Growth Factor Responses and Protooncogene Expression of Murine Mesothelial Cell Lines Derived

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1 Growth Factor Responses and Protooncogene Expression of Murine Mesothelial Cell Lines Derived from Asbestos-Induced Mesotheliomas* LEE A. GOODGLICK, CHARLES A. VASLET, NORMA J. MESSIER, AND AGNES B. KANE Department of Pathology and Laboratory Medicine, Brown University School of Medicine, Providence, Rhode Island ABSTRACT Repeated intraperitoneal injections of crocidolite asbestos fibers induced diffuse malignant mesotheliomas in mice. A series of mesothelial cell lines was isolated from mice at different stages in the development of these tumors. The cell lines isolated from mice with mesotheliomas recapitulated their growth pattern in vivo and were tumorigenic when reinjected into syngeneic mice. Similar to human mesothelial cells, growth of the murine cell lines was stimulated by epidermal growth factor. Reactive mesothelial cells and mesotheliomas expressed the receptor for this growth factor. Crocidolite asbestos fibers have been reported to induce sustained expression of the c-fos and c-jun protooncogenes in rat pleural mesothelial cells in vitro (Heintz et al, Proc. Natl. Acad. Sci. USA 90: , 1993). Human malignant mesotheliomas have been shown to express c-fos in situ (Ramael et al, Histol. Histopathol. 10: , 1995). Two of the cell lines derived from highly invasive murine mesotheliomas overexpressed c-fos and c-jun. This murine model recapitulates the histopathology, growth factor responses, and protooncogene expression of human malignant mesotheliomas. Keywords. Fibers; c-fos; c-jun ; epidermal growth factor; platelet-derived growth factor INTRODUCTION Diffuse malignant mesothelioma is a rare neoplasm arising from the pleural, pericardial, or peritoneal linings. The epidemiology, histopathology, and natural history of this neoplasm are distinctive (11). Most patients with malignant mesothelioma have a history of exposure to asbestos fibers, especially amphibole types of asbestos (11), or to naturally occurring erionite in regions of Turkey (4). Other cases are associated with chronic inflammation or irradiation (31). Although the mesothelium is embryologically derived from mesoderm, diffuse malignant mesotheliomas have different histopathologic patterns ranging from epithelioid or glandular forms to fibroblastic or sarcomatous forms. There is a long latent period preceding the clinical appearance of this neoplasm. Malignant mesotheliomas spread diffusely over the pleural, pericardial, and peritoneal linings and may obliterate these spaces. Deep invasion and metastases occur late in the clinical course (9). The diffuse growth pattern complicates complete surgical removal and most patients die within 6-18 mo despite irradiation or chemotherapy (1). Rodent models of diffuse malignant mesothelioma have been produced by direct intrapleural or intraperitoneal injection (13, 32, 39, 43) or, at a lower frequency, following inhalation of asbestos fibers (44). Although direct delivery of fibers into the pleural or peritoneal spaces is an unnatural route of exposure, the histopathology, pattern of growth, and natural history are identical to human diffuse malignant mesotheliomas (9, 10). We developed a murine model system using weekly intraperitoneal injections, rather than a single large dose of Union International Contre le Cancer (UICC) crocidolite asbestos fibers. A series of cell lines was isolated from mice at different morphologic stages in the progression of these tumors. Their response to exogenous growth factors in vitro and tumorigenicity in vivo were assessed. Acute exposure of peritoneal or pleural mesothelial cells to asbestos fibers stimulates proliferation. It is hypothesized that this acute proliferative response is a regenerative response to mesothelial cell injury (26). Alternatively, asbestos fibers may trigger proliferation indirectly, perhaps by activation of intracellular signal transduction pathways (reviewed in 28). Exposure of rat pleural mesothelial cells in vitro to crocidolite asbestos fibers, but not to nonfibrous particulates, causes a persistent induction of the protooncogenes c-fos and c-jun (18). We investigated whether these protooncogenes were constitutively induced and overexpressed in murine mesothelial