EXPERIMENTAL

CELL

RESEARCH

190,

91-98

(1990)

xpression of Growth Factor and Growth Factor in Rat Pleural Mesothelial Cells in EDILBERTOBERMUDEZ,~JEFFREYEVERITT, Chemical Industry

Institute

of Toxicology,

P.O. Box 12137, Research Triangle

Inc.

INTRODUCTION A relationship between the occurrence of malignant mesothelioma in man and exposure to asbestos was first established by Wagner et al. [l] and has been further substantiated by others. The majority of the cases of mesothelioma can be linked to asbestos exposure, but the significant incidence of apparently spontaneous cases [2] raises the possibility that there may also be an underlying genetic component to this disease. The mesothelium is generally thought of as the progenitor tis-

1 To whom dressed.

correspondence

and reprint

requests

should

Park, North

Carolina

27709

sue for mesothelioma. This tissue is composed of a mesoderm-derived monolayer of epit elial cells that lines the pleural and peritoneal cavities and ~~~ctio~s to reduce friction between organs and cavity walls. The pathogenesis of mineral fiber-~~duce~~ mesothelioma has not been defined. One proposed mechanism for the development of tumors posits that damage to the mesothelial cell DNA results from th ~~~erat~o~ of oxygen free radicals by macropbages t at have taken up fibers [3], with the subsequent stimulatio growth factors elaborated by the macr mesothelial cells themselves. Th tors on human mesothelial cells growth factors by these cells have been et al. have demonstrated that in norm thelial cells there is expression of trams factor /II 1 (TGF-6,) and ~~at@~et-derived growth factor A-chain (PDGF-A), but a lack of derived growth factor B-chain cells have also been shown to be genie effects of PDGF, TGF-& (EGF), and fibroblast growth larly, increased expression of PDG in cell lines derived from mesotheliomas [4, $1. Various chromosomal abnormalities have been reportedin mesotheliomas 191, including alterati some 22, on which the human P [lo]. These studies have led to the ~ly~otb~s~a that autocrine stimulation may play a role in the unregulate growth of mesothehomas. Exposure of rats to natural and ~~~-~ade fibers leads to the development of m mesotbehomas [l&13] similar in many ways to cm seen in man, The occurrence of chromosoma [14], the manner in which the tumor spreads in the [ 151, the morphologic characteristics of th and the pattern of keratin expression [I larities observed between fiber-induced ~~s~the~~ornas in rats and humans. Therefore, it appears that the rat may serve as a relevant model in the study of this disease. In this series of experiments we the similarities between human cells by the direct determination of

Mineral fiber-induced pleural mesothelioma in the rat is a suitable model for asbestos-induced mesothelioma in humans. A proposed mechanism for the genesis of mesotheliomas is the initiation of an autocrine pathway leading to unregulated growth of the mesothelium. To understand if changes in the expression of mRNA of critical growth factors and receptors occur in target mesothelial cells, it is first necessary to characterize the pattern of expression of these genes in normal mesothelial cells. Rat mesothelial cells were isolated from the parietal pleura and strains of these cells were propagated in vitro. The cells were diploid, had epithelial gross morphology and ultrastructure, and coexpressed keratins and vimentin. Northern blot analysis demonstrated that the cells expressed transforming growth factor @ 1 and fibroblast growth factor. Transcripts for transforming growth factor (Y, platelet-derived growth factor A-chain, and platelet-derived growth factor Bchain were not detected. Receptors for platelet-derived growth factor, epidermal growth factor, and insulin were detected. Although normal mesothelial cells express receptors for these growth factors, no production of their corresponding ligands by these cells could be detected, suggesting that autocrine stimulation of growth via the production of such factors may be specific to transformed mesothelial cells. 0 isso Academic Press,

WALKER

ANDCHERYL

be ad-

91

0014-4827190 53.00 Copyrig& 0 1990 by Academic Press, Inc. All

rights

of qmxluction

ia any

form

reserved.

