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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 6, June 2001 694-702

Paradoxical Effects of Platelet-Derived Growth Factor-A Overexpression in Malignant Mesothelioma
Antiproliferative Effects In Vitro and Tumorigenic Stimulation In Vivo

Linda J. Metheny-Barlow, Barbara Flynn, Hilde E. van Gijssel, Aizen Marrogi, and Brenda I. Gerwin

Laboratory of Human Carcinogenesis; and Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland



    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Malignant mesothelioma is associated with asbestos exposure and remains resistant to all therapeutic intervention. Previous studies have suggested an enhancing role for platelet-derived growth factor (PDGF) in mesothelial tumorigenicity, although the mechanism by which PDGF facilitates tumorigenicity is unknown. Here, we evaluate the contribution of PDGF-A expression to mesothelial tumorigenicity using ectopic modulation of PDGF-A expression. We find, in accordance with other reports, that the receptor for PDGF-A, although expressed at high levels in normal human mesothelial cells, is not easily detectable in mesothelioma. Further, we show that PDGF-A overexpression is responsible for autocrine downregulation of its receptor. Our data indicate, surprisingly, that for mesothelioma cells in vitro, high-level activation of a PDGF-A-PDGF receptor loop is antiproliferative whereas abrogation of PDGF-A expression stimulates growth. These data suggest that PDGF-A does not contribute to tumorigenicity by autocrine stimulation of proliferation. In contrast, increased PDGF-A expression in vivo increases tumor incidence and growth rate and decreases the latency period to tumor formation whereas abrogation of PDGF-A expression decreases tumor incidence and increases latency. Thus, the tumorigenic effect of PDGF-A must act through paracrine mechanisms relevant at early stages of tumor initiation.



    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Human malignant mesothelioma (MM) is a rare tumor type arising in mesothelial cells after exposure to asbestos. There is evidence that asbestos exerts its carcinogenic effects both by direct clastogenic interaction with mesothelial cells (1) and by stimulating a macrophage-induced inflammatory environment rich in cytokines and reactive oxygen species (2). These effects may act in concert, over a long latency period, to produce the molecular changes necessary for the initiation and progression of malignancy in mesothelial cells. One early response to asbestos observed in rat inhalation models is the rapid induction of both platelet-derived growth factor (PDGF) A and B chains in the bronchiolar-alveolar epithelium and underlying mesenchymal cells, which is sustained for at least 2 wk after exposure (5). Interestingly, the PDGF receptor (PDGFR)-alpha , but not PDGFR-beta , is also elevated in asbestos-exposed rat lung (6, 7), suggesting a possible role for autocrine stimulation in the early stages of asbestos-associated lung damage.

Early work by this lab showed that PDGF acts as a mitogen for normal mesothelial cells in vitro (8), with other reports confirming its activity in vivo (9). PDGF exists as a homo- or heterodimer consisting of two related subunits, the A and B chains. The ligand dimer exerts its mitogenic effects by stimulating two cell-surface receptors: PDGFR-alpha , which can bind all three dimers, and PDGFR-beta , which can bind only the BB isoform with high affinity (10). Binding of ligand results in receptor dimerization and activation by transphosphorylation. PDGFR-alpha and PDGFR-beta have been shown to activate both common as well as distinct signaling effectors (11); differential PDGF ligand-dimer/receptor-dimer combinations have been shown to mediate a diverse array of signaling pathways which control proliferation, cell survival, chemotaxis, and apoptosis (reviewed in ref. 12).

Work from this laboratory and others has demonstrated that the PDGF-A chain, and, somewhat less frequently, the PDGF-B chain, is highly overexpressed in MM compared with normal human mesothelial (NHM) cells (13, 14), suggesting a role for this growth factor in the genesis of mesothelioma. Further, we have shown that simian virus 40 T-antigen-immortalized NHM cells are not tumorigenic but become so after ectopic PDGF-A overexpression. In the same study, a spontaneous vector control tumor showed overexpression of endogenous PDGF-A (15). Together, these results suggest that constitutive PDGF-A production may be a critical step in malignant transformation of mesothelial cells.

The role of PDGF-A in this tumorigenic conversion, however, remains unclear. Although PDGF autocrine loops have been implicated in many malignancies (16), several reports suggest that MM cells demonstrate little or no expression of PDGFR-alpha (21, 22), although others suggest that MM cells in effusions may express the receptor and therefore possess an autocrine stimulatory loop (23).

In the present work, we evaluated the role of PDGF-A expression in mesothelial tumorigenicity by abrogation of PDGF-A through a knockdown antisense approach. We report here that, in vitro, overexpression of PDGF-A has a growth-inhibitory effect on mesothelioma cells whereas inhibition of PDGF-A expression confers a proliferative effect. Further, we show that high levels of ligand downregulate PDGFR-alpha and that this downregulation appears to be a necessary step for in vitro growth of the human mesothelioma cells. In contrast, when cells were inoculated into nude mice, we found that abrogation of PDGF-A expression both decreased tumor incidence and increased the latency period to tumor formation, whereas increased PDGF-A expression increased tumor incidence and growth rate and decreased the latent period for tumor formation. These results underscore the importance of PDGF-A overexpression in the genesis of MM and suggest that its primary contribution results from paracrine effects critical to tumor establishment.


