Affiliations of authors: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (HJC, HKK); Department of Oral Histology, College of Dentistry, Chosun University, Gwanju, Korea (MJJ).
Correspondence to: Hynda K. Kleinman, PhD, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892 (e-mail: hkleinman{at}dir.nidcr.nih.gov)
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ABSTRACT |
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INTRODUCTION |
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Recently, the angiogenic effects of several members of the thymosin family of peptides were studied in the chick chorioallantoic membrane model (6). Thymosin 4, prothymosin, and thymosin
1 were associated with enhancement of angiogenesis, whereas parathymosin, thymosin
9, and thymosin
10 were associated with inhibition of angiogenesis. However, the mechanisms responsible for the regulation of angiogenesis by thymosin
4 have not been defined. Thymosin
4 also promotes wound healing and decreases inflammation in corneas damaged by exposure to heptanol (7) or alkali (8). In the latter case, alkali-exposed mouse corneas topically treated with thymosin
4 demonstrated accelerated re-epithelialization and decreased inflammation when compared with alkali-exposed corneas not treated with thymosin
4 (8).
Thymosin 4 also appears to be involved in tumor cell metastasis. Expression of thymosin
4, thymosin
10, and thymosin
15 are associated with the metastatic potential of tumor cells (9-13). In addition, thymosin
4 expression is elevated in metastatic melanoma compared with non-metastatic melanoma counterparts (10), and fibrosarcoma cells possessing greater metastatic potential express high levels of thymosin
4, whereas fibrosarcoma cells of low metastatic potential express little or no thymosin
4 (14). However, little is known about the mechanisms by which thymosin
4 promotes metastasis. We used an adenovirus-based expression vector to examine the effects of overexpressing thymosin
4 on various malignant activities of mouse melanoma B16-F10 cells in vitro and in vivo.
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MATERIALS AND METHODS |
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Mouse melanoma B16-F1, B16-F10, and B16-BL6 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). Primary cultured B16-F10 cells derived from a lung tumor were also obtained from ATCC. All cells were grown in Dulbeccos modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). For some experiments, B16-F10 cells were cultured for 18 hours in serum-free DMEM containing a synthetic thymosin 4 peptide (1 ng to 10 µg; RegeneRx Biopharmaceuticals, Bethesda, MD).
Recombinant Adenoviral Constructs and Infection
We used a high-fidelity polymerase chain reaction (PCR) system (Roche, Indianapolis, IN) to amplify a thymosin 4 cDNA from total mouse embryo RNA (Stratagene, La Jolla, CA). Amplification conditions for thymosin
4 were 0.5 min at 95 °C, 0.5 min at 60 °C, and 0.5 min at 72 °C for 30 cycles. These cycles were followed by a 10-minute elongation step at 72 °C. The forward and reverse primers used for this amplification contained HindIII and EcoRI restriction sites (underlined), respectively (forward primer, 5'-GTCAGTAAGCTTCTCCTTCCAGCAACCATGTC-3'; reverse primer, 5'-GTCAGTGAATTCAATGTACAGTGCATATTGGC-3'). The amplified product (0.2 kilobase [kb]) was digested with HindIII and EcoRI and cloned into the HindIII and EcoRI sites of the adenoviral plasmid shuttle vector pAC-EF1, where its expression was under control of the elongation factor 1 promoter (15). The sequence of the cloned mouse thymosin
4 cDNA was confirmed by DNA sequence analysis (National Institute of Dental and Craniofacial Research [NIDCR] Core Sequence Facility, Bethesda, MD). The resulting adenoviral vector was transfected into human epithelial kidney A293 cells for adenovirus production, as previously described (15). The high-expressing virus was expanded by infecting A293 cells. Large batches of recombinant adenovirus were purified by centrifugation through two consecutive cesium chloride gradients (16). Adenovirus containing an empty shuttle vector was used as a control. For adenovirus infection, 106 B16-F10 cells were incubated in serum-free DMEM for 2 hours with different amounts of virus (2002500 plaque-forming units [pfu]), after which the cells were washed and cultured in DMEM with 10% FBS.
