Coexpression of Simple Epithelial Keratins and Vimentin by Human Mesothelium and Mesothelioma in Vivo and in Culture Paul J

[CANCER RESEARCH 44, 2991-2999, July 1984]

Coexpression of Simple Epithelial Keratins and Vimentin by Human Mesothelium and Mesothelioma in Vivo and in Culture Paul J. LaRocca1 and James G. Rheinwald2

Division of Cell Growth and Regulation, Dana-Farber Cancer Institute and Department of Physiology and Biophysics, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT wanted to determine whether vimentin is present in the cells of the intact mesothelium also, or whether it is only acquired after We have determined the intermediate filament proteins present cell detachment or cultivation, as has been suggested by others in normal and malignant mesothelium in vivo. Pure sheets of (12, 21, 32, 39). The set of keratin proteins synthesized by normal lung mesothelium were obtained by transfer to agar- normal human peritoneal mesothelial cells in culture is different coated slides or by gentle scraping and cytocentrifugation. Cy- from that of stratified squamous epithelial and many glandular toplasmic filament networks in the mesothelium were labeled via epithelial cell types (22, 27, 38, 43). We wished to determine indirect immunofluorescence both by anti-Mr 40,000 keratin and whether mesotheliomas express any keratins different from anti-vimentin antisera. Two-dimensional gel electrophoresis of those of normal mesothelium, inasmuch as squamous cell car the Triton:high-salt-insoluble proteins of normal lung mesothel cinomas often synthesize keratins inappropriate for their cell type ium disclosed the presence of vimentin and all but the largest of origin (23, 43, 44). We report here that normal mesothelial (M, 55,000) of the four simple epithelial keratins synthesized by cells in the intact tissue coexpress keratins and vimentin, that mesothelial cells in culture. Samples of three peritoneal and three the largest keratin M, 55,000 appears to be induced by conditions pleural mesotheliomas were found to contain either all four simple of culture, and that mesotheliomas express vimentin and the epithelial keratins or all but the M, 55,000 keratin. Notably, none same set of keratins as does normal mesothelium. of the keratins characteristic of stratified and many glandular epithelia and their malignant forms was present in these meso-, theliomas. Two mesothelioma samples from which the tumor MATERIALS AND METHODS cells could be obtained free of other cell types were found to Preparation of Mesothelial Tissues. Sheets of normal lung mesothe contain vimentin as well as simple epithelial keratins and to lium were obtained free of underlying connective tissue by a modification synthesize these same proteins during short-term culture. None of the imprinting method (42). A molten solution of 1% agarose (Sea of the mesotheliomas placed in culture grew progressively in Kem) in water was applied to glass slides and dried overnight. The medium optimal for the growth of normal mesothelial cells. These surface of freshly excised human fetal lung was rinsed in isotonic PBS3 data demonstrate that malignant mesothelial cells preserve the and exposed to the air. Just as the mesothelium began to appear dry, an agarose-coated slide was pressed against the lung for several sec normal pattern of intermediate filament protein synthesis, includ ing coexpression of simple epithelial keratins and vimentin, and onds. When the slide was pulled away, parts of the mesothelial cell layer adhered to the agar and were peeled off. Slides were examined under suggest the use of this characteristic as an aid in the identification an inverted microscope with phase contrast optics to identify areas of cells of mesothelial origin. containing large sheets of mesothelium. These were rinsed with PBS to remove any blood cells. Slides were then either briefly air-dried and immersed in cold (-20Â°) methanol to fix for immunofluorescence micros INTRODUCTION copy, or the agarose and cell material were scraped into Triton:high-salt The mesothelium is the simple squamous epithelium that lines extraction buffer and stored frozen for future electrophoretic analysis. the pleural, pericardial, and peritoneal cavities and covers the Tissues to be used as electrophoresis samples were collected on slides outer surfaces of the organs contained therein (6, 7). Unlike most that had been coated with electrophoretically purified agarose in order other epithelia, the mesothelium is of embryonic mesodermal to reduce the levels of contaminants which would be stained in the subsequent silver staining procedure. As an alternate method for obtain origin. Perhaps because of this the malignant form, mesothe ing small sheets and clusters of mesothelial cells for Â¡mmunofluores- lioma, can adopt either sarcomatous or carcinomatous forms, cence, the lung surface was gently scraped with a rubber