1993;53:2858-2864. Published online June 1, 1993.Cancer Res Theodore C. Lee, Yihong Zhang, Christopher Aston, et al. Express Insulin-like Growth Factor I and Associated MoleculesNormal Human Mesothelial Cells and Mesothelioma Cell Lines  

  

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[CANCER RESEARCH 53. 2858-2864. June 15. 1993]

Normal Human Mesothelial Cells and Mesothelioma Cell Lines Express Insulin-likeGrowth Factor I and Associated Molecules1

Theodore C. Lee2, Yihong Zhang, Christopher Aston, Raymond Hintz, Jaishree Jagirdar, Mary Ann Perle,

Michael Burt, and William N. RomChest Sen'ice, Beilevue Hospital Center,W. N. K.] and Pathology ¡J.3.. M. A. P.¡./Vf«-York University Medical (.enter. New York, New York lUUlo: Department 0} PetCalifornia V4305: und Division of Thoracic Surgen- IM. B.I. Memorial Sioan-Ketlerinì>Cancer Center, New York, AViv York 10021

?r. Division of Pulmonary anil Criticai Care Medicine. Departments of Medicine and Environmental Medicine IT. C. L. Y. Z..P.]. New York University Medical Center. Ne»' York, New York 10016: Department of Pediatrics /R. H.I. Stanford University, Sic,

/T. C. L. Y. Z.. C. A..Stanford.

ABSTRACT

Insulin-like growth factor (IGF) I has important growth regulatoryfunctions in normal growth and development. IGF-I is also a mitogen for

a number of cancer cell lines; however, its autocrine effect has not beenwell established. In this study, the expression of IGF-I, its receptor, and itsmajor serum-binding protein were examined in 5 normal human mesothe-

lial (NHM) cell samples and 11 pleura) mesothelioma cell lines. All MINIcells and mesothelioma cell lines expressed IGF-I, IGF-binding protein 3(IGFBP-3), and IGF-I receptor mRNA by either Northern blot or reversetranscription polymerase chain reaction analysis. IGF-I (0.136 ±0.024ng/ml, mean ±SEM) and IGFBP-3 (18.5 ±3.2 ng/ml) proteins were

readily detected in the conditioned medium of mesothelioma cell lines butwere not greater than corresponding measurements in that of NHM cells(IGF-I, 0.120 ±0.080 ng/ml; IGFBP-3, 15.9 ± 1.3 ng/ml). Exogenousrecombinant IGF-I stimulated cell proliferation of NHM cells, demonstrating the presence of a functional IGF-I receptor. Our results suggestthat IGF-I may function as an autocrine growth stimulus in normal pro

liferating mesothelial cells, which may contribute to their malignant transformation.

INTRODUCTION

Malignant mesothelioma has a unique association with asbestosexposure with approximately two-thirds of cases having an occupa

tional or environmental contact with asbestos (1.2). These rare tumorsarise from mesothelial cells which are of mesoderma! origin and linebody cavities, including the pleura and peritoneum. All asbestos fibertypes have been implicated in the pathogenesis of mesothelioma, butepidemiological studies suggest a risk gradient with amphiboles, especially crocidolite (1. 2). NHM' cells are exquisitely sensitive com

pared to human bronchial epithelial cells or fibroblasts to the cytotoxicand clastogenic effects of asbestos fibers (3. 4). The mechanisms bywhich asbestos fibers transform normal mesothelial cells have yet tobe defined: however, the altered expression of growth factors is prominent in this process and enhances tumor progression.

Exogenous growth factors stimulate the proliferation of normaldiploid cells in culture. Cultured malignant cells often demonstrateindependence from exogenous growth factors, a characteristic of malignant cells resulting from the autocrine production of growth factors(5). Human mesothelioma cell lines have been reported to expressboth PDGF-A and -B chain genes at higher levels than culturednormal mesothelial cells (6, 7), express the ß-receptorwhereas normalmesothelial cells express the a-receptor (8), and secrete PDGF-like

Received 12/15/92: accepted 4/8/93.The costs nl' publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by ES05498 and MR (XX)96-3I from the National Institutes of Health.-To whom requests for reprints should be addressed, at Chest Service, Bellevue

Hospital Center 7N24, Division of Pulmonary and Critical Care Medicine. New YorkUniversity Medical Center. 550 First Avenue, New York. NY HX116.

