- Email: [email protected]
High Expression of Macrophage Migration Inhibitory Factor in Human Melanoma Cells and Its Role in Tumor Cell Growth and Angiogenesis
Biochemical and Biophysical Research Communications 264, 751–758 (1999) Article ID bbrc.1999.1584, available online at http://www.idealibrary.com on
High Expression of Macrophage Migration Inhibitory Factor in Human Melanoma Cells and Its Role in Tumor Cell Growth and Angiogenesis Tadamichi Shimizu,* Riichiro Abe,* Hideki Nakamura,* Akira Ohkawara,* Masaki Suzuki,† and Jun Nishihira† ,1 *Department of Dermatology and †Central Research Institute, Hokkaido University School of Medicine, Sapporo 060, Japan
Received September 27, 1999
Macrophage migration inhibitory factor (MIF) is known to function as a cytokine, hormone, and glucocorticoid-induced immunoregulator. In this study, we reported for the first time that human melanocytes and melanoma cells express MIF mRNA and produce MIF protein. Immunohistochemical analysis demonstrated that MIF was mostly localized in the cytoplasm of melanocytes and G361 cells, a widely available human melanoma cell line. In particular, strong positive staining was observed at the dendrites of these cells. Expression of MIF mRNA and production of MIF protein were much higher in human melanoma cells such as G361, A375, and L32 than in normal cultured melanocytes. To assess the role of MIF overexpression in melanoma cells, G361 cells were transfected with an antisense human MIF plasmid. The results demonstrated that the cell growth rate of the transfected cells was markedly suppressed, suggesting that MIF participates in the mechanism of proliferation of melanoma cells. To further evaluate the function of MIF, we employed the Boyden chamber method to examine the effect on tumor cell migration and found that MIF enhanced the migration of G361 cells in a dose-dependent manner. Furthermore, we administered anti-MIF antibody into tumor (G361 cells in a Millipore chamber)-bearing mice to assess the effect on tumor-associated angiogenesis. The antiMIF antibody significantly suppressed tumor-induced angiogenesis. Taken together, these results indicated that it is likely that MIF may function as a novel growth factor that stimulates incessant growth and invasion of melanoma concomitant with neovascularization. © 1999 Academic Press Key Words: angiogenesis; cytokine; macrophage; melanocytes; antisense transfection.
1
To whom correspondence and reprint requests should be addressed. Fax: 181-11-706-7864. E-mail: [email protected].
Recent studies suggest that cytokines play an important role in regulation of cellular proliferation and immunological responses in the epidermis of skin tissue (1, 2). Melanocytes, which represent 2–5% of the total cell population in the epidermis, have been of interest because of their unique biological function in pigmentation and melanosomas (3). Melanocytes are target cells of several hormones and other biological modifiers (4). The cells are also affected by paracrine factors secreted by epidermal cells. For example, interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-a produced by keratinocytes are known to inhibit melanocyte proliferation (5). Melanocytes by themselves produce several cytokines, including IL-1 (6). IL-1 and TNF-a, which stimulate them to produce IL-8 and monocyte chemotactic and activating factor in paracrine and autocrine manners (7). However, the precise roles of melanocytes in cutaneous inflammation and immunity are still poorly understood. On the other hand, melanoma cells, the malignant transformant cells of melanocytes, have been widely used as a model to investigate the process of malignant transformation. Melanoma cells, similar to melanocytes, have the potential to produce and secrete a number of cytokines, including IL-1 (8, 9), IL-6 (10) and TNF-a (11). Biological activities of these melanomasecreted cytokines have important biological and prognostic implications for tumor cell growth. Macrophage migration inhibitory factor (MIF) was the first lymphokine reported to prevent random migration of macrophages and recruit them at inflammatory loci (12). Although MIF has long been considered to be exclusively expressed in activated T lymphocytes, it is clear at present that a variety of cells have the potential to produce the protein (13–19). Accordingly, various novel immunological and hormonal functions of MIF have been reported during the past few years. For example, MIF was found to be the major secretary
751
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein released by anterior pituitary cells, potentiating the lethality of endotoxin shock (14), and to be a glucocorticoid-induced immunomodulator, regulating the production of other inflammatory cytokines (15). We recently reported the presence of MIF protein in human epidermal keratinocytes, particularly in the proliferative basal cell layers (16). This finding indicates that MIF may function to regulate cellular proliferation and differentiation in the epidermis in addition to immunity and inflammation. Melanocytes are intimately associated with human keratinocytes as the epidermal-melanin unit; however, the precise mechanism of their interaction remains to be elucidated. In this study, we show for the first time that melanocytes and melanoma cells produce MIF protein. Next, we demonstrate the possible biological function of MIF in cell growth by means of anti-sense MIF plasmid transfection to melanoma cells. In addition, we show the function of MIF with regard to tumor cell invasion and angiogenesis. MATERIALS AND METHODS Materials. The following materials were obtained from commercial sources. Nitrocellulose membranes and Millipore chambers were from Millipore (Bedford, MA); Isogen RNA extraction kit from Nippon Gene (Tokyo, Japan); random primer DNA labeling kit and SspI from Takara (Kyoto, Japan); G418 from Sigma (St. Louis, MI); pBKCMV vector from Stratagene (La Jolla, CA); [a- 32P]dCTP from Du Pont NEN (Boston, MA); CellTiter 96 AQ (MTS) assay kit from Promega (Madison, WI); Dulbecco’s modified Eagle’s minimal medium (DMEM) from Gibco (Grand Island, NY); heat-inactivated fetal calf serum (FCS) from Hyclone Labs (Logan, UT); horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody from Pierce (Rockford, IL); Konica HRP-1000 immunostaining kit from Konica (Tokyo, Japan); FITC-conjugated goat anti-rabbit IgG antibody from Bio-rad (Hercules, CA); non-immune rabbit IgG from Zymed (San Francisco, CA); Cellulose acetate membrane filter (8 mm pore) from Kurabo (Osaka, Japan), and Protein A Sepharose from Pharmacia (Uppsala, Sweden). All other chemicals used were of analytical grade. Preparation of rabbit polyclonal antibodies against human MIF. Polyclonal anti-human MIF serum was generated by immunizing New Zealand White rabbits with recombinant human MIF as previously described (16). In brief, the rabbits were inoculated intradermally with 100 mg of MIF diluted in complete Freund’s adjuvant at weeks 1 and 2, and with 50 mg of MIF diluted in incomplete Freund’s adjuvant at week 4. Immune serum was collected 1 week after the last inoculation. The IgG fraction was prepared using Protein A Sepharose according to the manufacturer’s protocol. Cell culture. Primary cultured normal human melanocytes were obtained from Morinaga Co. (Tokyo, Japan). The melanocytes were grown in a HFM culture medium containing 10% FCS under 95% air and 5% CO 2 at 37°C according to the manufacturer’s protocol. The cells were plated in 100-mm culture dishes, grown to near confluence, and the complete melanocyte growth medium was removed. The medium was replaced with minimum growth medium consisting of MCDB 153 containing 2% FCS and 30 mg/ml bovine pituitary extract. Human malignant melanoma cell lines G361, A375, and L32 were obtained from the American Type Culture Collection, and maintained in DMEM supplemented with 10% FCS. After incubation for specific times, culture supernatants of melanocytes and melanoma cells were removed and stored at 280°C for enzyme-