92

BERMUDEZ,

growth factor receptor expression, in normal rat mesothelial cells. MATERIALS

EVERITT,

using RNA analysis,

AND METHODS

Animals. Male Fischer-344 rats were purchased from Charles River Laboratories (Raleigh, NC) and housed in temperature (22 + l”C)- and humidity (55 f 5%)-controlled rooms with a light/dark cycle of 12 h. The animals were fed NIH-07 rodent chow (Ziegler Brothers, Gardners, PA) and water ad libitum. Mesothelial cell isolation. Rats were deeply anesthetized with 60 mg/kg pentabarbital i.p. and perfused via the portal vein with calcium and magnesium-free HBSS (GIBCO, Grand Island, NY) for 5 min at 10 ml/min. The entire thoracic cavity with the diaphragm attached was then dissected out and the heart and lungs removed. The cavity was then rinsed with HBSS and treated for 30 min at 37°C with a 200 units/ml solution of collagenase (Sigma, St. Louis, MO) prepared in Ham’s F-12 medium (GIBCO). Following treatment the intracostal parietal pleurae were gently rubbed with a rubber policeman and the cells were collected in HBBS containing 0.5 mM EDTA. The cells were then centrifuged at 1OOgfor 4 min and resuspended in complete growth medium (FC,,) consisting of Ham’s F-12 medium supplemented with 10% heat-inactivated fetal bovine serum (GIBCO) and containing 100 U/ml penicillin, 100 Kg/ml streptomycin and 0.25 gg/ ml Fungizone (GIBCO). Mesothelial cell culture. Cells isolated from the animal were seeded into 60-mm dishes (Falcon, Lincoln Park, NJ), coated with Cell-TAK (3.5 pglcm’, BioPolymers, Farmington, CT), containing FC,, incubated in 5% CO, at 37°C in a humidified incubator, and observed for growth daily. When colonies with epithelial morphology grew to approximately 200 cells they were removed using glass cloning rings and a solution of dispase (1 unit/ml F12 medium, Collaborative Research, Bedford, MA). The cells were then continuously subcultured in FC,, using 0.5% trypsin-0.5 mM EDTA (GIBCO) to detach them from the culture vessels. Two cell strains (designated M12Cl and M12C2), derived from separate cultures from the same donor, were chosen for further analysis and to date have been in continuous culture for over a year. Cells from these strains have been frozen in FC,, containing 10% dimethylsulfoxide (Sigma) and stored in liquid nitrogen. Growth of the cell populations was examined by seeding 2.5 X lo5 cells in each of three 60-mm dishes containing 6 ml FC,, and enumerating the cells present at various time points. Cell counts were performed by counting the number of cells in situ with a calibrated reticule in 30 or more fields per dish using a phase microscope. Other cells. Line 4/4 RM-4 [17] was derived from rat visceral pleura mesothelium (American Type Culture Collection, Rockville, MD). LP9 was derived from human ascites fluid [18] and presumed to be normal peritoneal mesothelial cells (NIA Aging Cell Culture Repository, Institute for Medical Research, Camden, NJ). U-2 OS was derived from a human osteogenic sarcoma [19] (American Type Culture Collection). Cell line EGV5T [20] was isolated from a squamous cell carcinoma produced by transformed rat tracheal epithelial cells. NRK-52E is an epithelial-like cell line cloned from a culture of normal rat kidney cells [21] (American Type Culture Collection). Electron microscopy. Cells were grown as described except that they were attached to a plastic coverslip (Thermanox, Miles Laboratories, Naperville, IL) for scanning electron microscopy (SEM) or on a Teflon membrane in a Bionique chamber (Bionique Laboratories, Saranac Lake, NY) for transmission electron microscopy (TEM). The cells on the membrane were fixed in paraformaldehyde-glutaraldehyde-Pipes cell fixative, postfixed in 1% osmium tetroxide, and embedded in epon/araldite for TEM. Fixation of the cells on the coverslips was followed by critical point drying and coating with goldpalladium prior to examination with the SEM.

