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[Cancer Research 61, 8306-8316, November 15, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Transforming Growth Factor-ß1 Increases Survival of Human Melanoma through Stroma Remodeling1

Carola Berking, Richelle Takemoto, Helmut Schaider, Louise Showe, Kapaettu Satyamoorthy, Paul Robbins and Meenhard Herlyn2

The Wistar Institute, Philadelphia, Pennsylvania 19104 [C. B., R. T., H. S., L. S., K. S., M. H.], and the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 [P. R.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor (TGF)-ß is growth inhibitory for normal epithelial cells and melanocytes but can stimulate mesenchymal cells. Resistance to its inhibitory effects is characteristic of human melanoma, the growth of which may instead be promoted by TGF-ß, because its production is increased with melanoma progression. Whether TGF-ß has an autocrine function for melanoma cells or is important for paracrine stimulation of the tumor stroma is not known. In this study, TGF-ß1 was expressed in melanoma cells via adenoviral gene transfer, and tumor growth was analyzed in vitro, in human skin grafts, and in mixtures with fibroblasts that were injected s.c. into immunodeficient mice. The TGF-ß1 produced by the melanoma cells activated the fibroblasts to produce matrix within and around the tumor mass, whereas control tumors showed less stroma and more cell death. High expression of collagen, fibronectin, tenascin, and {alpha}2 integrin was detected in the TGF-ß1-expressing tumors by immunohistochemistry. Number and size of lung metastases were significantly increased. cDNA expression array analysis of TGF-ß1-transduced fibroblasts embedded in type I collagen and of TGF-ß1-transduced melanoma cells demonstrated induction of types XV, XVIII, and VI collagens, tenascin, plasminogen activator inhibitor-I, vascular endothelial growth factor, cysteine-rich fibroblast growth factor receptor-1, and platelet-derived growth factor receptor-ß, which could be linked to promotion of growth and survival in melanoma. These data suggest that remodeling of the neighboring stroma, which provides a supporting scaffolding and a positive feedback stimulation of tumor growth, is an important function of TGF-ß1 in melanoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TGF3 -ß is an almost ubiquitously expressed protein with diverse functions in embryogenesis and adult tissue homeostasis. There exist three isoforms of TGF-ß (TGF-ß1, TGF-ß2, and TGF-ß3) in mammals with 75–80% homology, which arise from proteolytic cleavage of longer precursors (1 , 2) . Mature biologically active TGF-ß results from dissociation of the latent inactive TGF-ß complex, which can be stored in the ECM. TGF-ß antagonizes the mitogenic activities of many other growth factors by interfering with cell cycle progression and is the most potent growth inhibitor known for epithelial cells and cells of the immune system. On the other hand, TGF-ß can stimulate mesenchymal cells, such as fibroblasts, smooth muscle cells, and chondrocytes, and induce the synthesis of proteins found in the ECM including collagens, fibronectin, tenascin, thrombospondins, osteopontin, osteonectin, and elastin (1 , 3 , 4) . Simultaneously, TGF-ß can reduce the synthesis of proteases, such as collagenases, and increase the synthesis of protease inhibitors, such as TIMP-1 and PAI-I (5) .

Carcinoma cells of the breast, prostate, lung, and colon produce TGF-ß and are resistant to its growth-inhibitory effects, in contrast with their benign precursor cells (6 , 7) . It has been hypothesized that at these advanced stages of transformation, TGF-ß can act as a tumor promoter. Likewise in melanoma, expression of all three isoforms of TGF-ß has been found in malignant cells in culture (8) and in situ (9, 10, 11) , and an association with progression has been proposed. Both high-affinity receptors for TGF-ß, which form a heteromeric complex upon activation and transmit signals to the cytoplasmic SMAD proteins (2) , are expressed in melanoma (12 , 13) . Upon exogenous TGF-ß stimulation, melanoma cells display various degrees of resistance to TGF-ß-induced inhibition of DNA synthesis, whereas melanocytes are highly sensitive (8 , 14) . However, in contrast with what has been found in some other TGF-ß-resistant carcinomas, no inactivating mutations in the TGF-ß receptor system or of the SMAD signaling cascade have been detected in melanoma, suggesting additional mechanisms for resistance to the growth-inhibitory functions of TGF-ß in this tumor type (15) .

The biological benefits for melanoma cells to constitutively produce TGF-ß remain unclear. An autocrine TGF-ß-mediated up-regulation of integrins and matrix metalloproteinase-9 as well as a down-regulation of E-cadherin has been described and may facilitate melanoma cell migration (16) and adhesion to the endothelium (13) . Paracrine effects of TGF-ß on host cells in the tumor microenvironment may also be advantageous for melanoma cells. Suppressive effects on the immune system may allow tumor cells to escape from immune surveillance (17 , 18) , and angiogenic properties of TGF-ß could support nutrition of the tumor and facilitate metastasis (3 , 19) . In addition, stimulation of stromal cells by TGF-ß could lead to increased production of reciprocally paracrine-acting growth factors and to ECM production, which could, in turn, provide a scaffolding for melanoma cells to adhere and migrate. Finally, the modulation of proteases and their inhibitors by TGF-ß could facilitate remodeling of the stroma and invasion (20, 21, 22) .

In this report, we provide evidence that melanoma cells can modulate their surrounding stroma for their own benefits through the paracrine activity of TGF-ß1. Stimulation of production of ECM proteins by stromal fibroblasts provided a scaffolding for the melanoma cells, which showed increased survival and metastasis formation compared with the controls. Global gene expression analyses of TGF-ß1-expressing melanoma cells and fibroblasts in organotypic culture indicated a complex interplay between matrix proteins, adhesion molecules, and growth factors, providing an optimal environment for tumor growth and progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Normal human keratinocytes and melanocytes were isolated from the epidermis, and fibroblasts were isolated from the dermis of neonatal human foreskins. Keratinocytes were cultured in SFM (Life Technologies, Inc., Rockville, MD) supplemented with human recombinant epidermal growth factor and bovine pituitary extract. Melanocytes were cultured in MCDB153 (Sigma Chemical Co., St. Louis, MO) supplemented with 2% FBS, 10% chelated FBS, 2 mM glutamine (Mediatech, Herndon, VA), 20 pM choleratoxin (Sigma Chemical Co.), 150 pM recombinant human basic fibroblast growth factor, 100 nM endothelin-3 peptide (Peninsula, Belmont, CA), and 10 ng/ml recombinant human stem cell factor (R&D Systems, Minneapolis, MN). Fibroblasts and human embryonic kidney-derived 293 cells used for adenovirus replication were cultured in DMEM with glutamine (Life Technologies, Inc.) and 10% FBS (Hyclone, Logan, UT). Human primary and metastatic melanoma cells were isolated from clinically and histologically defined lesions and cultured as described (23 , 24) . They were maintained in MCDB153 with 20% Leibovitz’s L-15 medium (Life Technologies, Inc.), 2% FBS, and 5 µg/ml insulin (Sigma Chemical Co.).