Subcutaneous Tumor Growth and Experimental Metastasis Assays
We injected the dorsal neck region of C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) subcutaneously with B16-F10 cells infected with either control adenovirus or adenovirus expressing mouse thymosin 4 (2 x 105 cells/0.2 mL; 20 mice per group) and used calipers to measure the size of the resulting tumors several times per week for 20 days (17).
We also examined the metastatic potential of the adenovirus-infected cells by using a lung colonization assay, as described previously (17). In brief, we injected B16-F10 cells infected with either control adenovirus or adenovirus expressing mouse thymosin 4 (1 x 105 cells/0.2 mL) into the tail veins of C57BL/6 mice (20 mice per group). Two weeks later, the mice were killed by asphyxiation with CO2 and their lungs were removed. The metastatic nodules on the surface of their lungs were counted. All animal work was performed under protocols approved by the NIDCR Animal Care and Use Committee.
Northern Blot Analysis
We used an RNA mini-prep kit (Stratagene) to purify total RNA from B16 cells. Ten micrograms of each RNA sample was separated by formaldehydeagarose gel electrophoresis, transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH), and hybridized to a [32P] dCTP-labeled thymosin 4 cDNA probe, as previously described (18).
Western Blot Analysis and Immunostaining
Western blot analysis was conducted as described (14). Briefly, 100 µg of cell lysate or conditioned media was separated by electrophoresis on a Novex 4-20% Trisglycine gel (Invitrogen). Conditioned media were prepared from 106 cells cultured in DMEM without FBS for 18 hours and were concentrated with the use of Microcon YM-3 filters (Millipore, Bedford, MA). The protein concentrations of the lysates and conditioned media were determined by the bicinchoninic acid protein assay system (Pierce, Rockford, IL), and equal amounts of each sample were separated by electrophoresis on Novex 4-20% Trisglycine gel. Equal protein loading was confirmed by Coomassie blue staining of duplicate gels after electrophoresis. The gels were incubated for 1 hour in phosphate-buffered saline (PBS) containing 10% glutaraldehyde (Sigma-Aldrich, St. Louis, MO), washed three times for 20 minutes in PBS, and further incubated in a blotting buffer containing 1x Novex Trisglycine transfer buffer (Invitrogen) and 20% methanol for 30 minutes at room temperature. Proteins were transferred to a polyvinylidene difluoride membrane (Invitrogen) by electrotransfer. The membrane was pre-incubated for 2 hours in PBS containing 5% skim milk and 0.05% Tween 20 (PBS-T). The membrane was incubated for 1 hour at room temperature in PBS-T plus antibodies (rabbit polyclonal thymosin 4, 1 : 1000 dilution; ALPCO Diagnostics, Windham, NH), goat polyclonal vascular endothelial growth factor (VEGF; 1 : 1000 dilution; R&D Systems, Minneapolis, MN), goat polyclonal fibroblast growth factor-1 (FGF-1; 1 : 100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal fibroblast growth factor (FGF-2; 1 : 1000 dilution; Upstate Biotechnology, Lake Placid, NY), mouse monoclonal insulin-like growth factor (IGF; 1 : 1000 dilution; R&D Systems), goat polyclonal platelet-derived growth factor (PDGF; 1 : 1000 dilution; Upstate Biotechnology), or rabbit polyclonal epidermal growth factor (EGF; 1 : 100 dilution; Upstate Biotechnology). The membranes were washed five times with PBS-T and then incubated with a species-appropriate horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. The membranes were washed five times with PBS-T, and bound antibody was detected with an enhanced chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK). Recombinant VEGF and FGF-1, used as positive controls in western blot, were purchased from R&D Systems and Santa Cruz Biotechnology, respectively.
For immunohistochemistry, mouse lungs containing tumors derived from B16-F10 cells were fixed with 4% formaldehyde (Sigma-Aldrich) and paraffinized. The sections were deparaffinized and immunostained with a rabbit polyclonal antibody to thymosin 4 (1 : 100 dilution; a gift from Allan Goldstein, George Washington University, Washington, DC); antibody binding was detected with the use of an EnVision+ peroxidase system (DAKO, Carpinteria, CA).
