Keratin Subtypes of Small Cell Lung Cancer Anthony D

(CANCER RESEARCH 48, 2724-2729, May 15, 1988] Keratin Subtypes of Small Cell Lung Cancer Anthony D. Elias,1 Bruce F. Cohen, and Samuel D. Bernal2

Division of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT with the 56.5 kDa (keratin 10) and 65-67 kDa (keratins 1-2) keratins that may be regarded as markers of skin-type differentiation (keratini- The molecular forms of keratin in small cell lung cancer (SCLC) cell zation). AE-3 reacts with the basic family of keratins (keratins 1-8). lines and tumors were examined with antikeratin monoclonal antibodies. The immunoreactivity of keratin 64 kDa (keratin 9) with AE-1, AE-2, Immunostaining of SCLC by antikeratin antibody and examination by and AE-3 is unknown. A mouse monoclonal IgG antibody (287) un- fluorescence microscopy indicates population heterogeneity in keratin reactive with carcinoma cells was used as a negative control. content. Intensity of immunostaining is often weak. However, polyacryl- Cell Lines and Tumor Specimens. All cell lines were grown in RPMI amide gel electrophoresis and immunoblotting reproducibly demonstrate 1640 with 2 HIMglutam ine and 10% calf serum. The variant cell line the presence of keratin and allow analysis of the keratin subtypes. The (low dopa-decarboxylade, few neurosecretory granules, loose suspen finding of keratin subtypes closely associated with the development of sion growth) SW2-S was established from metastatic tumor in the bone keratinizing epithelium (the 68 kDa basic keratin) in SCLC was unex marrow of a patient with SCLC. The attached subline, SW2-A, was pected in a tumor that is regarded as poorly differentiated. The cytoskel- isolated and subcloned from tissue cultures of SW2-S after 2 years in etal composition of SCLC suggests the presence of a heterogeneous culture. The classic SCLC line (high dopa-decarboxylase, tight colony population with a significant proportion of cells expressing highly differ morphology, and many neurosecretory granules) OH-1 has been pre entiated epithelial properties. viously characterized (13). The lung adenocarcinoma cell lines A549 and CAÃ•,U-3,and the lung squamous cell carcinoma CALU-1 were purchased from the American Type Culture Collection. The classic INTRODUCTION SCLC cell line NCI-H69 was obtained from Dr. A. Gazdar, National The keratins are a complex set of at least 19 related proteins Cancer Institute, Bethesda, MD (14). The undifferentiated lung carci which form the intermediate filaments of epithelial cells. Al noma LX-1, derived from a tumor with both small cell and squamous though their functions within the cell are not well understood, cell carcinoma characteristics (15), was provided by Dr. A. E. Bogden, these proteins seem to be expressed in specific combinations Mason Research Institute, Worcester, MA. The lung squamous cell related to the cell type and its state of differentiation (1-7). carcinomas, HOTZ and U1752, were provided by Dr. P. Groscurth, University of Zurich, Switzerland, and Dr. J. Bergli, Uppsala, Sweden, Different isoelectric points and gene structures characterize the respectively (16, 17). Other cell lines were supplied by investigators at two families of keratins, acidic and basic. All keratins contain the Dana-Farber Cancer Institute: squamous cell carcinomas of the a constant central Â«-helical rod leading to their filamentous head and neck SCC-15, SCC-25, SCC-68, SCC-78 (Dr. J. Rheinwald); structure and variable regions at both ends which presumably breast carcinoma MCF-7 (Dr. D. Kufe); melanoma G361 (Dr. M. allow specific functions within the cell. Coordinate expression Wick); and lymphoblastic leukemia CEM (Dr. H. Lazarus). A2182, an of pairs of keratins has been observed; within each keratin pair attached cell line with SCLC characteristics, was kindly donated by as defined by frequent coexpression, the basic keratin is usually Centocor, Inc. (Dr. S. Mai). Other cell lines were developed in this about 8 kDa larger than its paired acidic counterpart (4). laboratory. Tumor tissues were either extracted within l h of collection or were In the past few years, it has been recognized that in most flash-frozen in liquid nitrogen and stored until used. Four surgical cases carcinoma cells contain similar patterns of keratin sub specimens were obtained from Dr. C. O'Hara at New England Deacon types as their normal cellular counterparts. This suggests that ess Hospital. The SCLC tissue shown in Fig. 2 and Table 1 (SCC) was analysis of keratin content may help delineate the relationships a lymph node metastasis from a patient with oat cell histology. SW2-S between the lung carcinomas and the highly complex lower cells were passaged in nude mice and extracts were made of the solid respiratory epithelium (8, 9). We have previously reported the tumor nodules (SW2-M2). Normal bronchial epithelial cells were presence of keratins in SCLC,3 analyzed by two-dimensional grown from expiants by Dr. K. Weinberg (18). gel electrophoresis (10). In this report, we describe a character Indirect Immunofluorescence Staining. Suspension cells or cells grown on glass coverslips were washed with phosphate-buffered saline, pH istic set of keratins in SCLC cell lines and tumors by immuno 7.4, twice and then fixed with methanol at -20Â°Cfor 3 min. The fixed staining and immunoblotting with antikeratin antibodies. Many cells were washed with distilled water, then PBS, and overlaid with of the SCLC keratins were also found in NBE cells and in primary antibody for 45 min at 37"C (AE-1, 1:10; AE-2, 1:5; and AE- NSCLC, suggesting a close relationship between these different 3, 1:10). After washing with PBS, flunrest-ciliated goat anti-mouse IgG cell types. 1:50 (Cappel Laboratories) was incubated for 45 min. After washing in PBS and distilled water, the cells were mounted in 10% PBS, 90% MATERIALS AND METHODS glycerol, 100 mg/dl /vphenylenediamine, pH 8.0 (19), and photo graphed under a Zeiss epifluorescence microscope with the use of TRI- Antikeratin Antibodies. The murine anti-human keratin monoclonal X ASA 400 Kodak film. antibodies AIM, AE-2, and AE-3 have been previously characterized Keratin Extraction. Using a procedure modified from Wu et al. (20), (11,12) and were kindly donated by Dr. Tung-Tien Sun. AE-1 has been cells were washed with PBS and resuspended in 5 ml of high salt buffer shown to react with all of the acidic family of keratins (keratins 10-19) (20 mM Tris-HCl, pH 7.4; 0.6 M KC1; 1% Triton X-100; 1 mM [Moll classification (1)], except 45/46 kDa (keratin 17/18). AE-2 reacts phenylmethylsulfonyl fluoride; 1 mM ethyleneglycol tetraacetate; 1 mM EDTA) at 4Â°C.After homogenization in a Wheaton homog- Received 6/17/87; revised 12/29/87; accepted 1/15/88. enizer, the insoluble fraction was pelleted by centrifugal ion at 10,000 The costs of publication of this article were defrayed in part by the payment x g for 15 min, then incubated with nuclease solution [DNAse I (Sig of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ma Chemical Co.) 100 units/ml-RNAse A (Sigma) 0.05 mg/ml in 1American Cancer Society Physician's Training Research Fellow. PBS] for 30-60 min at 4Â°C.The pellet was then homogenized in 2Supported by Grant Ã‡A4SS28 from the National Cancer Institute. high salt buffer and centrifuged. Finally the pellet was washed in PBS 'The abbreviations used are: SCLC, small cell lung cancer; NBE, normal and stored at â€”70Â°C.Insome experiments, cells were metabolically bronchial epithelial; NSCLC, non-small cell lung cancers; PBS, phosphate butt labeled with 100 uC\/ml ["SJmethionine for 3 h at 37Â°Cprior to ered saline; SDS, sodium dodecyl sulfate; 2-ME, 2-mercaptoethanol; TBS, 50 mM Tris (pH 7.4)-0.15 M NaCl buffer. extraction with high salt buffer. 2724