Growth Factor Detection.
Melanoma cells, melanocytes, and fibroblasts were plated in six-well plates at 1.5 x 105 cells/well in their culture medium for 24 h. They were transduced with TGF-ß1 or LacZ via adenoviral vectors, washed with SFM after 24 h, and cultured in SFM for another 48 h. Cell supernatants were then analyzed for the presence of TGF-ß1 using a human TGF-ß1 immunoassay (R&D Systems) that uses TGF-ß soluble receptor type II, which binds TGF-ß1, as the coating reagent and an enzyme-linked polyclonal antibody to TGF-ß1 as the second reagent. The procedure followed the manufacturer’s instructions. The measurements represent the total of both active and latent forms of TGF-ß1 and are given in pg/105 cells. For analysis of VEGF protein, an ELISA was used according to the manufacturer’s instructions (R&D Systems). Melanoma cells and fibroblasts were transduced with TGF-ß1 or LacZ control, respectively, and conditioned medium was collected 72 h later. Samples were frozen at -70°C and analyzed within the following 1–2 days. Results are expressed as mean ± SD ng/ml per 106 cells. All experiments were performed in duplicates.

[3H]Thymidine Incorporation Assay.
Melanoma cells, fibroblasts, and melanocytes were seeded at 2–4 x 104 cells/well in 96-well plates and infected with adenoviral vectors. After 2 days, 1 µCi of [3H]thymidine was added per well, and 18 h later, cells were harvested, and the activity was counted with a beta counter. Experiments were performed twice and in triplicates. Results are expressed as average difference (in percentages) ± SD compared with the respective LacZ-transduced control cells.

Adenoviral Vectors for TGF-ß1 and LacZ.
The adenoviral vector TGF-ß1/Ad5 carrying the gene for the TGF-ß1 protein has been described (25) . The control adenoviral vector LacZ/Ad5 (Vector Core; University of Pennsylvania, Philadelphia, PA) induces expression of the reporter gene ß-galactosidase from Escherichia coli. The vectors were prepared, purified, and titered to 1–5 x 1010 pfu/ml.

Melanoma cells were infected with 2 or 20 pfu/cell, and fibroblasts were infected with 40 pfu/cell in serum-free base medium for 3–4 h. Medium was then changed to growth medium, and cells were used the next day for experiments.

Human Skin Grafting.
Human foreskins from newborns were kept in sterile transport media (HBSS supplemented with antibiotics) and grafted within 48 h of excision as described (26) . Female and male CB-17 SCID mice were bred at the Animal Facility of the Wistar Institute and housed under pathogen-free conditions in groups of up to five animals/isolator cage. Grafts were well healed after 4–6 weeks, and mice were then used for the experiments. Mice received injections intradermally with the adenoviral vectors using a 26-gauge needle at a concentration of 0.5–5 x 108 pfu in a total volume of 100 µl of sterile PBS. Melanoma cells (2–5 x 106 cells) were injected intradermally with a 23-gauge needle in 100 µl of cell culture medium. The Wistar Institutional Animal Care and Use Committee approved all protocols.

Tumor Growth in Vivo.
For in vivo growth studies, 451Lu melanoma cells were mixed with normal human fibroblasts in 100 µl of medium and 100 µl of Matrigel matrix (Collaborative Biomedical Products, Bedford, MA) and injected s.c. into SCID mice with a 23-gauge needle. One day before injection, melanoma cells were transduced with TGF-ß1 or LacZ using adenoviral vectors, respectively, at an infection dose of 20 pfu/cell. The total injected cell number per mouse was 2.2 x 106 to 4 x 106 cells after mixing melanoma cells with fibroblasts at a ratio of 1:10 (2 x 105 melanoma cells and 2 x 106 fibroblasts), 1:1 (2 x 106 and 2 x 106 cells), and 10:1 (2 x 106 and 2 x 105 cells). Six mice/group received injections and were sacrificed after 2 weeks, and 10 additional mice that received injections of TGF-ß1- or LacZ-transduced melanoma cells mixed with fibroblasts at a ratio of 1:1, respectively, were sacrificed after 5.5 weeks. Tumor growth was monitored twice weekly.

Western Blot.
451Lu tumors were harvested from the mouse and minced with RIPA buffer (TGF-ß1) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1.8 mg/ml iodoacetamide). After centrifugation at 4°C for 20 min at 12000 x g, protein in the supernatants was quantified using the BCA kit (Pierce, Rockford, IL). Equal amounts of total protein from each sample were resolved in a 15% SDS-polyacrylamide gel, electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Richmond, CA), and blocked with a 5% solution of dry milk and 0.05% Tween 20 in PBS at room temperature for 1 h. The membrane was incubated with rabbit polyclonal antihuman-TGF-ß1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by peroxidase-labeled secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Immunoreactive bands were developed using the ECL detection system (Amersham, Arlington Heights, IL) and exposed to Kodak Biomax film (Eastman Kodak, Rochester, NY).