    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell Culture

All cell lines were cultured in a 37°C incubator with 3.5% CO2. NHM cells were cultured from pleural effusions obtained from donors with no history of malignancy. Eighteen established MM cell lines characterized earlier (24) were used for these studies. All MM and NHM cells were cultured in Laboratory of Human Carcinogenesis (LHC)-MM medium (Biofluids, Bethesda, MD) supplemented with glutamine (2 mM) and gentamycin (50 µg/ml) (Biofluids). Porcine aortic endothelial parental (PAE) cells or subclones engineered to overexpress PDGFR-alpha (4alpha R) or PDGFR-beta (4beta R) (PAE system a kind gift of L. Claesson-Welsh; compare ref. 11) and A204 cells (kindly provided by W. LaRochelle, National Cancer Institute Laboratory of Cellular and Molecular Biology, National Institutes of Health, Bethesda, MD) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Biofluids).

Western Blot Analysis

Cells grown to 70 to 80% confluency in T-150 flasks in LHC-MM (Biofluids) were washed three times with Hanks' balanced salt solution (Biofluids), dissociated from the flask with nonenzymatic cell dissociation reagent (Sigma, St. Louis, MO), pelleted, and resuspended in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton-X-100; 0.1% sodium dodecyl sulfate [SDS], and 1% deoxycholic acid, plus a protease inhibitor cocktail of 10 µl/ml of 10 mg/ml phenylmethylsulfonyl fluoride, 10 µl/ml of 10 mg/ml aprotinin [Sigma], 10 µl/ml of 10 mg/ml leupeptin, and 10 µl/ml of 100 mM sodium orthovanadate). Samples were lysed on ice for 10 min, cell debris was pelleted, and supernatant was collected. Protein concentrations were measured by a bicinchoninic assay (Pierce, Rockford, IL) and 75 µg/lane NHM cells or 100 to 150 µg/lane of MM lysate boiled and separated on 8 to 16% polyacrylamide gels (NOVEX, San Diego, CA) and electrotransferred to a 0.45 mM polyvinylene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The membranes were blocked for 1 h in TBS-T (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 0.05% Tween 20) containing 5% (wt/vol) nonfat dry milk and probed with 1 µg/ml Santa Cruz rabbit anti-PDGFR-alpha (cat. #sc-338; Santa Cruz Biotechnologies, Santa Cruz, CA) in TBS-T for 3 h or with 2 µg/ml Upstate Biotechnology rabbit anti- PDGFR-beta (cat. #06-498; Upstate Biotechnology, Lake Placid, NY) in phosphate-buffered saline (PBS) for 2 h. After washing, the membrane was incubated for 45 min with a horseradish peroxidase (HRP)-conjugated donkey antirabbit secondary antibody (1:10,000 in blocking solution; Amersham, Piscataway, NJ), developed with Super Signal chemiluminescent substrate (Pierce), and exposed to Hyperfilm ECL film (Amersham). For the actin loading control, the membrane was probed with 2.5 µg/ml mouse anti-beta -actin (Boehringer-Mannheim, Indianapolis, IN) in TBS-T for 1 h, incubated with HRP-conjugated goat antimouse secondary (1:10,000 in blocking solution; Amersham), and developed as described earlier. Quantitation of bands was performed with a Molecular Dynamics Densitometer using ImageQuant software. Multiple exposures were compared to achieve linearity.

Immunocytochemistry

Cultured cells were suspended in PBS containing 10% bovine serum albumin for 30 min at 4°C, pelleted at 1,000 × g, processed through a graded series of alcohol and xylene, and embedded in paraffin. Sections (4 µm) were cut from paraffin blocks and mounted on electrically charged glass slides. Sections were heated at 60°C for 45 min, deparaffinized in three changes of xylene, and dehydrated in a decreasing ethanol series (100 to 50%) for 5 min per solution. Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide (H2O2) for 30 min. Antigen retrieval involved treatment with an antigen-retrieval solution (BioGenex, San Ramon, CA) in a microwave oven (140 J) for 30 min. Slides were incubated overnight at 4°C in moist chambers with anti-PDGF-A (ZP-214; Genzyme, Cambridge, MA) or anti- PDGFR-alpha (1264-00; R&D Systems, Minneapolis, MN). Binding of the primary antibody was detected by incubation for 45 min at room temperature with biotinylated secondary antibody (1:200) (VectaStain ABC Kit; Vector Laboratories, Burlingame, CA) followed by incubation with streptavidin peroxidase as directed by the manufacturer. Chromogenic development was obtained by immersion in 3-3' diaminobenzidine solution (0.25 mg/ml in 3% H2O2). Slides were counterstained with methyl green and coverslipped after the application of mounting media. Results were considered positive when membranous or cytoplasmic staining was detected.