For immunocytochemistry, B16-F10 cells infected with either thymosin 4expressing or control adenovirus were grown on cover slips and visualized by confocal microscopy, as previously described (19). Briefly, B16-F10 cells on glass cover slips were rinsed three times with PBS, fixed for 10 minutes at room temperature by incubation in 4% paraformaldehyde, and then permeabilized by incubation in PBS containing 0.1% Triton X-100 for 5 minutes. The cells were incubated with 1% bovine serum albumin in PBS for 1 hour to block nonspecific antibody binding and then for 1 hour at room temperature with an anti-thymosin
4 antibody (ALPCO Diagnostics) diluted 1 : 100 in PBS. The cover slips were washed three times with PBS and incubated with affinity-isolated tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit immunoglobulin G (Molecular Probes, Eugene, OR). For localization of F-actin, the fixed cells were washed three times with PBS for 15 minutes and incubated with fluorescein isothiocyanatelabeled phalloidin (Sigma-Aldrich). All labeled cells were rinsed three times with PBS and once with distilled water and were then mounted on glass slides with the use of a fluorescent mounting medium (DAKO). The cells were observed for epifluorescence with the use of a confocal laser-scanning microscope.
Cell Proliferation Assay
A methylthiazoletetrazolium (MTT)-based cell growth determination kit (Sigma-Aldrich) was used to measure the proliferation of the adenovirus-infected B16-F10 cells. Proliferation was measured every 24 hours for 3 days. The experiments were repeated three times, and samples were tested in triplicate. The mean values and 95% confidence intervals (CIs) were determined.
Gelatin-Based Zymography
Conditioned media were prepared from 106 adenovirus-infected B16-F10 cells cultured with DMEM without FBS for 18 hours and concentrated by Microcon YM-3 filters. Concentrated conditioned media were separated by Novex Zymogram (gelatin) gels (Invitrogen). After electrophoresis, the gels were rinsed with Novex Zymogram Renaturing Buffer (Invitrogen) for 30 minutes, and gels were equilibrated with Novex Zymogram Developing Buffer (Invitrogen) for 30 minutes at room temperature. The gels were then rinsed with fresh Novex Zymogram Developing Buffer and incubated at 37 °C for 18 hours. Gelatinase-like matrix metalloproteinases MMP-2 or MMP-9 were identified following staining of the gel in 0.25% Coomassie blue R250 and destaining with 7% acetic acid. The digested area appeared clear on a blue background, indicating the expression and activity of gelatinases.
In Vitro Migration and Invasion Assays
In vitro migration and invasion assays were performed as previously described (17,20). Briefly, we used HTS FluoroBlok 24-well chambers containing membranes with an 8-µm pore size (Fisher Scientific, Pittsburgh, PA) for both assays. For the migration assay, we placed conditioned medium obtained by culturing mouse NIH-3T3 cells for 18 hours in serum-free DMEM into the lower chambers of each well. We resuspended 105 adenovirus-infected B16-F10 cells in 100 µL of serum-free DMEM and placed them in the upper chambers of each well. The fluoro blocks were incubated for 16 hours at 37 °C, and the cells in the lower chambers were stained with 5 µg/mL calcein AM (Molecular Probes) for 30 minutes. The fluorescence of the cells in the lower part of the chamber was determined with a computer-based fluorescence reader (Wallac Victor 2; PerkinElmer, Wellesley, MA). The invasion assay was performed in a similar fashion, except that the upper surface of the fluoro block filter was coated with 20 µL of 0.5 mg/mL Matrigel (BD Biosciences, Bedford, MA) before the cells were added to the upper chambers. All experiments were repeated at least three times, and each data point was measured in triplicate. The mean values and 95% confidence intervals were determined.