Histology and Immunohistochemistry.
At the end of each experiment, mice were sacrificed by CO2 inhalation, and skin grafts or s.c. tumors were excised. Half of the samples were fixed in 10% neutral-buffered formalin (Fisher Scientific, Pittsburgh, PA) for 6–12 h at room temperature and embedded in paraffin. The other half was dehydrated by increasing concentrations of sucrose solutions (5, 10, and 20%) at 4°C overnight, embedded in OCT medium (Miles, Elkhart, IN), snap-frozen, and stored at -70°C until cryosectioning at 6–8 µm. Formalin-fixed sections were stained with H&E for histopathological evaluation. Masson’s trichrome stain was used for estimation of the amount and distribution of collagen in the tissues. The DNA-binding fluorochrome Hoechst 33258 (Sigma Chemical Co.) was used to distinguish human from murine cells.

Immunohistochemistry was performed on serial sections using an avidin-biotin-peroxidase system kit (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) or 3-amino-9-ethylcarbazole (Vector) as chromogens. Antigens in the formalin-fixed tissues were retrieved by trypsin digestion at 37°C or microwave heat treatment in citrate buffer. Cryostat sections of 6–8 µm were air-dried and fixed in ice-cold acetone for 10 min. Prior to incubation with the primary antibodies in a humidified chamber at 4°C overnight or at room temperature for 1–2 h, nonspecific binding was blocked with 10% normal horse or 10% normal goat serum. Primary mouse monoclonal antibodies against the following human antigens were used: Ki-67 (Immunotech, Westbrook, ME); HMB45 (Biogenex, San Ramon, CA); type IV collagen (hybridoma from ATCC, Manassas, VA); fibronectin (American Type Culture Collection); tenascin (27) ; {alpha}2 integrin (Chemicon, Temecula, CA); ß3 integrin (28) ; smooth muscle actin (Zymed, South San Francisco, CA); aminopeptidase N (29) ; and CD31 (PECAM; Dako Carpinteria, CA). Primary rabbit polyclonal antibodies used in this study were rabbit anticow S100 (Dako) and rabbit antihuman TGF-ß1 (Santa Cruz Biotechnology). Mouse IgG1 isotype antibody (P3) was used as negative control for each staining with mouse monoclonal antibodies and a rabbit antihuman involucrin antibody (Biomedical Technologies, Stoughton, MA) as negative control for each staining with rabbit polyclonal antibodies. Between each incubation step, slides were rinsed twice in PBS for 3–5 min. Endogenous peroxidase was quenched with 3% H2O2 in methanol for 20–30 min at room temperature. A biotin-labeled antimouse secondary antibody was applied for 30 min at room temperature, followed by incubation with a preformed avidin-biotinylated enzyme complex for 30 min. After color development by addition of the chromogen and counterstaining with Mayer’s hematoxylin (Sigma Chemical Co.), sections were mounted and evaluated under a light microscope.

TUNEL.
Detection of apoptosis was done on formalin-fixed, paraffin-embedded tumor sections with a commercially available TUNEL kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s directions with modifications. Briefly, after deparaffinization, rehydration, and quenching of endogenous peroxidase, sections were treated with 0.1% Triton X-100 (Sigma Chemical Co.) and 0.1% citrate buffer for cell permeabilization. After incubation with terminal deoxynucleotidyl transferase and fluorescein-labeled nucleotides for 60 min at 37°C and incubation with peroxidase-conjugated anti-fluorescein antibody Fab fragments for 40 min at 37°C, 3-amino-9-ethylcarbazole substrate and H2O2 were added for color development. Counterstain was done with Mayer’s hematoxylin. Positive labeled cells were scored in a blinded manner in randomly chosen fields at x200.

Skin Reconstruction.
Skin reconstructs were prepared essentially as described with modifications (30) . Human fibroblasts (FF2441) were added to neutralized bovine type I collagen (Organogenesis, Canton, MA) to a final concentration of 0.8–1 mg/ml of collagen in MEM (BioWhittaker, Walkersville, MA), 1.66 mM L-glutamine (Life Technologies, Inc.), 10% FBS, and 0.21% sodium bicarbonate (BioWhittaker). Three ml of fibroblast-containing collagen (2.5 x 104 cells/ml) were added to each insert of a six-well tissue-culture tray (Organogenesis) after precoating with 1 ml of acellular collagen. Mixtures were allowed to constrict in DMEM with 10% FBS for 5–7 days. The day before seeding, melanoma cells were infected with TGF-ß1/Ad5, and controls were infected with LacZ/Ad5 at 20 pfu/cell for 4 h in protein-free SFM and then incubated overnight in complete SFM. Keratinocytes were mixed with melanoma cells at a ratio of 5:1 to 10:1 in low-calcium epidermal growth medium containing DMEM, F-12 Ham’s (Life Technologies, Inc.), 1% newborn calf serum (Hyclone), 4 mM glutamine, 1.48 x 10-6 M hydrocortisone, 4 pM progesterone, 20 pM triiodothyronine, 0.1 mM O-phosphorylethanolamine, 0.18 mM adenine (Sigma Chemical Co.), 5 mg/ml insulin, 5 mg/ml transferrin, 5 mM ethanolamine, 5 g/ml selenium (BioWhittaker), and 50 µg/ml gentamicin (Mediatech, Herndon, VA). A total of 5–6 x 105 cells was seeded on each contracted collagen gel. Cultures were maintained submerged in low calcium growth medium for 2 days and in normal calcium (1.88 mM) growth medium for another 2 days and then raised to the air-liquid interface for 10–12 days with feeding from below with normal calcium and high-serum (20%) medium.

