophoresis studies. J Histochem Cytochem 33:415426 2. Bader BL. Magin TM, Hatzfeld M, Franke WW (1986)Amino acid sequence and gene organization of cytokeratin no. 19, an cxccptional tail-less intermediate filament protein. EMBO J 5:1865-1875 3. Banks-Schlegel S,Harris CC (1983)Tissue-spccific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes. Exp Cell Res 164: 271-280 4. Beham A, Weybora W, Lackinger E, Denk H, BjBrklund V, Bjorklund B (1986) Distribution of TPA and cytokeratins in gastrointestinal carcinomas as revealed by immunocytochemistry. Virchows Arch [Patho] Anat] 409:641-655 5. Bjorklund B (1981) Tissue polypeptide antigen. Review and r m n t progress. In: von Kleist S, Breucr H (eds) Critical evaluation of tumor markers. Karger, Basel, pp 73-87 6. Blobel GA, Moll R, Frankc WW, Vogt-Moykopf I (1984) Cytokeratins in normal lung and lung carcinomas. 1. Adenocarcinomas, squamous cell carcinomas and cultured cell lines. Virchows Arch [Cell Pathol] 45 :407429 7. Blobel GA, Could VE, Moll R, Lee I, Huszar M, Geiger B. Franke WW (1985) Coexpression of neuroendocrine markers and epithelial cytoskeletal proteins in bronchopulmonary neuroendocrine neoplasms. Lab Invest 52:39-51 8. Blobel GA. Moll R, Franke WW, Kayser KW, Could VE (1985) The intermediate filament cytoskeleton of malignant mesotheliomas and its diagnostic significance. Am J Pathol 1211235-247 9.Briilet P, Babinet C, Kemler R, Jacob F (1980) Monoclonal antibodies against trophectoderm-specific markers during mousc blastocyst formation. Proc Natl Acad Sci USA 77:4113-4117 10. Cooper D, Schcrmer A, Sun T-T (1985) Biology of disease. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Strategies, applications, and limitations. Lab Invest 52:243-256 11. Cox KH, DeLeon DV, Angerer LEI, Angerer RC (1984)Detection of mRNAs in sea urchin cmbryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101 :485-502 12. Crewther WG, Dowling LM, Steinert PM, Parry DAD (1983) Structure of intermediate filaments. Int J Biol Macromol 5 :267-274 13.Czernobilsky B. Moll R, Levy R. Franke WW (1985) Coexpression of cytokeratin and vimentin filaments in mesothelial, granulosa and rete ovarii cells of thc human ovary. Eur J Cell Biol 37:175-190 14.Czernobilsky B, Moll R, Leppien G, Schweikhart G, Franke WW (1 986)Desmosomal plaque-associated vimentin filaments in human ovarian gmnulosa cell tumors of various histologic patterns. Am J Pathol (in press) 15. Davanloo P, Rosenberg AH, Dunn JJ, Studicr FW (1984) Cloning and expression of thc gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 81 :2035-2039 16.Debus E, Wcber K, Osborn M (1982)Monoclonal cytokcratin antibodies that distinguish simple from stratified squamous epithelia: Characterization on human tissues. EMBO J 1:1641-1647 17.Debus E, Moll R, Franke WW, Webcr K, Osborn M (1984) lmmunohistochemical distinction of human carcinomas by cytokeratin typing with monoclonal antibodies. Am J Pathol 114:121-1 30 18. Denk H, Krepler R, Lackinger E, Artlieb U, Frankc WW (1982) Biochemical and immunological analysis of the intermediate filament cyioskeleton in human hepatocellular carcinomas and in hepatic neoplastic nodules of mice. Lab Invest 46 :584596

