Dr. Bhavesh Vaidya -
Insulin-like Growth Factor-I-induced Migration of Melanoma Cells Is Mediated by Interleukin-8 Induction1
Kapaettu Satyamoorthy2,3, Gang Li2, Bhavesh Vaidya, Jiri Kalabis and Meenhard Herlyn4
Wistar Institute [K. S., G. L., B. V., J. K., M. H.] and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [G. L., M. H.]
Abstract
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Abstract
Introduction
Results
Discussion
Materials and Methods
References
Successive events of growth factor-induced autocrine and paracrine activation promote tumor growth and metastasis. Insulin-like growth factor-I (IGF-I) stimulates melanoma cells to grow, survive, and migrate. Interleukin-8 (IL-8) is produced by melanoma cells and has been correlated with melanoma metastasis, but the biological functions of this cytokine have not been elucidated. We show here that IGF-I-induced migration of melanoma cells could be inhibited by neutralizing antibody against IL-8. IGF-I overexpression induced IL-8 production in melanoma cells, especially in biologically early melanomas by accelerating its transcription rate via activation of mitogen-activated protein kinase pathway. IGF-I treatment phosphorylated c-Jun and stimulated the binding of AP-1 but not NF-B to the IL-8 promoter. These data identify IL-8 as a new target of IGF-I in melanoma and suggest that some of the biological functions of IGF-I are mediated by IL-8.
Introduction
TOP
Abstract
Introduction
Results
Discussion
Materials and Methods
References
Melanoma arises through disruption of homeostatic events of normal melanocytes. During the progression from RGP5 (1) to VGP and metastatic melanoma, melanoma cells produce a variety of growth factors and cytokines that enables them to progress to a more aggressive phenotype (1) . Besides autocrine stimulation, the cross-talk between melanoma cells and their microenvironment via growth factors and cytokines modulates cell proliferation, differentiation, adhesion, migration, invasion, apoptosis, stromal formation, and angiogenesis (2 , 3) . Many growth factors/cytokines are involved in this intricate network. Primarily among them are basic fibroblast growth factor, IGF-I, PDGF-A and -B, transforming growth factor- and -ß, IL-8, VEGF, and hepatocyte growth factor/scatter factor (for review, see Ref. 3 ).
In human skin, IGF-I is mainly produced by stromal fibroblasts that are activated by melanoma-derived PDGF (4) . Previously, it has been shown that IGF-I is critical for survival, growth, and motility of biologically early melanoma cells (5, 6, 7, 8, 9, 10) . These actions are apparently mediated by the type 1 IGF-IR (11) , which is expressed by all melanocytic cells (10) , and the expression increases with progression (12) . Down-regulation of IGF-IR with antisense oligonucleotides inhibited melanoma growth in vivo (13) . Mice transgenic for IGF-I under a keratin 1 promoter showed marked thickening of the dermis and hypodermis and prone to transformation (14) , suggesting that IGF-I is a critical factor in tumor development and progression.
IL-8 is not expressed by normal melanocytes (15, 16, 17) but by all human melanoma cells with the nontumorigenic RGP melanoma cells secreting significantly lower levels of IL-8, compared with VGP or metastatic melanoma cells (15) . IL-8 may sustain an autocrine effect because melanoma cells express its receptors IL-8RA and IL-8RB (18) . In certain cancer types, IL-8 can stimulate growth (19) , migration (20) , proteolysis (21 , 22) , and angiogenesis (23 , 24) ; however, the functions of IL-8 in melanoma have not been well studied.
In the present study, we demonstrate a new nexus between paracrine and autocrine effects of IGF-I and IL-8. IL-8 was transcriptionally induced by IGF-I in melanoma cells. IGF-I-induced migration of melanoma cells could be inhibited by neutralizing antibody against IL-8, suggesting that some of the biological functions of IGF-I are mediated by IL-8.