Quantitation of Blood Vessels in Solid Tumors
We determined the number of blood vessels in eight solid tumors derived from subcutaneous injection of adenovirus-infected (control and thymosin 4expressing) B16-F10 cells by staining with a rat polyclonal anti-CD31/plateletendothelial cell adhesion molecule-1 (PECAM-1) antibody that recognizes PECAM-1 on endothelial cells (1 : 100 dilution; Pharmingen, San Diego, CA). Antibody binding was detected with the use of an EnVision+ peroxidase system (DAKO), and the number of blood vessels was counted in six fields per three randomly chosen sections.
Statistical Analysis
The size of the solid tumors, the number of metastatic nodules, and the number of blood vessels were measured for calculation of mean values and 95% confidence intervals. We calculated mean values and 95% confidence intervals for cell proliferation, migration, and invasion from data collected in at least three independent experiments. Statistical significance of differences among the groups was determined using a two-tailed Students t test. P values less than .05 were considered statistically significant.
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RESULTS |
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We analyzed thymosin 4 mRNA levels in B16 melanoma cell lines and in primary cultured B16-F10 cells derived from lung tumors using northern blot analysis. Thymosin
4 mRNA was detected in the primary cultured B16-F10 cells derived from lung tumors but not in the B16-F1, B16-F10, or B16-BL6 cell lines (Fig. 1, A). Immunohistochemical staining indicated that thymosin
4 was more highly expressed in the lung tumor tissue that was derived from the B16-F10 cells (which have distinctive large nuclei) than in surrounding non-tumor tissue (Fig. 1, B). These data suggest that thymosin
4 expression is elevated in metastatic tumor cells in vivo and in primary cultured tumor cells derived from metastatic lesions.
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To elucidate the role of thymosin 4 in tumor metastasis, we constructed a thymosin
4 adenoviral expression vector and used it to infect B16-F10 cells. We first determined the expression level of thymosin
4 in lysates made from infected cells and found that the level of thymosin
4 protein increased with the amount of virus used to infect the cells and that thymosin
4 was detectable for at least 6 days after infection, although it peaked at 23 days (Fig. 2). The amount of total protein in the cell lysates, analyzed by Coomassie blue staining of the protein gel, did not change after virus infection (data not shown). Thymosin
4 was not detected in the culture medium of infected cells, suggesting that thymosin
4 was not secreted by the infected cells. In all of the subsequent experiments, we infected cells with 200 pfu of adenovirus per cell, which has the minimum dose of virus necessary to induce detectable thymosin
4 expression. This dose of virus had no effect on cell proliferation or viability (data not shown).
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We examined the effects of thymosin 4 expression on tumor growth and metastasis by injecting separate groups of mice (n = 20 per test group) subcutaneously and intravenously, respectively, with B16-F10 cells infected with adenovirus containing control or thymosin
4 expression vector. At 20 days after subcutaneous injection, mean tumor size was 21.7 mm (95% CI = 17.7 to 25.7 mm) for mice injected with B16-F10 cells infected with thymosin
4expressing adenovirus and 13.3 mm (95% CI = 11.1 to 15.3 mm) for mice injected with B16-F10 cells infected with control adenovirus (difference = 8.4 mm; P = .036) (Fig. 3, A). Two weeks after mice were injected intravenously with adenovirus-infected cells, the mean number of metastatic lung nodules was 46.7 (95% CI = 35.0 to 57.7) for mice injected with B16-F10 cells infected with thymosin
4expressing adenovirus and 10.9 (95% CI = 6.2 to 15.6) for mice injected with B16-F10 cells infected with control adenovirus (difference = 35.8 metastatic lung nodules; P<.001) (Fig. 3, B). Mice in the two groups had metastatic lung nodules that were small and of similar size (data not shown), presumably because we dissected the mice only 2 weeks after the B16-F10 cell injections. These data suggest that overexpression of thymosin
4 is associated with increases in primary tumor growth and in the number of lung metastases.