policeman into often making differential diagnosis difficult (9, 18, 37). PBS. The cell suspension was then diluted 1:1 with serum and applied This laboratory has recently determined the cell type-specific to a glass slide by spinning in a cytocentrifuge. The slides were then air requirements of normal, diploid human mesothelial cells for rapid dried and fixed in cold methanol. For these experiments, lungs were growth in long-term, serial culture (4). Mesothelial cells are obtained from 18 to 22-week-old human fetuses or from adult rats. different from most other normal epithelial cell types in that they Surgical specimens of solid peritoneal and pleural mesotheliomas (kindly provided by Dr. J. M. Corson, Brigham and Women's Hospital) express in culture high levels of 2 different types of intermediate filament proteins: keratins and vimentin (42,44). Because normal were collected within 1 h after resection and minced into 1 cu mm pieces. mesothelial cells that detach from the mesothelium and float in Some fragments from each tumor were placed in culture, and some were frozen in Triton:high-salt buffer until extracted for electrophoresis. Me ascites fluid also contain both vimentin and keratins (4), we sothelioma cells were also obtained from a pleural effusion of mesothe lioma (MS-7). This effusion was found by cytopathological evaluation to 1 Recipient of National Research Service Awards T32 CA09361 and consist of >90% mesothelioma cells. The cells were concentrated by IF32AG05303 Â¡npartial support of this investigation. 1 Recipient of grants from the National Cancer Institute and the National Institute on Aging. 3The abbreviation used is: PBS, phosphate-buffered saline (137 mm NaCI:3 HIM Received July 25,1983; accepted March 30,1984. KCI:16 mw HajrtPQt:2muKHJPO*, pH 7.3).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. P. J. La/tocca and J. G. Rheinwald centrifugation from 200 ml of effusion. One fourth of the cell pellet was of pure tumor cells were placed into culture in the growth medium extracted for electrophoretic analysis, and the rest was used to initiate described above. cultures. Keratin Nomenclature. Keratins 40KM, 44KM, 52KM and 55KM, the "mesothelial keratins" described in Wu ef a/. (43), are keratins 19,18, 8, Frozen ampuls of cells concentrated from the ascites fluid of a patient and 7, respectively, independently identified as "simple epithelial keratins" with an ovarian surface epithelial neoplasia were thawed and applied to slides by cytocentrifugation for examination by immunofluorescence. This by Moll ef a/. (22). was the same ascites fluid from which the normal mesothelial strain LP- 9 have been derived (4, 43). Indirect Immunofluorescence Microscopy. Anti-M, 40,000 keratin RESULTS antiserum, raised in rabbits, is specific for this particular keratin (43, 44). Anti-stratum corneum keratins antiserum, raised in rabbits, reacts M, 40,000 Keratin Immunofluorescence of Mesothelia. Nor strongly with epidermal keratins and cross-reacts to various degrees mal mesothelium was transferred from the outer surface of a with all the "mesothelial" or "simple epithelial" keratins (43, 44). An anti- fetal human lung to an agarose-coated slide by an imprinting human fibroblast vimentin antiserum was raised in rabbits. An anti- technique described by Whitaker et al. (42). Cell shape, nuclear vimentin antiserum raised in guinea pigs was kindly provided by Dr. position, and intermediate filament content was examined by Werner Franke, German Cancer Research Center, Heidelberg, West phase-contrast microscopy (Fig. 1a) and indirect immunofluor Germany. Mouse monoclonal antibody OC125, which recognizes a sur escence with anti-40KM keratin. Favorable areas of the trans face antigen of ovarian carcinoma cells (1), was obtained from Dr. Robert Bast of this institute. Rhodamine- and fluorescein-conjugated secondary ferred mesothelium were found to contain this keratin in a fibrous antibodies were obtained from Miles and Cappel Laboratories. The network (Fig. 1b) similar in appearance to that of cultured epithe procedures used for indirect immunofluorescence microscopy and pho lial cells. Heterogeneity of staining presumably resulted from tography has been described (4,44). variability in the amount of cytoskeleton transferred to the slide. Cell Labeling, Extraction, and Electrophoresis. Methods were sim In these preparations, the apical portion of the plasma membrane ilar to those previously described (4, 43, 44). Proteins synthesized by adheres to the agarose, and the cells apparently fracture such cultured cells were labeled by a 4-hr incubation with [^SJmethionine (50 that variably amounts of the cytoplasmic contents are removed ÃŸd/ml) (specific activity, ~800 Ci/mmol, MEN Chemicals) in otherwise methionine-free minimal Eagle's medium (Flow Laboratories) supple to the slide, even though the nucleus (or its outline) can be seen in