Messenger RNA Analysis

Total messenger RNA (mRNA) was extracted with TRIzol reagent (GIBCO BRL, Bethesda, MD) and first-strand synthesis was performed on 2 µg RNA using Superscript II (GIBCO BRL) primed with random hexamers as per manufacturer's directions. Integrity of complementary DNA (cDNA) was established by polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using sense primer 5'-TGATCAA TGGAATTCCCATCACCA-3' and antisense primer 5'-CCTC CGACGCCTGCTTCACCAC-3'. PDGFR cDNA was amplified by the PCR using alpha - or beta -specific primers. Primer sequences used were PDGFRalpha sense, 5'-TGGAAGAAATCAAAGTCCCA TCC-3', antisense 5'-GGATCAGCATTAATTTGCAACG-3'; and PDGFR-beta sense 5'-CTGACTTCCTCTTGGATATGC-3' and antisense 5'-TTTCGCCAGCGCTGGAGTCG-3'. PCR amplification of 2.5 µl reverse transcriptase (RT) reactions were carried out using AmpliTaq (Perkin Elmer, Branchburg, NJ) with 1 × PCR buffer, 250 µM each deoxynucleotide triphosphate, and 15 pmol each primer. Cycling parameters used were: 95°C, 5 min, one cycle; 35 cycles of 95°C for 40 s, 55°C for 30 s, and 72°C for 60 s; and one cycle at 72°C for 5 min. A total of 10 µl of product was analyzed on 3.5% agarose gel, stained with ethidium bromide, and photographed. To enhance the detection of PDGFR-alpha , either nested PCR or PCR followed by Southern blotting was used. For nested PCR, 1 µl of RT-PCR product was amplified for 15 cycles using 7.5 pmol of each primer Nalpha L2 (sense) 5'-CACGGTGAA AGACAGTGGAG-3' and Nalpha R1 (antisense) 5'-CACATCAG TGGTGATCTCAG-3'. For Southern blotting, PCR products were transferred to 0.45 micron Hybond-N nylon membrane (Amersham) overnight by capillary action, and the blot ultraviolet crosslinked and hybridized to gamma 32P-end labeled Nalpha L2 primer. After washings, the blot was exposed to Hyperfilm-MP film (Amersham) at -80°C overnight.

Conditioned Media/PDGF-A Experiments

NHM cells were incubated overnight in LHC-MM lacking epidermal growth factor (EGF) and serum (deficient). Cells were then treated for 4 h with fresh LHC-MM lacking EGF and serum, LHC-MM lacking EGF and serum but containing recombinant PDGF-AA (R&D Systems), or LHC-MM lacking EGF and serum but conditioned overnight by incubation with an 80 to 90% confluent MM cell line.

Suramin and PDGF-AA Blocking Experiments

VAMT-1 cells were incubated for 24 h in LHC-MM with 3% serum or in medium with no added serum supplemented with 200 µg/ml suramin (Biomol, Plymouth Meeting, PA), or 7 µg/ml antihuman PDGF-AA (R&D Systems) or normal goat immunoglobulin (Ig) G (R&D Systems). M9K cells were incubated for 24 h in LCH-MM without serum with or without 200 µg/ml suramin. Suramin treatment of A204 cells, which have previously been demonstrated to have a PDGFR-alpha autocrine loop (25), served as a positive control.

PDGF-A Antisense Transfection

At 1 d before transfection, VAMT-1 cells were plated at 5 × 105 cells per 100-mm dish in LHC-MM. For transient transfection experiments, 5 µg of plasmid pD5 containing the PDGF-A long-splice variant (26) in the sense or antisense orientation was added using Lipofectamine (GIBCO BRL). Cell lysates in RIPA were harvested 48 h after transfection. For stable transfection, PDGF-A was cloned in the sense or antisense orientation behind the cytomegalovirus promoter in the plasmid pcDNA3.1(+) (Invitrogen, Carlsbad, CA) by inserting the BamHI fragment from pD5-PDGF-A into the BamHI site in the multicloning site. The day after transfection, the cells were selected in 400 µg/ml G418 (GIBCO BRL). After several passages, selected cells were cloned by limiting dilution and subclones were isolated and characterized.

Colony-Forming Efficiency Assay

Clonal populations were plated at 500 cells per 60-mm dish and cultured for 14 d with a media change every 4 d. Colonies were then fixed in 10% glutaraldehyde for 5 min, stained for 5 min in 0.3% crystal violet, rinsed, dried, and counted.

PDGFR-alpha Transfection and Evaluation of Proliferation

VAMT-1 cells were transfected with Lipofectamine (GIBCO BRL) or electroporated with 5 µg pLTR2-PDGFR-alpha or 5 µg pLTR2-hyg with a BTX ElectroSquarePorator (BTX, San Diego, CA) using 2 × 106 cells/0.2-cm cuvette with three pulses at 90 V for 16 msec each. Cells were plated in 10-cm dishes and selected 24 h after transfection.

Tumorigenicity

Athymic nude mice were irradiated with 350 rads and, 24 h later, inoculated subcutaneously with 5 × 106 cells. A total of 20 mice were injected for each cell line. Mice were examined weekly for 22 wk or until death. A nodule was scored as a tumor when it measured > 2 × 2 mm in diameter and regression did not occur. Tumors were measured using calipers and tumor volumes were calculated (tumor volume = length × width × depth). Upon excision, tumors were treated in several ways. Some tissue was fixed in Bouin's stain, 10% neutral buffered formalin, or OCT (Sakura, Florence, CA) for pathologic and immunohistochemical evaluation after hematoxylin and eosin staining, or was snap-frozen in liquid N2 for molecular analysis.