1The abbreviations used are: NHM. normal human mesothelia!; IGF, insulin-likegrowth factor; PDGF. platelet-derived growth factor; BP. serum-binding protein; IGFBP-3. insulin-like growth factor-binding protein 3; IGF-IR. insulin-like growth factor Ireceptor: DMEM: Dulbecco's modified Eagle's medium: cDNA. complementary DNA;

SDS. sodium dodecyl sulfate; RT. reverse transcription: PCR. polymerase chain reaction.

mitogenic activity into conditioned medium (6. 9). Although PDGF ismitogenic for NHM cells (10). this has yet to be demonstrated formalignant mesothelioma cells (II). However, the tumorigenic conversion by overexpression of PDGF-A chain of an SV40 T-antigen-

immortalized nontumorigenic human mesothelial cell line implicatesa role for the PDGF-A chain in mesothelial cell tumorigenesis (9).

NHM cells respond mitogenically to a number of growth factorsincluding epidermal growth factor, transforming growth factor-ß.PDGF-AB heterodimer and -BB homodimer. fibroblast growth factor,interlcukin-la and -ß,interferon-y, and interferon-ß (10. 12-14).

Growth factor responsiveness results in increased mesothelial celldivision which would predispose these cells to asbestos-induced ane-uploidy. Chrysotile-exposed cells in anaphase have been shown to

contain significant increases in the number of cells with anaphaseabnormalities including lagging, bridging, and sticky chromosomes(15). Asbestos fibers were found in mitotic cells to interact directlywith chromosomes (16). Asbestos-induced aneuploidy probably re

sults from the physical interruption by asbestos fibers of chromosomalsegregation during mitosis (15).

IGF-I acts synergistically with PDGF in promoting mesenchymalcell proliferation (17). IGF-I-mediated growth stimulation of tumor

cells has been reported for several neoplastic epithelial and mesenchymal cell types (18-30). Because mesotheliomas are rare tumors of

mesodermal origin with both epithelial and mesenchymal cell features, we hypothesized that IGF-I may play an important role in

mesothelial cell tumorigenesis.IGF-I, a 70-amino acid basic polypeptide, is produced from the

human IGF-I gene on chromosome 12 that contains five exons tran

scribed by alternate splicing to two mRNA species. The two precursorpeptides. IGF-IA (153 amino acids) and IGF-IB (195 amino acids),have identical amino-terminal amino acids but different carboxyl-terminal extensions (31, 32). The signal for IGF-I is mediated by theIGF-I receptor similarly to the insulin receptor composed of 2 a (M,-135,000) and 2 ß(M, -90.000) subunits connected by disulfidebonds in a functional ß-a-a-ß-heterotetrameric receptor complex.When IGF-I binds to the extracellular domain, it stimulates intracell-

ular tyrosine kinase activity producing ßsubunit autophosphorylationand presumably cytoplasmic substituents of an IGF-I-specific signal

transduction pathway (33. 34). The actions of IGFs in tissues aremodulated by IGFBPs. Recently, six distinct IGFBPs were isolatedand their cDNAs cloned (35-48). IGFBP-3 is the major BP in humanserum for IGF-I. The principal effect of IGFBP-3 in vitro is to inhibitthe action of IGF-I, although potentiation of IGF-I action has beendemonstrated as well under different culture conditions (49-52).

To investigate the role of IGF molecules in malignant mesothelioma. we evaluated the effect of recombinant IGF-I on mesothelial

cell growth, the mRNA expression of these IGF genes in normal andmalignant human mesothelial cells, and their secretion of IGF-I andIGFBP-3 peptide into conditioned medium. Our results suggest thatIGF-I may function as an autocrine growth stimulus in normal pro

liferating mesothelial cells, which may contribute to their malignanttransformation.