each transferred cell (Fig. 1a). mented with 20% dialyzed fetal calf serum. Labeled cultured cells, Small clusters to large sheets of mesothelial cells could also agarose slide-collected mesothelium, and minced tumor tissue were rinsed with PBS, Dounce homogenized in Triton:high-salt extraction be dislodged by scraping the lung surface with a rubber police buffer (20 mw Tris-HCI, pH 7.4:0.6 M KCI:1% Triton X-100:1 ITIMphenyl- man and depositing the PBS:serum suspension of these cells on methylsulfonylfluoride) at 0Â°, and then sonicated for 45 sec. Lysates a slide with the aid of a cytocentrifuge (Fig. 1c). Clusters of easily were then centrifuged at 10,000 x g for 45 min at 4Â°.The insoluble identified mesothelial cells stained positively for the 40KM keratin pellets (containing the keratins and vimentin) were then dissolved in and also revealed details of the cytoplasmic filament network O'Farrell 2-dimensional lysis buffer. The proteins were separated in the (Fig. 1d). This was consistent with the results of our preliminary first dimension by isoelectric focusing (29) and in the second dimension examination of paraffin sections of mesothelium by immunoper- by sodium dodecyl sulfate:polyacrylamide gel electrophoresis (10% ac- oxidase staining (44). The mesothelium was also stained strongly rylamide) (20). High-purity agarose (Sea Kern) was used to hold the by anti-total stratum corneum keratins, an antiserum which isoelectric focusing tube gel in place at the top of the sodium dodecyl cross-reacts with most of the keratins (43, 44) (data not shown), sulfate:polyacrylamide slab gel. The possible presence of basic (isoelec as had been reported previously (5, 36). tric points 7 to 8) keratins was examined by separating the proteins by nonequilibrium pH gradient electrophoresis for 3.5 hr in the first dimen The M, 40,000 keratin was also detected by immunofluores sion, according to the method of O'Farrell ef al. (30). Labeled proteins cence in secondary cultures of mesotheliomas MS-7 (Fig. 1ft) were detected by scintillation fluor-enhanced (Enhance; MEN Chemicals) and MS-10 (data not shown). The mesothelioma cells had a autoradiography. morphology in culture (Fig. 1g) distinct from that of normal Protein Detection in Gels by Silver Staining. An ultrasensitive silver mesothelial cells (Fig. 1e). MS-7 and MS-10 could not grow method (25) was used with the following modifications. After fixation in progressively in the culture medium permissive for long-term 10% glutaraldehyde, the gel was rinsed for at least 48 hr to more growth of normal pleural and peritoneal mesothelial cells and completely remove glutaraldehyde and consequently reduce the back ceased dividing in secondary culture. We initiated 5 other cultures ground staining. Ethanolamine (5 Â¿J/ml)was added to the citric acid of solid pleural and peritoneal mesotheliomas, but the only cells fixative to prevent the formaldehyde from overdeveloping the silver (10). that grew had morphologies and growth requirements of either Wet gels were then photographed over a light box with Kodak Technical Pan film (ASA 50), which was developed in a 1:36 dilution of HC-110 for normal mesothelial cells or of normal connective tissue fibro- 8 min. Gels were placed in a solution of 0.02% sodium carbonate and blasts and were not tumorigenic in nude mice. Apparently the stored in sealed plastic bags for future examination. mesothelioma cells present in those 5 tumor specimens could Cell Culture Methods. A population of normal, diploid, human pleural not grow at all under the conditions we used. mesothelial cells (strain HPM-2) was cultured from pleural effusion fluid Detection of Vimentin in Normal Mesothelial Cells by Im withdrawn from an adult female. The normal, diploid, human peritoneal munofluorescence. This laboratory previously had reported that mesothelial strain LP-9 has been described previously (4, 43). LP-9 and normal mesothelial cells present among the cells in an ascites HPM-2 were grown as described (4), in either M199 or M199/ fluid specimen, which had been allowed to attach to a coverslip, MCDB202:15% fetal calf serum (Sterile Systems):hydrocortisone (0.4 contained both keratin and vimentin (4). We wished to confirm Mg/ml; Calbiochem):epidermal growth factor (10 ng/ml; Collaborative Research). In order to maximize keratin synthesis (Fig. 3, b to d), cells this coexpression in ascites fluid mesothelial cells that never had were grown to confluence during one passage in the absence of epider been exposed, even briefly, to culture conditions, and also to mal growth factor. determine whether the cells of intact mesothelium contain vimen Cells from a pleural effusion mesothelioma (MS-7) and from a solid tin. We applied human ascites fluid cells and intact rat lung peritoneal mesothelioma (MS-10) which could be dislodged as clusters mesothelium (dislodged by scraping with a rubber policeman) to