    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PDGFR-alpha Expression Is Decreased in MM Compared with NHM Cells

To determine whether the ability of PDGF-A to induce tumorigenicity results from autocrine growth stimulation of mesothelial cells, we first screened a panel of NHM and MM cell lines for PDGF-A and PDGFR-alpha expression. Results are summarized in Table 1. We have previously shown that zero of four NHM but five of five MM cell lines tested overexpress PDGF-A (13); further work has identified PDGF-A overexpression in 13 of 13 MM cell lines tested (see Table 1), confirming that PDGF-A overexpression is a consistent alteration in MM cell lines. Western analysis of PDGFR-alpha indicates that although the protein is readily detected in NHM cells, it is significantly reduced or absent in all MM lines tested (Table 1; for representative blots, see Figures 1A and 1B). To determine whether this downregulation occurs at the RNA level, RT-PCR analysis was used to clearly detect PDGFR-alpha mRNA in nine out of 18 MM lines with faint product detected in an additional three lines (Table 1). PDGFR-alpha mRNA could be detected in all but one MM cell line with the increased sensitivity of nested PCR or RT-PCR followed by Southern blotting (Figure 1C). Importantly, these results demonstrate that although MM cell lines do express PDGFR-alpha mRNA, the level is greatly reduced compared to NHM cells. By contrast, there was little differential PDGFR-beta protein expression, with detection in about 60% of NHM and 70% of MM cells (Table 1; for representative blot, see Figure 1A); all cells tested expressed PDGFR-beta mRNA detectable by RT-PCR (Table 1 and Figure 1D). Amplification of GAPDH from all samples confirmed integrity of cDNAs. Amplification of cDNA from PDGFR-overexpressing PAE clones 4alpha R and 4beta R (11) demonstrated specificity of the primers for the PDGFR-alpha and PDGFR-beta , respectively (Figure 1).


                              
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TABLE 1
PDGF and PDGFR expression in NHM and MM



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  		Figure 1.   Characterization of PDGFR expression in NHM and MM cells. (A) Western blot analysis of PDGFR-alpha and PDGFR-beta expression in NHM and MM cells. Growing cells were lysed in RIPA buffer and 75 µg (NHM) or 100 to 150 µg (MM) total protein was loaded on an 8-to-16% SDS gel. Lanes A-D, cultured NHM cells from different donors. Lane 1, JMN; lane 2, Hut290; lane 3, M20; lane 4, VAMT-1. (B) Western blot analysis for PDGFR-alpha using total protein from various MM and NHM cells described. NHBE, cultured normal human bronchial epithelial cells; MEPV23F, NHM cells immortalized by HPV E6/E7; lane 1, VAMT-1; lane 2, JMN; lane 3, M33K; lane 4, M28; lane 5, M25K; lane 6, M24K; lane 7, M20; lane 8, M19; lane 9, M14M; lane 10, M14K; lane 11, DND; lane 12, Hut290; NHM, cultured NHM cells. (C) Total RNA was isolated from growing cells with TRIzol reagent, reverse transcribed, and subjected to PCR amplification using PDGFR-alpha -specific primers. Upper panel, PDGFR-alpha nested RT-PCR. A total of 1 µl of the PDGFR-alpha RT-PCR reaction was reamplified for 15 cycles using PDGFR-alpha primers internal to those used for the first amplification. PCR products were analyzed on 3.5% agarose gels. Lower panel, Southern blotting of RT-PCR products. PDGFR-alpha RT-PCR products were separated on 3.5% agarose gels, transferred to nylon membrane, and probed with a gamma 32P end-labeled 20-mer specific for PDGFR-alpha . Lane 1, Hut290; lane 2, DND; lane 3, M14K; lane 4, M14M; lane 5, M19; lane 6, M20; lane 7, M24; lane 8, M25; lane 9, M28; lane 10, JMN; lane 11, VAMT-1; lane 12, M9K; MEPV23F, human mesothelial cells immortalized with HPV E6/E7; NHBE, normal human bronchial epithelial cells; NHM, cultured NHM cells; PAE, porcine aortic endothelial cells; 4alpha R, PAE cells transfected with a PDGFR-alpha expression construct; 4beta R, PAE cells transfected with PDGFR-beta expression construct. (D) RT-PCR for PDGFR-beta and GAPDH. Total RNA was isolated from growing cells with TRIzol reagent, reverse transcribed, and subjected to PCR amplification with PDGFR-beta -specific primers. The cDNAs were also amplified using GAPDH-specific primers to demonstrate cDNA integrity. Lanes are the same as in C.

Autocrine Downregulation of PDGFR-alpha by PDGF-A

To establish whether the PDGFR-alpha downregulation occurs as an autocrine effect of PDGF-A overexpression, we attempted to downregulate expression in NHM and upregulate expression in MM cells. NHM cells were incubated overnight in media lacking serum and growth factors (deficient), then treated for 4 h with deficient media that had been conditioned by MM cell lines for 24 h (CM) or with 10 to 100 ng/ml of PDGF-A; untreated cells cultured in regular growth media served as control. Representative results are shown in Figure 2A. Both high levels of PDGF-AA as well as the CM from several MM cell lines led to decreased PDGFR-alpha detection in NHM cells from several individual donors, indicating that the MM cells secrete a factor which leads to PDGFR-alpha downregulation. Treatment of NHM with less than 10 ng/ml PDGF-AA resulted in no change or a slight increase in PDGFR-alpha detection (data not shown).