linked immunosorbent assay (ELISA), and the cells were harvested and subjected to Northern and Western blot analyses. Northern blot analysis. Northern blot analysis was performed as previously described (20). In brief, total RNA was extracted from cell pellets with an Isogen total RNA extraction kit. RNA was resuspended in TE (10 mM Tris–HCl, 1 mM EDTA, pH 7.4). Total RNA (20 mg) was denatured and electrophoresed on 1% agarose formaldehyde gel. The RNA was then transferred to nylon membranes, and crosslinked by UV irradiation. Prehybridization was carried out in 0.75 M NaCl, 0.02 M Tris–HCl (pH 7.5), 2.5 mM EDTA, 0.53 Denhardt’s solution, 1% sodium dodecyl sulfate (SDS) and 50% formamide at 42°C for 4 h. Then hybridization was performed in the same buffer containing 10% dextran sulfate, salmon sperm DNA (250 mg/ml) and a radiolabeled probe at 42°C for 20 h. The radiolabeled probe was prepared using human MIF cDNA as a template and labeled with a random primer labeling kit using [a- 32P]dCTP. The membrane was washed twice with 23 SSC (16.7 mM NaCl, 16.7 mM sodium citrate) at 22°C for 5 min, twice with 0.23 SSC containing 0.1% SDS 65°C for 15 min, and 23 SSC at 22°C for 20 min prior to autoradiography. Quantitative densitometric analysis was performed using an MCID Image Analyzer (Fuji Film, Tokyo, Japan). Western blot analysis. Cell lysates prepared from the cultured melanocytes, G361, A375 and L32 melanoma cells were treated with 4 volumes of SDS-reducing buffer (60 mM Tris/HCl, pH 6.8, 2% SDS, 10% glycerol, 5% b-mercaptoethanol, and 0.025% bromophenol blue), and subjected to SDS–polyacrylamide gel electrophoresis (PAGE) as described (21). Proteins on the gel were electrophoretically transferred to a nitrocellulose membrane according to the procedure of Towbin et al. with minor modifications (22). The membrane was intensively washed with phosphate-buffered saline (PBS), and incubated with the polyclonal anti-human MIF antibody (1000-fold dilution of 4 mg/ml IgG) at room temperature for 1 h. After washing, the membrane was incubated with HRP-conjugated goat antibody against rabbit IgG at room temperature for 1 h. After the incubation, proteins were visualized with a Konica immunostaining kit as recommended in the manufacturer’s protocol. Immunohistochemistry. Immunohistochemical analysis of the cultured normal human melanocytes and G361 melanoma cells was carried out using the anti-human MIF antibody and fluorescenceconjugated anti-rabbit IgG antibody as described (16). In brief, the cultures of cells grown on coverslips were rinsed with PBS and immersed in 220°C acetone for 5 min. The fixed cell samples were incubated with the anti-human MIF antibody (1000-fold dilution of 4 mg/ml IgG) at 37°C for 1 h, followed by incubation with FITC-labeled goat-anti-rabbit IgG at 37°C for 30 min. Microscopic observation was carried out using a fluorescence microscope (Nikon, Tokyo, Japan). Assay for MIF. To measure the content of MIF secreted from the cultured melanocytes or melanoma cells, supernatants of these cells were measured essentially as described previously (17). In brief, the anti-human MIF antibody (4 mg/ml) dissolved in 50 ml of PBS was immobilized on each well of a 96-well microtiter plate. After incubation for 1 h at room temperature, the plate was washed three times with PBS. All wells were filled with PBS containing bovine serum albumin (1%) and left for 1 h at room temperature to block nonspecific binding. After removal of the blocking solution, the plate was washed three times with PBS containing 0.05% Tween 20 (washing buffer), samples of the supernatants were added to individual wells and incubated for a further 1 h at room temperature. After the plate was washed three times with the washing buffer, 50 ml of biotinconjugated anti-human MIF antibody was added. Following incubation for 1 h at room temperature, the plate was again washed three times with the washing buffer. Then avidin-conjugated HRP was added to individual wells, which were further incubated for 1 h at room temperature, and the plate was again washed with the washing buffer. Next, 50 ml of substrate solution containing 200 mg of o-phenylenediamine and 10 ml of 30% hydrogen peroxide in 10 ml of citrate-phosphate buffer (pH 5.0) was added. After incubation for 20
752
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS each assay, five fields were counted on each filter, and the counts were expressed as means with standard deviation. Angiogenesis assay. An angiogenesis assay using the dorsal air sac method was performed to evaluate the inhibitory effect of the anti-MIF antibody on the angiogenesis induced by melanoma cells as described (25). A Millipore chamber, which passes only soluble factors, was filled with 1 3 10 7 G361 cells in 200 ml of medium, and inoculated into the right flank of BALB/c mice. We divided mice into three groups; (i) negative control, no tumor cells in the chamber, (ii) positive control, G361 melanoma cells in the chamber, and (iii) anti-MIF antibody-treated test samples, tumor cells in the chamber and mice treated with the anti-rat MIF antibody. Animals in the treatment group received 200 ml of anti-MIF antibody (200 mg/body/ day) ip on days 1, 3, and 5, and those in the control group received the same amount of non-immune rabbit IgG. Mice were sacrificed on day 6. The area of neovascularization was measured using an MCID image analyzer, and the degree of tumor-induced angiogenesis was decided by the ratio of the area of blood vessels to the area in contact with the chamber.
SCHEME 1. Construction of the anti-sense MIF plasmid (pBKMIFAS). The full-length cDNA for human MIF was subcloned into the BamHI restriction site of pBK-CMV vector, a eukaryotic expression vector driven by the human cytomegalovirus (CMV) promoter. min at room temperature, the reaction was terminated with 50 ml of 1 N sulfuric acid. The absorbance at 492 nm was measured with an ELISA plate reader (Bio-Rad Model 3550). Construction of anti-sense MIF plasmid. The full-length cDNA for human MIF was prepared as described (23), and subcloned into the BamHI site of pBK-CMV vector, a eukaryotic expression vector driven by the human cytomegalovirus promoter. Subcloning into the BamHI restriction site yielded an insert in the anti-sense orientation (pBK-MIFAS) (Scheme 1). A plasmid, which contained a fragment of a human cDNA insert with 21 nucleotides deleted from the initiation coden at the 59-end in sense orientation, was also prepared as a control (pBK-MIFST). The orientation and proof of completeness of the insert were determined by means of restriction enzyme analyses and DNA sequencing using a DNA sequencer (ABI Model 373A). The G361 melanoma cells were transfected with either the pBKMIFAS or pBK-MIFST or the pBK-CMV vector alone linearized with SspI digestion by electroporation using a T820 optimizer (BTX, San Diego, CA) at 200 mV with a 99-ms pulse length and a single pulse. Drug selection was then started 24 h after the transfection by the addition of G418 (400 mg/ml) to the culture medium of DMEM containing 10% FCS. The cells were fed every three days with fresh medium for three weeks, G418-resistant colonies were separately transferred to individual wells of a 48-well plate and colonies were expanded.
RESULTS Northern blot analysis of MIF in melanocytes and G361 cells. We examined the level of MIF mRNA in melanocytes and melanoma cells by Northern blot analysis. Both the normal melanocytes, G361, A375 and L32 cells showed expression of MIF mRNA. The expression levels of G361, A375 and L32 cells were 13.6-, 10.6-, and 12.8-fold greater than that of the melanocytes, respectively, when MIF mRNA levels were normalized by each b-actin mRNA level (Fig. 1). These results suggested that MIF mRNA expression was exceedingly upregulated by malignant transformation. Western blot analysis of MIF in melanocytes and melanoma cells. To investigate the presence of MIF protein in the melanocytes and melanoma cells, Western blot analysis was performed using the antibody against human MIF. The immunoblot analysis revealed a specific positive band, migrating to about 12.5 kDa, corresponding to the molecular weight of recombinant human MIF (Fig. 2). The intensity of the bands of G361, A375, and L32 cells visualized by the immunostaining kit showed 3.1-, 2.5-, and 2.9-fold increase compared to that of the melanocytes, respectively. This
Cell proliferation assay. MTS assay was performed according to the manufacturer’s protocol. In brief, G361 melanoma cells and the anti-sense MIF transfected cells were seeded on each well of a 96well microtiter plate at the initial density of 5 3 10 3 cells/well and incubated for 96 h. Two hundred microliters of 2.5 mg/ml solution of MTS was added and incubated for 2 h at 37°C. The absorbance was determined on a plate reader read at 570 nm. Migration assay. To evaluate the effect of MIF on migration of melanoma cells, the Boyden chamber assay was performed as described (24). Briefly, the lower well was filled with 600 ml of medium (DMEM containing 10% FCS) in the presence of various amounts of MIF. The upper well contained G361 cell suspension (400 ml, 1 3 10 5 cell/ml medium). A cellulose acetate membrane filter was interposed between the two chambers. The chambers were kept in a humidified atmosphere of 5% CO 2 at 37°C for 4 h. Filters were subsequently fixed with methanol and acetone and stained with crystal violet. The number of cells that migrated into the filter and reached its lower side was determined microscopically. Three chambers were set up for
FIG. 1. Northern blot analysis of MIF mRNA expression in cultured melanocytes, and melanoma cells. Total RNA (15 mg) was electrophoresed through 1% agarose gel, transferred to a nylon membrane, and hybridized with a 32P-labeled full-length human MIF cDNA insert. Lane 1, melanocytes; lane 2, G361 cells; lane 3, A375 cells; lane 4, L32 cells. mRNA levels of b-actin were shown at the bottom of each lane.