AND

WALKER

RNA analysis. RNA was extracted from normal rat kidney and log phase cultures of mesothelial cells and cell lines using the method of Chirgwin et al. [22]. Poly (A)* RNA was isolated by oligo-(dT)-cellulose chromatography [23]. Poly (A)+ RNA was separatedby electrophoresis in 0.85% formaldehyde/agarose gels, and transferred to nitrocellulose using standard methods [24]. Nitrocellulose filters were baked, prehybridized, and hybridized at 42°C for 48 h in 40% formamide/3X SSC (0.15 M NaCl and 0.015 M sodium citrate) to specific c-DNA probes labeled with [32P]dCTP to a specific activity of >1 X 10’ cpm/pg by nick translation or random priming [24]. Filters were washed with four changes of 1X SSC/O.l% SDS at 30°C for 2 h, dried, and exposed to X-ray film with an intensifying screen. The following probes were used: murine keratin probes pKB204 (endo B) and pKA56 (endo A) [25] from A. Royal; human vimentin cHuVim1 andinsulin receptor pHIR/PlB-1 from ATCC; human actin pHF 1261 from K. Nelson; rat bFGF RObFGF503 [27] from S. Shimasaki; rat TGF-ol prTGF,, [28] from D. Lee; murine TGF-0, sp65Murbas 1291 from R. Derynck; human PDGF-A PDGFaD-1 [30] from C. Betsholtz, human PDGF-B PSM-1[31] from M. F. Clarke; a subclone (pGR102) of the murine type B PDGF-R [32] from L. T. Williams; and rat EGF receptor pJH 10.1 [33] from H. S. Earp. Karyotype analysis. The number of chromosomes present in the cell strains was determined by treating cells in log growth with 0.05 pg colcemid (Boehringer-Mannheim, Indianapolis, IN) for 2 h at 37°C. The cells were removed from the dish with trypsin-EDTA and collected in FC,,. Following centrifugation, the cell pellet was resuspended in 0.075 M KC1 for 15 min at 37°C and the cells were fixed with three changes of fresh methanolacetic acid (3/l, v/v). Metaphase spreads were prepared and stained with Giemsa and the chromosomes were counted. Keratin immunohistochemical stain. Cells were grown in plastic dishes, fixed with methanol and air-dried. Monoclonal antibodies directed against human keratins (AEl, AE3, or AlfAE3 pool, Boehringer-Mannheim) were used in an indirect peroxidase-antiperoxidase method as per the manufacturer’s instructions.

RESULTS Isolation and Characterization Pleural Mesothelial Cells

of Normal

Rat Parietal

Our initial attempts at isolating the cells of the pleural mesothelium centered on the diaphragm; however, it proved difficult to remove sufficient numbers of mesothelial cells without contamination by cells resembling myoblasts. The intracostal region of the parietal pleura proved to be more amenable to removal of the mesothelial cells. Digestion with high specific activity collagenase followed by gentle removal of the cells from the surface of the wall gave better results, in terms of the ratio of mesothelial cells to fibroblast-like cells, than did short treatment with pronase or longer treatment with dispase. The cells obtained in this manner were then used to initiate primary cultures. Generally a lag of 2 to 4 days was experienced before colonies of cells with distinct polygonal morphology were observed (Fig. 1). Cells from these colonies were then expanded and frozen. Our current inventory of these cell lines includes nine lines isolated in the manner described above. Confluent cultures showed no overlapping of cells (Fig. 1). Scanning electron microscopy of subconfluent cells re-

GROWTH

FACTOR

mRNA

EXPRESSlON

PIG. 1. Mesothelial cells in culture. Cells in subconfluent indicated. Phase contrast (A, 275X; B, 138~).