Results
TOP
Abstract
Introduction
Results
Discussion
Materials and Methods
References
IGF-I is critical for survival, growth, and motility of melanocytic cells (5 , 10) , and its multiple roles in melanoma progression might be mediated by a number of downstream pathways and effectors. In a modified Boyden chamber assay, SBcl2 melanoma cells were allowed to migrate through 8-µm pores of polycarbonate filters after treatment with IGF-I and compared with control cells. IGF-I was able to induce 3-fold increase in the migration (Fig. 1A) . This migratory effect was inhibited by >90% by neutralizing antibodies to either IGF-I or IL-8 (Fig. 1A) , suggesting that the pro-migratory effect of IGF-I is mediated by secreted IL-8. The specificity of the blocking experiments was checked by including a nonspecific antibody, which showed no effect on IGF-I-stimulated migration. In support of this observation, we evaluated the expression of IL-8 receptors, IL-8RA and IL-8RB, by Western blotting. All melanoma cells examined expressed both receptors in comparable amounts (Fig. 1B) , suggesting a role of IL-8 as an autocrine factor.
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Fig. 1. IGF-I-induced cell migration is mediated by IL-8. In A, SBcl2 cells with or without IGF-I treatment were tested for migration using NeuroProbe chamber as described in "Materials and Methods." Neutralizing antibodies against IGF-I (10 µg/ml) or IL-8 (20 µg/ml) was included to determine the functional contribution of individual molecules on cell migration. Nonspecific antibody was used to determine the blocking specificity. Data were gathered from five independent assays for each treatment. B, expression of IL-8 receptor in melanoma cells. Total cell extracts were revolved on 12% SDS-PAGE gel, and monoclonal antibodies against IL-8RA and IL-8RB were used to detect both receptors.
By comparing mRNA expression profile of IGF-I-treated SBcl2 cells with untreated cells using cDNA microarray analysis, we identified IL-8 as one of the genes whose mRNA levels were substantially increased (15-fold) after IGF-I treatment. Along with IL-8, VEGF expression was also increased after IGF-I treatment (data not shown), which has been shown in other cell types (25, 26, 27) . To confirm the microarray results, SBcl2 cells were treated with IGF-I either by IGF-I adenoviral transduction at 20 pfu/cell (Fig. 2) or using recombinant IGF-I added to culture media at 100 ng/ml (Fig. 3) . Our previous studies showed that under either condition, the same signaling pathways were activated, and the same biological effects were observed (10) . Increased expression of IL-8 was detected at both mRNA (Fig. 2, A and B) and protein (Fig. 2C) levels after IGF-I adenoviral transduction. The increased IL-8 expression was maintained for >96 h (Fig. 2C) because of continuous stimulation by IGF-I adenoviruses. mRNA induction was also observed in recombinant IGF-I-treated (100 ng/ml) SBcl2 cells (Fig. 3A) . A concentration of IGF-I as low as 10 ng/ml induced 2-fold increase in IL-8 production after 24 h, whereas 100 ng/ml IGF-I induced 8-fold increase over endogenous level (Fig. 3B) . However, 500 ng/ml IGF-I did not substantially induce more IL-8 than 100 ng/ml, possibly because of saturation effect.
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Fig. 2. Induction of IL-8 by IGF-I adenoviral transduction in melanoma. In A, SBcl2 melanoma cells were transduced with IGF-I or LacZ adenoviruses at 20 pfu/cell. Total RNA was isolated at 0, 12, 24, 48, and 72 h after gene transduction. Total RNA (10 µg/lane) was separated on a formaldehyde-agarose gel, transferred to nylon membrane, and probed with radiolabeled IL-8 and GAPDH cDNA. In B, the data in A were quantitated using a densitometer and are reported in arbitrary units relative to IL-8 mRNA expression at time 0. Densities were corrected for loading differences by normalizing for variances in GAPDH expression. In C, time course of IL-8 secretion was tested by ELISA. SBcl2 melanoma cells with different treatments were cultured in PF media for indicated times. The supernatants in triplicates were collected, and IL-8 released into the medium was measured by ELISA. Control, no treatment; IGF-I, IGF-I/Ad5 at 20 pfu/cell; LacZ, LacZ/Ad5 at 20 pfu/cell.