753
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Western blotting analysis of the cultured human melanocytes and melanoma cells for MIF. Each cell sample was collected, electrophoresed, transferred to a nitrocellulose membrane, and visualized using a Konica immunostaining kit as described under Materials and Methods. Lane 1, prestained molecular marker (New England Biolabs); lane 2, recombinant human MIF (50 ng); lane 3, G361 cells (1 3 10 5 cells); lane 4, A375 cells (1 3 10 5 cells); lane 5, L32 cells (1 3 10 5 cells); lane 6, melanocytes (1 3 10 5 cells).
result indicated that much more MIF protein was produced in melanoma cells than in the melanocytes. Immunohistochemistry. The primary cultured human melanocytes were positively stained using the antibody against human MIF. The immunofluorescence study revealed that MIF was predominantly identified in the cytoplasm of melanocytes. In particular, strong positive staining was observed at the dendrites (Fig. 3A). Similarly, more enhanced positive cytoplasmic staining was observed in G361 melanoma cells (Fig. 3C). The nucleus was also clearly stained. No specific positive staining was observed in either the melanocytes (Fig. 3B) or G361 melanoma cells (Fig. 3D) reacted with nonimmune rabbit IgG.
cells by Western blot analysis (Fig. 5). To examine the anti-sense MIF effect on the cell proliferation rate, MTS assay was carried out. For cell growth, the proliferation rate of the transfectant G361 cells was markedly suppressed (35.2 6 11.5%) compared to nontreated control cells (100%) and those transfected with the vector alone (93.6 6 6.1%) or pBK-MIFST (95.2 6 5.1%) (Fig. 6). This indicated that MIF contributed to the cell growth.
Detection of MIF in the culture supernatants of melanocytes and melanoma cells. Contents of secreted MIF protein in the supernatants of cultured melanocytes and melanoma cells (1 3 10 5 cells/ml for each cells) were measured by ELISA after 48 h incubation. The MIF contents in the supernatants of the G361, A375, and L32 cells, 27.5 6 4.8, 20.4 6 2.5, and 18.6 6 1.8 ng/ml, respectively, were significantly higher than that of the melanocytes (6.8 6 2.2 ng/ml) (Fig. 4). The result showed that melanoma cells produced and secreted more MIF than the melanocytes.
Effect of MIF on melanoma cell migration. Migration of tumor cells is an important event for invasiveness and metastasis. To assess the role of MIF in tumorigenesis, migration of G361 melanoma cells in response to MIF was examined by the Boyden chamber method. We added various doses of MIF from 1 to 100 ng/ml in the lower chamber. Enhancement of G361 cell migration was observed in a dose-dependent manner (Fig. 7). Cell counts per field were 12 6 1.5, 13.7 6 0.9, 28.3 6 2.6, and 63.7 6 6.6 at the doses of 0, 1, 10, and 100 ng/ml of MIF, respectively. Since the MIF concentration can be up-regulated at the level of more than 80 ng/ml at loci, e.g., synovial fluids and follicular fluids (26, 27), it is considered that the local MIF concentration at the site of tumor lesions might reach such levels as to induce melanoma cell migration in vivo.
Effect of anti-sense MIF transfection on cell growth. The transfectant G361 melanoma cells with the pBKMIFAS were prepared as described under Materials and Methods. We isolated three different clonal cells in the presence of G418, and confirmed a decrease of the intracellular MIF protein level compared to the control
Effect of anti-MIF antibody on angiogenesis in vivo. Angiogenesis within the subcutaneous fascia of antiMIF antibody-treated mice was compared with that of the control. In the positive control mice bearing G361 cells in the chambers, we found that the cells significantly promoted angiogenesis of the subcutaneous
754
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. MIF expression on melanocytes and G361 melanoma cells. An immunofluorescence photograph shows human melanocytes and G361 cells stained with anti-human MIF antibody as described under Materials and Methods. MIF protein was observed in the cytoplasm of both melanocytes (A) and G361 cells (C). Note the strong positive staining at the dendrites and accentuation at the tips of dendrite processes on normal melanocytes, and nuclei were also clearly stained in G361 cells. Pre-immune IgG showed no specific positive reaction (B, D). (A, B, C, D; 3100.)
vessels (Fig. 8A). By contrast, tumor-associatedangiogenesis was significantly suppressed in the antiMIF antibody-treated group in comparison with the positive control (Fig. 8B). The ratios of the area of blood vessels to the areas in contact with the chamber were 13.5 6 4.0%, 51.2 6 5.0%, and 24.2 6 0.7%, in the negative control with medium alone, positive control with the tumor cells, and anti-MIF antibody-treated groups, respectively. DISCUSSION Melanocytes are localized in the basal layers of the epidermis. They interact with the surrounding keratinocytes via their dendrites, and exert their biological functions such as pigmentation. In this event, melanocytes act in concert with keratinocytes to pigment the skin. There is emerging evidence indicating the involvement of melanocytes in the events of cutaneous inflammation and immunity via secretion of a variety of immune and inflammatory mediators. In this study,
we demonstrated the expression of MIF mRNA and production of MIF protein in human melanocytes and melanoma cells. This fact suggested that MIF, a putative inflammatory cytokine (28), affected or regulated the pathophysiological states of the skin tissue. In particular, it should be noted that MIF mRNA is highly expressed and that very much MIF protein was present in the cytoplasm and nucleus of melanoma cells, suggesting its role in tumor cell growth. MIF was originally identified as a lymphokine concentrating macrophages at inflammatory sites and is a potent activator of macrophages (12, 28). This protein has been detected in various tissues, including cells in the cornea and reproductive organs, and in osteoblasts (17–19). These cells release MIF protein into extracellular spaces in response to various stimuli such as lipopolysaccharide and TNF-a (13, 15). The immunohistochemical results in this study demonstrated the presence of a large amount of MIF in dendrites of melanocytes. This suggested the possibility that MIF protein may be readily released to the epidermis by
755
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. MIF content in the supernatants of cultured normal human melanocytes and melanoma cells. The MIF content in the culture supernatants (1 3 10 5 cells/ml for each cells) was measured by ELISA as described under Materials and Methods. The values were means 6 SD of at least six experiments. Lane 1, melanocytes; lane 2, G361 cells; lane 3, A375 cells; lane 4, L32 cells. *P , 0.01; **P , 0.005.
various environmental stimuli, such as ultraviolet irradiation, which in consequence may affect the pathophysiological state of the epidermis. Indeed, we demonstrated that MIF secretion was markedly stimulated by low doses of ultraviolet irradiation (29). Accordingly, it is speculated that MIF in melanocytes may play a pivotal role in cutaneous immunity and inflammatory processes. It is of great interest that exceedingly high expression of MIF was observed in melanoma cells compared to that of normal melanocytes. This finding indicated that MIF may also participate in the regulation of cell growth as a novel growth factor. From the data available to date, cultured melanoma cells have been reported to produce a variety of growth factors and cytokines, including IL-1 and platelet-derived growth factor (8, 9). These biological mediators are known to
FIG. 5. Production of MIF protein in G361 melanoma cells and anti-sense MIF plasmid transfected cells. Western blot analysis was carried out to examine the expression of MIF protein by each cell type. The MIF protein bands were visualized by a Konica HRPimmunostaining kit. Lane 1, molecular marker; lane 2, recombinant human MIF (100 ng); lane 3, G361 cells (2 3 10 5 cells) transfected pBK vector alone; lane 4, G361 cells (2 3 10 5 cells) transfected pBK-MIFST; lanes 5–7, G361 cells (2 3 10 5 cells) transfected with pBK-MIFAS.
FIG. 6. Effect of anti-sense MIF plasmid transfection on cell growth of G361 melanoma cells. Growth of G361 cells (initial count, 5 3 10 3 cells) transfected with anti-sense MIF plasmid was examined by MTS assay. Lane 1, control (G361 cells); lane 2, the cells transfected with pBK vector alone; lane 3, the cell transfected with pBKMIFST; lane 4, the cell transfected with pBK-MIFAS. The result is the mean 6 SD of at least three different experiments. *P , 0.01.
be critical for malignant transformation through their direct effects on tumor cell growth. For example, IL-1 has been reported to be mitogenic for a number of cell types. However, the biological effects of IL-1 released by melanoma cells are not uniform, showing both positive and negative effects on cell proliferation (9, 30). Nonetheless, it is conceivable that MIF may stimulate cell proliferation via these cytokines because MIF has the potential to induce cytokines stimulating cell growth such as IL-1 and TNF-a (13, 15). In association with cell growth, it was reported that MIF mRNA expression is correlated with cell differen-
FIG. 7. Migration of G361 cells in response to MIF. The culturing method and migration assay are described under Materials and Methods. The doses of MIF examined were from 0 to 100 ng/ml. The values were means 6 SD of at least 5 experiments. Lane 1, control; lane 2, 1 ng/ml; lane 3, 10 ng/ml; lane 4, 100 ng/ml. *P , 0.01; **P , 0.005.