19.Dixon IS, Stanley MA (1984) lmmunofluorescent studies of human cervical epithelia in vivo and in vitro using antibodies against specific keratin components. Mol Biol Med 2:37-51 20. Duprey P, Morello D, Vasseur M, Babinet C, Condiimine H, Briilet P, Jacob F (1985) Expression of the cytokeratin Endo A gene during early mouse embryogenesis. Proc Natl Acad Sci USA 82:8535-8539 21. Eckert RL, Green H (1984) Cloning of cDNAs specifying vitamin A-responsive human keratins. Proc Natl Acad Sci USA 81 :43214325 22. Fabricant RN. Delarco JE, Todaro G (1977) Nerve growth factor receptors on human melanoma cells in cultures. Proc Natl Acad Sci USA 74:565-569 23. Fisher DZ, Chaudhary N, Blobel G (1986)cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediatc filament proteins. Proc Natl Acad Sci USA 83:645W54 24. Franke WW, Schmid E, Osborn M, Weber K (1978)Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci USA 75 :503&5038 25. Franke WW, Weber K, Osborn M, Schmid E, Freudenstein C (1 978)Antibody to prekeratin. Decoration of tonofilamentlike arrays in various cells of epithelial character. Exp Cell Res 116:42-5 26. Franke WW, Appelhans B, Schmid E, Freudenstein C, Osborn M, Weber K (1979) Identification and characterization of epithelial cells in mammalian tissues by immunofluorescence microscopy using antibodies to prekeratin. Differentiation 15:7-25 27. Frankc WW, Schmid E, Osborn M (1979)HeLa cells contain intermediate-sized filaments of the prekeratin type. Exp Cell Res 118:9S109 28. Franke WW, Schmid E, Winter S,Osborn M, Weber K (1979) Widespread Occurrence of intermediate-si7d filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp Cell Res 123 :25-46 29. Franke WW, Denk H, Kalt R, Schmid E (1981) Biochemical and immunological identification of cytokeratin proteins in hepatocytes and hepatoma cells. Exp Cell Res 131 :299-318 30. Franke WW, Mayer D, Schmid E, Denk H, Borenfreund E (1981) Differences of expression of cytoskeletal proteins in cultured rat hepatocytes and hepatoma cells. Exp Cell Res

134:345-365 31. Franke WW, Schiller DL, Moll R, Winter S, Schmid E, Engelbrecht I, Denk H, Krepler R, Platzer B (1981) Diversity of cytokeratins : Differentiation-specific expression of cytokeratin polypeptides in epithelial cells and tissues. J Mol Biol

153~933-959 32. Franke WW, Winter S, Grund C, Schmid E, Schiller DL, Jarasch E-D (1981) Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border. J Cell Biol 90:116127 33. Franke WW, Grund C, Kuhn C, Jackson BW, Illmensee K (1982) Formation of cytoskeletal elements during mouse embryogenesis. Ill. Primary mesenchymal cells and the first appearance of vimentin filaments. Differentiation 23 :43-49 34. Franke WW, Schmid E, Grund C, Geiger B (1982)Intermediate filament protcins and nonfilamentous structures: Transicnt disintegration and inclusion of subunit proteins in granular aggregates. Cell 30 :103-1 13 35. Franke WW, Schmid E, Schiller DL, Winter S, Jarasch E-D, Moll R, Denk H, Jackson B, Illmensee K (1982) Differentiation-related patterns of expression of proteins of intermediatesized filaments in tissues and cultured cells. Cold Spring Harbor Symp Quant Biol46:431453 36. Franke WW, Schiller DL, Hatzfeld M, Winter S (1983) Protein complexes of intermediate-sized filaments : Melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics. Proc Natl Acad Sci USA 80:7113-7117 37. Franke WW, Schmid E. Wellsteed J, Grund C, Gigi 0, Geiger B (1 983) Change of cytokeratin filament organization during

