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Coexpression of Simple Epithelial Keratins and Vimentin by Human

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					[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-,
                                                                                          MATERIALS        AND    METHODS
theliomas. Two mesothelioma samples from which the tumor
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
                                                                                          onds. When the slide was pulled away, parts of the mesothelial cell layer
ing coexpression of simple epithelial keratins and vimentin, and
                                                                                          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 (-20°) 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
                                                                                          to reduce the levels of contaminants which would be stained in the
other epithelia, the mesothelium is of embryonic mesodermal
                                                                                          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 ¡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.
                                                                                          Some fragments from each tumor were placed in culture, and some were
mesothelial cells that detach from the mesothelium and float in
                                                                                          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 ¡n   partial 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).



JULY 1984                                                                                                                                                          2991
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
ßd/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,
agarose slide-collected mesothelium, and minced tumor tissue were
                                                                                      Small clusters to large sheets of mesothelial cells could also
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 0°, 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 4°.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 ¿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).
Wet gels were then photographed over a light box with Kodak Technical
                                                                                  that grew had morphologies and growth requirements of either
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

2992                                                                                                                        CANCER     RESEARCH       VOL. 44
                                                                              Intermediate Filament Proteins of Mesothelium and Mesothelioma

                                                                         Table 1
                                        Keratin types and vimentin coexpression in normal and malignant mesothelia
                                                                                    Keratins
                                                            (40KM/19)   (44KH/18)     (46K>,)   (52IW8)   (55KM/7)   Vimentin
                         Normal mesothelium8
                          Pleural                v6            +°          +          -          ++                  +
                          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 summarized from 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 summarized from Figs. 3 and 4.
                           e Amount in mesotheliomacells indeterminate because 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


JULY 1984                                                                                                                                  2993
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
cessfully analyzed by 2-dimensional gel electrophoretic separa          technical assistance with cell culture and preparation of vimentin antiserum. We
                                                                        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. We appreciate Lynne Dillon's
quantitative estimation of the M, 52,000 simple epithelial keratin      skillful processing of the manuscript.
and vimentin was often prevented by substantial amounts of
proteolysis. These 2 intermediate filament proteins are particu         REFERENCES
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      different types of intermediate sized filaments in mouse keratinocytes prolif       32.   Ramaekers, F. C. S., Hagg, D., Kant, A., Moesker, O., Jap, P. H. K., and
      erating m vitro. Differentiation, 14: 35-50,1979.                                         Vooijs, G. P. Coexpression of keratin- and vimentin-type intermediate filaments
12.   Franke, W. W., Schmid, E., Osbom, M., and Weber, K. Different intermediate-               in human metastatic carcinoma cells. Proc. Nati. Acad. Sci. USA, 80: 2618-
      sized filaments distinguished by immunofluorescence microscopy. Proc. Nati.               2622, 1983.
      Acad. Sci. USA, 75: 5034-5038, 1978.                                                33.   Ramaekers, F. C. S., Puts, J. G., Kant, A., Moesker, O., Jap, P. H. K., and
13.   Franke, W. W., Schmid, E., Winter, S., Osbom, M., and Weber, K. Widespread                Vooijs, G. P. Use of antibodies to intermediate filaments in the characterization
      occurrence of intermediate-sized filaments of the vimentin-type in cultured               of human tumors. In'. Cold Spring Harbor Symposia on Quantitative Biology,
      cells from diverse vertebrates. Exp. Cell Res., 723: 25-46,1979.                          Vol. 46, pp. 331 -340. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory,
14.   Granger, B. L, and Lazarides, E. Desmin and vimentin coexist at the periphery             1981.
      of the myofibril Z disc. Cell, 78: 1053-1063, 1979.                                 34.   Rheinwald, J. G., O'Connell, T. M., Connell, N. D., Rybak, S. M., Allen-
15.   Granger, B. L., Repasky, E. A., and Lazarides, E. Synemin and vimentin are                Hoffmann, B. L., LaRocca, P. J., Wu, Y.^J., and Rehwoldt, S. M. Expression
      components of intermediate filaments in avian erythrocytes. J. Cell Bid., 92:             of specific keratin subsets and vimentin in normal human epithelial cells—a
      299-312,1982.                                                                             function of cell type and condition of growth during serial culture. In: Cancer
16.   Herman, C., Moesker, 0., Kant, A., Huysmans, A., Vooijs, G. P., and Ramaek-               Cells 1/The Transformed Phenotype, pp. 217-228. Cold Spring Harbor, NY:
      ers, F. C. S. Is renal cell (Grawitz) tumor a carcinosarcoma? Evidence from               Cold Spring Harbor Laboratory, 1984.
      analysis of intermediate filament types. Virchows Arch. B Cell Pathd., 44: 73-      35.   Schiller, D. L., and Franke, W. W. Limited proteolysis of cytokeratin A by an
      83, 1983.                                                                                 endogenous protease: removal of positively charged terminal sequences. Cell
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      Pathol. (Lond.), 30: 138-140, 1977.                                                       H., and Franke, W. W. Differential location of different types of intermediate-
18.   Klemperer, P., and Rabin, C. B. Primary neoplasms of the pleura. Arch. Pathol.,           sized filaments in various tissues of the chicken embryo. Differentiation, 75:
      77:385-412,1931.                                                                          27-40,1979.
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      R., and Boon, M. E. Histologie distinction between malignant mesothelioma,                34:951-962,      1942.
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      morphometry combined with histochemistry and immunostaining. Virchows                     Mitchell, J., and Sun, T.-T. Correlation of specific keratins with different types
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      1983.                                                                               42.   Whitaker, D., Papadimitriou, J. M., and Walters, M. N.-l. The mesothelium:
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      1981.                                                                                     epithelia. Cell, 37: 693-703, 1982.
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      molecular markers of neoplasms with a stratified epithelial origin. Cancer Res.,          made by some cultured human squamous cell carcinomas. Cell, 25: 627-635,
      44: 1600-1603, 1984.                                                                      1981.




JULY     1984                                                                                                                                                              2995
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    Fig. 1. Morphology and M, 40,000 keratin immunofluoresœnce 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, ¡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 »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
                                                                                        Intermediate Filament Proteins of Mesothelium and Mesothelioma




    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.




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2998                                                         CANCER   RESEARCH   VOL. 44
                                                                                        Intermediate Filament Proteins of Mesothelium and Mesothelioma




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                                                                               c-
   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; £>,   pleural mesothelioma 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 52K«
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, 40«M/19;b, 44KM/18; c, 52K«/8;d, 55KM/7; e, 46KM. Arrows, actin. c' is presumed to be a
proteolytic digestion product of keratin 52KM/8. v, vimentin.



JULY    1984                                                                                                                                                            2999

				
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