756
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
est that high expression of MIF was discovered in metastatic prostate cancer (33). Moreover, in the present study, we found that anti-sense MIF cDNA transfection markedly suppressed the cell growth of melanoma cells. Recently, consistent with our results, it was reported that MIF stimulated the proliferation of NIH/3T3 cells with the activation of mitogenactivated protein kinase (34). Based on these facts, it is speculated that MIF, present in a large amount in melanoma cells, might be involved in the mechanism of cell proliferation. It has been suggested that MIF affects cellular proliferation as a growth factor-induced delayed earlyresponse gene (35). We identified the increased expression of MIF mRNA during corneal wound healing after penetrating injury concomitant with keratocytes migration (36). Because corneal injury can induce immediate-early-response genes (c-fos and c-jun), the upregulation of MIF after the injury as a delayedearly-response gene appears to be reasonable. This fact supports the idea that MIF is profoundly involved in cell growth at the gene-transcription level. Moreover, we demonstrated that MIF possessed the potential to enhance invasiveness of melanoma cells and stimulated angiogenesis. Judging from these observations, it is conceivable that MIF could be involved not only in non-tumor cell growth in the event of wound healing but also in tumor cell invasion and neovascularization. Finally, it is widely accepted that cytokines play an important role in responses of the host with a malignant tumor. It is very likely that MIF functions as a pivotal element for cellular proliferation and differentiation in autocrine and paracrine manners. Very recently, consistent with our results, MIF was reported to contribute to growth and angiogenesis of murine lymphoma (37). These findings, including ours, could be a new starting point to investigate the broadspectrum pathophysiological roles of MIF in tumorigenesis beyond the immunological system. ACKNOWLEDGMENTS FIG. 8. Histological evidence for the effect of anti-MIF antibody on tumor-associated angiogenesis in vivo. Effectiveness of the antirat MIF polyclonal antibody in mediating suppression of G361 cellinduced angiogenesis was examined by the subcutaneous dorsal air sac technique. On day 6, angiogenesis within the subcutaneous fascia of anti-MIF antibody-treated mice was compared with that of the control (n 5 5). Typical pictures for neovascularization of nonimmune IgG-treated (A) and anti-rat MIF antibody-treated (B) mice are shown. Arrows show typical tumor-associated neovascularization.
This work was supported by a Grant-in-Aid for Research (09670144) from the Ministry of Education, Science, and Culture of Japan. We are grateful to Yuka Mizue of Sapporo Immunodiagnostic Laboratory for her technical assistance.
REFERENCES
tiation of lens cells (31), suggesting the involvement of this protein in cellular proliferation and differentiation. We demonstrated that MIF was predominantly expressed in highly proliferative basal cells of human epidermis and in leukemia cells (16, 32). It is of inter757
1. Gordon, P. R., Mansur, C. P., and Gilchrest, B. A. (1989) J. Invest. Dermatol. 92, 565–572. 2. Morelli, J. G., and Norris, D. A. (1993) J. Invest. Dermatol. 100, 191–195. 3. Ramirez-Bosca, A., Bernd, A., Werner, R., Dold, K., and Holzmann, H. (1992) Arch. Dermatol. Res. 284, 358 –362. 4. De Kuca, M., D’Anna, F., Bondanza, S., Franzi, A. T., and Cancedda, R. (1988) J. Cell. Biol. 107, 1919 –1926. 5. Swope, V. P., Abdel-Malek, Z. A., Kassem, L. M., and Nordlund, J. J. (1991) J. Invest. Dermatol. 96, 180 –185.
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6. Swope, V. P., Sauder, D. N., McKenzie, R. C., Sramkoski, R. M., Krug, K. A., Babcock, G. F., Nordlund, J. J., and Abdel-Malek, Z. A. (1994) J. Invest. Dermatol. 102, 749 –753. 7. Zachariae, C. O. C., Thestrump-Pedersen, K., and Matsushima, K. (1991) J. Invest. Dermatol. 97, 593–599. 8. Ko¨ck, A., Schwarz, T., Urbanski, A., Peng, Z., Vetterlein, M., Micksche, M., Ansel, J. C., Kung, H. F., and Luger, T. A. (1989) J. Natl. Cancer Inst. 81, 36 – 42. 9. Bennicelli, J. L., Elias, J., Kern, J., and Guerry, D. (1989) Cancer Res. 49, 930 –935. 10. Armstrong, C. A., Tara, D. D., Hart, C. E., Ko¨ck, A., Luger, T. A., and Ansel, J. C. (1992) Exp. Dermatol. 1, 37– 45. 11. Mattei, S., Colombo, M. P., Melani, C., Silvani, A., Parmiani, G., and Herlyn, M. (1994) Int. J. Cancer 56, 853– 857. 12. Bloom, B. R., and Bennett, B. (1966) Science 153, 80 – 82. 13. Calandra, T., Bernhagen, J., Mitchell, R. A., and Bucala, R. (1994) J. Exp. Med. 179, 1895–1902. 14. Bernhagen, J., Calandra, T., Mitchel, R. A., Martin, S. B., Tracey, K. J., Voelter, W., Manogue, K. R., Cerami, A., and Bucala, R. (1993) Nature 365, 756 –759. 15. Calandra, T., Bernhagen, J., Mets, C. N., Spiegel, L. A., Bacher, M., Donnely, T., Cerami, A., and Bucala, R. (1995) Nature 377, 68 –71. 16. Shimizu, T., Ohkawara, A., Nishihira, J., and Sakamoto, W. (1996) FEBS Lett. 381, 188 –202. 17. Matsuda, A., Kotake, S., Tagawa, Y., Matsuda, H., and Nishihira, J. (1996) Immunol. Lett. 53, 1–5. 18. Suzuki, H., Kanagawa, H., and Nishihira, J. (1996) Immunol. Lett. 51, 141–147. 19. Onodera, S., Suzuki, K., Matsuno, T., Kaneda, K., and Nishihira, J. (1996) Immunology 89, 430 – 435. 20. Sakai, M., Nishihira, J., Hibiya, Y., Koyama, Y., and Nishi, S. (1994) Biochem. Mol. Biol. Int. 33, 439 – 446. 21. Schagger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368 –379.
22. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 23. Sugimoto, H., Suzuki, M., Nakagawa, A., Tanaka, I., and Nishihira, J. (1996) FEBS Lett. 389, 145–148. 24. Michel, G., Kemeny, L., Peter, R. U., Beetz, A., Ried, C., Arenberger, P., and Ruzicka, T. (1992) FEBS Lett. 305, 241–243. 25. Folkman, J., Merler, E., Abernathy, C., and Williams, G. (1971) J. Exp. Med. 133, 275–288. 26. Onodera, S., Tanji, H., Suzuki, K., Kaneda, K., Mizue, Y., Sagawa, A., and Nishihira, J. (1999) Cytokine 11, 163–167. 27. Wada, S., Kudo, T., Kudo, M., Sakuragi, N., Hareyama, H., Nishihira, J., and Fujimoto, S. (1999) Hum. Reprod. 14, 395– 399. 28. Bucala, R. (1996) FASEB J. 10, 1607–1613. 29. Shimizu, T., Abe, R., Ohkawara, A., and Nishihira, J. (1999) J. Invest. Dermatol. 112, 210 –215. 30. Lachman, L. B., Dinarello, C. A., Llanca, N. D., and Fildler, I. J. (1986) J. Immunol. 136, 3098 –3102. 31. Wistow, G. J., Shaughnessy, M. P., Lee, D. C., Hodin, J., and Zelenka, P. S. (1993) Proc. Natl. Acad. Sci. USA 90, 1272– 1275. 32. Nishihira, J., Koyama, Y., and Mizue, Y. (1996) Biochem. Mol. Biol. Int. 40, 861– 869. 33. Meyer-Siegler, K., and Hudson, P. B. (1996) Urology 48, 448 – 452. 34. Mitchell, R. A., Metz, C. N., Peng, T., and Bucala, R. (1999) J. Biol. Chem. 18, 18100 –18106. 35. Lanahan, A., Williams, J. B., Sanders, L. K., and Nathans, D. (1992) Mol. Cell. Biol. 12, 3919 –3929. 36. Matsuda, A., Tagawa, Y., Matsuda, H., and Nishihira, J. (1997) Invest. Ophthalmol. Vis. Sci. 38, 1555–1562. 37. Chesney, J., Metz, C., Bacher, M., Peng, T., Meinhardt, A., and Bucala, R. (1999) Mol. Med. 5, 181–191.