cell lines isolated at different times after exposure to crocidolite asbestos fibers in vivo. MATERIALS AND METHODS Preparation and Characterization of Crocidolite Asbestos Fibers. Crocidolite asbestos, which was prepared and characterized according to the UICC, was purchased from Duke Scientific (Palo Alto, CA). UICC crocidolite asbestos (referred to in this report as &dquo;native&dquo; or &dquo;mixed&dquo; crocidolite) was used as the starting material to prepare samples enriched in short and long crocidolite fibers. Short and long crocidolite samples were prepared and characterized as previously described (26). Stock suspensions of 200 )JLg/ml of long or mixed crocidolite and 120 )JLg/ml of short crocidolite were prepared in phosphate-buffered saline (PBS) without calcium, ph 7.4 (Life Sciences, Grand Island, NY), and stored at 4 C. The stock suspensions of mixed or short crocidolite contained * Address correspondence to: Agnes B. Kane, Department of Pathology and Laboratory Medicine, Brown University School of Medicine, Providence, Rhode Island equal numbers of fibers (5.8 X 108/ml). Mice injected c 565

2 566 with equal numbers of long crocidolite fibers developed extensive fibrosis and intestinal obstruction after 12 weekly injections; therefore, a lower dose (2.9 X 108 fibers/ml) was used to induce mesotheliomas. Injection Protocol. Male C57BI/6 mice (1-2 mo old; Charles River Laboratories, North Wilmington, MA) were injected intraperitoneally with crocidolite asbestos under ether anesthesia. Groups of mice were injected weekly with 200 ~g/ml of long crocidolite, 200 >g/ml of mixed crocidolite, or 120 (Jbg/ml of short crocidolite fibers. Fiber suspensions were warmed to 37 C and sonicated for 10 min prior to injection. Mice were housed in filter-top plastic cages under the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The mice were examined daily and sacrificed immediately if they developed massive ascites or weight loss. Histopathologic Analysis. Mice were sacrificed with an overdose of ether at the times described in the text. Complete necropsies were performed on all mice. The following tissue samples were fixed in Carnoy s fixative (90% methanol, 10% glacial acetic acid) at room temperature : diaphragm, liver, pancreas, spleen, intestines, stomach, kidneys, testes, epididymis, seminal vesicles, parietal peritoneal lining, mesenteries, lungs, heart, and rib cage. Tissues sections were embedded in paraffin, cut in 6-)JLm sections, and stained with hematoxylin and eosin (H&E). Four to 16 sections were cut and stained for each tissue sample. Stained sections were examined and photographed using a Zeiss Axioplan photomicroscope equipped for brightfield, darkfield, and phase-contrast viewing. Malignant mesotheliomas were diagnosed using the histopathologic criteria described by Davis et al (12). Immunohistochemical Assays. Unstained paraffin-embedded sections were deparaffinized by sequentially incubating slides in xylene (3 times, 3 min each), absolute ethanol (2 times, 3 min each), 95% ethanol (2 times, 3 min each), and PBS (1 time, 5 min). Deparaffinized sections were stained for cytokeratin by a modified procedure of Bolen and Thorning (5) using a rabbit polyclonal antikeratin, wide-spectrum screening antibody (#Z622; Dako Corporation, Santa Barbara, CA; 1:600 dilution) and a Dako peroxidase-antiperoxidase kit (#K528). According to the manufacturer, this polyclonal cytokeratin antibody reacts with bovine muzzle epidermal keratins 58, 56, 52, 60, 51, and 48 kd. A sheep polyclonal antibody directed against the epidermal growth factor receptor (Upstate Biotechnology, Lake Placid, NY) was used at a dilution of 1:500. Isolation of Murine Mesothelial Cell Lines. Mesothelial cells were isolated using modifications of the technique described previously (10). Mice were sacrificed with an overdose of ether and the diaphragm attached to the rib cage and abdominal wall was removed en bloc. was rinsed twice The inferior surface of the diaphragm with Hanks balanced salt solution and treated with Dispase (a neutral protease isolated from bacteria supplied by Collaborative Biomedical Products, Bedford, MA) diluted 1:10 in Hanks balanced salt solution at 37 C. After 30 min, the surface of the diaphragm was scraped gently with a sterile rubber policeman. After centrifugation at 100 X g