2S5S

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IGF GENE EXPRESSION IN HUMAN PLEURAL MESOTHELIAL CELLS

MATERIALS AND METHODS

Cells and Culture Conditions. Eleven malignant mesothelioma cell lineswere isolated and developed from pleural effusions, biopsy, or resected mate

rial from patients with cytologically, histologically. immunohistochemically.and ultrastructurally confirmed malignant pleural mesotheliomas. Cytogeneticstudies on short-term chromosome harvests of tumors demonstrated aneup-

loidy. Tissue specimens were minced and incubated in growth medium consisting of a 1:1 composition of DMEM and Ham's F12 medium (Whittaker

Bioproducts. Walkersville. MD). supplemented with 10% fetal calf serum(GIBCO BRL. Grand Island. NY), penicillin (0.1 mg/ml). and streptomycin(0.1 mg/ml). Cell cultures were incubated at 37°Cin a humidified atmosphere

of 5% CO? for 14 days to achieve 75% confluence. Mesothelioma pleuraleffusions were prepared by initially centrifuging at 1000 X g for 10 min.rinsing of the pellet with growth medium, recentrifuging, and seeding cellsfrom 200 ml of pleural fluid onto one 100-mm Primaria culture dish (Falcon).Near-confluent cultures were dissociated using trypsin and either expanded byinoculating 2 x IO5 cells/775 flask, cryopreserved. or used in experimental

protocols. Two normal human mesothelial cell lines. LP-3 and LP-9. were

purchased from the National Institute on Aging Cell Repository (Camden. NJ).Normal mesothelial cells were also isolated from transudative pleural effusionsobtained from patients (n = 3) with congestive heart failure and no history of

malignancy and prepared as described above. These cells were cytologicallyidentified as mesothelial cells and expressed vimentin and cytokeratins byimmunocytochemistry. The cells were cultured in the same growth mediumsupplemented with epidermal growth factor (10 ng/ml; Collaborative Research, Bedford. MA). Human non-small cell lung cancer cell lines (SK-

MES-1, A549, and Calu-3) were purchased from American Type CultureCollection (Rockville. MD) and cultured in DMEM and Ham's F12 supple

mented with 10% fetal calf serum and antibiotics as above.Cell Growth Assay. Normal mesothelial cells ( 1 X IO4) were placed into

96-well plates in serum-free medium and incubated at 37°Cin a 5% CO2

atmosphere. Within the first 24 h after plating, the medium was changed toeither serum-free medium alone or that containing additives: recombinanthuman IGF-I (100 ng/ml; a generous gift from Ciba-Geigy. Suffern, NY),

recombinant human PDGF-AB (10 ng/ml; Upstate Biotechnology. Inc.. Lake

Placid. NY), or 107r FCS. The cells were allowed to grow for 3 days. Theplates were removed from the incubator, and the medium was rapidly decanted.The cells were fixed for 15 min with 10% formalin in 9% acetic acid-0.1 M

sodium acetate buffer. The cells were then stained with a solution of 0.5%naphthol blue black (Aldrich Chemical Co.. Milwaukee. WI) in 9% acetic acidwith 0.1 Msodium acetate. The plates were then washed with H20. and the dyewas eluted by adding 50 m.si NaOH (150 ^.I/well). Absorbance of each wellwas read at 630 nm in a Dynatech Minireader 5000 (53).

IGF-I and IGFBP-3 Radioimmunoassay. Cells were seeded into T75

flasks and cultured until approximately 75% confluent. The monolayers werewashed twice with phosphate-buffered saline, and medium was replaced with10 ml of serum-free medium. After a 24-h incubation, the medium was col

lected and centrifuged at 1000 x g for 5 min. and the supernatant was storedat -70°C. Immunoreactivity for IGF-I and IGFBP-3 was determined from

conditioned media by radioimmunoassay as previously described (54).Northern Analysis. RNA was extracted from subconfluent cultures of me

sothelioma cell lines using a modified method of Chirgwin et al. (55). Poly-adenylated RNA was selected over oligo(dT)-cellulose column chromatogra-phy, separated by electrophoresis in a 1% agarose-6% formaldehyde

denaturing gel. transferred onto nylon filters (BA 85; Schleicher & Schnell),and baked at 80°Cfor 2 h. The filters were incubated at 42°Cfor 2-4 h inprehybridization buffer (50% formamide-5X SSPE-5X Denhardt's reagent-