RNA Preparation and Labeling.
Total RNA was isolated with Trizol Reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. After DNase I treatment (Roche Diagnostics, Mannheim, Germany) for 15 min at 37°C and ethanol precipitation, the RNA quality and quantity was visualized on a 1% agarose gel with ethidium bromide. One to 2 µg of total RNA were reverse transcribed with 300 units of Superscript II reverse transcriptase (Life Technologies, Inc.) in the presence of 2 µg of oligo(dT)15 primer (Promega Corp., Madison, WI), 1 µl of 10 x decamers (Ambion, Austin, TX), 1 mM dATP, dGTP, and dTTP (Amersham Pharmacia, Piscataway, NJ), respectively, 0.1 mCi of [33P]dCTP (ICN Biomedicals, Costa Mesa, CA), and 3.5 mM DTT at 39°C for 90 min. The labeled cDNA targets were separated from the unincorporated [33P]dCTP through Sephadex G-50 Quick Spin columns (Roche Diagnostics, Indianapolis, IN) and denatured at 100°C before hybridization to the filter arrays. [33P]CTP incorporation was quantitated by scintillation counting.

cDNA Expression Array.
Human arrays from the Genomics Core of the Wistar Institute were used. Each array consisted of a nylon membrane (2.5 x 7.5 cm) spotted with 200–600-bp cDNA fragments from sequence-validated human clones (Research Genetics, Huntsville, AL) representing 2280 different genes, 9 housekeeping genes, and negative controls. Arrays were produced with a GM417 array station (Genetic MicroSystems, Bedford, MA) using 300-µm pins with 750-µm center-to-center spacing.

The labeled cDNA targets were hybridized to the arrays in Church buffer with boiled and chilled Cot-1 DNA and sheared salmon sperm DNA in plastic hybridization bags at 65°C for 18 h. The membranes were washed at 65°C in 2x SSC/1% lauryl sulfate sodium salt (SDS) for 30 min 3 times in 0.1x SSC/0.5% SDS for 30 min once and then exposed to a Phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 24 h to 5 days. Duplicate hybridizations were performed with RNA from two independent experiments. Sequential filters from the same printing were used for the analyses. Phosphorscreens were scanned with a Storm Phosphorimager (Molecular Dynamics) at a resolution of 50 µm. Imagequant files from scans were imported into ArrayVision (Imaging Research, St. Catharine, Ontario, Canada) for quantification. Spot intensities were background subtracted, globally normalized, and reported as median pixel densities by the Wistar Genomics Core.

Statistics.
For ELISA, TUNEL assay, proliferation, and tumor growth experiments, the arithmetic mean and SDs were calculated. Statistical differences to LacZ-treated controls were validated by the two-sided Student’s t test. P <= 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive TGF-ß1 Production in Melanoma and Sensitivity to Induced TGF-ß1 Expression.
Constitutive TGF-ß1 production in 21 human melanoma cell lines from different progression stages ranged between 0 and 248 pg/105 cells with an average of 49 ± 70 pg/105 cells as measured by ELISA (Fig. 1A)Citation . There was a tendency of higher TGF-ß1 levels in late progression stages but not exclusively. More than 40 pg of TGF-ß1 per 105 cells were found in 3 of 7 (43%) metastatic melanoma lines, 2 of 9 (22%) primary VGP, and 1 of 5 (20%) primary RGP melanoma lines, whereas <5 pg/105 cells were found in 1 of 7 (14%) metastatic lines, 3 of 9 (33%) primary VGP, and 3 of 5 (60%) primary RGP melanoma lines. Metastatic lines WM373, WM1617, and 1205Lu secreted higher levels of TGF-ß1 than their primary melanoma counterparts WM75, WM278, and WM793 established from the same patient, respectively. The metastatic line WM239A, on the contrary, showed little or no TGF-ß1 production (2 ± 2 pg/105 cells), whereas the respective primary tumor line WM115 produced 36 ± 35 pg/105 cells. Normal human melanocytes produced negligible levels of TGF-ß1 (2 pg/105 cells), whereas neonatal foreskin fibroblasts produced 64 pg/105 cells.



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  		Fig. 1. Constitutive production of TGF-ß1 and proliferative response to transduction with TGF-ß1. A, TGF-ß1 production in 21 human melanoma cell lines, in foreskin melanocytes (FM), and in foreskin fibroblasts (FF) analyzed by ELISA of cell culture supernatants 72 h after seeding. Progression stage decreases from left to right. No., normal cells. Bars, SD. B, growth of the same melanoma cell lines as above after transduction with TGF-ß1. Given is the percentage relative to the [3H]thymidine uptake after transduction of each respective line with LacZ control. Bars, SD.

 
The sensitivity of 21 melanoma cell lines to TGF-ß1 was analyzed after transduction with an adenoviral vector expressing TGF-ß1 (Fig. 1B)Citation . Cell proliferation was measured by [3H]thymidine incorporation and compared with LacZ control vector-transduced cells. Resistance or a <30% inhibition upon TGF-ß1 transduction was found in 12 of 21 lines (57%). Inhibition defined as a >30% reduction compared with the controls was observed in 7 of 21 lines (33%), and stimulation was found in 2 of 21 lines (10%). Melanocytes were inhibited, and fibroblasts were resistant. In general, cell lines with high constitutive TGF-ß1 levels were resistant to TGF-ß1 transduction, and cell lines with low or no endogenous production of TGF-ß1 showed either stimulation, resistance, or inhibition. The strongest inhibition was observed for WM239A, which was found to be the lowest TGF-ß1 producer among the metastatic lines analyzed.

The dose-dependent protein production of TGF-ß1 after adenoviral transduction was analyzed in six selected melanoma cell lines by ELISA (Fig. 2A)Citation . An infection dose of 2 pfu/cell led to a 1.2–4-fold increase in TGF-ß1 protein production in five of six cell lines and a 153-fold increase in cell line WM239A. An infection dose of 20 pfu led to a 9–23-fold increase in TGF-ß1 protein production in four of six cell lines and an 115-fold and 333-fold increase in the cell lines 1205Lu and WM239A, respectively. This dose was therefore used in the following in vivo studies.



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  		Fig. 2. Induction of TGF-ß1 production by adenoviral gene transfer and morphological effect on melanoma. A, TGF-ß1 production in six different melanoma cell lines after transduction with TGF-ß1 at 2 and 20 pfu, LacZ control vector (20 pfu), or without transduction. Bars, SD. B, morphology of 1205Lu melanoma cells 3 days after transduction with LacZ control (A) or TGF-ß1 (B). Note the TGF-ß1-induced, elongated spindle shape of the cells.

 
The effect of TGF-ß1 on cell morphology after adenoviral transduction was tested in 24 melanoma cell lines. Three of the 24 cell lines, WM1552c, WM793, and 1205Lu, which had an epitheloid morphology, displayed a more spindle-shaped, fibroblastoid morphology starting 3 days after transduction (Fig. 2B)Citation . It should be noted that the VGP primary melanoma cell line WM793 and the metastatic melanoma cell line 1205Lu were derived from the same patient (31) .