758
High Expression of Macrophage Migration Inhibitory Factor in Human Melanoma Cells and Its Role in Tumor Cell Growth and Angiogenesis Tadamichi Shimizu,* Riichiro Abe,* Hideki Nakamura,* Akira Ohkawara,* Masaki Suzuki,† and Jun Nishihira† ,1 *Department of Dermatology and †Central Research Institute, Hokkaido University School of Medicine, Sapporo 060, Japan
Received September 27, 1999
Macrophage migration inhibitory factor (MIF) is known to function as a cytokine, hormone, and glucocorticoid-induced immunoregulator. In this study, we reported for the first time that human melanocytes and melanoma cells express MIF mRNA and produce MIF protein. Immunohistochemical analysis demonstrated that MIF was mostly localized in the cytoplasm of melanocytes and G361 cells, a widely available human melanoma cell line. In particular, strong positive staining was observed at the dendrites of these cells. Expression of MIF mRNA and production of MIF protein were much higher in human melanoma cells such as G361, A375, and L32 than in normal cultured melanocytes. To assess the role of MIF overexpression in melanoma cells, G361 cells were transfected with an antisense human MIF plasmid. The results demonstrated that the cell growth rate of the transfected cells was markedly suppressed, suggesting that MIF participates in the mechanism of proliferation of melanoma cells. To further evaluate the function of MIF, we employed the Boyden chamber method to examine the effect on tumor cell migration and found that MIF enhanced the migration of G361 cells in a dose-dependent manner. Furthermore, we administered anti-MIF antibody into tumor (G361 cells in a Millipore chamber)-bearing mice to assess the effect on tumor-associated angiogenesis. The antiMIF antibody significantly suppressed tumor-induced angiogenesis. Taken together, these results indicated that it is likely that MIF may function as a novel growth factor that stimulates incessant growth and invasion of melanoma concomitant with neovascularization. © 1999 Academic Press Key Words: angiogenesis; cytokine; macrophage; melanocytes; antisense transfection.
1
To whom correspondence and reprint requests should be addressed. Fax: 181-11-706-7864. E-mail: [email protected].
Recent studies suggest that cytokines play an important role in regulation of cellular proliferation and immunological responses in the epidermis of skin tissue (1, 2). Melanocytes, which represent 2–5% of the total cell population in the epidermis, have been of interest because of their unique biological function in pigmentation and melanosomas (3). Melanocytes are target cells of several hormones and other biological modifiers (4). The cells are also affected by paracrine factors secreted by epidermal cells. For example, interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-a produced by keratinocytes are known to inhibit melanocyte proliferation (5). Melanocytes by themselves produce several cytokines, including IL-1 (6). IL-1 and TNF-a, which stimulate them to produce IL-8 and monocyte chemotactic and activating factor in paracrine and autocrine manners (7). However, the precise roles of melanocytes in cutaneous inflammation and immunity are still poorly understood. On the other hand, melanoma cells, the malignant transformant cells of melanocytes, have been widely used as a model to investigate the process of malignant transformation. Melanoma cells, similar to melanocytes, have the potential to produce and secrete a number of cytokines, including IL-1 (8, 9), IL-6 (10) and TNF-a (11). Biological activities of these melanomasecreted cytokines have important biological and prognostic implications for tumor cell growth. Macrophage migration inhibitory factor (MIF) was the first lymphokine reported to prevent random migration of macrophages and recruit them at inflammatory loci (12). Although MIF has long been considered to be exclusively expressed in activated T lymphocytes, it is clear at present that a variety of cells have the potential to produce the protein (13–19). Accordingly, various novel immunological and hormonal functions of MIF have been reported during the past few years. For example, MIF was found to be the major secretary
751
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein released by anterior pituitary cells, potentiating the lethality of endotoxin shock (14), and to be a glucocorticoid-induced immunomodulator, regulating the production of other inflammatory cytokines (15). We recently reported the presence of MIF protein in human epidermal keratinocytes, particularly in the proliferative basal cell layers (16). This finding indicates that MIF may function to regulate cellular proliferation and differentiation in the epidermis in addition to immunity and inflammation. Melanocytes are intimately associated with human keratinocytes as the epidermal-melanin unit; however, the precise mechanism of their interaction remains to be elucidated. In this study, we show for the first time that melanocytes and melanoma cells produce MIF protein. Next, we demonstrate the possible biological function of MIF in cell growth by means of anti-sense MIF plasmid transfection to melanoma cells. In addition, we show the function of MIF with regard to tumor cell invasion and angiogenesis. MATERIALS AND METHODS Materials. The following materials were obtained from commercial sources. Nitrocellulose membranes and Millipore chambers were from Millipore (Bedford, MA); Isogen RNA extraction kit from Nippon Gene (Tokyo, Japan); random primer DNA labeling kit and SspI from Takara (Kyoto, Japan); G418 from Sigma (St. Louis, MI); pBKCMV vector from Stratagene (La Jolla, CA); [a- 32P]dCTP from Du Pont NEN (Boston, MA); CellTiter 96 AQ (MTS) assay kit from Promega (Madison, WI); Dulbecco’s modified Eagle’s minimal medium (DMEM) from Gibco (Grand Island, NY); heat-inactivated fetal calf serum (FCS) from Hyclone Labs (Logan, UT); horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody from Pierce (Rockford, IL); Konica HRP-1000 immunostaining kit from Konica (Tokyo, Japan); FITC-conjugated goat anti-rabbit IgG antibody from Bio-rad (Hercules, CA); non-immune rabbit IgG from Zymed (San Francisco, CA); Cellulose acetate membrane filter (8 mm pore) from Kurabo (Osaka, Japan), and Protein A Sepharose from Pharmacia (Uppsala, Sweden). All other chemicals used were of analytical grade. Preparation of rabbit polyclonal antibodies against human MIF. Polyclonal anti-human MIF serum was generated by immunizing New Zealand White rabbits with recombinant human MIF as previously described (16). In brief, the rabbits were inoculated intradermally with 100 mg of MIF diluted in complete Freund’s adjuvant at weeks 1 and 2, and with 50 mg of MIF diluted in incomplete Freund’s adjuvant at week 4. Immune serum was collected 1 week after the last inoculation. The IgG fraction was prepared using Protein A Sepharose according to the manufacturer’s protocol. Cell culture. Primary cultured normal human melanocytes were obtained from Morinaga Co. (Tokyo, Japan). The melanocytes were grown in a HFM culture medium containing 10% FCS under 95% air and 5% CO 2 at 37°C according to the manufacturer’s protocol. The cells were plated in 100-mm culture dishes, grown to near confluence, and the complete melanocyte growth medium was removed. The medium was replaced with minimum growth medium consisting of MCDB 153 containing 2% FCS and 30 mg/ml bovine pituitary extract. Human malignant melanoma cell lines G361, A375, and L32 were obtained from the American Type Culture Collection, and maintained in DMEM supplemented with 10% FCS. After incubation for specific times, culture supernatants of melanocytes and melanoma cells were removed and stored at 280°C for enzyme-