2.5% herring sperm DNA-0.1% SDS) and hybridized to cDNA probes labeledwith [3-P]dCTP (3000 Ci/mmol: New England Nuclear, Boston. MA) to aspecific activity of >1 X 10" cpm/^g by the random primer method. The

following cDNA probes were used: IGF-I. provided by P. Rotwein. WashingtonUniversity. St. Louis. MO: IGFBP-3. a kind gift from D. Powell. BaylorUniversity. Houston, TX; IGF-IR. kindly provided by Genentech. Inc.. SanFrancisco. CA. Filters were washed for 20 min at 22°Cin 2x standard saline

citrate (1 x standard saline-citrate is 150 M NaCl-1.5 mM sodium citrate)-0.1% SDS. followed by 60 min at 65°Cin 0.5 x standard saline citrate-0.1%

SDS. Autoradiography was performed on Kodak X-omat AR X-ray film withan intensifying screen at -70°C.

RT-PCR Analysis. Reverse transcription of RNA into cDNA was performed by incubating 1 fj.g of total cellular RNA with 50 /¿Iof 1x reversetranscription buffer (GIBCO BRL)-100 /j.g/ml bovine serum albumin-0.5 mMdeoxyribonucleoside 5'-triphosphates-250 fig/ml oligo(dT)¡2_i8 (Pharmacia.

Piscataway. NJ)-200 units Moloney murine leukemia virus reverse tran-scriptase (GIBCO BRL) at 37°Cfor 60 min. PCR was performed at a finalconcentration of IX PCR buffer-50 JUMdeoxyribonucleoside 5'-triphos-phates-1 (J.Meach 5' and 3' primers-2.5 units thermostable Taq polymerase

(Perkin-Elmer Cetus. Norwalk, CT)-5 fil cDNA template in a total volume of

50 fi\. The mixture was overlaid with 75 fxl mineral oil and then amplified witha thermal cycler (Perkin-Elmer Cetus). The amplification profile consisted ofdenaturation at 94°Cfor 30 s, primer annealing at 55°Cfor 60 s, and extension

at 72°Cfor 40 s. The number of amplification cycles were as follows: for

IGF-I, 35: IGF-IR. 35; IGFBP-3. 30; ß-actin,30. Oligonucleotide primers were

synthesized on an Eppendorf DNA synthesizer (General Clinical ResearchCenter, New York University Medical Center). The IGF-IA primers were fromnucleotides 2399-3926 which include a portion of exon 3, producing a 297-base pair product (56). The IGF-IR primers were from nucleotides 3997—4263,producing a 266-base pair product (57). The IGFBP-3 primers were from8391-8761. producing a 370-base pair product (58). The human ß-actinprimers were from 3076-3425. producing a 349-base pair product (59). Each PCR

product mixture (15 /¿I)was electrophoresed in 2% agarose gels in Trisborate/EDTA buffer. Molecular weight markers ( 123-base pair ladder; GIBCO

BRL) were run with each gel. Gels were stained with ethidium bromide andphotographed under UV light. For Southern blot analysis, gels were blottedonto nylon filters (BA 85; Schleicher & Schuell) electrophoretically with IXTris borate/EDTA as the transfer buffer. Blots were baked at 80°Cfor 2 h to fixthe DNA. The specific PCR products were labeled with ["P]dCTP by random

priming. Prehybridization/hybridization and washes were performed as described previously (60).