Stroma Activation and Decreased Tumor Cell Death through TGF-ß1 in Melanoma.
To analyze melanoma-stroma interactions mediated by TGF-ß1, we used an in vivo model of close proximity of human melanoma cells with fibroblasts. Melanoma cell line 451Lu (32) was chosen, because the cells showed low constitutive levels of TGF-ß1, were resistant to TGF-ß1 transduction (see Fig. 1Citation ), produced 23-fold higher levels of TGF-ß1 than controls after transduction (see Fig. 2Citation ), and were highly tumorigenic in immunodeficient mice (33) . After transduction with TGF-ß1 in vitro, 451Lu melanoma cells were mixed with normal human skin fibroblasts in ratios of 1:10, 1:1, and 10:1 and injected together with Matrigel matrix s.c. into SCID mice. After 2 weeks, solid tumors were palpable in all groups with no significant difference in tumor volume from controls (data not shown). Increased protein expression of TGF-ß1 in tumors of TGF-ß1-transduced melanoma cells was confirmed by Western blot analysis (Fig. 3)Citation . Histologically, thick stroma septae were found throughout and around the TGF-ß1-expressing tumors (Fig. 4A)Citation , whereas there was only little stroma in the control tumors (Fig. 4B)Citation . Control tumors were characterized by a higher proportion of necrosis with blood extravasation (Fig. 4B)Citation , and TUNEL assay demonstrated a significantly higher number of apoptotic cells (Fig. 4D)Citation compared with the TGF-ß1-expressing tumors (Fig. 4C)Citation . Decreased cell death in melanoma by TGF-ß1 was also observed in organotypic cultures. Melanoma cell line WM793, which showed low constitutive levels of TGF-ß1 and growth resistance as well as spindle-shaped morphology after TGF-ß1 transduction, was incorporated into human skin reconstructs after TGF-ß1 and LacZ transduction, respectively (Fig. 4, E and F)Citation . In this organotypic culture model, fibroblasts embedded in type I collagen form the dermis, and keratinocytes seeded on top form a stratified epithelium, i.e., epidermis. After 2 weeks, melanoma cell clusters were found in the epidermis and upper dermis in both groups; however, much less cell death was observed in the tumors formed by TGF-ß1-transduced melanoma cells (Fig. 4E)Citation compared with the controls (Fig. 4F)Citation .



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  		Fig. 3. Detection of TGF-ß1 precursor forms by Western blot analysis of 2-week-old 451Lu melanomas after transduction of melanoma cells with TGF-ß1 or LacZ in vitro and injection s.c. into SCID mice together with human fibroblasts in a ratio 1:1. Equal amounts of protein were loaded.

 


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  		Fig. 4. Stroma activation and decreased tumor cell death through TGF-ß1 in melanoma. A–D, 451Lu melanoma 19 days after s.c. injection into SCID mice. Before injection, melanoma cells were transduced with TGF-ß1 (A and C) or LacZ (B and D) and mixed together with normal human fibroblasts in Matrigel at a ratio 1:1. A and B, increase in stroma around the TGF-ß1-transduced (A) versus LacZ-transduced (B) melanoma cells (H&E, x100). The control tumors (B) show more necrosis and blood extravasation. C and D, TUNEL assay for evaluation of apoptosis (red) shows only few positive cells in the TGF-ß1-transduced melanoma cells (C) but high positivity in the control tumors (D; x100). E and F, histological section of 16-day-old human skin reconstructs with normal human fibroblasts, keratinocytes, and WM793 primary melanoma cells (arrowheads) transduced with TGF-ß1 (E) or LacZ control (F). Note the higher degree of cell death in the control tumor (F; H&E, x200). G and H, WM3248 melanoma after transduction with TGF-ß1 (G) or LacZ control (H) and intradermal injection into human skin grafts. Shown is an H&E-stained histological section of a sample 3 weeks after injection (x200). Note the capsule-forming stroma reaction around the tumor WM3248 transduced with TGF-ß1 (G).

 
Paracrine stroma activation by TGF-ß1 expression in melanoma cells was also detected in an orthotopic melanoma model. Primary melanoma cell line WM3248, which was stimulated by TGF-ß transduction in vitro, was transduced with TGF-ß1 or LacZ in vitro and then injected intradermally into human foreskins grafted to SCID mice. After 2 weeks, solid dermal tumors were palpable in both the TGF-ß1 and LacZ groups without a significant difference in volume until sacrifice 3–4 weeks after injection. A capsule-like stromal matrix had formed around the TGF-ß1-expressing melanoma with increased numbers of fibroblasts (Fig. 4G)Citation when compared with the controls (Fig. 4H)Citation . The stromal cells produced high levels of collagen as demonstrated by Masson’s trichrome stain (not shown), and stroma capsules directly surrounding tumor cell clusters contained type IV collagen, which was not seen in the controls (not shown). Numerous vessels were detected in this stroma; however, there seemed to be no significant difference in vascularity compared with the LacZ controls.

Induction of ECM Proteins by TGF-ß1 in Melanoma.
The stroma activation by TGF-ß1 produced by melanoma cells was further characterized in 2-week-old tumors of TGF-ß1-transduced 451Lu melanoma cells, which had been mixed with fibroblasts 1:1 and s.c. injected into SCID mice. Masson’s trichrome stain revealed an increase in collagens (Fig. 5A)Citation when compared with LacZ controls (Fig. 5B)Citation . Immunohistochemical analyses demonstrated an induction of fibronectin (Fig. 5C)Citation and {alpha}2 integrin (Fig. 5E)Citation expression by TGF-ß1, whereas they were only weak or undetectable in the controls (Fig. 5, D and F)Citation . Tenascin expression was not stronger in intensity but more widely distributed throughout the TGF-ß1-expressing tumors (not shown). Detection of CD13 (aminopeptidase N) as a human fibroblast marker revealed high positivity in the interstitium of the TGF-ß1-expressing tumors (Fig. 5G)Citation in contrast with the controls (Fig. 5H)Citation , suggesting the close proximity of human fibroblasts to the melanoma cells.