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We used adenovirus-infected B16-F10 cells to examine the functional role(s) of thymosin 4 in tumor metastasis. Cells infected with thymosin
4expressing adenovirus displayed the same level of cell proliferation as cells infected with the control adenovirus (Fig. 4). To analyze the effect of thymosin
4 on the expression or activity of MMPs, i.e., enzymes that degrade the extracellular matrix, we conducted gelatin-based zymography with the conditioned media prepared from adenovirus-infected B16-F10 cells. Thymosin
4 overexpression was not associated with the expression or activity of MMP-2 or MMP-9 (data not shown).
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We next used confocal laser-scanning immunofluorescence microscopy to examine the subcellular distribution of thymosin 4 and actin in adenovirus-infected cells. Cells were co-stained with an anti-thymosin
4 antibody (red) and FITC-labeled phalloidin (green), which binds to F-actin. Thymosin
4 was detected in the cytoplasm and in the nucleus of cells infected with thymosin
4expressing adenovirus (Fig. 5) but was not detectable in cells infected with control adenovirus (data not shown). F-actin was localized to the cell surface in cells infected with control adenovirus and in cells infected with thymosin
4expressing adenovirus (data not shown), in agreement with previously published results (24). When we merged the images for thymosin
4 and actin staining, we found that thymosin
4 co-localized with F-actin only at the leading edges of the cell (Fig. 5).
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We previously reported that thymosin 4 expression is associated with the induction of endothelial cell differentiation and the promotion of angiogenesis (18,25). To analyze the angiogenic activity of thymosin
4, we counted the number of blood vessels in the solid tumors formed by injecting mice with B16-F10 cells infected with control or thymosin
4expressing adenovirus. The fold increase in the number of blood vessels was determined by comparison with the number of blood vessels formed by injecting mice with control adenovirusinfected B16-F10 cells. Thymosin
4 overexpression was associated with a 4.4-fold mean increase (95% CI = 3.3- to 5.5-fold mean increase; P<.001) in the number of blood vessels in solid tumors derived from injected B16-F10 cells (Fig. 4).
Although these data suggest that thymosin 4 stimulates angiogenesis, it is possible that this effect may be an indirect reflection of changes in the levels of growth factors that are known to regulate tumor angiogenesis. We therefore measured the levels of VEGF, FGF-1, FGF-2, IGF, PDGF, and EGF in B16-F10 cells at different times after infection with control or thymosin
4expressing adenovirus (26-30). We found that VEGF levels in cells infected with thymosin
4expressing adenovirus peaked at 2 days after infection. The increased VEGF expression pattern paralleled that of thymosin
4 in cells infected with thymosin
4expressing adenovirus (Fig. 6). By contrast, the levels of FGF-1, PDGF, and EGF did not change after the cells were infected with thymosin
4expressing adenovirus (Fig. 6). Neither FGF-2 nor IGF was detectable by western blotting in cells infected with the control or the thymosin
4expressing adenovirus (data not shown).
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DISCUSSION |
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We previously reported that thymosin 4 has strong angiogenic activity (25). To confirm the effect of thymosin
4 on angiogenesis, we counted blood vessels in the solid tumors derived from B16-F10 cells infected with control or thymosin
4expressing adenovirus. Thymosin
4 overexpression was associated with the stimulation of blood vessel formation, as shown in Fig. 4. We addressed the possible mechanism for the angiogenic effect of thymosin
4 by showing that VEGF expression was increased upon infection of cells with a thymosin
4expressing adenovirus or by treatment of cells with synthetic thymosin
4 peptide. Thymosin
4 is not secreted, but we have found that exogenously added thymosin
4 localizes in the cytoplasm and nucleus of cells (data not shown). However, it is not clear whether thymosin
4 alone is angiogenic, whether all of the angiogenic potential associated with thymosin
4 is actually mediated by VEGF, or whether both VEGF and thymosin
4 are functionally important. Thymosin
4 is also reported to have anti-inflammatory activity (8,33); thus, it may also act in promoting tumor growth by inhibiting immune surveillance.
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Manuscript received April 10, 2003; revised September 8, 2003; accepted September 15, 2003.
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