for 5 min, the cells were resuspended in Dulbecco s Modified Eagle s high-glucose medium (DMEM) supplemented with 2.5% Medium 199, ITSIM Premix (Collaborative Biomedical Products), 0.02 (JLg/ml epidermal growth factor (Collaborative Biomedical Products), 2 mm L-glutamine, 1 mm sodium pyruvate, penicillin and streptomycin (10 units/ml each), gentamicin (0.05 mg/ml), and 10% heat-inactivated fetal bovine serum (FBS). After repeated injections of crocidolite asbestos fibers, cell lines were isolated from ascitic fluid, if present. The peritoneal cavity was then rinsed 3 times with Hanks balanced salt solution. Small sections of the pa- were dissected under sterile conditions rietal peritoneum and placed in 60-mm tissue culture dishes (Falcon, Lincoln Park, NJ). Cells obtained from ascitic or lavage fluid and the explant cultures were placed in DMEM supplemented with 10% FBS, L-glutamine, sodium pyruvate, and antibiotics as described above and incubated at 37 C in 6% COZ/94% air. The medium was changed twice a week. After 3-4 wk, the cultures became confluent and were then subcultured once or twice weekly using 0.5% trypsin-0.5 mm EDTA. Assay for Tumorigenicity. C57BI/6 mice 4-6 wk old were injected subcutaneously or intraperitoneally with 2 X 106 cells; at least 4 mice were injected with cells from each cell line. The mice were examined weekly for tumors, ascites, or weight loss for 9 mo. After the subcutaneous tumors reached 1 cm in diameter or ascites appeared, the mice were sacrificed. All tumors and abdominal organs were sampled and examined histologically. Scanning and Transmission Electron Microscopy. Mesothelial cell lines were plated on glass coverslips and grown to confluency. Prior to fixation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate at 4 C for min, the cultures were rinsed twice in 0.2 M sodium cacodylate, ph 7.2 at 4 C. The cultures were dehydrated in a graded series of ethanol, dried in a Bomar 900 Critical Point Dryer using liquid CO2, and coated with gold. The cultures were examined in an AMR 1000 scanning electron microscope at 30 kv and X 6,000 magnification. For transmission electron microscopy, cells were plated on 60-mm plastic petri dishes and grown to confluency. The cultures were fixed in 2.5% glutaraldehyde in 0.1 I M sodium cacodylate at 4 C for 1 hr. The monolayers were scraped with a rubber policeman and the cells collected by centrifugation at 500 X g for 5 min. The cell pellets were postfixed in 2% osmium in 0.2 M sodium cacodylate, then rinsed and dehydrated in a graded ethanol series. The pellets were infiltrated for 6 hr in Spurr s resin, embedded, and cured at 70 C overnight. Sections were viewed in a Philips 410 transmission electron microscope at X6,000-18,000 magnification. Effects of Growth Factors on Murine Mesothelial Cells. Cells were plated at a density of 5 X 10=~/96-well microtiter plate in DMEM + 10% FBS and allowed to at 37 C. The cells were rinsed twice with attach overnight DMEM, then placed in 0.4 ml of DMEM containing or the following growth factors: murine epidermal growth factor (EGF; Collaborative Biomedical Products) or platelet-derived growth factor (PDGF) purified from porcine platelets (R&D Systems) at 5-50 ng/ml. At 24

3 567 hr, 1 VLCI of 3H-thymidine was added per well. After 48 hr, cells were solubilized in 1 % Nonidet P-40 for 15 min and collected onto glass filter paper using a Mini-MASH II harvester. 3H-thymidine incorporation was quantitated by liquid scintillation counting. The mean ± SD of 6 replicate wells was determined and expressed as fold stimulation of growth relative to growth in DMEM + The standard deviation was 11.5% of the mean for this assay. Similar results were obtained using a colorimetric microtiter plate assay (6) to determine cell number ; this assay confirmed growth stimulation of cell lines 5, 7, 9, and 41 by EGE Northern Blot Analysis. Total RNA was isolated from each cultured cell line at 75% confluency as described previously (38). Cells stored frozen in liquid nitrogen were lysed in a 4 M guanidinium solution and genomic DNA was sheared by several high-speed polytron bursts. The RNA was pelleted through a 5.7 M CsCl pad to separate it from contaminating DNA and protein. The RNA pellet was dissolved and further concentrated by ethanol precipitation. Total RNA yields and integrity were