linked immunosorbent assay (ELISA), and the cells were harvested and subjected to Northern and Western blot analyses. Northern blot analysis. Northern blot analysis was performed as previously described (20). In brief, total RNA was extracted from cell pellets with an Isogen total RNA extraction kit. RNA was resuspended in TE (10 mM Tris–HCl, 1 mM EDTA, pH 7.4). Total RNA (20 mg) was denatured and electrophoresed on 1% agarose formaldehyde gel. The RNA was then transferred to nylon membranes, and crosslinked by UV irradiation. Prehybridization was carried out in 0.75 M NaCl, 0.02 M Tris–HCl (pH 7.5), 2.5 mM EDTA, 0.53 Denhardt’s solution, 1% sodium dodecyl sulfate (SDS) and 50% formamide at 42°C for 4 h. Then hybridization was performed in the same buffer containing 10% dextran sulfate, salmon sperm DNA (250 mg/ml) and a radiolabeled probe at 42°C for 20 h. The radiolabeled probe was prepared using human MIF cDNA as a template and labeled with a random primer labeling kit using [a- 32P]dCTP. The membrane was washed twice with 23 SSC (16.7 mM NaCl, 16.7 mM sodium citrate) at 22°C for 5 min, twice with 0.23 SSC containing 0.1% SDS 65°C for 15 min, and 23 SSC at 22°C for 20 min prior to autoradiography. Quantitative densitometric analysis was performed using an MCID Image Analyzer (Fuji Film, Tokyo, Japan). Western blot analysis. Cell lysates prepared from the cultured melanocytes, G361, A375 and L32 melanoma cells were treated with 4 volumes of SDS-reducing buffer (60 mM Tris/HCl, pH 6.8, 2% SDS, 10% glycerol, 5% b-mercaptoethanol, and 0.025% bromophenol blue), and subjected to SDS–polyacrylamide gel electrophoresis (PAGE) as described (21). Proteins on the gel were electrophoretically transferred to a nitrocellulose membrane according to the procedure of Towbin et al. with minor modifications (22). The membrane was intensively washed with phosphate-buffered saline (PBS), and incubated with the polyclonal anti-human MIF antibody (1000-fold dilution of 4 mg/ml IgG) at room temperature for 1 h. After washing, the membrane was incubated with HRP-conjugated goat antibody against rabbit IgG at room temperature for 1 h. After the incubation, proteins were visualized with a Konica immunostaining kit as recommended in the manufacturer’s protocol. Immunohistochemistry. Immunohistochemical analysis of the cultured normal human melanocytes and G361 melanoma cells was carried out using the anti-human MIF antibody and fluorescenceconjugated anti-rabbit IgG antibody as described (16). In brief, the cultures of cells grown on coverslips were rinsed with PBS and immersed in 220°C acetone for 5 min. The fixed cell samples were incubated with the anti-human MIF antibody (1000-fold dilution of 4 mg/ml IgG) at 37°C for 1 h, followed by incubation with FITC-labeled goat-anti-rabbit IgG at 37°C for 30 min. Microscopic observation was carried out using a fluorescence microscope (Nikon, Tokyo, Japan). Assay for MIF. To measure the content of MIF secreted from the cultured melanocytes or melanoma cells, supernatants of these cells were measured essentially as described previously (17). In brief, the anti-human MIF antibody (4 mg/ml) dissolved in 50 ml of PBS was immobilized on each well of a 96-well microtiter plate. After incubation for 1 h at room temperature, the plate was washed three times with PBS. All wells were filled with PBS containing bovine serum albumin (1%) and left for 1 h at room temperature to block nonspecific binding. After removal of the blocking solution, the plate was washed three times with PBS containing 0.05% Tween 20 (washing buffer), samples of the supernatants were added to individual wells and incubated for a further 1 h at room temperature. After the plate was washed three times with the washing buffer, 50 ml of biotinconjugated anti-human MIF antibody was added. Following incubation for 1 h at room temperature, the plate was again washed three times with the washing buffer. Then avidin-conjugated HRP was added to individual wells, which were further incubated for 1 h at room temperature, and the plate was again washed with the washing buffer. Next, 50 ml of substrate solution containing 200 mg of o-phenylenediamine and 10 ml of 30% hydrogen peroxide in 10 ml of citrate-phosphate buffer (pH 5.0) was added. After incubation for 20
752
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS each assay, five fields were counted on each filter, and the counts were expressed as means with standard deviation. Angiogenesis assay. An angiogenesis assay using the dorsal air sac method was performed to evaluate the inhibitory effect of the anti-MIF antibody on the angiogenesis induced by melanoma cells as described (25). A Millipore chamber, which passes only soluble factors, was filled with 1 3 10 7 G361 cells in 200 ml of medium, and inoculated into the right flank of BALB/c mice. We divided mice into three groups; (i) negative control, no tumor cells in the chamber, (ii) positive control, G361 melanoma cells in the chamber, and (iii) anti-MIF antibody-treated test samples, tumor cells in the chamber and mice treated with the anti-rat MIF antibody. Animals in the treatment group received 200 ml of anti-MIF antibody (200 mg/body/ day) ip on days 1, 3, and 5, and those in the control group received the same amount of non-immune rabbit IgG. Mice were sacrificed on day 6. The area of neovascularization was measured using an MCID image analyzer, and the degree of tumor-induced angiogenesis was decided by the ratio of the area of blood vessels to the area in contact with the chamber.
SCHEME 1. Construction of the anti-sense MIF plasmid (pBKMIFAS). The full-length cDNA for human MIF was subcloned into the BamHI restriction site of pBK-CMV vector, a eukaryotic expression vector driven by the human cytomegalovirus (CMV) promoter. min at room temperature, the reaction was terminated with 50 ml of 1 N sulfuric acid. The absorbance at 492 nm was measured with an ELISA plate reader (Bio-Rad Model 3550). Construction of anti-sense MIF plasmid. The full-length cDNA for human MIF was prepared as described (23), and subcloned into the BamHI site of pBK-CMV vector, a eukaryotic expression vector driven by the human cytomegalovirus promoter. Subcloning into the BamHI restriction site yielded an insert in the anti-sense orientation (pBK-MIFAS) (Scheme 1). A plasmid, which contained a fragment of a human cDNA insert with 21 nucleotides deleted from the initiation coden at the 59-end in sense orientation, was also prepared as a control (pBK-MIFST). The orientation and proof of completeness of the insert were determined by means of restriction enzyme analyses and DNA sequencing using a DNA sequencer (ABI Model 373A). The G361 melanoma cells were transfected with either the pBKMIFAS or pBK-MIFST or the pBK-CMV vector alone linearized with SspI digestion by electroporation using a T820 optimizer (BTX, San Diego, CA) at 200 mV with a 99-ms pulse length and a single pulse. Drug selection was then started 24 h after the transfection by the addition of G418 (400 mg/ml) to the culture medium of DMEM containing 10% FCS. The cells were fed every three days with fresh medium for three weeks, G418-resistant colonies were separately transferred to individual wells of a 48-well plate and colonies were expanded.
RESULTS Northern blot analysis of MIF in melanocytes and G361 cells. We examined the level of MIF mRNA in melanocytes and melanoma cells by Northern blot analysis. Both the normal melanocytes, G361, A375 and L32 cells showed expression of MIF mRNA. The expression levels of G361, A375 and L32 cells were 13.6-, 10.6-, and 12.8-fold greater than that of the melanocytes, respectively, when MIF mRNA levels were normalized by each b-actin mRNA level (Fig. 1). These results suggested that MIF mRNA expression was exceedingly upregulated by malignant transformation. Western blot analysis of MIF in melanocytes and melanoma cells. To investigate the presence of MIF protein in the melanocytes and melanoma cells, Western blot analysis was performed using the antibody against human MIF. The immunoblot analysis revealed a specific positive band, migrating to about 12.5 kDa, corresponding to the molecular weight of recombinant human MIF (Fig. 2). The intensity of the bands of G361, A375, and L32 cells visualized by the immunostaining kit showed 3.1-, 2.5-, and 2.9-fold increase compared to that of the melanocytes, respectively. This
Cell proliferation assay. MTS assay was performed according to the manufacturer’s protocol. In brief, G361 melanoma cells and the anti-sense MIF transfected cells were seeded on each well of a 96well microtiter plate at the initial density of 5 3 10 3 cells/well and incubated for 96 h. Two hundred microliters of 2.5 mg/ml solution of MTS was added and incubated for 2 h at 37°C. The absorbance was determined on a plate reader read at 570 nm. Migration assay. To evaluate the effect of MIF on migration of melanoma cells, the Boyden chamber assay was performed as described (24). Briefly, the lower well was filled with 600 ml of medium (DMEM containing 10% FCS) in the presence of various amounts of MIF. The upper well contained G361 cell suspension (400 ml, 1 3 10 5 cell/ml medium). A cellulose acetate membrane filter was interposed between the two chambers. The chambers were kept in a humidified atmosphere of 5% CO 2 at 37°C for 4 h. Filters were subsequently fixed with methanol and acetone and stained with crystal violet. The number of cells that migrated into the filter and reached its lower side was determined microscopically. Three chambers were set up for
FIG. 1. Northern blot analysis of MIF mRNA expression in cultured melanocytes, and melanoma cells. Total RNA (15 mg) was electrophoresed through 1% agarose gel, transferred to a nylon membrane, and hybridized with a 32P-labeled full-length human MIF cDNA insert. Lane 1, melanocytes; lane 2, G361 cells; lane 3, A375 cells; lane 4, L32 cells. mRNA levels of b-actin were shown at the bottom of each lane.