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Table 1 Keratin types and vimentin coexpressionin normal and malignant mesothelia Keratins

(40KM/19) (44KH/18) (46K>,) (52IW8) (55KM/7) Vimentin Normal mesothelium8 Pleural v6 +Â° + - ++ + Pleural(HPM-2) c + ++/-++ + Peritoneal(LP-9) c + + +/- ++ +

Malignant mesothelium Pleural(MS-2) Pleural(MS-11) Pleural(MS-7)

Peritoneal(MS-4) Peritoneal(MS-5) Peritoneal(MS-10)

" Data summarizedfrom Fig. 3 and Refs. 4 and 43. v, in vivo',c, in culture. 0 +, moderate amounts; ++, relatively high amounts; +/-, variable amounts among experiments; -, none present. " Data summarizedfrom Figs. 3 and 4. eAmount in mesotheliomacells indeterminatebecause of connective tissue present in tumor specimen. ' Relative amounts unknown due to partial proteolysis. slides by cytocentrifugation and double labeled them with anti- synthesized these same proteins in secondary culture (Fig. 3, e vimentin and anti-keratin antisera. As shown in Fig. 2, a to c, all and g). MS-10 contained vimentin, keratins 40KM and 44KM, a mesothelial cells in rat lung mesothelium coexpressed vimentin small amount of keratin 52KM, and what appeared to be proteo- and keratin. Consistent with the previous report from this labo lytic digestion products of 52KM (Fig. 3f). Cultured MS-10 cells ratory (4), some of the ascites fluid cells coexpressed vimentin synthesized vimentin and all 4 simple epithelial keratins (Fig. 3h). and keratin (Fig. 2, d to f). Vimentin-positive, keratin-negative Thus, malignant mesothelial cells may also increase their expres cells in the ascites fluid were assumed to be macrophages (3, sion of 55KM in culture. 11); these cells attached and spread under culture conditions Samples of 3 other solid peritoneal mesotheliomas and 4 other but did not divide (4). The keratin-positive, vimentin-negative pleural mesotheliomas were extracted and analyzed by 2-dimen cells were presumed to be neoplastic cells of ovarian surface sional gel electrophoresis. Three samples gave no interpretable epithelial origin because cells in the sample which stained with spots due to failures of isoelectric focusing or to paucity of the ovarian surface-specific monoclonal antibody OC125 (1) were mesothelioma cells in a predominantly fibrous tumor. Four sam vimentin negative (Fig. 2, g to /) and keratin positive (data not ples could be scored (Fig. 4). Keratins 40KM, 44KM, and 52KM shown). Thus, keratin and vimentin coexpression is a normal were present in all samples, with keratin 55KM present in all but characteristic of mesothelial cells and is retained by cells that one sample (Fig. 4o). One tumor (Fig. 4d) also contained a become detached from the mesothelium. protein migrating in the position of keratin 46KM. Vimentin was Electrophoretic Analyses of Mesothelial and Mesothelioma detected in 3 of the samples, but since these contained connec Cytoskeletal Proteins. The Triton:high-salt-insoluble protein tive tissue, the vimentin content of the tumor cells themselves fraction extracted from normal and malignant mesothelial sam was indeterminate. ples were analyzed by 2-dimensional gel electrophoresis to Proteolysis of vimentin (14, 28) and of keratin 52KM (35) was determine precisely the intermediate filament proteins present extensive in some tumor extracts (Fig. 3f; Fig. 4, a, b, and d), (Table 1). Fetal human pleural mesothelium collected by stripping precluding quantitative estimation of these proteins when it with an agar-coated slide, precluding contamination with under occurred. Evaluation of gels made from extracts of small tissue lying connective tissue, contained vimentin, keratins 40KM and samples was also complicated because the silver staining 44Km, and, in greatest abundance, keratin 52KM (Fig. 3a). Keratin method used to detect very low levels of protein also stained 55KM, synthesized by adult peritoneal mesothelial cells in culture minor impurities in the buffers and in the agarose used to (4, 43) (see Fig. 3, c and d) was not detectable. Normal pleural immobilize the tube gel during the second dimension of electro mesothelial cells (HPM-2) synthesized vimentin and all 4 simple phoresis. These impurities were most common in the M, 56,000 epithelial keratins in culture, negating the possibility that cells of to 70,000 range (eg., cf. Fig. 3 c and d, and see Fig. 3 a and Fig. pleural origin are unable to synthesize keratin 55KM. A rapidly 4 b, c, and d). Despite the staining background, no proteins turning over, basic M, 46,000 keratin which is expressed at migrating at the position of any known keratin (22,43) other than variable levels by mesothelial cells growing in culture (4, 43) (see the simple epithelial keratins were identified in any of the me Fig. 3cO,also was not detected in the in vivo mesothelium. sothelioma extracts. A relatively pure cell suspension of pleural mesothelioma (MS- 7) and clusters of cells teased from a solid peritoneal mesothe DISCUSSION lioma (MS-10) were extracted directly and their intermediate filament proteins compared with those synthesized by cells from We previously had identified vimentin in the normal mesothelial these same tumors after 1 to 2 weeks in culture. MS-7 contained cells present in ascites fluid and rising to higher levels in these vimentin and keratins 40KM, 44KM, and 52KM in vivo and also cells during serial culture (4). Nevertheless, it was necessary to