determined by spectrophotometry at 260 nm/280 nm and by monitoring 28S/18S rrna on denaturing agarose gels. Total RNA (10 Rg) was separated on a 1 % formaldehyde agarose gel, transferred to a nitrocellulose filter, and hybridized to P32-labeled, random primed DNA probes. Equal loading was confirmed by ethidium bromide staining of the gel prior to transfer. The c-myc probe was a 711-base pair (bp) PCR amplification product of rat c- myc exon 3. The c-jun probe was a 311-bp polymerase chain reaction (PCR) amplification product generated from a human c jun cdna coding sequence. Other probes included a 1.0-kilobase (kb) Pst 1 v-i fragment from vfos (American Type Culture Collection, Rockville, MD) and a 1.3 kb human GAPDH cdna sequence (from Dr. Michael Seidman, Otsuka Pharmaceutical, Gaithersburg, MD). Hybridizations were done at 42 C for 18 hr in 5 X salt/sodium citrate (SSC), 1 X Denhardt s solution, 20 mm NaP04, ph 6.8, 100 >g/ml denatured salmon sperm DNA, 10% dextran sulfate, and 50% formamide. The filter was washed twice in 2X SSC, 0.1% sodium dodecyl sulphate (SDS) at room temperature, and once in O.lx SSC, 0.1% SDS, at 55 C for 1 hr followed by exposure to Kodak XAR-5 film. Each probe was stripped from the filter in 1 % glycerol at 80 C for 3 min before rehybridization. Reverse Transcriptase (RT)-PCR Analysis. Preconfluent, exponentially growing cultures of mesothelial cell lines were rinsed in ice-cold PBS, then lysed directly in RNAzo1 & B. Total RNA was extracted in chloroform, precipitated with isopropanol, and washed in 75% ethanol as described previously (7). Equal amounts of total RNA were treated with DNasel in the presence of RNase inhibitor to eliminate genomic DNA contamination and used as template for random primed cdna synthesis. Reactions using 0.5 Rg of treated RNA were performed with and without reverse transcriptase for 15 min at 42 C. PCR amplification of 5.0 jjd reactions were performed as follows: 95 C for 5 min; 95 C for 1 min; 60 C for 2 min; 72 C for 3 min, 35 cycles; 72 C for 10 min. Reaction conditions were optimized to assure generation of specific product during the exponential phase of the reaction. PCR products were visualized on 3% agarose (TAE) gels containing ethidium bromide and compared to known size standards. Positive and negative controls were included in each reaction as safeguards for nonspecific products and carryover contamination. The oligonucleotide primers for WT1 were described in Walker et al (47); murine B-actin oligonucleotide primers were purchased from CLONTECH Laboratories (Palo Alto, CA). RESULTS Induction of Mesotheliomas by Crocidolite Asbestos Fibers Malignant mesotheliomas were identified histologically at necropsy of mice that presented with massive ascites or weight loss after weekly injections of long, short, or mixed crocidolite fiber preparations. The earliest malignant mesotheliomas were identified histologically after 35 weekly injections of long fibers. These tumors had a variable histologic appearance, ranging from epithelioid to fibroblastic or sarcomatous, as illustrated in Fig. 1. The malignant cells expressed cytokeratins as demonstrated by immunohistochemistry. One malignant mesothelioma induced after 60 weekly injections of short fibers had areas of osteoid and bone formation. Cystic areas containing hyaluronic acid (stained by Alcian blue after hyaluronidase digestion) were present, especially in the mixed tumors. Few asbestos fibers were seen within these tumors when examined under darkfield illumination. Angiogenesis was not a common feature of these superficial tumors. Small detached tumorlets or spheroids of malignant mesothelioma cells were found in some mice. The central areas of these spheroids often contained abundant hyaluronic acid. Rarely, sheets and masses of malignant mesothelioma cells invaded locally into skeletal muscle or mesenteric fat. In contrast to the tumors spreading over the serosal surfaces, these locally invasive mesotheliomas elicited a prominent angiogenic response. Invasive mesotheliomas in this murine system were frequently less differentiated, fibroblastic, or mixed tumors. No spontaneous mesotheliomas have been found in uninjected or saline-injected mice during the last 10 yr in this laboratory. Isolation of Murine Mesothelial Cell Lines Cell lines were established from C57B1/6 