RESULTS

Growth-stimulating Effect of IGF-I. In order to assess the effectof IGF-I on cell proliferation, we performed 3-day culture experiments in serum-free medium or with growth factors (IGF-I. PDGF-

AB. or fetal calf serum). The results of these studies displayed aspercentages of growth during the 3-day growth period revealed increases in cell growth when serum-free medium was supplementedwith fetal calf serum (94 ±15%. mean ±SEM. P < 0.01 ). PDGF-AB(104 ±18%, P < 0.01). or IGF-I (76 ±26%, P < 0.05) (Fig. 1). In

contrast, cells in control medium demonstrated a 24 ±8% decrementin cell density, probably reflecting cell death during 3 days of growthfactor deprivation. The addition of exogenous IGF-I to 2 different

human mesothelioma cell lines did not enhance their proliferativeresponse (data not shown).

IGF-I mRNA Expression. Hybridization of an IGF-I cDNA probewhich detects both IGF-IA and IGF-IB to human liver polyadenylated

RNA demonstrated 3 mRNA species (1.1. 1.3. and 7.6 kilobases) asreported by others (31, 61). In contrast, however, cultured normalmesothelial cells and 3 mesothelioma cell lines expressed IGF-ImRNA as a single 1.3-kilobase mRNA by Northern analysis (Fig. 2).A sarcomatous mesothelioma cell line (lane 4) expressed IGF-I

mRNA at levels higher than two epithelial mesothelioma cell lines(lanes 2 and 3) and normal mesothelial cells (lane 1). The relativeamounts of RNA loaded into each lane are indicated in Fig. 2, bottom,demonstrating hybridization to the control gene, pHe7. a non-cellcycle-regulated gene (62). Detection of IGF-I gene expression re

quired long exposure times of up to 9 days, reflecting the relativelylow abundance of this mRNA. We then examined by RT-PCR 5

normal and 11 malignant mesothelial cell lines, including the onesstudied by Northern analysis. The PCR product after Southern blottingdemonstrated the presence of the expected 297-base pair IGF-I am

plification product in all normal mesothelial cell samples (Fig. 3, lanes1-5: lanes 1 and 2. normal mesothelial cell lines: lanes 3, 4, and 5,

normal mesothelial cells from pleural effusions) and mesothelioma2859

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IGF GENE EXPRESSION IN HUMAN PLEURAL MESOTHELIAL CELLS

200

gO

-100

CONTROLMEDIUM

PDGF

STIMULUS

Fig. 1. Growth response of normal human mesothelial cells to exogenous IGF-I.Three-day cultures of cells were grown in serum-free media, fetal calf serum (10%). IGF-I( 1(K)ng/ml). or PDGF-AB (10 ng/ml). Cell density was measured at AA.Wnmafter naptholblue black staining as described in "Materials and Methods." The data are expressed as

percentages of growth during the 3-day period. Column (bari, mean (±SEM) of triplicatewells. *P < 0.05 compared to medium alone; **P < 0.01 compared to medium alone.

medium (Fig. 7/4). Conditioned media from malignant mesothelialcells did not contain more IGF-I (0.136 ±0.024 ng/ml, mean ±SEM)

than that of normal cells (0.120 ±0.080 ng/ml). Conditioned mediafrom non-small cell lung cancer cell lines (SK-MES-1. A549, andCalu-3) were used for comparison and contained a mean IGF-I protein

content (0.233 ±0.088 ng/ml) comparable to that reported by others(64-66) and was only slightly higher than that for both normal andmalignant mesothelial cells. IGFBP-3 was also readily detected in

conditioned media from normal (15.9 ± 1.3 ng/ml) and malignant(18.5 ±3.2 ng/ml) mesothelial cells (Fig. IB) at levels comparable tothat of non-small cell lung cancer cell lines (24.0 ±7.3 ng/ml) whichhave been reported to release both IGFBP-1 and -3 proteins into

conditioned medium detected by immunoblotting (67).