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. P. J. LaRocca and J. G. Rheinwald examine mesothelial cells in situ, since it has been proposed that medium permissive for rapid, long-term growth of normal me vimentin may become expressed as a consequence of loss of sothelial cells. This suggests that mesothelioma cells have sig cell-cell contact after detachment from an epithelial sheet (21, nificantly altered growth requirements which are difficult to sat 32). It is also known that some epithelial cell types (12, 39) but isfy in culture. We suspect that this is the reason that we have not others (4, 34) abnormally synthesize vimentin under certain been unable to find any published reports of mesothelioma cell conditions of culture. lines. Normal mesothelial cells are anchorage independent, form We have found by indirect immunofluorescence microscopy of ing large colonies in semisolid medium.4 Thus, there is no basis scraped mesothelium and by 2-dimensional gel electrophoretic at present for culturing mesothelioma cells or selectively identi analysis of pure, agar slide-transfer preparations of mesothelium fying them. that this tissue contains both keratins and vimentin in vivo. In addition to the coexpression of keratins and vimentin in Previous reports of an absence of detectable vimentin in cross- mesothelioma, the specific subset of keratins expressed by sections of chick (36) and mammalian (33) mesothelium by mesotheliomas could be used to aid in the differential diagnosis immunofluorescence microscopy may have been due to the of tumors in the pleural and peritoneal cavities. Tumors of relatively low vimentin content in mesothelial cells compared to stratified epithelial and mammary epithelial origin contain 2 or the high levels present in fibroblasts and endothelial cells and more keratins from a set of more basic (M, 58,000 and 56,000) because the mesothelium is very thin and easily damaged during and more acidic (M, 52,000, 50,000, and 46,000) keratins (ker handling and fixation. Mesothelial cells may express keratins and atins 5 and 6 and keratins 13, 14, and 17, respectively) (22-24, vimentin because they are derived from the embryonic meso- 26, 44). Carcinomas of the colon, stomach, and ovary would not derm (this subject is reviewed in Refs. 9, 18, and 37), inasmuch be distinguishable from mesothelioma by the keratin pattern as most other cell types of mesodermal origin, including mes- alone because these tumors also are restricted to simple epithe enchyme-derived cells and cells of some hematopoietic lineages, lial keratins (22-24). Nevertheless, mesotheliomas are carci- express vimentin (2, 8, 12, 13, 15, 31, 36). noembryonic antigen negative or very weakly positive (5,19, 40, The types of keratins in normal mesothelial tissue are restricted 41 ), while adenocarcinomas of the colon are strongly carcinoem- to the simple epithelial keratins, as we had predicted from the bryonic antigen-positive (17). Ovarian epithelial neoplasms can previous results of this laboratory on peritoneal mesothelial cells be recognized by a specific antibody against a surface antigen in culture (4, 43). Samples of human