IN RAT

(A) and confluent

vealed a relatively sparse complement of short microvilli. The microvilh were also evident by transmission electron microscopy but they were fewer in number than observed in the rat in uivo (Fig. 2). Transmission electron microscopy of the cultured cells revealed numerous mitochondria and a well-developed dilated rough endoplasmic reticulum, which contained abundant amorphous material (Fig. 2). Pinocytotic vesicles

MESQTHELIAL

CELLS

(B) cultures are shown. The nuclei @Jr) and nucleoli

(n) are

were observed on both the apical an basilar surfaces of cells. Bundles of intermediate rnicrQ~~arne~t$ were present in perinuclear areas. Desmosomes and tight junctions were noted between adjacent cells (Fig. 2) in culture, as has been observed in situ. The growth characteristics sf two mesothehal cell strains, M42CI and M12C2, were ako examined. The cell strains maintained their epithelial morphology and

.-

94

GROWTH

FACTOR

mRNA

EXPRESSION

IN RAT

MESQTHELEAL

95

CELLS

time been continuously cul (approximately 80 passages) sidered continuous or “immo

can therefore

be con-

Gene Expression in Normal Pleural Mesothelial Cells Gene expression was analyzed in cultures by hybridizing a panel of c(A)+ RNA isolated from the cell lial cells expressed receptors for lin (Fig. 4). The PDGF receptor

~esotbelial

cell

esothelial

cell

which contains

0

20

40

60

a0

TIME

100

120

140

160

iao

bls~

FIG. 3. Growtth curves for rat mesothelial strains M12Cl(U, passage 15) and M12C2 (0; passage 12). Cells were seeded into plastic culture vessels and the change in cell numbers with time was determined by counting cells in situ with the phase microscope.

continued to grow as a monolayer throughout the culture period. The population doubling time was determined for both M12Cl and M12C2 at passage 15 and 12, respectively. As shown in Fig. 3, the average population doubling time was 36 h for M12Cl and 33 h for M12C2. Fewer cells of the Ml261 strain than of the M12C2 strain were present initially after 20 h in culture, possibly due to less efficient attachment of the cells (Fig. 3). The lag in growth after seeding of the cells was about 1.5 days for both strains. Immunohistochemical staining with anti-keratin monoclonal antibodies indicated that the cells contained acidic and basic keratins (data not shown). Cytogenetic analysis for Ml2Cl and M12C2 (passage 13 and 10, respectively) indicated that the cells were normal with a modal chromosome number of 42. Of the 100 M12Cl metaphases examined 78,13, and 9% had 42, 43, and 84 chromosomes, respectively. In contrast, M12C2 cells had 93% with 42 and 7% with 43 chromosomes. Lines Ml261 and M12C2 have at this

served in various rat cells [33f. Low levels of insulin receptor transcripts were also present (Fig. 4); two transcripts of 9.6 and 7.5 kb were observed Several growth factors were also expressed mesothelial cells TGF-P, was expressed as a 2.3 transcript by the mesothelial cells at low levels relat to the rat kidney cell line NRK 52E (Fig. 4). The mesothelial cells also expresse bFGF (Fig. 4). This probably 6.0-kb transcript previously mus and brain cortex 1271. PDGF-A or PDGF-B (Fig. 4 sothelial cells, whereas the etranscripts of2.9,2.3, and 1.8 in control cultures of t S (Fig. 4). TGF-a trams served in transformed cells such as line EGV-5T [ZO], were not cells (Fig. 4). In addition, Nortbern analysis was the expression of cytoskeletal ~orn~o~e tured mesothelial cells. The cells cant and basic (1.7 kb) keratins for both acidic (1.6 k (Table l), consistent munohistocbemistry. Th scripts for vimentin (1.9 kb Gene Expression in Other Mesothelial Cell Strains Two other mesothelial cell strains estab rat pleura (RM 4) 1173 and human ascites

FIG. 2. Transmission electron micrograph of rat mesothelial cells grown in culture. (A, B) Microvilli (mv) normally found on mesothelial cells in vim are shown. Pinocytotic vesicles (pv) can be seen on the basal and apical surfaces. Rough endoplasmic reticulum (RER), mitochondria (m), nucleus (N), and nucleolus (n) are also indicated. (C) A tight junction (arrow) and a desmosome (arrowhead) between ceils are shown. A, 14,400X; B, 20,800x; 6,57,200X.