DISCUSSION

IGFs are produced in the liver and exhibit insulin-like and growth-

promoting activities. Local activity of IGFs has been detected in manytissues, suggesting that these growth factors are involved in autocrineor paracrine growth regulation (32, 68-70). IGF-I stimulation of tu

mor cell growth has been reported for breast carcinoma (18, 19). coloncarcinoma (20), liposarcoma (20), rat medullary thyroid carcinoma(21), leiomyoma and leiomyosarcoma (22), pancreatic carcinoma(23), lung carcinoma (24-27), ovarian carcinoma (28), fibrosarcoma(29), and osteosarcoma (30). These observations suggest that IGF-I is

an important growth factor in tumorigenesis.In order to test this hypothesis in mesotheliomas, we first defined

whether exogenous IGF-I is mitogenic for mesothelial cells. Our cellproliferation studies demonstrated that exogenous IGF-I was indeed

cell lines (lanes 6-16: lanes 7 and //, sarcomatous mesothelioma celllines; lanes 6 and 16, biphasic mesothelioma cell lines; lanes 8-10,12-15, epithelial mesothelioma cell lines). Fig. 3, bottom, demonstrates amplification of the control gene ß-actin.

IGF-I Receptor mRNA Expression. The IGF-I receptor is a ty-

rosine kinase receptor distinct from the insulin receptor despite extensive homology and has a higher binding affinity for IGF-I thanIGF-II. IGF-IR mRNA expression was detected by Northern analysis

in only one patient sample of normal mesothelial cells and not inmesothelioma cell lines (data not shown). However, the expectedamplification product of 266-base pair was detected in all normal andmalignant mesothelial cells by using RT-PCR (Fig. 4). The corresponding amplification of ß-actinis the same as that in Fig. 3.

IGFBP-3 mRNA Expression. The main carrier of IGFs in thehuman vascular compartment is a MT 150,000 binding protein complex, including the protein IGFBP-3 and the IGF-I ligand (58, 63).IGFBP-3 may modulate the biological activity of IGF-I both enhanc

ing or inhibiting cellular proliferation depending on culture conditions(49-52). Northern blot analysis of IGFBP-3 mRNA expression ofnormal and malignant mesothelial cells revealed a single 2.5-kilobasegene transcript (Fig. 5; lane 1, normal; lanes 2-6, malignant mesothe

lioma). One sarcomatous mesothelioma cell line (lane 5) produced astriking amount of IGFBP-3 mRNA. Interestingly, the same cell linedemonstrated increased IGF-I mRNA levels (Fig. 2). Five normal and

11 malignant mesothelial cell samples revealed the expected amplification product in all normal mesothelial cell samples and mesothelioma cell lines when RT-PCR was used (Fig. 6).

IGF-I and IGFBP-3 Protein Expression. Since normal and malignant mesothelial cells express mRNAs for IGF-I, IGF-I receptor,and IGFBP-3, we next examined the conditioned media from culturednormal and malignant mesothelial cells for the presence of IGF-I andIGFBP-3 by specific radioimmunoassay. IGF-I was detected in normal (3 of 5) and malignant (10 of 11) mesothelial cell-conditioned

2860

1.3kb

pHe7 III!Fig. 2. Northern blot analysis of IGF-I mRNA expression. Fractionated polyadenylated

mRNA (7 ¿ig)was hybridized with a radiolabeled IGF-I cDNA probe. A single mRNAtranscript of 1.3 kilobases was identified. A sarcomatous mesothelioma cell line (lane 4)expressed IGF-I mRNA at levels higher than two epithelial mesothelioma cell lines (lanes

2 and 3) and normal mesothelial cells (lane I}. Botlnm. hybridization to a control gene,pHe?.

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IGF GENE EXPRESSION IN HUMAN PLEURAL MESOTHELIAL CELLS

Fig. 3. Top. IGF-I gene expression in normaland malignant mesothelial cell lines by RT-PCRand Southern blotting. A 297-base pair IGF-I DNAprobe was used. Bottom, ethidium bromi de-sta ined2% agarose gel demonstrating amplification of thecontrol gene ß-actin.Lanes 1-5, normal mesothelial cells; lanes 6-16, malignant mesothelioma celllines (lanes 7 and //, sarcomatous mesotheliomacell lines; lanes 6 and 16, biphasic mesotheliomacell lines; lanes 8-10, 12-15, epithelial mesothe

lioma cell lines).