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  		Figure 2.   PDGFR-alpha is downregulated by PDGF-A in MM cells. (A) NHM cells were lysed after treatment for 4 h with PDGF-A or deficient media that had been conditioned for 24 h by VAMT, Hut290 (H290), or M9K mesothelioma cell lines. Total protein (75 µg) from each sample was loaded on 8-to-16% SDS-polyacrylamide gels and Western blot analysis was performed using PDGFR-alpha and actin-specific antibodies. Bands were quantified by densitometry. NHM, cultured NHM cells; CM, conditioned media. (B) VAMT-1, A204, and M9K cells were lysed after 24 h treatment with 200 µg/ml suramin or deficient media. Total protein (100 to 150 µg) was separated on 8-to-16% polyacrylamide gels and immunoblotted with antibodies specific for PDGFR-alpha and actin. Deficient media has no serum or growth factors. (C) Western blot of PDGFR-alpha expression after modulation of PDGF-A. VAMT-1 cells were lysed after incubation with 7 µg/ml PDGF-AA neutralizing antibody or control IgG, or after transient transfection with pD5-PDGF-A sense or antisense vectors. Total protein (100 to 150 µg) was separated on 8-to-16% polyacrylamide gels and Western analysis was performed using antibodies specific for PDGFR-alpha and actin.

To examine this autocrine regulatory loop in MM tumor cells, VAMT-1 and M9K cells were treated with suramin, an agent that binds growth factors and prevents ligand/ receptor association; the A204 rhabdomyosarcoma cell line, which has an established PDGF autocrine loop interruptible by suramin (25), was used as a positive control. Western analysis of the treated cells (Figure 2B) showed increased PDGFR-alpha expression, confirming that a secreted suramin-binding factor is active in an extracellular autocrine loop that leads to decreased PDGFR-alpha expression. This interpretation is further supported by the observation that incubation of MM cells in fresh serum-deficient media also led to increased detection of PDGFR-alpha protein (Figure 2B). The PDGFR-alpha protein began to be detectable 4 h after the addition of deficient media, peaked around 18 h, and began to decrease again by 24 h (data not shown).

To determine whether PDGF-AA was the suramin-sensitive agent responsible for decreased PDGFR-alpha detection in MM, VAMT-1 cells were treated with PDGF-AA neutralizing antibodies or transiently transfected with a PDGF-A antisense expression construct. Analysis of PDGFR-alpha expression after these manipulations demonstrated increased protein expression (Figure 2C), confirming that PDGF-A effects PDGFR-alpha downregulation in VAMT-1 cells.

To test the possibility that increased PDGFR-alpha expression would further increase the proliferation rate of PDGF-A-overexpressing MM cells, we introduced PDGFR-alpha under control of a Moloney leukemia virus Long Terminal Repeat into VAMT-1 cells. In multiple attempts using both lipofection and electroporation, we were unable to obtain VAMT-1 clones that stably express PDGFR-alpha in the presence of the excess ligand, although a vector control line was obtained, suggesting that a high level of signaling through PDGFR-alpha by PDGF-A confers a proliferative disadvantage to MM.

The PDGF-A Antisense-Induced Increase in PDGFR-alpha Correlates with Increased Proliferative Potential In Vitro

In another approach to evaluate a possible autocrine stimulation of proliferation by PDGF-A, stable PDGF-A sense, antisense, or vector control PDGF clones were generated in the VAMT-1 MM cell line. As was seen in the transient PDGF-A antisense experiments, individual PDGF-A antisense-expressing clonal isolates responded with significant increases in PDGFR-alpha expression. Whereas vector control clones expressed variable receptor levels, the antisense clones expressed between two and 12 times more PDGFR-alpha protein than the vector clones (Figure 3, top panel). Interestingly, there was an inverse correlation between the level of PDGFR-alpha expressed and the clonogenic potential of selected clones.



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  		Figure 3.   PDGF-A antisense-induced increase in PDGFR-alpha expression correlates with increased proliferative potential. Upper panel, Western blot analysis of PDGFR-alpha expression in PDGF-A sense, antisense, or vector control stable transfectants. Growing clonal isolates were lysed and total protein (100 µg) was separated on 8-to-16% SDS-polyacrylamide. Blots were probed with PDGFR-alpha and actin-specific antibodies. Bands were quantified by densitometry. Lower panel, colony-forming efficiency of PDGF-modulating clones. A total of 500 cells of each clone were incubated in triplicate 60-mm dishes for 14 d and colonies were stained with crystal violet and counted. Values represent the mean of three experiments ± standard error of the mean (SEM).