pleural mesothelium con (1). Our results indicate that vimentin is likely to be present in tained the 40 KM, 44 KM, and 52 KM simple epithelial keratins, mesotheliomas, in contrast to its absence in tumors of all other but very little or no keratin 55 KM.This lack of keratin 55 KMwas epithelial cell types (31). Vimentin has also been found recently not simply a regional difference between pleural and peritoneal in several renal carcinomas (16) which originate from another mesothelial cells, however, because pleural mesothelial cells mesoderm-derived epithelial cell type. The data we have pre expressed the keratin 55 KM in culture. We do not know the sented here suggest that coexpression of simple epithelial ker mechanism of this specific regulation of keratin 55 KMbut it could atins and vimentin is a useful marker for distinguishing normal be the result of very rapid cell division or the abnormal culture and malignant cells of mesothelial origin from those of most other substratum. We had found previously that synthesis and content cell types. of all 4 keratins are controlled coordinately in mesothelial cells in culture as a function of growth rate (4, 34). ACKNOWLEDGMENTS Solid and effusion mesotheliomas and 2 short-term cultured We are very grateful to Dr. J. M. Corson, Brigham and Women's Hospital, for mesothelioma populations contained 3 or 4 of the simple epithe providing mesothelioma samples and pathology reports and for helpful comments on the manuscript. Susan Rehwoldt and Terese O'Connell provided excellent lial keratins and vimentin. Six of 9 surgical samples were suc technical assistance with cell culture and preparation of vimentin antiserum. We cessfully analyzed by 2-dimensional gel electrophoretic separa thank Dr. Oswaldo Alberti, Brigham and Women's Hospital, for providing fetal tion and silver staining. Qualitative determination of the inter human lung samples, Dr. Werner Franke, German Cancer Research Institute, Heidelberg, West Germany, for his gift of guinea pig anti-vimentin, and Dr. Robert mediate filament proteins was possible by this method, but Bast of this institute for a sample of OC125 antibody. 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JULY 1984 2995

, . >-v% ; f, ^'. 1Â¿vâ€¢#M / ?'.' -"iâ€¢Ã¤ * â€¢Â»il, r4 ' f<4V

Fig. 1. Morphology and M, 40,000 keratin immunofluoresÅ“nce of human mesothelium in vivo and of mesothelioma cells in culture, a, fetal lung mesothelium transferred to an agarose-coated slide; b, immunofluorescence of same field as a; c, scraped and cytocentrifuged fetal lung mesothelium; cf, immunofluorescence of same field as c; e, normal peritoneal mesothelial strain LP-9 in secondary culture; I, Â¡mmunofluorescence of a different LP-9 culture; g, pleural mesothelioma strain MS-7 in secondary culture; h, immunofluorescence of a different culture of MS-7. a, c, e, and g are phase contrast; b, d, f, and h are anti-M, 40,000 keratin immunofluorescence. Bar in a is 50 urn and is the same magnification for 6; bar in c is 50 Â»mand is the same magnification for d; bar in e is 200 urn and is the same magnification for g; bar in f is 50 ^m and is the same magnification for h.