266bp

IGF-I

297bp -»- f»»»t ••§§•t t«

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

349bp

ß-actin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fig. 4. IGF-IR gene expression in normal andmesothelial tumor cell lines by RT-PCR, followedby Southern blotting. A 266-base pair IGF-I receptor DNA probe was used. The corresponding amplification of the control gene ß-actinis the same asthat in Fig. 3. Lanes as in Fig. 3.

8 9 10 11 12 13 14 15 16

mitogenic for NHM cells; its relative potency was less than that ofPDGF-AB, however. Since NHM cells have been reported to respond

to a broad spectrum of growth factors (10), an autocrine mechanismmay be part of the mechanism that leads to transformation of thesecells by asbestos. Asbestos fibers interact directly with chromosomesand interfere physically with the mitotic apparatus, causing misseg-

regation of chromosomes which results in aneuploidy (15). SinceIGF-I stimulates NHM cells to increase cell division, these cellswould be more susceptible to asbestos-induced aneuploidy (4, 15).Thus. IGF-I may be an important factor leading to mesothelial cell

transformation.

Although IGF-I is believed to be an autocrine growth factor invarious cancers, expression of IGF-I mRNA, in contrast to immunore-active IGF-I, by malignant epithelial cells has not been frequentlydescribed. In this study we have shown that both IGF-I gene transcript

and protein were expressed by normal and malignant mesothelialcells. We have also shown that normal and malignant mesothelialcells, like several other cell lines, expressed the IGF-I receptor

mRNA. Malignant mesothelioma can now be added to a very limitedlist of malignancies which have been reported in the literature toexpress IGF-I receptors. Most of the evidence in the literature hasbeen based on cross-linking, ligand-binding, and receptor blockade

2.5kbFig. 5. Northern blot analysis of IGFBP-3

gene expression. Top, 2.5-kilobase IGFBP-3

transcript; bottom, housekeeping gene. Lane I,normal mesothelial cells; lanes 2-4, epithelialmalignant mesothelioma; lanes 5 and 6, sarcomatous mesothelioma.

pHe7 - * * S *2861

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IGF GENE EXPRESSION IN HUMAN PLEURAL MESOTHELIAL CELLS

Fig. 6. IGFBP-3 gene expression in normal andmalignant mesothelial cell lines by RT-PCR analysis. Top. ethidium bromide-stained 2c7cagarose geldemonstrating the 370-base pair IGFBP-3 amplification product; bottom, amplification of a 349-basepair fragment of the control gene ß-actin.Lanes as

in Fig. 3.

370bp —

349bp —

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

o.oNORMAL

MESOTHELIALCELLS

MALIGNANTMESOTHELIAL

CELLS

LUNGCANCERCELLS

B

nà.fflILO

NORMALMESOTHELIAL

CELLS

MALIGNANTMESOTHELIAL

CELLS

LUNGCANCERCELLS

Fig. 7. ÕGF-Iand IGFBP-3 protein content in conditioned medium determined byradioimmunoassay. Cells (normal mesothelial cells, n = 5; malignant mesothelial cells.n = 11; lung cancer cells, n = 3) were cultured in T75 flasks until near confluence andwashed twice with phosphate-buffered saline, and then the medium was replaced with 10ml serum-free DMEM/Ham's FI2 medium. After a 24-h incubation (37°C.57r CO:), the

conditioned medium was collected and centrifuged. and the supernatant was frozen at-70°C until assay. A. IGF-I; B. IGFBP-3.