PDGF-A Confers Tumorigenic Advantages In Vivo

We have previously established that overexpression of PDGF-A is sufficient to allow for tumorigenic conversion of an immortalized NHM cell line (15). These data appear to conflict with the antiproliferative autocrine effects of PDGF expression reported here. However, paracrine effects operative only in an animal tumorigenicity experiment might explain this incompatibility. Therefore, we tested the possibility that downregulation of PDGF-A expression would inhibit tumor formation whereas increased expression would stimulate the tumorigenic process. One representative clone each of VAMT-1 transfected with pcDNA3.1-PDGF-A sense, antisense, or vector alone was selected for further study. Because of low levels of receptor expression in sense-expressing clones, immunocytochemical titration was used to compare PDGF-A and PDGFR-alpha expression in these cell lines. Figure 4 shows cells at the last positive dilution for each protein or, in the case of negative results, at the highest antibody concentration tested. Figure 4A shows the VAMT-1 clone expressing the PDGF-A sense vector stained for PDGF-A. Cells were positive to the 1:800 dilution shown here. The VAMT-1 clone containing the vector control (Figure 4E) was positive only to the 1:100 dilution. Strikingly, the VAMT-1 PDGF-A antisense-expressing clone did not stain positively even at the 1:50 dilution (Figure 4C). Results for PDGFR-alpha expression were as predicted by previous data. The PDGF-A sense-expressing clone required the highest antibody concentration for detection (Figure 4B; 1:40 dilution), whereas the antisense-expressing clone remained positive at a dilution of 1:320 (Figure 4D). The vector control clone was closer to the antisense-expressing cells, showing some positivity for the receptor to a dilution of 1:160 (Figure 4F). Thus, as expected, the cells used for inoculation expressed either high ligand and low receptor (PDGF-A sense clone), undetectable ligand and high receptor (PDGF-A antisense clone), or intermediate levels of both proteins (vector control).



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  		Figure 4.   Immunocytochemical analysis of PDGF-A and PDGFR-alpha expression in ectopically modulated VAMT-1 mesothelioma cells. Paraffin-embedded cells were stained with antibodies to PDGF-A (A, C, E, and G) or PDGFR-alpha (B, D, F, and H) as described in MATERIALS AND METHODS. The VAMT-1 clone stably transfected with vector containing PDGF-A in sense orientation was positive for PDGF-A at a 1:800 antibody dilution (A) and for PDGFR-alpha at a 1:40 dilution (B). The VAMT-1 clone containing the antisense orientation of PDGF-A was negative for PDGF-A detection even at a 1:50 dilution (C) but showed PDGFR-alpha staining at a 1:320 dilution (D). The VAMT-1 clone containing the vector control was positive for PDGF-A at a 1:100 dilution (E) and for PDGFR-alpha at 1:160 (F ). Cells from an explant of an antisense-expressing tumor were negative for PDGF-A at 1:50 (G) and positive for PDGFR-alpha at 1:160 (H).

As shown in Figure 5A, inoculation of athymic nude mice with VAMT-1 cells expressing PDGF-A in the sense orientation demonstrated an increase in the probability of tumor formation (77.8%) by these cells as compared with the vector control (64.3%). This finding contrasts with the decrease in in vitro colony-forming efficiency shown by this PDGF-A-overexpressing clone (Figure 3). VAMT-1 cells expressing PDGF-A antisense demonstrated a decreased probability of tumor formation (47.1%) relative to the vector control, although the antisense-expressing cells exhibited the highest colony-forming efficiency in vitro (Figure 3). These data suggest that PDGF-A promotes tumorigenicity in vivo by acting as a paracrine factor to facilitate tumor establishment.



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  		Figure 5.   Effect of PDGF-A modulation on tumor formation. Athymic nude mice (20/group) were injected subcutaneously with 106 VAMT-1 cells transfected with PDGF-A sense (open triangles), antisense ( filled triangles), or vector control ( filled circles). (A) Appearance of tumors was assessed once per wk for a period of 20 wk or until the animal's death. A nodule was scored as a tumor when it measured > 2 × 2 mm in diameter and did not regress. (B) Tumors were measured using calipers and tumor volumes were calculated (tumor volume = length × width × diameter). The data shown are mean values ± SEM.

PDGF-A Overexpression Decreases Latency Period to Tumor Formation

As shown in Figure 5A, VAMT vector control cells have a mean latency period of 12.1 ± 2.6 wk from the time of injection to the development of a measurable tumor. This latency period decreased to 8.4 ± 1.4 wk in the PDGF-A sense-expressing cells; by contrast, the latency period increased to 13.8 ± 1.8 in the PDGF-A antisense-expressing cells. This increase in latency is statistically significant (P < 0.0001, 95% confidence interval > 3.9, two-tailed t test). Overexpression of PDGF-A, therefore, led to both increased incidence as well as decreased latency of tumors, whereas abrogation of PDGF-A by antisense led to decreased incidence of tumors as well as increased latency of tumor formation.