83

the cell cycle: Selective masking of an immunologic determinant in PtK, cells. J Cell Biol97:1255-1260 38. Franke WW, Grund C, Achtstatter T (1986) Co-expression of cytokeratins and eurofilament proteins in a permanent cell line: Cultured rat PC12 cells combine neuronal and epithelial features. J Cell Biol (in press) 39. Franke WW, Moll R, Achtstatter T, Kuhn C (1986) Cell t y p ing of epithelial and carcinomas of the female genital tract using cytoskeletal proteins as markers. In Banbury Report 21 : Viral etiology of cervical cancer. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 121-148 40.Franz JK, Franke WW (1986) Cloning of cDNA and amino acid sequence of a cytokeratin expressed in oocytes of Xenopus lueuis. Proc Natl Acad Sci USA 83 :6475-6479 41. Fuchs E, Green H (1 978) The expression of keratin genes in epidermis and cultured epidermal cells. Cell 15 :887-897 42.Fuchs EV, Coppock SM, Green H, Cleveland DW (1981) Two distinct classes of keratin genes and their evolutionary significance. Cell 27: 75-84 43. Gabbiani G , Kapanci Y, Barazone P, Franke WW (1981) lmmunochemical identification of intermediate-sized filaments in human neoplastic cells. Am J Path01 104:2&216 44. Gall IG, Pardue M L (1971) Nucleic acid hybridization in cytological preparations. Methods Enzymol 21 :47&480 45. Geisler N, Weber K (1981) Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: Desmin and vimentin. Proc Natl Acad Sci USA 78:4120-4123 46. Glass C, Kim KH, Fuchs E (1985) Sequence and expression of a human type 11 mesothelial keratin. J Cell Biol 101:23662373 47. Gown AM, Vogel AM (1984) Monoclonal antibodies to human intermediate filament proteins. 11. Distribution of filament proteins in normal human tissues. Am J Pathol 1141309-321 48. Hafen ML. Levine M, Garber RL, Gehring WJ (1983) Improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex. EMBO J 2:617-623 49. Hanukoglu 1, Fuchs E (1982) The cDNA of a human epidermal keratin: Divergence of sequence but conservation of structure among intermediate-filament proteins. Cell 31 :24S252 50. Hanukoglu I, Fuchs E (1983) The cDNA sequence of a type-11 cytoskeletal keratin reveals constant and variable structural domains among keratins. Cell 33 :915-924 51. Hatzfeld M, Franke WW (1985) Pair formation and promiscuity of cytokeratins: Formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol 101 : 18261841 52. Hazan R, Denk H, Franke WW, Lackinger E, Schiller DL (1986) Change of cytokeratin organization during development of Mallory bodies as revealed by a monoclonal antibody. Lab Invest 54: 54S553 53. Hoefler H, Denk H, Lackinger E, Helleis G, Polak JM, Heitz PU (1986) Immunocytochemical demonstration of i n t m e d i ate filament cytoskeleton proteins in human endocrine tissucs and (neuro-) endocrine tumors. Virchows Arch [Pathol Anat] 409 :609-626 54. Hoefler H, Childers H, Montminy MR, M a n RM, Goodman RH. Wolfe HJ (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem J (in press) 55. Huszar M, Gigi-Leitner 0, Moll R, Franke WW, Geiger B (1986) Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia. Selective markers for stratification and squamous cell carcinomas. Differentiation 31~141-153 56. Huynh TV, Young RA, Davis RW (1985) Constructing and screening cDNA libraries in lambda gtlO and lambda g t l l .