96

BERMUDEZ, Growth

Factor

I

I

EVERITT,

AND

WALKER Growth

Receptors I

I

EGF-R

INS-R

PDGF-R

bFGF

iiz

EC

5zp s

68

I TGFa

iJg!z 5

Factors I

TGFP,

q

I PDGF A

r;$$

I PDGF B

r;gg r

2

FIG. 4. Northern blot analysis of poly (A)+ RNA (5 pg/lane) isolated from rat mesothelial cells (M12Cl and M12C2) and controls (rat kidney, and the cell lines EGV5T, NRK 52E, and U-2 OS). RNA blotted onto nitrocellulose was probed with radiolabeled DNA probes. Transcript sizes in kilobases for the markers in an RNA ladder standard are indicated by arrowheads. Occasionally a very diffuse background staining was observed in Cl and C2 lanes hybridized to the PDGF A-chain probe. Due to the absence of discrete bands for specific transcripts, the Cl and C2 cells were scored as negative in this assay for PDGF-A expression.

[ 181 were examined to compare gene expression in cells derived in this culture system to other mesothelial cell models. As shown in Table 1, the pattern of expression of cytoskeletal elements was similar in all strains examined. The expression of growth factor receptors was also qualitatively similar between the various cell strains (Table 1). Insulin receptor transcript levels showed some slight difference between rat and human cells, most likely due to species-specific differences in probe

TABLE1 Gene Expression

in

M12C2

RM4

LP9

nd nd + + + -

+ + + + + -

+ nd + + + -

nd +/fl+

+/+ +/+

+ nd +/+ +

+ nd nd + + +/+ + nd + + +

M12Cl Endo A (keratin) Endo B (keratin) Vimentin Actin FGF TGF-a b TGF-8,’ PDGF-Ad PDGF-Bd PDGF receptore Insulin receptor EGF receptor

Mesothelial Cell Strains”

“Northern analysis was performed on poly(A)+ RNA (5 pg) isolated from the indicated cells. The detection of the appropriate transcript size is indicated by f (appropriate transcript size observed), +/- (low levels detected), or - (no transcripts detected). nd, not determined. b Positive control, EGVST. ’ Positive control, NRK-52E. d Positive control, U-2 OS. e Positive control, rat kidney.

homology. Transcript sizes also were species specific; rat cells displayed two transcripts of 9.6 and 7.5 kb whereas the human cells contained several transcripts ranging from 9.1 to 3.1 kb. PDGF and EGF receptor levels were similar in both rat and human cells; EGF receptor transcripts in LP9 cells were 9.7,6.7, and 3.0 kb and PDGF receptor transcripts were 6.1 and 5.0 kb. Growth factor expression did show some variability between the different strains examined. FGF was expressed as a 6.3-kb transcript by RM4 cells and as multiple transcripts of 6.8, 4.1, 2.0, and 1.2 kb in the human LP 9 cells. The size of the human mesothelial FGF transcripts correlates closely with the report of two major (7.0 and 3.7 kb) and several minor transcripts in other human cells [35]. TGF-& mRNA that was transcribed at a very low level in M12C2 cells was more abundant in both the RM4 and LP9 cells. Both RM4 and LP9 expressed TGF-& as a 2.3-kb transcript. No TGF-a! expression was detected in the rat RM4 cells; however, in contrast to the rat mesothelial cells, the human LP9 cells expressed low but detectable amounts of a 4.4-kb TGF-a transcript. PDGF A-chain mRNA (1.8 and 2.1 kb) was also expressed at very low levels in the LP9 cells, but could not be detected in any of the rat mesothelial cultures. DISCUSSION We have isolated cells of the parietal pleura and established cell strains from these isolates. As a specific marker for the cells of the mesothelium has yet to be found, we relied on various cellular characteristics to substantiate the mesothelial cell origin of these strains. Observation of the cells at the light and electron micro-