PDGF-A Overexpression Leads to More Rapid Growth of Tumors

Weekly manual measurements of tumor size allowed an estimation of growth rate after the establishment of a tumor. The data in Figure 5B demonstrate that PDGF-A- overexpressing tumors have a more rapid growth rate than either vector control or PDGF-A antisense-expressing tumors. This increased growth rate is significant at later weeks, as shown in Figure 5B. Surprisingly, there was little difference between the growth rate of tumors arising from vector control or PDGF-A antisense-expressing cells. These data might indicate that any tumors formed had suppressed the abrogation of PDGF-A expression. This possibility was examined by establishing tumor explant cell lines from tumors arising after inoculation of PDGF-A sense, antisense, or vector control containing cell lines and examining expression of PDGF-A and PDGFR-alpha by immunohistochemistry. Staining of a tumor-explant cell line from the PDGF-A antisense-expressing inoculum is shown in Figure 4. The cells were negative for PDGF-A expression even at a 1:50 dilution (Figure 4G) as were the inoculated cultures (Figure 4C). PDGFR-alpha expression was detected at 1:160 (Figure 4H), within a dilution of the inoculated cells (Figure 4D, 1:320). These data demonstrate continued repression of PDGF-A expression and upregulation of PDGR-alpha , indicating that tumor formation is inhibited but possible in this situation.


    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have previously shown that overexpression of PDGF-A is sufficient to cause tumorigenic conversion of T antigen- immortalized human mesothelial cells (15). We report here that this contribution of PDGF-A to mesothelial tumorigenesis results largely from paracrine effects. We also find evidence for the presence of an autocrine loop which leads to the downregulation of PDGFR-alpha , and suggest that modulation of PDGF-A/PDGFR-alpha signaling by PDGFR-alpha downregulation may be crucial to mesothelioma survival.

We first report, in extension of our earlier observations (13), that PDGF-A is consistently overexpressed in MM compared with normal mesothelial cells. Examination of PDGFR-alpha in these cells, however, demonstrates severely decreased PDGFR-alpha expression compared with NHM cells (Figure 1, Table 1). These results, coupled with previous reports (13, 14, 21), suggest that overexpression of PDGF-A and decreased expression of its receptor, PDGFR-alpha , are characteristic of MM. However, the data indicate that the PDGF-A/PDGFR-alpha autocrine loop does exist but is not a source of proliferative signals. Indeed, we show that ectopically increased PDGF-A expression results in a decrease in mesothelioma cell line growth in vitro whereas inhibition of PDGF-A by antisense results in an increase in cell growth. Although PDGF-A is stimulatory for a normal mesothelial cell, such high levels of autocrine ligand expression as seen in the tumors appear to be growth inhibitory in vitro, as evidenced by the inverse correlation between the level of PDGFR-alpha and the clonogenic potential of transfected cell lines. These data, coupled with the inability to produce clones that overexpress PDGFR-alpha in the presence of PDGF-A expression, led us to postulate that uncontrolled PDGF-A/ PDGFR-alpha signaling is antiproliferative in vitro. This interpretation is further supported by the decreased colony-forming efficiency observed in the PDGF-A sense-expressing cells (Figure 3, lower panel). Because the VAMT-1 parental cells overexpress PDGF-B as well as PDGF-A, we postulate that the increased proliferation in the antisense clones results from the elimination of PDGF-A/PDGFR-alpha -induced antiproliferative signals or from stimulation of the PDGFR-alpha by PDGF-B.

Exposure to high levels of PDGF-AA leads to downregulation of PDGFR-alpha (Figure 2). The existence of this regulatory loop in MM indicates that a low level of PDGFR-alpha protein is being produced which was detectable only by immunocytochemistry at high antibody concentration (Figure 4). PDGF autocrine loops have been implicated in many human malignancies, including meningioma (16, 17), esophageal carcinoma (18), pancreatic cancer (19), and malignant astrocytoma (20). Although PDGFRs are overexpressed in many tumors that contain an autocrine loop (19, 27), downregulation of receptor as a consequence of a high level of chronic stimulation has been documented previously for PDGFR as well as EGF receptor (25, 28).

Although our data suggest that a PDGF-A-stimulated autocrine loop does not play a positive role in mesothelioma proliferation in vitro, we cannot preclude the possibility that PDGF-A signaling confers some biologic advantage to the mesothelioma cell. PDGF has been shown to be a potent survival agent in many cell types (18, 31); it will be of interest to determine whether the low level of signaling maintained by the autocrine loop contributes to the resistance of mesothelioma to apoptotic stimuli (35, 36).

In contrast to its growth inhibitory role in vitro, PDGF-A expression confers significant tumorigenic advantage in vivo. Our data demonstrate that an antisense-mediated decrease in PDGF-A expression in VAMT-1 cells leads to a decreased incidence and an increased latency of tumor formation. We showed here that ectopic overexpression of PDGF-A in the VAMT-1 cell line further increases tumor incidence, decreases latency, and increases tumor growth rate relative to vector control (Figure 5). These findings, coupled with our previous observation that overexpression of PDGF-A leads to tumorigenic conversion in T antigen-immortalized human mesothelial cells (15), underscores the importance of PDGF-A in mesothelial tumorigenicity. Because the growth rate of tumors is not significantly altered by abrogation of PDGF-A (Figure 5B), it is likely that PDGF-A acts primarily in processes involved in tumor establishment.

In light of the fact that autocrine signaling is downregulated, PDGF-A may provide its considerable advantage by acting in a paracrine fashion to generate a more favorable environment for tumor growth. PDGF has also been associated directly (37, 38) or indirectly (39) with angiogenesis, possibly by the induction of VEGF expression in endothelial cells (40) or vascular smooth-muscle cells (41). The paracrine advantages conferred by PDGF-A expression may be selected for in vivo in spite of a decrease in proliferative potential induced by autocrine PDGFR-alpha signaling in vitro.