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  		Fig. 5. Induction of ECM proteins by TGF-ß1 in melanoma. A–H, 451Lu melanoma 19 days after s.c. injection into SCID mice. Before injection, melanoma cells were transduced with TGF-ß1 (A, C, E, and G) or LacZ (B, D, F, and H) and mixed together with normal human fibroblasts in Matrigel at a ratio 1:1. A and B, Masson’s trichrome stain illustrates an increase in collagen (blue) induced by TGF-ß1-expressing melanoma cells (A) compared with the controls (B; x100). C and D, immunohistochemical detection of fibronectin (red) reveals an increase in the stroma septae around the TGF-ß1-transduced (C) compared with the LacZ-transduced (D) melanoma cells (x50). E and F, {alpha}2 integrin expression in the interstitium of TGF-ß1-transduced melanoma cells (E) is not seen in the LacZ-transduced controls (F). G and H, immunohistochemical detection of human fibroblasts by CD13 (aminopeptidase N; red) in the TGF-ß1-transduced (G) compared with the LacZ-transduced (H) control group (x100). I and J, human foreskin graft 10 days after intradermal injection of 5 x 108 pfu of TGF-ß1/Ad5 (I) or LacZ/Ad5 control vector (J) in 100 µl of sterile PBS. Masson’s trichrome stain reveals an increase in collagen in blue (I) compared with the control (J; x100).

 
The described remodeling of stroma was obviously mediated by paracrine effects of TGF-ß1 produced by melanoma cells. Therefore, we tested direct expression of TGF-ß1 in human skin by intradermal injection of the adenoviral vectors into human skin xenografts, which results in a highly efficient gene transduction of fibroblasts in the dermis.4 As early as 1 week, a strong thickening of the skin was visible and palpable, whereas there was no change in LacZ-injected skins. TGF-ß1 production in the dermis led to the formation of a strong collagen fiber network, as determined by H&E (not shown) and Masson’s trichrome stain (Fig. 5I)Citation , which was not seen in the controls (Fig. 5J)Citation .

The TGF-ß1/Ad5-treated animals suffered from systemic effects in the second week after injection. They became weak and apathic and eventually died. Autopsy showed normal lung and liver tissue but myeloid metaplasia in the spleen and acute tubular necrosis in the kidney. Lethal effects of the TGF-ß1/Ad5 treatment were also observed with 10-fold lower injection doses. The systemic serum levels of circulating TGF-ß1 were at ng/ml levels, as analyzed by ELISA.

Increased Metastasis Development by TGF-ß1-transduced Melanoma Cells.
For metastasis studies, SCID mice were injected s.c. with TGF-ß1- or LacZ-transduced 451Lu melanoma cells mixed with normal human skin fibroblasts in Matrigel matrix in a ratio of 1:1 and observed for 39–41 days. Tumor volume was not significantly different between groups during the first 32 days. At day 39, TGF-ß1-transduced tumors were 1.7-fold larger than LacZ controls (1.46 ± 0.95 cm3) versus 0.8 ± 0.55 cm3). S100-positive micrometastases in the lungs were found in 8 of 10 mice with TGF-ß1-transduced melanomas (Fig. 6A)Citation and in 7 of 10 mice with LacZ-transduced control melanomas (Fig. 6B)Citation . Metastases in 10 randomly chosen fields at x100 were counted in each lung. The average number of micrometastases/microscopic lung field was significantly (P < 0.03) higher in the TGF-ß1 group (4.1 ± 4.7) compared with the controls (0.4 ± 0.7), and the average size of each metastasis was bigger as well. The average diameter of each metastasis was in the TGF-ß1 group (85 ± 44 µm), 1.8-fold larger than in the LacZ group (47 ± 29 µm; Table 1Citation ). The metastases were found to express TGF-ß1 (Fig. 6C)Citation , whereas the controls showed no or only weak staining for TGF-ß1 (Fig. 6D)Citation . Smooth muscle actin, which was detected in the vessel walls of the murine lungs, was commonly expressed around the micrometastases in the TGF-ß1 group (Fig. 6E)Citation but only rarely in the control group (Fig. 6F)Citation .



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  		Fig. 6. Increased metastasis formation by TGF-ß1-transduced melanoma cells. A and B, S100-positive micrometastases in lungs from SCID mice 39 days after s.c. injection of TGF-ß1-transduced (A) or LacZ-transduced (B) 451Lu melanoma cells mixed in a ratio 1:1 with normal human fibroblasts in Matrigel matrix. Number and size of metastases were significantly increased in the TGF-ß group. Metastases stained positive for TGF-ß1 (C), whereas controls showed no or only weak stain for TGF-ß1 (D; x100). E and F, smooth muscle actin expression (red) in the lung in vessel walls (arrowheads) and around melanoma metastases (arrows) after TGF-ß1 (E) and LacZ (F) transduction of s.c.-injected 451Lu melanoma cells (x200). G and H, smooth muscle actin expression (immunohistochemical detection in brown) is induced by injection of adenoviral vectors for TGF-ß1 (G) in human skin grafts but not by injection of LacZ control vectors (H; x50).

 

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  		Table 1 Lung metastases of TGF-ß1-transduced melanoma in SCID mice

 
Induction of smooth muscle actin expression was also observed in fibroblasts in human skin grafts when injected with adenoviral vectors for TGF-ß1 (Fig. 6G)Citation . This indicated that TGF-ß1 can induce transdifferentiation of fibroblasts in the dermis into myofibroblasts, suggesting that they are also induced by TGF-ß1-derived from melanoma cells.