In: Glover DM (ed) DNA Cloning. I. A practical approach. IRL Press, Oxford Washington, pp 49-78 57. Jackson BW, Grund C, Schmid E, Biirki K, Franke WW, Illmensee K (1980) Formation of cytoskeletal elements during mouse embryogenesis. I. Intermediate filaments of the cytokeratin type and desmosomes in preimplantation embryos. Differentiation 17: 161-179 58. Jackson BW, Grund C, Winter S, Franke WW, Illmensee K (1981) Formation of cytoskeletal elements during mouse embryogenesis. 11. Epithelial differentiation and intermediatesized filaments in early postimplantation embryos. Differentiation 20:203-216 59. Jonas E, Sargent TD, Dawid IB (1985) Epidermal keratin gene expressed in embryos of Xenopus lamis. Proc Natl Acad Sci USA 82 :541 S5417 60. Jorcano JL, Franz JK, Franke WW (1984) Amino acid sequence diversity betwccn bovine epidermal cytokeratin polypeptides of the basic (type TI) subfamily as determined from cDNA clones. Differentiation 28: 155163 61. Jorcano JL, Magin TM, Franke WW (1984) Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones. J Mol Biol 176:21-37 62. Jorcano JL, Rieger M, Franz JK,Schiller DL, Moll R, Franke WW (1984) Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I. J Mol Biol 179:257-281 63. Kim KH, Rheinwald JG, Fuchs EV (1983) Tissue specificity of epithelial keratins : Differential expression of mRNAs from two multigene families. Mol Cell Biol3:495-502 64. Knapp B, Rentrop M, Schweizer J, Winter H (1986) Nonepidermal members of the keratin multigene family: cDNA sequences and in situ localization of the mRNAs. Nucleic Acids Rm14:751-763 65. Lane EB (1982) Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J Cell Biol92:665-673 66. Lane EB, Bartek J. Purkis PE, Leigh IM (1985) Keratin antigens in differentiating skin. Ann NY Acad Sci 455 :241-258 67. Lapeyre B, Amalric F, Ghdffari SH, Venkatarama Rao SV, Dumbar TS, Olson MOJ (1986) Protein and cDNA sequence of a glycine-rich, dimethylarginine-containingregion located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa). J Biol Chem 261 :9167-9173 68. Lawrence JB, Singer R H (1986) lntracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45: 407-41 5 69. Lewis SA, Cowan NJ (1985) Temporal expression of mouse glial fibrillary acidic protein mRNA studied by a rapid in situ hybridization procedure. J Neurochem 45:913-919 70. Luening B, Nilsson U (1983) Sequence homology between tissue polypeptide antigen (TPA) and intermediate filament (IF) proteins. Acta Chem Scand 37:731-733 71. Luening B, Wiklund B, Redelius P, Bjiirklund B (1980) Biochemical properties of tissue polypeptide antigen. Biochim Biophys Acta 624:90-101 72. Magin TM, Jorcano JL, Franke WW (1983) Translational products of mRNAs coding for non-cpidermal cytokeratins. EMBO J 2:1387-1392 73. Magin TM, Jorcano JL, Franke WW (1986) Cytokeratin expression in simple epithelia. 11. cDNA cloning and sequence characteristics of bovine cytokeratin A (no. 8). Differentiation 30: 254-264 74. Manidtis T, Frisch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New Y ork 75. Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65 :499-560 76. McKeon FD, Kirschner MW, Caput D (1986) Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature 319:463-468

84 77. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101 :2&78 78. Miettinen M, Clark R, Virtanen 1 (1986) Intermediate filament proteins in choroid plexus and ependyma and their tumors. Am J Pathol 123:231-240 79. Moll R. Franke WW (1985) Cytoskeletal differences between human neuroendocrine tumors: A cytoskeletal protein of molecular weight 46,OOOdistinguishes cutaneous from pulmonary neuroendocrine neoplasms. Differentiation 30: 1 6 5 175 80. Moll R, Franke WW (1986) Cytochemical cell typing of metastatic tumors according to their cytoskelctal proteins. In: Lapis K, Liotta LA, Rabson AS (eds) Biochemistry and moleculargenetics ofcancer metastasis. M. Nijhoff, Boston, pp 101-1 14 81. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratin polypeptides: Patterns of expression of specific cytokeratins in normal epithelia, tumors, and cultured cells. Cell 31 :11-24 82. Moll R, Krepler R, Franke WW (1983) Complex cytokeratin polypeptide patterns observed in certain human carcinomas. Differentiation 23 :256-269 83. Moll R, Levy R, Czernobilsky B. Hohlweg-Majcrt P, Dallenbach-Hellweg G, Franke WW (1983) Cytokeratins of normal epithelia and some neoplasms of thc female genital tract. Lab Invest 49 :599-61 0 84. Moll R, Moll I, Franke WW (1984) Identification of Merkel cells in human skin by specific cytokeratin antibodies: Changes of cell density and distribution in fetal and adult plantar epidermis. Differentiation 28 :136 1 5 4 85. Nagle RB, Moll R, Weidauer H, Ncmetschek H, Franke WW (1985) Different patterns of cytokeratin expression in thc normal epithelia of the upper respiratory tract. Differentiation 30: 130-140 86. Nathrath W BJ, Heidenkummer P, Bjorklund V, Bjorklund B (1985) Distribution of tissue polypeptide antigen (TPA) in normal human tissues: lmmunohistochemical study of unfixed, methanol-. ethanol-, and formalin-fixed tissues. J Histochem Cytochem 33:99-109 87. Osborn M, Webcr K (1983) Tumor diagnosis by intermediate filament typing: A novel tool for surgical pathology. Lab Invest 48: 372-394 88. Oshima RG (1981) Identification and immunoprecipitation of cytoskeletal proteins from murine extra-embryonic endodermal cells. J Biol Chem 256:8124-8133 89. Oshima RG, Howe WE, Klier FG, Adamson ED, Shevinsky LH (1983) lntcrmediate filament protein synthesis in preimplantation murine embryos. Dev Biol99:447+55 90. Ouhayoun J-P, Gosselin F, Forest N, Winter S, Franke WW (1985) Cytokeratin patterns of human oral epithelia: Differe n m in cytokeratin synthesis in gingival epithclium and the adjacent alvcolar mucosa. Differentiation 30: 123-1 29 91. Quinlan RA, Cohlberg JA, Schiller DL, Hatzfeld M, Franke WW (1984) Heterotypic tetramer (A,D,) complexes of nonepithelial keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells. J Mol Biol 178:365 -388 92. Quinlan RA, Schiller DL, Hatzfeld M, Achtstiitter T, Moll R, Jorcano JL, Magin TM, Franke WW (1985) Pattcms of exprcssion and organiiation of cytokeratin intcrmediatc filamcnts. Ann NY Acad Sci 455 :282-306 93. Ramaekcrs F, Huysmans A, Moesker 0, Kant A, Jap P, Herman C, Vooijs P (1983) Monoclonal antibody to keratin filaments, specific for glandular epithelia and their tumors. Lab Invest 49:353 -361 94. Ramaekers FCS, Puts JJG, Moeskcr 0, Kant A, Huysmans A, Ilaag D. Jap PHK, Herman CJ, Vooijs G P (1983) Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: An overview. Histochem J 15:691-713 95. Redelius P, Luening B, Bjoerklund B (1980) Chemical studies of tissue polypeptide antigcn (TPA). 11. Partial amino acid sequences of cyanogen bromide fragments of TPA subunit B,. Acta Chem S a n d [B] 34:265-273

96. Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237251 97. Riva S, Morandi C, Tsoulfas P, Pandolfo M, Biamonti G, Merrill B, Williams KR, Multhaupt G, Beyreuther K, Werr H, Henrich B, Schifer K P (1986) Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1 . EMBO J 5 :2267-2273 98. Robakis NK, Sawh PR, Wolfe GC, Rubenstein R, Carp RI, lnnis MA (1986) Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues. Proc Natl Acad Sci USA 83:63774381 99. Romano V, Hatzfeld M, Magin TM, Franke WW, Maier G, Ponstingl H (1 986) Cytokeratin expression in simple epithelia. I. Identification of mRNA coding for human cytokeratin no. 18 by a cDNA clone. Differentiation 30:244-253 100. Roop DR, Hawley-Nelson P, Cheng CK, Yuspa SH (1983) Keratin gene expression in mouse epidermis and cultured epidermal cells. Proc Natl Acad Sci USA 80:716720 101. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:54635467 102. Schiller DL, Franke WW, Geiger B (1982) A subfamily of relatively largc and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells. EMBO J 1 :761-769 103. Singcr PA, Trevor K, Oshima R G (1986) Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos. J Biol Chem 261 :538-547 104. Soule HD, Vazquez J, Long A, Albert S, Brennan M (1973) A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer lnst 51 :1409-1416 105. Steinert PM, Parry DAD, Idler WW, Johnson LD, Steven AC, Roop DR (1985) Amino acid sequences of mouse and human epidermal type I I keratins of M,67,000 provide a systematic basis for the structural and functional diversity of the end domains of keratin intermediate filament subunits. J Biol Chem 260:7142-7149 106. Steinert PM, Steven AC, Roop D R (1985) The molecular biology of intermediate filaments. Cell 42:411--419 107.Summerhayes IC, Chen LB (1982) Localization of an M, 52,000 keratin in basal epithelial cells of the mouse bladder and expression throughout neoplastic progression. Cancer Res 42:4098-4109 108. Sun T-T, Shih C, Green H (1979) Keratin cytoskeletons in epithelial cells of internal organs. Proc Natl Acad Sci USA 76:2813-2817 109. Sun T-T, Eichner R, Schermer A, Cooper D, Nelson WG, Wciss RA (1984) Classification, expression and possible mechanisms of evolution of mammalian epithelial keratins: A unifying model. In: The transformed phenotype, cancer cells, vol 1 . Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 169-176 110. Tseng SCG, Jarvinen MJ, Nelson WG, Huang J-W, Woodcock-Mitchell J, Sun T-T (1982) Correlation of specific keratins with different types of epithelial differentiation : Monoclonal antibody studies. Cell 30: 361 -372 111. Ullrich A, Coussens JS, Dull TJ, Gray A, Tam AW. Lee J. Yarden Y, Libermann TA, Schlessinger J, Downward J, Mayes ELV, Whittle N, Waterfield MD, Seeburg PH (1984) Human epidermal growth factor receptor cDNA sequcnce and aberrant expression of the amplified genes in A431 epidermoid carcinoma cells. Nature 309: 41 8 4 2 5 112. Van Muijen GNP, Ruiter DJ, Franke WW, Achtstaetter T, Haasnoot WHB, Ponec M, Warnaar SO (1986) Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13. Exp Cell Rcs 162 :97-1 13 113. Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-

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derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268 114. Ward WS, Schmidt WN, Schmidt CA, Hnilica LS (1985) Cross-linking of Novikoff ascites hepatoma cytokeratin filaments. Biochemistry 24 :4 4 2 W 3 4 115. Weber K, Geisler N (1984) Intermediate filaments from wool a-keratins to neurofilaments: A structural overview. In: The transformed phenotype, cancer cells, vol 1 . Cold Spring Habor Laboratories, Cold Spring Harbor, pp 153-1 59 116. Weber K, Osbom M, Moll R, Wiklund B, Luening B (1984) Tissue polypeptide antigen (TPA) is related to the non-epiderma1 keratins 8, 18 and 19 typical of simple and non-squamous epithelia: Reevaluation of a human tumor marker. EMBO J 3:2101-2714 117. Wiklund B, Liining B, Bjorklund B (1981) Chemical studies of tissue polypeptide antigen (TPA). 111. On the nature of

the antigenic determinant(s) of TPA subfraction B1. Acta Chem Scand [B] 35: 325-336 118. Wild GA, Mischke D (1986) Variation and frequency of cytokeratin polypeptide patterns in human squamous non-keratinizing epithelium. Exp Cell Res 162 :114-126 119. Woodcock-Mitchell J, Eichner R, Nelson WG, Sun T-T (1982) Immunolocalization of keratin polypeptides in human epiderm i s using monoclonal antibodies. J Cell Biol95:58&588 120. Wu Y-J,Parker LM, Binder NE, Beckett MA, Sinard JH, Griffiths CT, Rheinwald JG (1982) The mesothelial keratins: A new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell 31~693-I03 Received August 1986 / Accepted in revised form September 30, 1986