mice injected weekly with crocidolite asbestos at intervals between 1 and 60 wk. After 1 wk, cells were isolated from the in- as described ferior surface of diaphragm using Dispase in Materials and Methods. After 3-4 wk in culture, cell lines were established from only 10% of these mice; these lines required enriched media for growth in vitro. One of these lines, D9, was selected for further characterization. These cells showed ultrastructural characteristics of mesothelial cells: surface microvilli (Fig. 2A, B) and formation of tight junctions (Fig. 2C). Complex interdigitations and branching of surface microvilli characteristic of mesothelioma cells in situ (reviewed in 8) were absent in this mesothelial cell line in vitro. In contrast to isolation of nontumorigenic mesothelial

4 568 FIG. 1.-Histologic appearances of an epithelioid malignant mesothelioma (A) or a sarcomatous malignant mesothelioma (B) induced in mice by weekly intraperitoneal injections of crocidolite asbestos fibers. H&E. X400. cells, neoplastic mesothelial cell lines were readily established from mice after weekly injections of crocidolite asbestos fibers. Cell lines were derived from ascites, peritoneal lavage fluid, or peritoneal explants with a success rate of 100%. These lines were established rapidly and did not require enriched media for growth in vitro. As illustrated in Fig. 3, the morphologic patterns of the tumors in the donor mouse were recapitulated by the cell lines grown in vitro. In contrast to human and rodent mesothelioma cell lines isolated previously, we successfully established neoplastic mesothelial cell lines from mice with early, focal mesotheliomas (cell lines 19, 41, 70, and 71 in Table I). Two of the donor mice had more extensive tumor growth with formation of tumorlets or spheroids-round masses of tumor cells growing within the peritoneal space (cell lines 7 and 40 in Table I). Cell line 40 recapitulated this growth pattern in vitro FIG. 2.-Ultrastructural features of a nontumorigenic murine mesothelial cell line grown in monolayer culture. A) Transmission electron micrograph of surface microvilli. X6,000. B) Scanning electron micrograph of surface microvilli. X6,000. C) Transmission electron micrograph of tight junctions between mesothelial cells. X 18,000.

5 569 I j FIG. 4.-Expression of WTI and actin mrna by murine mesothelial cell lines using RT-PCR analysis. The cell lines corresponding to each lane are: lane 1, D9; lane 2, 5; lane 3, 7; lane 4, 9; lane 5, 19; lane 6, 40; lane 7, NIH 3T3 fibroblasts; lane 8, marker. lavage fluid, they grew as attached monolayers, rather than suspension cultures, under these conditions in vitro. Rat (47) and human (2) mesothelioma cell lines have been shown to express the Wilms tumor suppressor gene (WT~. As shown in Fig. 4, with one exception (cell line 19), these murine mesothelial cell lines expressed variable levels of WTI mrna as determined by RT-PCR. NIH 3T3 fibroblasts did not express the WTI gene product. FIG. 3.-Morphology of an epithelioid (A) and sarcomatous (B) malignant mesothelial cell line in vitro. Phase contrast. X775. when grown in serum-free medium. With the exception of cell line 41, all of these cell lines derived from mice with mesotheliomas formed foci of disarrayed growth and multiple cell layers in dense cultures. Although 8 of these cell lines were derived from ascitic or peritoneal Assessment of Tumorigenicity All of the cell lines derived from mice with mesotheliomas were tumorigenic after subcutaneous or intraperitoneal injection in syngeneic mice. In contrast, the D9 cell line did not form tumors after subcutaneous or intraperitoneal injection (Table I). Tumors produced by subcutaneous reinjection were histologically similar to the morphology of the cell lines in vitro and the original tumor in the donor mouse. The cell lines derived from mice with early focal mesotheliomas had a long latent period before tumor formation after subcutaneous or intraperitoneal reinjection ( days) In contrast, cell lines established from mice with mesotheliomas growing diffusely over the visceral and parietal peritoneal linings formed tumors more quickly after subcutaneous injection (7-80 days). The subcutaneous tumors were detected ini- TABLE I.-Origin and tumorigenicity of murine mesothelial cell lines. No tumor formation after 250 days.