Gene Expression Profiling of TGF-ß1-transduced Fibroblasts and Melanoma Cells.
The mRNA expression of 2280 different genes in melanoma cells and fibroblasts after transduction with TGF-ß1 was analyzed by cDNA microarray. Melanoma lines 1205Lu, 451Lu, and WM793 were tested 3 days after adenoviral infection with 20 pfu of TGF-ß1/Ad5 or LacZ/Ad5 per cell. At this harvesting time point, 1205Lu and WM793 displayed in 40–50% of all cells a TGF-ß1-induced fibroblastoid phenotype, which was not seen in the LacZ-transduced or nontransduced controls. Normal human foreskin fibroblasts were analyzed after adenoviral infection with 40 pfu of TGF-ß1/Ad5 or LacZ/Ad5 per cell and organotypic culture in type I collagen for 4 days. Table 2Citation summarizes the expression results of selected genes encoding for ECM proteins, adhesion receptors, growth factors, their binding proteins and receptors, as well as protease inhibitors.5


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  		Table 2 Modulation of gene expression by TGF-ß1 in fibroblasts and melanoma cells

 
Among the different collagen subtypes, expression of the {alpha}1 chain of type XVIII and type XV was increased up to 12-fold in both melanoma cells and fibroblasts. Tenascin and osteonectin expression increased 5- and 6-fold, respectively, in TGF-ß1-transduced fibroblasts, whereas it was unchanged or 2-fold reduced in melanoma cells. Integrins ß1, ß5, {alpha}V, {alpha}3, and {alpha}6 showed a 2–6-fold reduction in TGF-ß1-transduced fibroblasts. In at least 2 melanoma cell lines, a 2–4-fold increase in {alpha}v and {alpha}3 integrin after TGF-ß1 transduction was detected, whereas other subtypes were not changed in more than one of the three analyzed lines. VEGF-A increased after TGF-ß1 transduction up to 35-fold in all analyzed cell lines and was therefore the growth factor that exhibited the strongest induction. The induction of VEGF was confirmed at the protein level by ELISA (Fig. 7)Citation . Although WM793 melanoma cells and dermal fibroblasts were strongly induced to secrete VEGF by TGF-ß1 transduction, VEGF levels in 451Lu melanoma cells were already relatively high in the controls (1.53 ng/ml/106 cells) and stayed at similar levels after TGF-ß1 transduction.



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  		Fig. 7. Production of VEGF protein by human melanoma cells (WM793 and 451Lu) and dermal fibroblasts (FF2441) 72 h after transduction with TGF-ß1 ({blacksquare}) or LacZ control () via adenoviral vectors. Results are expressed in average ng/ml per 106 cells; bars, SD.

 
PDGF receptor-ß expression was induced by TGF-ß1 transduction 5- and 21-fold in two of three analyzed melanoma lines, and CFR-1 was increased up to 6-fold in TGF-ß1-transduced melanoma cells and 4-fold in fibroblasts. PA1-I was, with 14- to 43-fold increase in expression in all analyzed TGF-ß1-transduced cell lines, the highest induced protease inhibitor.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Interactions of tumor cells with their microenvironment and the influence of stroma on tumor and vice versa have been increasingly recognized to be essential for tumor survival and progression and have been primarily studied in carcinomas of the breast (33) , pancreas (34) , prostate (35) , skin (36) , and cervix (37) . In melanoma, a wide variety of different cytokines and growth factors are expressed (38) , which often act in an autocrine way, but also may influence the tumor environment via paracrine loop (39) . Most studies have hereby focused on the induction of angiogenesis by VEGF, basic fibroblast growth factor, PDGFs, and IL-8 (40) . In this study, it is shown that stroma can be remodeled by the paracrine effects of TGF-ß1 produced by melanoma cells, which results in an increased deposition of ECM proteins in the interstitium of the tumor. The previously described induction of collagen, fibronectin, tenascin, and {alpha}2 integrin by TGF-ß (41, 42, 43) could be demonstrated in the human fibroblast-containing stroma surrounding TGF-ß-producing melanoma cells in an in vivo model. Microarray studies of TGF-ß1-transduced fibroblasts in organotypic culture, in which tenascin and types VI, XV, and XVIII collagen were up to 12-fold increased, partly mirrored the in vivo data. However, other collagen subtypes, {alpha}2 integrin, or fibronectin precursor were not increased, which might be because of limitations of the in vitro culture system, degradation of RNA, missed transient time points of induction, or the fact that fibroblasts were transduced with TGF-ß and not stimulated by exogenous TGF-ß.

Concomitant with the stroma reaction, the absence of larger necrotic areas and the fewer number of apoptotic cells in the TGF-ß1-transduced tumors were the most striking differences to the controls. The fact that this did not result in a greater volume of the ECM-rich melanomas in the first 4 weeks was most likely attributable to increased edema and blood content in the more necrotic control tumors, which, however, at later time points were found to be smaller than the TGF-ß1-transduced tumors, possibly as a consequence of resorption of the edema and necrotic cells. Decreased death attributable to TGF-ß1 transduction was also seen in human melanoma skin reconstructs. TGF-ß obviously conferred a selective survival advantage to the melanoma cells, which culminated in the significant increase in number and size of lung metastases in mice injected with TGF-ß1-transduced 451Lu melanoma cells. No growth induction by TGF-ß1 was seen in 451Lu melanoma cells in vitro, indicating that direct autocrine effects of TGF-ß1 were not responsible for this phenomenon, but that neighboring stroma cells in vivo were a prerequisite for these beneficial effects of TGF-ß1 on the tumor. The stroma formation might have provided the melanoma cells a scaffolding, to which they can adhere and along which they can migrate. The stimulation of fibroblasts might have induced in turn other growth factors, which positively regulate survival and growth of melanoma. Microarray and ELISA analyses demonstrated VEGF to be strongly induced by TGF-ß1 in both fibroblasts and melanoma cells, confirming previous in vitro data (44) . However, angiogenesis was not significantly increased in tumors from TGF-ß1-transduced 451Lu melanoma cells. This may have been attributable to the already high constitutive production of VEGF protein in 451Lu cells and the already high vascularization of 451Lu control tumors. On the other hand, TGF-ß1-induced VEGF in fibroblasts might have contributed to the survival advantage of the TGF-ß1-transduced tumors by acting as an antiapoptotic factor both for the fibroblasts themselves and for the melanoma cells via reciprocal paracrine routes.