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1988 American Association for Cancer Research. KERATIN SUBTYPES OF SCLC Gel Electrophoresis and Western Blotting. For one-dimensional gel kDa, and 65.5 kDa (keratins 2-4 and 13-16) were also ob electrophoresis, each lane contained high salt extract from 2x10" served. These findings are summarized in Table 1 and shown cells. These extracts were dissolved in one-dimensional loading buffer in Fig. 2. The 68 kDa antigen reactive with AE-2 and AE-3 is (20 HIMTris-HCl, pH 7.4; 5% SDS; 5% 2-ME) at 100'C for 5 min and expressed in most SCLC tumors by immunoblot (Fig. 2), vari clarified by centrifugal ion at 10,000 x g for 5 min. The proteins were separated electrophoretically on a SDS-polyacrylamide (12.9%) slab ably in NBE cultures, and in trace amounts in squamous cell gel by the Laemmli technique (21). High molecular weight protein carcinomas, but not seen in other lung histologies (data not standards were obtained from Bethesda Research Laboratories, Be- shown). The classic SCLC cell lines OH-1 and NCI-H69 con thesda, MD. The gels were equilibrated in transblot buffer (20 HIMTris- tain relatively large amounts of this keratin, whereas only a 67 m,\i glycine in 20% methanol) for 30 min, then sandwiched with small amount is detected in variant cell line SW2-S. The 59 nitrocellulose paper and run on a transphor electrophoresis unit at 70 kDa keratin was present in OH-1 and in SCLC tissue specimens V for 3 h at 4"C. The molecular weight lanes were removed and stained but not in the other two cell lines. The 54 kDa keratin was not with amido black. After incubation with TBS and 1% Triton X-100 to detectable in SW2-S cultivated in vitro, even though it was remove SDS, nonspecific binding sites on the paper were blocked by found in low quantities in SW2-S grown in vivo. The 40 kDa incubation for 2 h with 5% bovine serum albumin and 3% normal goat AE-1-reactive keratin has been found to react with the anti-40 serum in TBS. The nitrocellulose strips were incubated with primary antibody in 5% bovine serum albumin and 3% normal goat serum (AE- kDa monoclonal antibody of Wu and Rheinwald (22) (data not 1 or AE-3, 1:200; AE-2, 1:100) for 16 h at 4Â°C.After washing with shown). SCLC tissue specimens, whether fresh tumor from TBS buffer, peroxidase-conjugated rabbit anti-mouse immunoglobulin lymph node biopsy or xenografts in nude mice, revealed similar (RAMpox; Cappel) was added and incubated for 2 h at 20Â°C.After keratin patterns compared with SCLC cell lines. SW2-A con rewashing, filtered 3,3'-diaminobenzidine hydrochloride (Sigma) 1 mg/ tains all of the keratin species found in SW2-S. However, SW2- ml in TBS with 0.05% H2O2was added to develop the brown peroxidase A appears to contain higher amounts of keratin per mg protein reaction for 3-5 min before quenching with water. Standard molecular compared to SW2-S. Similarly, the attached subline 69A con weight protein markers in adjacent lanes were used to determine tains the same subtypes as its parent cell line NCI-H69, but molecular weights of individual bands. Immunoblots were also per also shows increased keratins. The attached sublines, SW2-A formed with the use of the anti-40 kDa keratin antibody described and and 69A, contain a 56.5 kDa keratin, not detected by immu kindly supplied by Wu and Rheinwald (22). Two-Dimensional Gel Electrophoresis. Isoelectric focusing was per noblot in their parent cell lines, but observed in NBE cells. The formed according to the method of O'Farrell (23). A high salt extract major antikeratin-reactive proteins of 69A, 46 kDa and 49 kDa prepared from [35S]methionine metabolically labeled cells was dissolved (AE-3), are also detected in the majority of adenocarcinomas in two-dimensional lysis buffer [9.5 M urea; 2% 2-ME (Sigma); 25 HIM (data not shown). All the keratin species found in SCLC are Tris, pH 7.4; and 2% LKB ampholytes (pH 4-6.5:pH 5-8:pH 3.5-9.5; also detected