6 570 TABLE II.-Growth factor responses of murine mesothelial cell lines. tially as raised lesions 2-5 mm in diameter and grew to mm in diameter at the time of sacrifice. Subcutaneous tumors formed by cell lines 7 and 40 showed invasive growth into underlying skeletal muscle. Growth Factor Responses of Murine Mesothelial Cell Lines Previous studies of normal human mesothelial cell lines established that this cell type required enriched medium for sustained proliferation in vitro (29) and responded for at least one cycle of cell proliferation to a wide range of growth factors including EGF, PDGF, and transforming growth factor-beta (TGF-f31) (16). In contrast, growth of normal rat pleural mesothelial cells is inhibited by EGF (41). Therefore, we compared the responses of these murine nontumorigenic and tumorigenic cell lines to these growth factors. After initial plating in medium containing 10% FBS, exponentially growing cultures were transferred to serum-free medium supplemented with insulin, transferrin, selenium, and exogenous growth factors. After 24 hr, cultures were pulse-labeled with 3Hthymidine and harvested after an additional 24 hr as described in Materials and Methods. EGF at 5 ng/ml significantly stimulated growth (1.3 to 2.0-fold) of all cell lines except 19 (Table II). This line was established from ascitic fluid obtained from a mouse with a focal mesothelioma with a fibroblastic morphology. This was also the only cell line stimulated by exogenous PDGF Expression of Epidermal Growth Factor Receptor Neoplastic and nonneoplastic human mesothelial cells have been shown to express the receptor for epidermal growth factor, EGF-R (14, 33). We examined expression of EGF-R in murine mesothelium using immunohistochemistry. As illustrated in Fig. 5A, reactive mesothelial cells 3 days after a single intraperitoneal injection of 200 jjbg of crocidolite asbestos fibers show diffuse cytoplasmic reactivity for EGF-R. Diffuse malignant mesotheliomas induced by repeated intraperitoneal injections of crocidolite asbestos fibers also showed cytoplasmic reactivity for EGF-R, especially in the epithelioid component of these tumors (Fig. 5B). Protooncogene Expression by Murine Mesothelial Cell Lines Acute exposure of rat pleural mesothelial cells to crocidolite asbestos fibers in vitro induces persistent expres- 1 tv..-1l1l1l1ullulll~lu~1lc;1l1l -i:11 GJ~~J1GJJ1V11 Vl C;PIUC;1111i:11 1:;1 UW Ll1 Id - lvl u receptor by mesothelial cells (A) and malignant mesothelioma cells (B). Immunoperoxidase reaction with hematoxylin counterstain. X400. sion of the protooncogenes c fo.r and c-jun. These authors hypothesize that persistent elevation of Fos and Jun proteins activates activator protein-1 (AP-1 ) binding to DNA and stimulates transcription of genes leading to cell proliferation (18). Therefore, we examined steady-state mrna levels of these protooncogenes in nontumorigenic and tumorigenic murine mesothelial cell lines using northern blot hybridization. As illustrated in Fig. 6, 2 of the tumorigenic cell lines, 7 and 40, showed over-expression of c-fos and c-jun relative to expression of c- myc or GAPDH. Both of these lines produced invasive tumors when reinjected subcutaneously into syngeneic mice. DISCUSSION We have developed a murine model of malignant mesothelioma that appears to progress through distinct morphologic stages. In this paper, we describe the isolation, characterization, and growth factor responses of a series

7 571 FIG. 6.-Expression of protooncogenes c-fos, c-jun, and c-myc by murine mesothelial cell lines using northern blot analysis. The cell lines corresponding to each lane are: lane 1, D9; lane 2, 5; lane 3, 7; lane 4, 9; lane 5, 19; and lane 6, 40. of mesothelial cell lines isolated from mice at different stages in the development of malignant mesothelioma induced by weekly injection of asbestos fibers. In this model, proliferating mesothelial cells were obtained from the parietal peritoneal surface of the diaphragm 1 wk after injection of crocidolite asbestos fibers at the peak of mesothelial cell DNA synthesis (26). Human mesothelial cell lines have also been established from patients with pleural or