Microarray analyses further revealed an induction of PDGF receptor-ß by TGF-ß1 in melanoma cells, which normally do not express this receptor (45) . Also an increase in CFR-1 was detected, indicating that melanoma cells might have become increasingly responsive to growth factors produced by stromal cells through up-regulation of growth factor receptors. Increased survival and motility were additionally observed by an increase in systemic metastases. This might have been either attributable to an altered expression of adhesion molecules, proteases, or plasminogen activators on the melanoma cells themselves (13 , 16 , 21 , 22) or to the altered microenvironment enhancing the chance of surviving melanoma cells to migrate along the stromal septae and enter the lymphatic or blood system.

Although TGF-ß1 had stimulatory effects in melanoma in vivo, it does not seem to be essential for tumor growth and progression in general. This is reflected in the heterogeneous pattern of endogenous TGF-ß1 production in melanoma, which included advanced stages of melanoma that showed no or low levels of TGF-ß1. It cannot be excluded that the ratio between the active and the latent form of TGF-ß1 differed among the tested cell lines, because only the total levels of TGF-ß1 were measured in the ELISA used. However, in selected cases, constitutive TGF-ß1 production was associated with progression with highest concentrations found in single metastatic and advanced primary cell lines (WM373, WM1617, and WM902B) and higher concentrations in advanced cell lines when compared with their primary counterparts derived from the same patient (WM373-WM75, WM278-WM1617, and 1205Lu-WM793). The levels of TGF-ß1 production in transduced melanoma cells were within the range of constitutive TGF-ß1 levels in selected "high producer" melanoma cell lines.

Induced expression of TGF-ß1 in melanoma cells had different effects on their phenotype and proliferation capability. Many melanoma cell lines were resistant to the growth-inhibitory effects of TGF-ß1, which was in line with previously reported effects of exogenous TGF-ß on melanoma cells (8 , 14) . For a more detailed analysis of resistance of melanoma cells to TGF-ß1, both the active and the latent form of TGF-ß1 need to be determined separately before and after TGF-ß1 transduction of each cell line and different infection doses need to be tested to be able to determine the threshold concentration of sensitivity, which was, however, not the aim of this study. Resistance to TGF-ß has been linked in several tumors to mutations in genes involved in the TGF-ß signaling pathway, such as TGF-ß receptors I and II, Smad2, or Smad4 (6) ; however, none of these mutations could be demonstrated in melanoma (15) .

Three of 24 cell lines displayed a morphological transdifferentiation toward a spindle cell-like fibroblastoid phenotype. This has been reported previously after addition of exogenous TGF-ß and was associated with increased metastatic capacity (16) . Metastatic capacity of the transdifferentiated cell lines was not tested in this study; however, in skin reconstructions, increased tumor cell survival was found. Transdifferentiation was also detected in fibroblasts, when TGF-ß1 was overexpressed in human skins. The widespread detection of smooth muscle actin in the dermis indicated a differentiation of the skin fibroblasts into myofibroblasts, which has been described only in vitro (46) and not yet in human skin in vivo. The myofibroblast phenotype has been discussed to be the main source of increased ECM deposition in fibrosis of the kidney, liver, and lung (47) , and the data of this study suggest that the same holds true for fibrosis of the skin. A strong collagen fiber network was induced by TGF-ß1, which was stronger than for any other growth factor studied.6

In summary, we have shown that TGF-ß1 expression in human melanoma cells can lead to stimulation of the neighboring stroma cells with increased production and deposition of ECM proteins. The activation of stroma in turn leads to a survival advantage and increased metastasis formation of the melanoma cells. A TGF-ß1-triggered complex interplay between matrix proteins, proteases, integrins, and growth factors is suggested by global gene expression studies and excludes attempts to limit the characterization of melanoma-stroma interactions to just single genes or a single gene group.


   ACKNOWLEDGMENTS
 
We thank Sylvia Major, Katerina Chruma, Adrien Jarvis, and Dr. Ling Li for technical assistance with the in vitro cell and immunohistochemistry studies; Dr. Jonathan Garlick for invaluable support for the skin reconstruction model; Emma DeJesus and Rena Finko for technical assistance with cell cultures and skin reconstruction; Elsa Aglow for excellent histological processing of the samples; Dr. Dirk Ruiter for helpful discussions about the histopathological sections; and Wen Hwai Horng for biostatistical analysis of the microarray data.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA80999, CA25874, and CA10815 (to M. H.) and a postdoctoral research fellowship BE2189/1-1 from the Deutsche Forschungsgemeinschaft (to C. B.). Back

2 To whom requests for reprints should be addressed, at The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: (215) 898-3950; Fax: (215) 898-0980; E-mail: herlynm{at}wistar.upenn.edu Back

3 The abbreviations used are: TGF, transforming growth factor; ECM, extracellular matrix; PAI, plasminogen activator inhibitor; TIMP, tissue inhibitor of metalloproteinase; VEGF, vascular endothelial growth factor; CFR, cysteine-rich fibroblast growth factor receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; SFM, serum-free medium; FBS, fetal bovine serum; SCID, severe combined immunodeficient; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling; Ad5, adenovirus serotype 5; pfu, plaque-forming unit; VGP, vertical growth phase; RGP, radial growth phase. Back

4 C. J. Gruss, K. Satyamoorthy, C. Berking, J. Lininger, M. Nesbit, H. Schaider, Z-J. Liu, M. Oka, M-Y. Hsu, T. Shirakawa, G. Li, P. Carmeliet, W. El-Deiry, S. L. Eck, J. S. Rao, A. H. Baker, J. Bennett, T. Crombleholme, J. Karmacharya, D. J. Margolis, J. M. Wilson, S. Werner, M. Detmar, M. Skobe, P. D. Robbins, C. Johnson, D. Carbone, C. Buck, and M. Herlyn. Re-modeling of the human skin architecture in vivo by adenovirus-mediated gene transfer of growth factors, adhesion molecules, proteolytic enzymes, oncogenes and tumor suppressor genes, submitted for publication. Back

5 The detailed results of all 2280 genes can be found on our Web site, http://www.wistar.upenn.edu/herlyn. Back

6 Unpublished data. Back

Received 6/ 8/01. Accepted 9/19/01.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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