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Fig. 3. Induction of IL-8 by recombinant IGF-I in melanoma. In A, SBcl2 melanoma cells were treated with recombinant IGF-I (100 ng/ml) or vehicle. Total RNA was isolated at 0, 4, and 24 h. Total RNA (10 µg/lane) was separated on a formaldehyde-agarose gel, transferred to nylon membrane, and probed with radiolabeled IL-8. GAPDH and 28S rRNA were used as loading control. In B, concentration dependence of IL-8 induction. SBcl2 cells were incubated with various concentrations of IGF-I for 24 h. IL-8 secretion in triplicates was quantitated by ELISA.
To exclude the possibility that IL-8 induction by IGF-I is restricted to SBcl2 cells, conditioned media (24 h) from seven melanoma cell lines treated with IGF-I (100 ng/ml) were measured by ELISA (Fig. 4) . The cell lines used included primary RGP/VGP melanoma (SBcl2, WM1552C, and WM35) and metastatic melanoma (WM852, WM1158, WM9, and 1205Lu). Endogenously secreted IL-8 levels ranged from 1 to 35 ng/105cells/24 h, and there was no clear correlation between levels of IL-8 secretion and progression stages (Fig. 4 , dotted bars). RGP melanoma cells (SBcl2, WM1552C, and WM35) responded to IGF-I treatment and induced the secretion of IL-8 levels 2–4-fold after 24 h (Fig. 4 , shaded bars). On the contrary, VGP and metastatic melanoma cells did not respond to IGF-I treatment with the exception of WM9.
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Fig. 4. IL-8 induction by IGF-I in melanoma cell lines. Melanoma cells were treated with IGF-I (100 ng/ml) or vehicle (Control). IL-8 levels were measured in triplicates after 24 h by ELISA.
It has been shown that IGF-I exert its effects through both MAP kinase and phosphatidylinositol 3'-kinase pathways (28) . To test which of the two pathways is involved in IL-8 induction, specific inhibitors were included in the assays. As shown in Fig. 5 , in SBcl2 cells, the induction of IL-8 secretion can be completely inhibited by MAP kinase inhibitor PD98059 but not by phosphatidylinositol 3'-kinase inhibitor Wortmannin. Similar effects were observed in WM35 RGP primary melanoma cell line (data not shown). Noticeably, PD98059 inhibited basal level of IL-8, suggesting that besides IGF-I, another MAP kinase-dependent process is responsible for basal expression of IL-8. To determine whether IL-8 induction is a specific effect of IGF-I, we also tested the ability of basic fibroblast growth factor and PDGF to induce IL-8. Neither of them could increase IL-8 production (data not shown).
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Fig. 5. Effect of Wortmannin and PD98059 on IGF-I-induced IL-8 expression. SBcl2 cells were transduced with IGF-I (20 pfu/cell) or LacZ (20 pfu/cell) and treated with indicated drugs (Wortmannin, 40 µM; PD98059, 10 µM) for 24 h, and IL-8 levels were measured by ELISA and expressed in percentages of IL-8 secretion by cells without adenoviral treatment.
The mechanisms by which IGF-I increased IL-8 expression were then defined. To examine whether IGF-I affected the stability of IL-8 mRNA, SBcl2 cells were exposed to 100 ng/ml IGF-I or vehicle for 16 h before addition of 5 µg/ml actinomycin D. Total RNA was extracted from the cells at various times and subject to Northern blotting. As shown in Fig. 6A , the half-life of IL-8 mRNA was not significantly different in IGF-I treated cells from the control, suggesting that increased IL-8 production is not because of increased IL-8 mRNA stability. Nuclear run-on assay was then used to determine whether IGF-I increased the rate of transcription for IL-8 mRNA (Fig. 6B) . IGF-I induced IL-8 mRNA transcription rate by 4-fold within 4 h. Thereafter, the rate remained significantly above the control levels. Similar observations were also made in WM35 cells (data not shown). These data suggest that induction of IL-8 by IGF-I is transcriptionally controlled.