peritoneal effusions secondary to other conditions such as congestive heart failure or autoimmune diseases. In these cases, mesothelial cells exfoliate into the are maintained in pleural or peritoneal spaces where they suspension, frequently mixed with inflammatory cells. The growth factor responses of these normal human mesothelial cell lines have been characterized in detail (16). These cells require an enriched growth media, are not established readily, and express and respond to multiple growth factors, including EGF, PDGF, and TGF-f31 (17). There is considerable variation between individual donors in their growth rates and growth factor requirements in vitro (23). Because these cell lines were established from exfoliated cells isolated from patients with other underlying diseases, it is not known whether they are suitable models for asbestos-induced mesothelial diseases. Most of the rodent and human mesothelioma cell lines described previously have been isolated from advanced pleural or peritoneal malignant mesotheliomas. These mesothelioma cell lines also express the messages for multiple growth factors and their receptors (17). The effects of exogenous growth factors on these malignant mesothelioma cell lines has not been thoroughly characterized. However, it is apparent that human (17, 42) and rat (45) mesothelioma cell lines show different patterns of expression of at least one growth factor and its receptor, PDGF In this series of murine mesothelial cell lines, the pattern of growth factor responses was similar to human mesothelial cell lines. The complex relationship between autocrine and paracrine production of growth factors and cytokines in the development and progression of diffuse malignant mesothelioma has not yet been unraveled (15). Despite the different responses of rat (41) and human mesothelial cells to exogenous EGF (29), there is evidence that TGF-a, a growth factor that binds to EGF-R, is an autocrine growth factor for rat (46) and human (27) malignant mesothelioma cells. Additional evidence for this autocrine mitogenic pathway is the demonstration that human neoplastic and nonneoplastic mesothelial cells in situ express EGF-R (14, 33). Both of these studies found increased EGF-R immunoreactivity in the epithelioid subtype of malignant mesotheliomas; low immunoreactivity was seen in mixed or sarcomatous malignant mesotheliomas. A similar immunohistochemical staining pattern was seen in murine malignant mesotheliomas. These observations suggest that this autocrine mitogenic pathway is more active in malignant mesothelioma cells with epithelial differentiation. EGF is a potent mitogen; however, it also influences mesothelial cell differentiation. A human mesothelial cell line exposed to EGF in vitro assumed a fibroblastic morphology with enhanced expression of vimentin and decreased expression of cytokeratins (21). In this in vitro model, intercellular interactions were also required for sustained cytokeratin expression. EGF and TGF-o. may modulate the growth and differentiation of normal and malignant mesothelial cells differently depending on the in vitro culture conditions, cell density, and cell-cell interactions. The Wilms tumor susceptibility gene (WTI) is expressed by normal and malignant human (2), rat (47), and murine (30) mesothelial cells. Expression of WTI was found to be higher in rat epithelioid mesotheliomas than in sarcomatous tumors (47); this pattern was confirmed in this series of murine mesothelial cell lines. In embryonic mesenchyme and kidney, WTI expression accompanies differentiation from mesenchymal elements to epithelium (35). Maintenance of WTI expression in malignant mesotheliomas may reflect their potential for epithelioid or sarcomatous differentiation (47). WTI is a transcription factor that modulates expression of several growth factors, including insulin-like growth factor (IGF)-II and its receptor, PDGF-A, and TGF-B1 (35). These growth factors are expressed by human malignant mesothelioma cell lines; however, Langerak et al (22) did not find any correlation between the levels of WTI mrna expression and PDGF-A or IGF-II expression in their series of human cell lines. The transcriptional regulatory activity of WTI is modulated by the p53 tumor suppressor gene product (24). Since the p53 protein is overexpressed in greater than 50% of human malignant mesotheliomas (20, 25), this is another potential modulator of the effects of WTI growth factor expression (35). Expression of c-fos and c-myc proteins has been detected in both nonneoplastic mesothelium and in malignant mesotheliomas arising in the human pleura using immunohistochemistry (34). Northern blot analysis