in NBE cells. NBE cells express the major anti 1:1:0.33)] and clarified by centrifugation at 10,000 x g for 10 min. The gens 38 kDa [previously described in esophageal epithelium high salt extract from 5 x IO5cells was loaded per tube gel and run at (24)], 40 kDa, 54 kDa, 58 kDa, and 59 kDa, with minor bands 400 V for 16 h, then boosted to 1000 V for 2-3 h. After a 20-min at 48 kDa, 50 kDa, 52.5 kDa, 56.5 kDa, 63 kDa, and 65.6 kDa incubation with SDS equilibration buffer [125 mM Tris (pH 6.8)-1 % (Fig. 2; Table 1). Immunoblot bands outside the usual molecular SDS-10% glycerol-5% 2-ME], the second dimension was run overnight weights for keratins are observed, but are quite variable and at 8 mA/gel. The gels were fixed in methanol:acetic acid:lM) may represent protein aggregates or proteolytic products. (7.5:7.5:85), placed in Enhance (New England Nuclear) for 30 min and dried. Autoradiograms were exposed for 1-3 weeks. Two-Dimensional Gel Electrophoresis. The keratin subtypes in SCLC cells were also analyzed by two-dimensional gel elec trophoresis of [35S]methionine-labeled extracts (Fig. 3, Table RESULTS 2). The classic SCLC cell line OH-1 contained keratins 65.5/ Immunofluorescence. Immunofluorescence staining of SCLC 68 kDa (keratin 1/2), 59 and 54 kDa (keratins 4 and 13), 58 and 50/50' kDa (keratins 5 and 14/15), and 52.5 and 40 kDa cells with antikeratin monoclonal antibody was generally weak and diffuse. AE-2 did not reveal significant immunofluores- (keratins 8 and 19) by two-dimensional gels. Trace amounts of cence staining of SCLC cells compared with a control mouse 63 kDa (keratin 3) and 64 kDa (keratin 9) are observed with monoclonal antibody. Heterogeneous staining was observed in metabolic labeling, but could not be distinguished by one- the different SCLC cell lines (including classical, variant, and dimensional immunoblot. The 58 kDa keratin is detected by attached sublines), using AE-1 and AE-3 antikeratin mono two-dimensional gel but appears as a broad band not readily clonal antibodies as well as rabbit antiserum raised against total resolved from the 59 kDa band on one-dimensional blots. keratin (courtesy of T-T. Sun). The staining of SCLC cells by Comparison of the results of the two-dimensional gels with AE-1 (Fig. 1, left and middle), AE-3, and antikeratin antiserum one-dimensional immunoblots and the known antibody reactiv was weak and diffuse in contrast to the strong filamentous ities of AE-1, AE-2, and AE-3 (Table 2) allowed correlation cytoplasmic staining generally observed with NSCLC cells by with the keratin classification system of Moll et al.(\) and Sun using these antibodies. Unlike the suspension SCLC cells, the et al. (4). Two-dimensional immunoblots using AE-1 correlate attached subline SW2-A had greater amounts of cytoplasm, well with these assignments (3 bands are observed with NCI- and showed more intense filamentous keratin immunofluores- H69: 43 kDa, pi 5.4, 3+; 51 kDa, pi 5.1, 1-2+; and 56 kDa, cence with AE-1 (Fig. 1, right). pi 5.3, 1+; data not shown). Gel Electrophoresis and Western Blotting. Unlike immunofluorescence staining of intact cells, the presence of keratin was DISCUSSION more easily and reliably demonstrated by immunoblots of high salt extracts. SCLC cells and tumors expressed a pattern of Small cell lung cancer cells contain a characteristic set of keratin subtypes that was characteristic of this histology. The keratins. The 40/52.5 kDa pair, the 50, 54/58, 59 kDa pairs, major keratin subtypes observed by immunoblotting of small and the 65.5, 68 kDa proteins constitute the major keratins cell lung cancer extracts were 40 kDa, 52.5 kDa, and 68 kDa expressed in SCLC. This keratin pattern has been detected in corresponding to keratins 19, 8, and 1. Minor bands with SCLC cells and tissues and in classic and variant SCLC his molecular weights of 48 kDa, 50 kDa, 54 kDa, 59 kDa, 63 tologies, but not in NSCLC or non-lung tumors, although some 2725