Our results suggest that PDGF-A overexpression is critical during early stages of tumor establishment. Indeed, examination of tumor specimens for microvessel density by CD31 immunohistochemistry or necrosis by histochemistry indicated no significant differences among tumors from the three mesothelioma inocula (data not shown), suggesting that any significant PDGF-A-mediated effects on these parameters preceded formation of palpable tumors. Consistent with this theory, asbestos exposure has been shown to induce PDGF-A expression and proliferation in lung fibroblasts and tracheal explants (7) in vitro and bronchiolar-alveolar epithelial and underlying mesenchymal cells in vivo (5). In vitro treatment of mesothelial cells with asbestos leads to increased proliferation as well as an initial round of apoptosis (36, 42, 43). It is possible that a PDGF-A-rich environment plays a role in these early responses through both autocrine and paracrine signaling. Once these early events have led to an increased mesothelial cell proliferation and life span, surviving cells may undergo changes which supercede the need for a PDGF-A/ PDGFR-alpha autocrine loop, and PDGFR-alpha expression is downregulated or even selected against. PDGF-A expression would then act in a paracrine fashion to facilitate establishment of the tumor. It should be noted that PDGF-A is only one of many factors that may contribute to the genesis of mesothelioma. It has been previously shown that mesothelioma cells express a wide variety of growth factors and cytokines, including PDGF-BB (13, 22), VEGF (44), FGF-1 and -2 (46), and transforming growth factor-beta (13, 46). In the subset of PDGF-A antisense-expressing cells which progress to form tumors, these and other factors may cooperate in autocrine and paracrine fashions to facilitate the establishment and growth of tumors in a fashion that may be independent of PDGF-A expression.

What biologic situation would lead to downregulation of signaling by a factor that usually stimulates proliferation? One explanation is that decreased expression of PDGFR-alpha in MM cells would prevent autocrine competition for PDGF-A, which we demonstrate is a critical paracrine factor in vivo for the initiation of tumor formation. Another possibility is suggested by a number of recent studies which show that, under certain conditions, activation of PDGFR-alpha can have negative consequences for cellular proliferation. PDGF signaling has been implicated in the induction of apoptosis in quiescent fibroblasts and smooth-muscle cells (49, 50), and has been shown to confer sensitivity to ionizing radiation-induced apoptosis in human prostate cancer cells (51). In addition, PDGFR-alpha has recently been identified as a growth-arrest gene (52). Further, a recent study demonstrates that signaling through PDGFR-alpha produces inhibitory signals mediated by activation of Jun amino-terminal kinase-1 (53). This activation of PDGFR-alpha was shown to antagonize PDGF-B-induced transformation of NIH 3T3 cells. Although the overexpression of PDGF-A by MM cells assists in the establishment of tumors by paracrine interactions, it is possible that downregulation of inhibitory signals from PDGFR-alpha may enhance the malignancy of MM cells induced by other autocrine pathways.

Our early work demonstrating that overexpression of PDGF-A causes tumorigenic conversion of immortalized mesothelial cells implicated PDGF-A as an attractive target for therapeutic intervention. However, our present results suggest that the role of PDGF-A in mesothelioma is complex. Although therapies targeting growth factor- mediated tumor growth, such as immunotherapy with Herceptin to treat erbB-2-positive breast cancers, can be quite effective, use of such treatments must be approached with caution until the precise role of the factor in question is established. In the case of mesothelioma, therapy aimed at blocking effects of PDGF-A would likely be ineffective in established tumors.

In summary, we provide evidence that PDGF-A exerts differential effects on mesothelioma cells, stimulating tumorgenesis in vivo but inhibiting cell growth in vitro. These data demonstrate that PDGF-A is an important paracrine factor for the early stages of tumor formation but that purely autocrine effects of PDGF-A play a complex growth regulatory role for both normal and malignant mesothelial cells.


    Footnotes

Address correspondence to: Dr. L. J. Metheny-Barlow, Georgetown University, Lomabardi Cancer Center, The Research Building E301, 3970 Reservoir Road NW, Washington, DC 20007. E-mail: ljm{at}gunet.georgetown.edu

(Received in original form August 21, 2000 and in revised form January 2, 2001).

Abbreviations: complementary DNA, cDNA; epidermal growth factor, EGF; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; Laboratory of Human Carcinogenesis, LHC; malignant mesothelioma, MM; messenger RNA, mRNA; normal human mesothelial, NHM; porcine aortic endothelial parental, PAE; polymerase chain reaction, PCR; platelet-derived growth factor, PDGF; PDGF receptor, PDGFR; reverse transcriptase, RT.

Acknowledgments: The authors are grateful to Dr. Curtis Harris for valuable discussions and support for this work. They also thank Tim Gannon for technical assistance, Dr. William LaRochelle for providing the A204 cells and pLTR- PDGFR-alpha construct, and Dr. Lena Claesson-Welsh for providing the PAE cell system.
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Abstract
Introduction
Materials and Methods
Results
Discussion
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