753
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Western blotting analysis of the cultured human melanocytes and melanoma cells for MIF. Each cell sample was collected, electrophoresed, transferred to a nitrocellulose membrane, and visualized using a Konica immunostaining kit as described under Materials and Methods. Lane 1, prestained molecular marker (New England Biolabs); lane 2, recombinant human MIF (50 ng); lane 3, G361 cells (1 3 10 5 cells); lane 4, A375 cells (1 3 10 5 cells); lane 5, L32 cells (1 3 10 5 cells); lane 6, melanocytes (1 3 10 5 cells).
result indicated that much more MIF protein was produced in melanoma cells than in the melanocytes. Immunohistochemistry. The primary cultured human melanocytes were positively stained using the antibody against human MIF. The immunofluorescence study revealed that MIF was predominantly identified in the cytoplasm of melanocytes. In particular, strong positive staining was observed at the dendrites (Fig. 3A). Similarly, more enhanced positive cytoplasmic staining was observed in G361 melanoma cells (Fig. 3C). The nucleus was also clearly stained. No specific positive staining was observed in either the melanocytes (Fig. 3B) or G361 melanoma cells (Fig. 3D) reacted with nonimmune rabbit IgG.
cells by Western blot analysis (Fig. 5). To examine the anti-sense MIF effect on the cell proliferation rate, MTS assay was carried out. For cell growth, the proliferation rate of the transfectant G361 cells was markedly suppressed (35.2 6 11.5%) compared to nontreated control cells (100%) and those transfected with the vector alone (93.6 6 6.1%) or pBK-MIFST (95.2 6 5.1%) (Fig. 6). This indicated that MIF contributed to the cell growth.
Detection of MIF in the culture supernatants of melanocytes and melanoma cells. Contents of secreted MIF protein in the supernatants of cultured melanocytes and melanoma cells (1 3 10 5 cells/ml for each cells) were measured by ELISA after 48 h incubation. The MIF contents in the supernatants of the G361, A375, and L32 cells, 27.5 6 4.8, 20.4 6 2.5, and 18.6 6 1.8 ng/ml, respectively, were significantly higher than that of the melanocytes (6.8 6 2.2 ng/ml) (Fig. 4). The result showed that melanoma cells produced and secreted more MIF than the melanocytes.
Effect of MIF on melanoma cell migration. Migration of tumor cells is an important event for invasiveness and metastasis. To assess the role of MIF in tumorigenesis, migration of G361 melanoma cells in response to MIF was examined by the Boyden chamber method. We added various doses of MIF from 1 to 100 ng/ml in the lower chamber. Enhancement of G361 cell migration was observed in a dose-dependent manner (Fig. 7). Cell counts per field were 12 6 1.5, 13.7 6 0.9, 28.3 6 2.6, and 63.7 6 6.6 at the doses of 0, 1, 10, and 100 ng/ml of MIF, respectively. Since the MIF concentration can be up-regulated at the level of more than 80 ng/ml at loci, e.g., synovial fluids and follicular fluids (26, 27), it is considered that the local MIF concentration at the site of tumor lesions might reach such levels as to induce melanoma cell migration in vivo.
Effect of anti-sense MIF transfection on cell growth. The transfectant G361 melanoma cells with the pBKMIFAS were prepared as described under Materials and Methods. We isolated three different clonal cells in the presence of G418, and confirmed a decrease of the intracellular MIF protein level compared to the control
Effect of anti-MIF antibody on angiogenesis in vivo. Angiogenesis within the subcutaneous fascia of antiMIF antibody-treated mice was compared with that of the control. In the positive control mice bearing G361 cells in the chambers, we found that the cells significantly promoted angiogenesis of the subcutaneous
754
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. MIF expression on melanocytes and G361 melanoma cells. An immunofluorescence photograph shows human melanocytes and G361 cells stained with anti-human MIF antibody as described under Materials and Methods. MIF protein was observed in the cytoplasm of both melanocytes (A) and G361 cells (C). Note the strong positive staining at the dendrites and accentuation at the tips of dendrite processes on normal melanocytes, and nuclei were also clearly stained in G361 cells. Pre-immune IgG showed no specific positive reaction (B, D). (A, B, C, D; 3100.)
vessels (Fig. 8A). By contrast, tumor-associatedangiogenesis was significantly suppressed in the antiMIF antibody-treated group in comparison with the positive control (Fig. 8B). The ratios of the area of blood vessels to the areas in contact with the chamber were 13.5 6 4.0%, 51.2 6 5.0%, and 24.2 6 0.7%, in the negative control with medium alone, positive control with the tumor cells, and anti-MIF antibody-treated groups, respectively. DISCUSSION Melanocytes are localized in the basal layers of the epidermis. They interact with the surrounding keratinocytes via their dendrites, and exert their biological functions such as pigmentation. In this event, melanocytes act in concert with keratinocytes to pigment the skin. There is emerging evidence indicating the involvement of melanocytes in the events of cutaneous inflammation and immunity via secretion of a variety of immune and inflammatory mediators. In this study,
we demonstrated the expression of MIF mRNA and production of MIF protein in human melanocytes and melanoma cells. This fact suggested that MIF, a putative inflammatory cytokine (28), affected or regulated the pathophysiological states of the skin tissue. In particular, it should be noted that MIF mRNA is highly expressed and that very much MIF protein was present in the cytoplasm and nucleus of melanoma cells, suggesting its role in tumor cell growth. MIF was originally identified as a lymphokine concentrating macrophages at inflammatory sites and is a potent activator of macrophages (12, 28). This protein has been detected in various tissues, including cells in the cornea and reproductive organs, and in osteoblasts (17–19). These cells release MIF protein into extracellular spaces in response to various stimuli such as lipopolysaccharide and TNF-a (13, 15). The immunohistochemical results in this study demonstrated the presence of a large amount of MIF in dendrites of melanocytes. This suggested the possibility that MIF protein may be readily released to the epidermis by
755
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. MIF content in the supernatants of cultured normal human melanocytes and melanoma cells. The MIF content in the culture supernatants (1 3 10 5 cells/ml for each cells) was measured by ELISA as described under Materials and Methods. The values were means 6 SD of at least six experiments. Lane 1, melanocytes; lane 2, G361 cells; lane 3, A375 cells; lane 4, L32 cells. *P , 0.01; **P , 0.005.
various environmental stimuli, such as ultraviolet irradiation, which in consequence may affect the pathophysiological state of the epidermis. Indeed, we demonstrated that MIF secretion was markedly stimulated by low doses of ultraviolet irradiation (29). Accordingly, it is speculated that MIF in melanocytes may play a pivotal role in cutaneous immunity and inflammatory processes. It is of great interest that exceedingly high expression of MIF was observed in melanoma cells compared to that of normal melanocytes. This finding indicated that MIF may also participate in the regulation of cell growth as a novel growth factor. From the data available to date, cultured melanoma cells have been reported to produce a variety of growth factors and cytokines, including IL-1 and platelet-derived growth factor (8, 9). These biological mediators are known to
FIG. 5. Production of MIF protein in G361 melanoma cells and anti-sense MIF plasmid transfected cells. Western blot analysis was carried out to examine the expression of MIF protein by each cell type. The MIF protein bands were visualized by a Konica HRPimmunostaining kit. Lane 1, molecular marker; lane 2, recombinant human MIF (100 ng); lane 3, G361 cells (2 3 10 5 cells) transfected pBK vector alone; lane 4, G361 cells (2 3 10 5 cells) transfected pBK-MIFST; lanes 5–7, G361 cells (2 3 10 5 cells) transfected with pBK-MIFAS.
FIG. 6. Effect of anti-sense MIF plasmid transfection on cell growth of G361 melanoma cells. Growth of G361 cells (initial count, 5 3 10 3 cells) transfected with anti-sense MIF plasmid was examined by MTS assay. Lane 1, control (G361 cells); lane 2, the cells transfected with pBK vector alone; lane 3, the cell transfected with pBKMIFST; lane 4, the cell transfected with pBK-MIFAS. The result is the mean 6 SD of at least three different experiments. *P , 0.01.
be critical for malignant transformation through their direct effects on tumor cell growth. For example, IL-1 has been reported to be mitogenic for a number of cell types. However, the biological effects of IL-1 released by melanoma cells are not uniform, showing both positive and negative effects on cell proliferation (9, 30). Nonetheless, it is conceivable that MIF may stimulate cell proliferation via these cytokines because MIF has the potential to induce cytokines stimulating cell growth such as IL-1 and TNF-a (13, 15). In association with cell growth, it was reported that MIF mRNA expression is correlated with cell differen-
FIG. 7. Migration of G361 cells in response to MIF. The culturing method and migration assay are described under Materials and Methods. The doses of MIF examined were from 0 to 100 ng/ml. The values were means 6 SD of at least 5 experiments. Lane 1, control; lane 2, 1 ng/ml; lane 3, 10 ng/ml; lane 4, 100 ng/ml. *P , 0.01; **P , 0.005.