Fig. 1. AE-1 immunofluorescence; Left, SW2-S, a variant SCLC cell line; middle, OH-1, a classic SCLC cell line; right, SW2-10A, an attached subline of SW2-S. Immunofluorescence with AE-3 is identical and immunostaining with AE-2 is negative.

Table 1 Keratin analysis of SCLC and normal bronchial epithelium by immunoblot Classic Attached Tissue Molecular Variant NBE AE Moll wt (kDa) OH-1 NC1-H69 SW2-S SW2-A 69-A SCC SW2-M2 cells RxnÂ° class* lia\ic keratins

68 +var 2,3 65.5 2,3 63 2, (3) 59 2,3 58 3 52.5 49 3" 46 3"

Acidic keratins 56.554 +1,2+ +++ ++50 + 1(+) (+) (+) ++++ (+)48 + ++ 1+ (+) (+) ++ +40 + + 1+++ (+) ++ ++38+++ ++ ++ ++ -t- (+) +++1++ 1"101314/1516194 " AE Rxn, reactivity with each antikeratin monoclonal antibody, 1 AE-1; 2 = AE-2; 3 = AE-3. * Moll class, keratin number by Moll's classification (1). ' (+), (3) weak. * Previously unreported reactivities.

individual keratins are shared. In particular, SCLC expresses transformed cells because of their rounded shape and poorly the high molecular weight keratins not detected in large cell or spread cytoplasm (36). adenocarcinomas of the lung. Conversely, in SCLC, we did not The keratin subtypes we have identified in SCLC by immu- observe the 56 kDa keratin detected in squamous or large cell noblotting include those associated with simple, stratified, and carcinoma, nor the 38 kDa keratin detected in adenocarcinomas even terminally differentiated squamous epithelia. Certain pat of the lung." terns of keratin expression have been associated with different Some investigators were not able to detect keratin by im states of epithelial differentiation (1, 37). The three lowest munostaining of SCLC (25, 26), but others have detected molecular weight acidic (40, 45, 46 kDa) and the two lowest keratin in a few SCLC specimens (10, 27-35). Our results show molecular weight basic keratins (52.5 and 54 kDa) were iden that even when immunostaining is negative with an antikeratin tified in simple epithelia and the basal layer of skin (7, 20, 35, antibody such as AE-2, keratins can be reliably identified by 37). The 48 and 56 kDa keratins were found in hyperprolifer- biochemical techniques. The scanty cytoplasm in SCLC adds ative keratinocytes (38). Nonkeratinized stratified epithelium to the difficulty in observing cytoplasmic components by im and corneal epithelium contained the pairs 54/59 kDa, and 55/ munostaining. When staining is observed, it is often diffuse and 63 kDa, respectively (39, 40). The highest molecular weight does not show discrete filaments. This may be related to the pairs, 56.5/65.5 and 68 kDa, are found in terminally differen rounded morphology of SCLC cells. By electron microscopy, tiated stratified squamous epithelium (4, 11, 39, 41). the low amount of cytokeratin in SCLC tends to be poorly The finding of keratin subtypes related to keratinizing epi thelium (the 65.5-68 kDa basic keratins reactive with AE-2 organized into tonofilaments (29). Similar difficulty in dem and AE-3) in SCLC was unexpected in a carcinoma which is onstrating microtubular networks was encountered in some regarded as so poorly differentiated. These keratins have been 4 Manuscript in preparation. closely associated with the process of cornification and terminal 2726

65. Â»-64

52.5 ^-54

50'T 50 86.6 84.0 80.0 40 4B.O

40.O

6 5

Fig. 3. Two-dimensional gel electrophoresis of OH-l ["Sjmethionine-labeled OH-l 8W-2 8W-2A H-89 SCO HBB high salt extract.