8 572 showed expression of c-fos and c-myc mrna by nontumorigenic and tumorigenic murine mesothelial cell lines. Coordinated overexpression of c-fos and c-jun mrna relative to expression of the housekeeping gene, GAPDH, was found in only two of the murine mesothelial cell lines examined. Both of these lines (7 and 40) were invasive when reinjected subcutaneously. Higher expression of c-fos protein was described previously in invasive areas of murine and human skin tumors studied by immunohistochemistry (40). Fos protein is a component of the AP-1 transcription factor complex. Activation of gene expression by AP-1 binding is involved in stimulation of cell proliferation, differentiation, and transformation (reviewed in 3, 19). Exposure of rat pleural mesothelial cells to crocidolite asbestos fibers in vitro stimulates phosphorylation of the EGF-R, elevates c-fos and c-jun expression, and increases AP-1 binding to DNA. Activation of these cell signaling pathways may be responsible for the proliferative effects of asbestos on mesothelial cells (reviewed in 28). Constitutive overexpression of the c-fos and c-jun protooncogenes may lead to unregulated cell growth and transformation of mesothelial cells (3). The role of c-fos in multistage skin carcinogenesis was recently analyzed in c-fos knockout mice. In this model system, papillomas did develop in c-fos-deficient mice; however, they did not progress into malignant carcinomas (37). These c fos-deficient mice did not express certain AP-1 regulated genes, such as stromelysin, type 1 collagenase, and vascular endothelial growth factor which are important for tumor angiogenesis and invasion (37). Overexpression of c-fos and c-jun in these invasive murine mesothelial cell lines may be associated with increased expression of this set of AP-1-regulated genes. Overexpression of c-fos alone or in combination with c-jun in transgenic mice leads to development of osteosarcomas and chondrosarcomas (36, 48). Similar to malignant mesotheliomas (9), these tumors are of mesenchymal origin. Malignant mesotheliomas induced by repeated injections of crocidolite asbestos fibers in this murine model system show a range of morphologic patterns, including sarcomatous morphology. Rarely, these tumors show foci of bone formation when reinjected intraperitoneally. Expression of c-fos and c-jun by these murine mesothelial cell lines may reflect their mesenchymal origin and ability to differentiate along multiple pathways. Additional experiments using genetically engineered mice are required to explore the role of c fos in the development and differentiation of malignant mesotheliomas. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance of Lynn J. Garant, and the skilled photographic assistance of Janice L. Macdonald. This research was supported by research grant ROl ES03189 from the National Institute of Environmental Health Sciences and a training grant T32 GM from the National Institutes of Health. REFERENCES 1. Alberts AS, Falkson G, Goedhals L, Vorobiof DA, and van der Merwe CA (1988). Malignant pleural mesothelioma: A disease unaffected by current therapeutic maneuvers. J. Clin. Oncol. 6: Amin KM, Litzky LA, Smythe WR, Mooney AM, Morris JM, Mews DJY, Pass HI, Kari C, Rodeck U, Fauscher FJ III, Kaiser LR, and Albelda SM (1995). Wilms tumor/susceptibility (WT1) gene products are selectively expressed in malignant mesothelioma. Am. J. Pathol. 146: Angel P and Karin M (1991). The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim. Biophys. Acta 1072: Baris I, Simonato L, Artvinli M, Pooley F, Saracci R, Skidmore J, and Wagner C (1987). 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