2996

Fig. 2. Vimentin and keratin immunofluorescence of intact lung mesothelium and of mesothelial cells In ascites fluid, a to c, intact mesothelium scraped from rat lung. Double labeling with guinea pig antivimentin and fluorescein (b) and with rabbit anti-M, 40,000 keratin and rhodamine (c). Small, retractile cells in a are erythrocytes. d to f. human ascites fluid cells from patient with ovarian epithelial neoplasia, double labeled with guinea pig antivimentin and fluorescein (e) and with rabbit anti-stratum comeum keratins and rhodamine (f). g to i, human ascites fluid cells from the same patient, double labeled with rabbit antivimentin and rhodamine (h) and with mouse monoclonal anti-ovarian carcinoma cell surface and fluorescein (/). Bar in a is 50 pm and is the same magnification for b to i. In d, arrows indicate mesothelial cells. In d and g, arrowheads indicate neoplastic ovarian epithelial cells. Vimentin-positive, keratin-negative cells in d to t are presumably macrophages.

JULY 1984 2997

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2998 CANCER RESEARCH VOL. 44

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Fig. 4. Two-dimensional gel electrophoretic separation of mesothelioma cytoskeletal proteins. Triton:high-salt-insoluble proteins of solid tumor samples separated horizontally by charge (loll, basic; right, acidic) and vertically by sodium dodecyl sulfate:polyacrylamide (10%) gel electrophoresis. Gels a and c were focused isoelectrically between pH 6.5 and 5.0. Gels b and d were separated by nonequilibrium pH gradient gel electrophoresis to permit detection of more basic proteins, a, peritoneal mesothelioma MS-4; Â£>,pleuralmesothelioma MS-2; c, peritoneal mesothelioma MS-5; d, pleural mesothelioma MS-11. All gels are silver stained. Lower case letters and arrows within each frame, position of keratins, vimentin, and actin, as in the legend of Fig. 3; arrowheads, proteolytic breakdown products of vimentin and the 52KÂ« keratin. * in frame a, prominent species of size and charge different from that of any known keratin, v, vimentin.

Fig. 3. Two-dimensional gel electrophoretic separation of the cytoskeletal proteins of normal human mesothelium and mesotheliomas. Triton:high-salt-insolubte protein fractions of tissues, tumors, and cultured cells separated horizontally by their charge (left, basic; right, acidic) and vertically by sodium dodecyl sulfate;polyacrylamide (10%) gel electrophoresis. Gels a, c, e, and g were isoelectrically focused between pH 6.5 and 5.0 in the horizontal dimension. Gels b, d, f, and h were separated by nonequilibrium pH gradient gel electrophoresis in the horizontal dimension in order to permit detection of more basic proteins, a, human fetal lung mesothelium collected by slide transfer (spots marked with * are agarose contaminants detected by silver staining); b, cultured adult lung mesothelial strain HPM-2; c and d. cultured adult peritoneal mesothelial cell strain LP-9; e, pleural effusion mesothelioma MS-7; f, peritoneal mesothelioma MS-10; g, secondary culture of MS-7; h, primary culture of MS- 10. Proteins in a, d, and e were revealed by silver staining, in frame f by Coomassie blue staining, and in b, c, g, and h by autoradiography of [^Slmethionine-proteins. Lower case letters within each frame, positions of keratins and vimentin: a, 40Â«M/19;b, 44KM/18; c, 52KÂ«/8;d, 55KM/7; e, 46KM. Arrows, actin. c' is presumed to be a proteolytic digestion product of keratin 52KM/8. v, vimentin.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Coexpression of Simple Epithelial Keratins and Vimentin by Human Mesothelium and Mesothelioma in Vivo and in Culture

Paul J. LaRocca and James G. Rheinwald

Cancer Res 1984;44:2991-2999.

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