Table 2 Two-dimensional gel electrophoresis of OH-l: correlation with keratin classification mo Molecular lecular wt wt(kDa)6865.5635958565452.56456.556555450'5048464540pirange7.8-7.07.8-7.47.5-7.27.3-6.47.4-6.67.8-7.06.0-5.86.1-5.95.4-5.25.3-5.15.3-5.14.9-4.55.1-4.85.3-5.14.95.1-4.95.1-4.95.7-5.55.2-4.9AERx"2,32,32,32,3333311,21,2NoneNone1Mollclass*12345678910111213141516171819OH-l(kDa)6865.563595852.5645450'5040pi7.87.77.26.17.66.15.45.35.34.95.3Amount3+3+<+)'1+2+<+)2+2+2+3+(+)

68.0- 65.5- 63.0-

86.8-

" AE Rx, reactivity with each antikeratin antibody, I = AE-1; 2 = AE-2; 3 = OH-1 SW-8 SW-2A H-69 SCO NBE AE-3. 0 Moll class, keratin number by Moll's classification (1). <(+),weak.

differentiation of stratified squamous cells (4, 11, 39, 41), including the development of a granular and a cornified layer in skin and with "pearl formation" in well differentiated squa mous cell carcinomas. However, several other investigators have found similar keratin content in other respiratory epithe 68.0- lium by a variety of techniques and reagents. Emura et al. (42) 08.6- 63.0- grew fetal trachÃ©alcellsin culture and found markedly positive 89.0- immunofluorescence with antibodies raised against human stra 82.8- tum corneum and with antimerokeratin antibody reactive with superficial epidermal keratins between 60 and 70 kDa without observing squamous differentiation. The basal, ciliated, and intermediate cells in normal bronchial epithelium have reacted with specific antisera to stratum corneum keratins including 68 kDa by immunohistochemical staining (25, 33). In these stud ies, 2/16 SCLC specimens were also reactive. The detection of the high molecular weight keratins in SCLC described in this paper is unlikely to represent artifacts produced by the poten Fig. 2. A, AE-2 immunoblot of SCLC and NBE; B, AE-2 immunoblot of tially ubiquitous stratum corneum of experimenters' hands. SCLC and NBE; C, AE-3 immunoblot of SCLC and NBE. 2727