756
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
est that high expression of MIF was discovered in metastatic prostate cancer (33). Moreover, in the present study, we found that anti-sense MIF cDNA transfection markedly suppressed the cell growth of melanoma cells. Recently, consistent with our results, it was reported that MIF stimulated the proliferation of NIH/3T3 cells with the activation of mitogenactivated protein kinase (34). Based on these facts, it is speculated that MIF, present in a large amount in melanoma cells, might be involved in the mechanism of cell proliferation. It has been suggested that MIF affects cellular proliferation as a growth factor-induced delayed earlyresponse gene (35). We identified the increased expression of MIF mRNA during corneal wound healing after penetrating injury concomitant with keratocytes migration (36). Because corneal injury can induce immediate-early-response genes (c-fos and c-jun), the upregulation of MIF after the injury as a delayedearly-response gene appears to be reasonable. This fact supports the idea that MIF is profoundly involved in cell growth at the gene-transcription level. Moreover, we demonstrated that MIF possessed the potential to enhance invasiveness of melanoma cells and stimulated angiogenesis. Judging from these observations, it is conceivable that MIF could be involved not only in non-tumor cell growth in the event of wound healing but also in tumor cell invasion and neovascularization. Finally, it is widely accepted that cytokines play an important role in responses of the host with a malignant tumor. It is very likely that MIF functions as a pivotal element for cellular proliferation and differentiation in autocrine and paracrine manners. Very recently, consistent with our results, MIF was reported to contribute to growth and angiogenesis of murine lymphoma (37). These findings, including ours, could be a new starting point to investigate the broadspectrum pathophysiological roles of MIF in tumorigenesis beyond the immunological system. ACKNOWLEDGMENTS FIG. 8. Histological evidence for the effect of anti-MIF antibody on tumor-associated angiogenesis in vivo. Effectiveness of the antirat MIF polyclonal antibody in mediating suppression of G361 cellinduced angiogenesis was examined by the subcutaneous dorsal air sac technique. On day 6, angiogenesis within the subcutaneous fascia of anti-MIF antibody-treated mice was compared with that of the control (n 5 5). Typical pictures for neovascularization of nonimmune IgG-treated (A) and anti-rat MIF antibody-treated (B) mice are shown. Arrows show typical tumor-associated neovascularization.
This work was supported by a Grant-in-Aid for Research (09670144) from the Ministry of Education, Science, and Culture of Japan. We are grateful to Yuka Mizue of Sapporo Immunodiagnostic Laboratory for her technical assistance.
REFERENCES
tiation of lens cells (31), suggesting the involvement of this protein in cellular proliferation and differentiation. We demonstrated that MIF was predominantly expressed in highly proliferative basal cells of human epidermis and in leukemia cells (16, 32). It is of inter757
1. Gordon, P. R., Mansur, C. P., and Gilchrest, B. A. (1989) J. Invest. Dermatol. 92, 565–572. 2. Morelli, J. G., and Norris, D. A. (1993) J. Invest. Dermatol. 100, 191–195. 3. Ramirez-Bosca, A., Bernd, A., Werner, R., Dold, K., and Holzmann, H. (1992) Arch. Dermatol. Res. 284, 358 –362. 4. De Kuca, M., D’Anna, F., Bondanza, S., Franzi, A. T., and Cancedda, R. (1988) J. Cell. Biol. 107, 1919 –1926. 5. Swope, V. P., Abdel-Malek, Z. A., Kassem, L. M., and Nordlund, J. J. (1991) J. Invest. Dermatol. 96, 180 –185.
Vol. 264, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6. Swope, V. P., Sauder, D. N., McKenzie, R. C., Sramkoski, R. M., Krug, K. A., Babcock, G. F., Nordlund, J. J., and Abdel-Malek, Z. A. (1994) J. Invest. Dermatol. 102, 749 –753. 7. Zachariae, C. O. C., Thestrump-Pedersen, K., and Matsushima, K. (1991) J. Invest. Dermatol. 97, 593–599. 8. Ko¨ck, A., Schwarz, T., Urbanski, A., Peng, Z., Vetterlein, M., Micksche, M., Ansel, J. C., Kung, H. F., and Luger, T. A. (1989) J. Natl. Cancer Inst. 81, 36 – 42. 9. Bennicelli, J. L., Elias, J., Kern, J., and Guerry, D. (1989) Cancer Res. 49, 930 –935. 10. Armstrong, C. A., Tara, D. D., Hart, C. E., Ko¨ck, A., Luger, T. A., and Ansel, J. C. (1992) Exp. Dermatol. 1, 37– 45. 11. Mattei, S., Colombo, M. P., Melani, C., Silvani, A., Parmiani, G., and Herlyn, M. (1994) Int. J. Cancer 56, 853– 857. 12. Bloom, B. R., and Bennett, B. (1966) Science 153, 80 – 82. 13. Calandra, T., Bernhagen, J., Mitchell, R. A., and Bucala, R. (1994) J. Exp. Med. 179, 1895–1902. 14. Bernhagen, J., Calandra, T., Mitchel, R. A., Martin, S. B., Tracey, K. J., Voelter, W., Manogue, K. R., Cerami, A., and Bucala, R. (1993) Nature 365, 756 –759. 15. Calandra, T., Bernhagen, J., Mets, C. N., Spiegel, L. A., Bacher, M., Donnely, T., Cerami, A., and Bucala, R. (1995) Nature 377, 68 –71. 16. Shimizu, T., Ohkawara, A., Nishihira, J., and Sakamoto, W. (1996) FEBS Lett. 381, 188 –202. 17. Matsuda, A., Kotake, S., Tagawa, Y., Matsuda, H., and Nishihira, J. (1996) Immunol. Lett. 53, 1–5. 18. Suzuki, H., Kanagawa, H., and Nishihira, J. (1996) Immunol. Lett. 51, 141–147. 19. Onodera, S., Suzuki, K., Matsuno, T., Kaneda, K., and Nishihira, J. (1996) Immunology 89, 430 – 435. 20. Sakai, M., Nishihira, J., Hibiya, Y., Koyama, Y., and Nishi, S. (1994) Biochem. Mol. Biol. Int. 33, 439 – 446. 21. Schagger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368 –379.
22. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 23. Sugimoto, H., Suzuki, M., Nakagawa, A., Tanaka, I., and Nishihira, J. (1996) FEBS Lett. 389, 145–148. 24. Michel, G., Kemeny, L., Peter, R. U., Beetz, A., Ried, C., Arenberger, P., and Ruzicka, T. (1992) FEBS Lett. 305, 241–243. 25. Folkman, J., Merler, E., Abernathy, C., and Williams, G. (1971) J. Exp. Med. 133, 275–288. 26. Onodera, S., Tanji, H., Suzuki, K., Kaneda, K., Mizue, Y., Sagawa, A., and Nishihira, J. (1999) Cytokine 11, 163–167. 27. Wada, S., Kudo, T., Kudo, M., Sakuragi, N., Hareyama, H., Nishihira, J., and Fujimoto, S. (1999) Hum. Reprod. 14, 395– 399. 28. Bucala, R. (1996) FASEB J. 10, 1607–1613. 29. Shimizu, T., Abe, R., Ohkawara, A., and Nishihira, J. (1999) J. Invest. Dermatol. 112, 210 –215. 30. Lachman, L. B., Dinarello, C. A., Llanca, N. D., and Fildler, I. J. (1986) J. Immunol. 136, 3098 –3102. 31. Wistow, G. J., Shaughnessy, M. P., Lee, D. C., Hodin, J., and Zelenka, P. S. (1993) Proc. Natl. Acad. Sci. USA 90, 1272– 1275. 32. Nishihira, J., Koyama, Y., and Mizue, Y. (1996) Biochem. Mol. Biol. Int. 40, 861– 869. 33. Meyer-Siegler, K., and Hudson, P. B. (1996) Urology 48, 448 – 452. 34. Mitchell, R. A., Metz, C. N., Peng, T., and Bucala, R. (1999) J. Biol. Chem. 18, 18100 –18106. 35. Lanahan, A., Williams, J. B., Sanders, L. K., and Nathans, D. (1992) Mol. Cell. Biol. 12, 3919 –3929. 36. Matsuda, A., Tagawa, Y., Matsuda, H., and Nishihira, J. (1997) Invest. Ophthalmol. Vis. Sci. 38, 1555–1562. 37. Chesney, J., Metz, C., Bacher, M., Peng, T., Meinhardt, A., and Bucala, R. (1999) Mol. Med. 5, 181–191.
758