studies as reviewed by Cullen et al. (71). IGF-IR niRNA expression

has been described in only a few malignancies thus far: primitiveneuroectodermal tumors (72), breast cancer cell lines and tumors (73),ovarian carcinoma (28). In our studies, not only was the receptor

expressed at the mRNA level but the presence of a functional IGF-I

receptor on NHM cells was suggested by our cell proliferation studies.Furthermore, we detected IGFBP-3 gene expression by Northern

analysis in cultured normal mesothelial cells and mesothelioma celllines. By RT-PCR analysis, all normal and mesothelioma cell linesdemonstrated expression of the IGFBP-3 mRNA transcript. The pep-

tide was found in significant concentrations in the conditioned mediaof all normal and mesothelioma cell lines like other cells in cultureincluding human fibroblasts (74) and human umbilical vein endothe-lial cells (75). The biological significance of IGF-binding proteins

produced by NHM and mesothelioma cells is not clear at this time.However, a number of studies have demonstrated that these proteinsmodulate cellular responses to IGF-I stimulation. The predominant invitro effect of purified IGFBP-3 is to inhibit the actions of exogenousor endogenous IGF-I. This inhibition is mediated by the effectiveremoval of IGF-I availability to IGF-I receptors by the binding ofIGF-I to IGFBP-3 under conditions of coincubation (49, 50). Poten-tiation of IGF-I action in vilro by IGFBP-3 has also been reported

under particular experimental conditions. In contrast to the finding ofDeMellow and Baxter (49), Elgin et al. (51) demonstrated underdifferent coincubation conditions that a marked enhancement of DNAsynthesis and cell proliferation resulted. Potentiation of IGF-I action

is also observed when it is added as a stoichiometric complex, presumably because it allows a continuous slow release of IGF-I similarto an in vitro model in which IGF-I is added intermittently, therebyavoiding down-regulating the IGF-I receptor (52). Release of IGF-I

may occur by mass action or upon association with cells. Potentiationof IGF-I-stimulated thymidine incorporation was also noted whencells were preincubated with IGFBP-3 for 8-48 h before adding IGF-Ito the system (49). This effect of IGFBP-3 is what we observed when

NHM cells were preincubated for 8 h with increasing concentrationsof IGFBP-3. The effect was dose dependent with optimal potentiationof IGF-I-stimulated cell proliferation occurring with 100 ng/ml ofrecombinant IGFBP-3. In contrast, coincubation of IGFBP-3 andIGF-I inhibited IGF-I-stimulated NHM cell proliferation. An expla

nation for the biological activities of the IGFBPs is suggested fromvarious reports: cell surface-associated IGFBPs can enhance IGF-Iaction, whereas soluble IGFBPs inhibit IGF-I action (76). Pretreatment with IGFBP-3 may increase the cell surface-associated component, which could then enhance IGF-I action, whereas coincubation ofIGFBP-3 and IGF-I would tend to result in preferential sequestering ofIGF-I peptide, thus decreasing its bioavailability (49).

For IGF-I to be considered an autocrine growth factor, the peptide

must be synthesized by the cell that is affected by its presence, and thecell must possess all the necessary functional components for theautocrine loop, such as the corresponding receptor and the necessarybinding protein in the case of IGF-I. Our results suggest that IGF-Iexerts its growth-promoting effects on mesothelial cells through anautocrine pathway. The precise mechanisms of IGF-I-stimulated autocrine action in mesothelial cells are not clear. Whether IGF-I inter

acts with its receptor in the extracellular or intracellular compartmentwill help delineate the pathway for the autocrine effects of IGF-I. The

2862

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IGF GENE EXPRESSION IN HUMAN PLEURAL MESOTHELIAL CELLS

cellular location of the interaction of IGF-I with its receptor is ofparticular interest since concurrent synthesis of IGF-I and IGF-I re

ceptor protein could permit their intracellular interaction. Recent evidence from studies in a rat thyroid cell line, FRTL-5, suggests that thedominant mechanism of the autocrine action of IGF-I is the secretionof IGF-I and its interaction with surface receptor molecules (77).

This study is the first to identify IGF-I, IGFBP-3, and IGF-IR gene

expression by normal and malignant mesothelial cells and to demonstrate a functional mitogenic response to IGF-I in normal humanmesothelial cells. These findings suggest that IGF-I may function as

an autocrine growth factor in proliferating mesothelial cells whichthrough enhanced cell division may make these cells susceptible toaneuploidy and cell transformation by asbestos fibers.

ACKNOWLEDGMENTS

We offer special thanks to the Tumor Procurement Service of MemorialSloan-Kettering Cancer Center for its assistance.

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