Control cell lines such as G361, a melanoma cell line, or CEM, a certain keratin subtype may indicate a commitment toward a a I cull lymphoblastic leukemia line, did not demonstrate sig certain program of differentiation. The potential differentiation nificant reactivity with antikeratin antibodies (neither did im pathways of stem cells in SCLC tumors may include squamous munoblots with irrevelant mouse anti-human IgG as primary and adenocarcinomatous differentiation in addition to the neu- antibody). These negative controls are important for keratin roendocrine SCLC. After long term tissue culture (more than studies since cell extracts may be contaminated with stratum 2 years), SCLC cell lines develop variants that resemble non- corneum present in laboratory equipment (6). small cell histology (46). Further evidence for this model stems The observation that SCLC expressed a subset of the keratins from the biological behavior of SCLC in vivo. Mixed histolo found in normal bronchial epithelial cells is consistent with a gies, small cell and non-small cell, are recognized relatively pulmonary epithelial lineage for SCLC. Normal bronchial epi frequently, either accompanying the initial presentation, de thelial cells contain all the keratins expressed by SCLC, but in tected in residual disease after appropriate therapy for small addition exhibit keratins that are observed in NSCLC histolo cell, or seen at autopsy (29, 46-51). The most common histo- gies. The 68 kDa keratin can also be detected in NBE cells, logical mixture contains large cell or undifferentiated carci particularly after differentiation is induced by high calcium in noma with SCLC, but adenocarcinoma or squamous cell mix vitro* However, NBE cells contain other keratins not found in tures also occur. Further differentiation of the tumor cells of SCLC such as the 38 kDa band. These lower molecular weight SCLC to NSCLC elements has been observed to occur in vivo keratins were also detected in NSCLC cell lines. Blobel et al. (52). These observations have led to proposals that small cell (8) have also detected the 40,45/52.5 kDa, the 46/54 kDa, and carcinoma is derived from an endoderma! stem cell that is the 50-58 kDa pairs by using 2 dimensional nonequilibrium capable of differentiating along multiple pathways (51 ). pH gel electrophoresis in NBE from surgical specimens. The Identification of the molecular forms of keratins may be presence of keratins associated with simple epithelia, stratified helpful to analyze the changes that occur during SCLC differ epithelia, and differentiated epithelia in NBE implies an ability entiation. Development of molecular probes specific for indi of the progenitor cell(s) of bronchial epithelium to differentiate vidual keratins may identify unique populations within a SCLC along multiple pathways, expressing different pairs of keratins tumor and population shifts caused by alteration of growth during the process of maturation. This conclusion is supported conditions or addition of differentiation agents such as dibu- by the heterogeneity in keratin content detected by immunoflu- tyryl-cAMP or a-interferon. Correlation with other potential orescence. markers of SCLC differentiation (neurosecretory granules, sur The presence of keratin, a fundamental marker of epithelial face membrane antigens, morphology, and decarboxylase en differentiation, casts serious doubt on the interpretation that zymes) may allow definition of the differentiation pathways of SCLC has a monocytic origin simply due to the presence of SCLC and its relationship to the normal bronchial epithelium. surface antigens shared with monocytes (9). Many monoclonal antibodies, including those recognizing natural killer cells and monocyte "differentiation" antigens cross-react not only with ACKNOWLEDGMENTS SCLC, but also with NSCLC and a variety of tissues such as Many thanks to Dr. Tung-Tien Sun for his generous gift of antiker neural or hematopoietic histology (43). Analysis of intermediate atin antibodies and his invaluable criticism and careful reading of the filament composition may be more useful in determining tissue manuscript. I wish to thank Jane Mayotte for her technical help. derivation since it had been shown that cancer cells generally express the same distinct intermediate filament type as their REFERENCES normal tissue counterparts. Thus, normal and neoplastic epi thelial cells express keratin. Neuroendocrine tissues and tumors 1. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B., and Krepier, R. The contain neurofilament proteins. Cells of hematopoietic origin catalog of human cytokeratins: patterns of expression in normal epithelia, tumors, and cultured cells. Cell, .*/: 11-24, 1982. express vimentin, but not keratin. We have previously analyzed 2. Franke, W. W., Schmid, E., and Moll, R. The intermediate filament cyto the proteins associated with the cytoskeleton of human hema skeleton in tissues and in cultured cells: differentiation specificity of expres topoietic cells (44). In addition to vimentin, 10 other proteins sion of cell architectural elements. In: C. C. Harris and H. N. Amrup (eds.), Human Carcinogenesis, Chap. 1, pp. 3-34. New York: Academic Press, Inc., were identified in monocytes which were also present in the 1983. cytoskeleton of other hematopoietic cells, including peripheral 3. Osborn, M.. and Weber, K. Tumor diagnosis by intermediate filament typing: a novel tool for surgical pathology. Lab. Invest., 48: 372-394, 1983. blood granulocytes and myeloid leukemic cells. None of these 4. Sun, T-T., Eichner, R., Schermer, A., Cooper, D., Nelson, W. G., and Weiss, proteins were shared with SCLC or other lung cancers. Recently R. A. Classification, expression, and possible mechanisms of evolution of Zauli et al. (45) have observed immunofluorescent staining of mammalian epithelial keratins: a unifying model. Cancer Cells, 1: 169-176, 1984. keratin and vimentin in the K562 cell line which was originally 5. Bernal, S. D., and Stahel, R. S. Cytoskeleton-associated proteins: their role derived from a patient with chronic myelogenous leukemia in as cellular integrators in the neoplastic process. CRC Crii. Rev. Oncol. Hematol., 3: 191-204, 1984. blast crisis. About 30% of the cells were diffusely stained by 6. Cooper, D., Schermer, A., and Sun, T-T. Classification of human epithelia AE-1 but all were negative for AE-3. It would be important to and their neoplasms using monoclonal antibodies to keratins: strategies, perform immunoblots of keratin extracts to rule out nonspecific applications, and limitations. Lab. Invest., 52:243-2S6, 198S. 7. Franke, W. W., Schiller, D. L., Moll, R., et al. Diversity of cytokeratins: staining, especially since it is unlikely for cells to contain only differentiation specific expression of cytokeratin polypeptides in epithelial acidic keratins (recognized by AE-1) but not basic keratins cells and tissues. J. Mol. Biol., 153:933-959, 1981. (recognized by AE-3). 8. Blobel, G. A., Moll, R., Franke, W. W., and Vogt-Moykopf, I. Cytokeratins in normal lung and lung carcinomas. 1. Adenocarcinomas, squamous cell Our results with antikeratin immunoblots and immunofluo- carcinomas and cultured cell lines. Virchows Arch. B Cell Pathol., 45: 407- rescence suggest that SCLC exists as a heterogeneous popula 429, 1984. 9. Ruff, M. R., and Pert, C. B. Small cell carcinoma of the lung: macrophage- tion with the major proportion expressing highly differentiated specific antigens suggest hemopoietic stem cell origin. Science (Wash. DC), epithelial antigens. A minor population may include a relatively 225: 1034-1036, 1984. undifferentiated and multipotent stem cell which can progress 10. Bernai, S. D., Baylin, S. B., Shaper, J. Hâ€žGazdar, A. F., and Chen, L. B. Cytoskeletal-associated proteins of human lung cancer cells. Cancer Res., 43: along various pathways of differentiation during continuous 1798-1808,1983. tissue culture or during in vivo selection. The ability to express 11. Woodcock-Mitchell, J., Eichner, R., Nelson, W. G., and Sun, T-T. Immu- 2728