CROW’I’H FACTOR mRNA EXPRESSION IN RAT MESQTHELIAL scopic levels showed the cells to be very similar in morphology and ultrastructure to mesothelial cells in situ [36, 371. The observed extended population doubling time, density-inhibited growth, normal modal chromosome number, and prolonged lifespan in culture are in agreement with previous reports on normal rat visceral [17] and parietal [38, 391 pleural mesothelial cells in culture. Production of acidic and basic keratins is characteristic of epithelial cells [4Q] and the production of the 4O- to 55kd keratin proteins by nonkeratinizing epithelial cells has been shown to be distinct from those synthesized by keratinocytes. The presence of these proteins in normal rat mesothelial cells was demonstrated by immunohistochemical staining and Northern analysis. Coexpression of keratins and vimentin is an unusual feature of mesothelial cells that has been observed in human cell lines [41]; we also found coexpression of these cytoskeletal elements in the rat strains. All of the above findings indicate a probable mesothelial cell origin for the cell strains examined. Aberrant growth is a major feature in neoplasia and the discovery of structural similarities between normal growth factor genes and viral oncogenes has led to the examination of alterations in growth factor expression and regulation as possible components of the neoplastic process [42,43]. We surveyed the expression of mRNA in rat mesothelial cells of several of these factors for which there is evidence of involvement in the neoplastic process in human cells. Basic FGF was expressed in both rat and human mesothelial cells. Mitogenic activity of bFGF in mesoderm-derived cells, including human mesothelial cells, has been reported [5]. The presence of this mitogen has been reported in various normal and transformed tissues and cultured cells [44]. The possibility of this factor playing a role in transformation is supported by the recent reports of Quart0 et al. and Sasada et al., who transformed NIH 3T3 [45] and BALB/c 3T3 [46] cells with a human basic FGF c-DNA. Transcripts for the EGF receptor, which is recognized by EGF and TGF-a, were observed in both the rat and human epithelial cells. The presence of this receptor has been reported for human mesothelioma cells [47]. TGF~11 transcripts were not observed in the three rat mesothelial strains examined. In contrast, a low level of message for TGF-a! was observed in the human LP9 cells. TGF-(U is expressed by a limited number of normal cells and its enhanced expression is commonly associated with neoplasia [48,49]. Production of this growth factor by mesothelioma but not by normal mesothelial cells has been reported [5]. TGF-& was also expressed at variable levels by rat and human mesothelial cells. Expression of TGF-P, has been reported for normal and transformed human mesothelial cells [4]. Normal mesothelial cells are unusual among epithelial cells in that TGF-& elicits a mitogenic response 15, 501 whereas in other normal cells ofe ithelial origin it acts as an inhibitor of growth [5P, 521.

CELLS

7

DGF-B-specific tra~s~~~~ts were not or detected in the rat MUGI were observed in have been shown to express low els of PDGF-A and PDGF-B in normal in mesothelioma ccl the mitogenic effects of PDGF. Va recently reported that transformed esothelial cells produced EGF-like ma1 cell growth was not stimul pression of the PDGF receptor cell strains and in LP9 cells. PDGF receptor was mesothelial cells, no as to whether the this study have translated this m insulin receptor is of receptors for w were detectable levels of message for this receptor in both the rat and human cells. The utility of mesothelial cells as a model system to study the toxic and carcinogenic effects of mineral other workers fibers has been previously recognize [54]. These cells have been used to st be phagocytosis and degradation [553 of asbestos em, the interaction of asbestos with chromosomes [56], an of asbestos to mediate cell transformation IS?]. present study we have isolated ~o~~a~ mesotheli from the rat by methodology district from the previously reported metbods of ~a~ra~~ et ak. [38] and Aronson and Cristofalo [17]. Ns attem parison of these methodologie apparent we have isolated cell vitro characteristics to those p importantly, in this study we determination of growth factor ceptor expression of these normal rat me$otbe~ial cells. as a basis to The results of this work can no rat mesothelianalyze gene expression in fiber-i oma cells with the aims of elucidating important molecud, ultimately, unin the mesothelial cell lar targ ar basis for fiberg the cellular and mole derstan induced disease. The authors thank J. Martin for his expertise in electron microscopy, J. Ginsler and W. Stewart for the preparation of the various probes used in this analysis, and K. Fun&i for the karyotype ana:ysis.

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Received January 2,199O Revised version received May 7, 1990

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23.

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