nolocalization of keratin polypeptides in human epidermis using monoclonal tumors: patterns of localization of different-molecular-weight keratin pro antibodies. J. Cell Biol., 95: 580-588, 1982. teins. Am. J. Pathol., 113: 27-32, 1983. 12. Eichner, R., Bonitz, P., and Sun, T-T. Classification of epidermal keratins 33. Gusterson, B., Mitchell, D., Warburton, M., and Sloane, J. Immunohisto according to their immunoreactivity, isoelectric point, and mode of expres chemical localisation of keratin in human lung tumours. Virchows Arch. sion. J. Cell Biol., 98:1388-1396, 1984. Pathol. Anat., 394: 269-277, 1982. 13. Francis, J., Thompson, R. A., Bernal, S. D., et al. Effect of dibutyryl cyclic 34. Banks-Schlegel, S. P., Gazdar, A. F., and Harris, C. C. Intermediate filament adenosine 3':5'-monophosphate on the growth of cultured human small cell and cross-linked envelope expression in human lung tumor cell lines. Cancer lung carcinoma and specific cellular activity of L-Dopa decarboxylase. Cancer Res.,Â«: 1187-1197, 1985. Res., 43: 639-645, 1983. 35. Banks-Schlegel, S. P., McDowell, E. M., Wilson, T. S., Trump, B. F., and 14. Gadzar, A. F., Carney, D. N., RÃ¼ssel,E. K., et al. Establishment of contin Harris, C. C. Keratin proteins in human lung carcinomas: combined use of uous, clonable cultures of small-cell carcinoma of the lung which have amine morphology, keratin immunocytochemistry, and keratin immunoprecipita- precursor uptake and decarboxylation cell properties. Cancer Res., 40:3502- tion. Am. J. Pathol., 114: 273-286, 1984. 3507, 1980. 36. Osborn, M., and Weber. K. The display of microtubules in transformed cells. 15. Giovanella, B. ( '., Stehlin, J. S., Williams, L. J., et al. Heterotransplantation Cell, /2.-561, 1975. of human cancers into nude mice. Cancer (I'llila.), 42: 2269-2281, 1978. 37. Debus, E., Weber, K., and Osborn, M. Monoclonal cytokeratin antibodies 16. Peter, H. J., and Kistler, G. S. Isolation und in vitro Kult ivat Â¡onvon that distinguish simple from stratified squamous epithelia: characterization Tumorzellen aus Menschlichen in der nu/nu Maus transplantierten Broii- on human tissues. EMBO J., ;.- 1641-1647, 1982. chuskarzinomen. Schweiz. Med. Wochenschr., 109:830-832, 1979. 38. Weiss, R. A., Eichner, R., and Sun, T-T. Monoclonal antibody analysis of 17. Bergh, J., Nilsson, K., Zech, L., and Giovanella, B. Establishment and keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as characterization of a continuous lung squamous cell carcinoma cell line (U- molecular markers for hyperproliferative keratinocytes. J. Cell Biol., 98: 1752). Anticancer Res., /: 317-322, 1981. 1397-1406, 1984. 18. Bemal, S., Weinberg, K., Kakefuda, M., Stahel, R., O'Hara, C, and Wong, 39. Tseng, S. C. G., Jarvinen, M. J., Nelson, W. G., et al. Correlation of specific Y. C. Membrane antigens of normal bronchial epithelial cells identified by keratins with different types of epithelial differentiation: monoclonal anti monoclonal antibodies. In Vitro Cell. Dev. Biol., in press, 1988. body studies. Cell, 30: 361-372, 1982. 19. Johnson, G. IX. Araujo, G. M. de C. N. A simple method of reducing the 40. Nelson, W. G., Battifora, H., Santana, H., and Sun, T-T. Specific keratins fading of immunofluoresccnce during microscopy. J. 1nun uno I. Methods, 43: as molecular markers for neoplasms with a stratified epithelial origin. Cancer 349-350, 1981. Res., 44:1600-1603, 1984. 20. Wu, Y-J., Parker, L. M., Binder, N. E., et al. The mesothelial keratins: a 41. Sun, T-T., and Green, H. Keratin filaments of cultured human epidermal new family of cytoskeletal proteins identified in cultured mesothelial cells cells: formation of intermolecular disulfide bonds during terminal differen and nonkeratinizing epithelia. Cell, 31:693-703, 1982. tiation. J. Biol. Chem., 253: 2053-2060, 1978. 21. Laemmli, U. K. Cleavage of structural proteins during the assembly of the 42. Emura, M., Mohr, U., Kakunaga, T., and HilfrÃch,J. Growth inhibition and head of bacteriophage T4. Nature (Lond.), 277.-680-685, 1970. transformation of a human fetal trachÃ©alepithelial cell line by long-term 22. Wu, Y-J., and Rheinwald, J. G. A new small (40 kD) keratin filament protein exposure to diethylnitrosamine. Carcinogenesis (Lond.), 6:1079-1085,1985. made by some cultured human squamous cell carcinomas. Cell, 25:627-635, 43. International Workshop on Small Cell Lung Cancer Antigens: London, 1987. 1981. Lung Cancer, 4, in press, 1988. 23. O'Farrell, P. H. High resolution two-dimensional electrophoresis of proteins. 44. Bernal, S.D., and Chen, L. B. Induction of cytoskeleton-associated proteins J. Biol. Chem., 250:4007-4021, 1975. during differentiation of human myeloid leukemic cell lines. Cancer Res., 42: 24. Banks-Schlegel, S., and Harris, C. C. Tissue specific expression of keratin 5106-5116, 1982. proteins in human esophageal and epidermal epithelium and their cultured 45. Zauli, D., Gobbi, M., Crespi, C., et al. Vimentin and keratin intermediate keratinocytes. Exp. Cell Res., 146: 271-280, 1983. filaments expression by K562 leukemic celi line. I euk Res., 10:29-33,1986. 25. Bejui-Thivolet, F., Viac, J., Thivolet, J., and Paure, M. Intracellular keratins 46. Gazdar, A. F., Carney, D. N., Guccion, J. G., and Baylin, S. B. Small cell in normal and pathological bronchial mucosa. Virchows Arch. Pathol. Anat. carcinoma of the lung: cellular origin and relationship to other pulmonary Â¿95:87-98, 1982. tumors. In: F. A. Greco, R. K. Oldham, and P. A. Bunn (eds.), Small Cell 26. Lehto, V-P., Stenman, S., Miettinen, M., et al. Expression of a neural type Lung Cancer, pp. 145-175. New York: GruÃ±e& Stratton, Inc., 1981. of intermediate filament as a distinguishing feature between oat cell carci 47. Gazdar, A. F., Cohen, M. H., Idhe, D. C., et al. Bronchial epithelial changes noma and other lung cancers. Am. J. Pathol., 110: 113-118, 1983. in association with small cell carcinoma of the lung. In: F. Muggia and M. 27. Broers, J. L. V., Carney, D. N., De Ley, L., Vooijs, G. P., and Ramaekers, Rozencweig (eds.). Lung Cancer: Progress in Therapeutic Research, pp. 167- F. C. S. Differential expression of intermediate filament proteins distin 174. New York: Raven Press, 1979. guishes classic from variant small-cell lung cancer cell lines. Proc. Nati. Acad. 48. Abeloff, M. D., Eggleston, J. C., Mendelsohn, G., et al. Changes in morpho Sci. USA, 82: 4409-4413, 1985. logic and biochemical characteristics of small cell carcinoma of the lung: a 28. Sappine, A-P., Ellison, M. L., and Gusterson, B. A. Immunohistochemical clinicopathologic study. Am. J. Med., 66: 757-764, 1979. localisation of keratin in small cell carcinoma of the lung: correlation with 49. Brereton, H. D., Mathews, M. M., Costa, J., et al. Mixed anaplastic small- response to combination chemotherapy. Eur. J. Cancer Clin. Oncol., 19: cell and squamous-cell carcinoma of the lung. Ann. Int. Med., 88:805-806, 1365-1370,1983. 1978. 29. McDowell, E. M., McLaughlin, J. S., Merenyl, D. K., et al. The respiratory 50. Radice, P. A., Matthews, M. J., Ihde, D. C. et al. The clinical behavior of epithelium. V. Histogenesis of lung carcinomas in the human. J. Nati. Cancer "mixed" small cell/large cell bronchogenic carcinoma compared to "pure" Inst., 61: 587-606, 1978. small cell subtypes. Cancer (Phila.), 50:2894-2902, 1982. 30. Van Muijen, G. N. P., Ruiter, D. J., Van Leeuwen, C., et al. Cytokeratin 51. Matthews, M. J. Effects of therapy on the morphology and behavior of small and neurofilament in lung carcinomas. Am. J. Pathol., 116: 363-369, 1984. cell carcinoma of the lungâ€”a clinicopathologic study. In: F. Muggia and M. 31. Said, J. W., Nash, G., Tepper, G., and Banks-Schlegel, S. Keratin proteins Rozencweig (eds.), Lung Cancer: Progress in Therapeutic Research, pp. 155- and carcinoembryonic antigen in lung carcinoma: an immunoperoxidase 165. New York: Raven Press, 1979. study of fifty-four cases, with ultrastructural correlations. Hum. Pathol., 14: 52. Bepler, G., Jaques, G., 1Invernanti. K., ci al. Characterization of two cell 70-76, 1982. lines with distinct phenotypes established from a patient with small cell lung 32. Said, J. W., Nash, G., Banks-Schlegel, S., et al. Keratin in human lung cancer. Cancer Res., 47:1883-1891, 1987.

2729

Anthony D. Elias, Bruce F. Cohen and Samuel D. Bernal

Cancer Res 1988;48:2724-2729.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/10/2724

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/48/10/2724. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.