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Fig. 6. Induction of IL-8 mRNA by IGF-I is dependent on increased transcription. In A, IGF-I treatment does not change stability of IL-8 mRNA. SBcl2 cells were exposed to 100 ng/ml IGF-I or vehicle (Control) for 16 h before the addition of 5 µg/ml actinomycin D. Total RNA was extracted from the cells at the indicated times after actinomycin D administration, fractionated by agarose gel electrophoresis, and transferred to a nylon membrane. The Northern blot was then hybridized to cDNA probes for IL-8 and GAPDH (loading control). The corrected densities are expressed as percentages of the value at time 0 and plotted on a logarithmic scale. One representative of three replicates is shown. B, nuclear run-on assay. After a 24-h serum starvation, SBcl2 cells were treated with or without IGF-I (100 ng/ml). Cells were harvested at different time points. Nuclei were purified from the cells, and nuclear run-on assays were carried out as described in "Materials and Methods." The resulting autoradiographs were quantitated using a densitometer and represented as arbitrary units.
Because it has been shown that IL-8 expression is mediated by differential activation and binding of inducible transcription factors, such as AP-1 and NF-B, to the promoter of the IL-8 gene (29) , the involvement of AP-1 and NF-B in IGF-I-mediated IL-8 induction was tested by EMSA. As shown in Fig. 7A , AP-1 binding was considerably increased in IGF-I-treated SBcl2 cells compared with control. On the other hand, 1205Lu cells constitutively showed high binding capacity to AP-1, which correlated with high levels of endogenous IL-8 production, and did not show significant changes between the groups in binding to the AP-1 element. Previous studies have shown that AP-1-induced transcription is dependent on phosphorylation of serine residues in the transactivation domain of c-Jun (30) . Interestingly, 1205Lu cells showed constitutive phosphorylation of c-Jun proteins regardless of IGF-I treatment, whereas in SBcl2 cells, IGF-I induced phosphorylation of c-Jun (Fig. 7B) . These data suggest that induction of AP-1 DNA-binding activity is mediated by the phosphorylation of c-Jun in SBcl2 cells. In contrast to AP-1, no difference in the binding pattern of NF-B was observed in either SBcl2 (Fig. 7C) or 1205Lu cells (data not shown) after IGF-I treatment.
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Fig. 7. EMSA analyses of AP-1 and NF-B binding to IL-8 promoter elements. In A, nuclear proteins from SBcl2 and 1205Lu melanoma cells were extracted from control or IGF-I-treated (100 ng/ml, 30 min) cells. Radiolabeled AP-1 binding element was incubated with the nuclear extracts, separated on a 5% nondenaturing polyacrylamide gel, and analyzed by autoradiography. Unlabeled wild-type AP-1-binding element (wtAP-1) was used to confirm the specificity of the protein-DNA binding. B, c-Jun phosphorylation after IGF-I treatment. SBcl2 and 1205Lu cells were serum starved for 24 h and treated with IGF-I (100 ng/ml) for 30 min, and equal amounts of protein extracts were separated on 10% SDS-polyacrylamide gel. Western blotting was performed using phosphorylation-specific c-Jun antibody and total c-Jun antibody. ß-Actin was probed as loading control. In C, nuclear extracts were analyzed by EMSA for NF-B-binding activity after IGF-I treatment. SBcl2 and 1205Lu showed the same pattern of mobility shift; therefore, only SBcl2 is shown. NS, nonspecific binding.
Discussion
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Abstract
Introduction
Results
Discussion
Materials and Methods
References
Oncogenic transformation appears as the result of multiple mechanisms by which growth, differentiation, and apoptosis pathways are perturbed and deregulated. Sequential activation of growth factors is crucial for melanoma growth and invasion and may provide growth advantage by interacting with the microenvironment. In this study, we describe a role for IGF-I in the regulation of IL-8 in human melanoma cells. We show that: (a) IGF-I induces IL-8 expression in various melanoma cells, especially in early stage melanomas; (b) the induction is because of increased transcription rate, not enhanced stability; (c) IGF-I functions through MAP kinase/c-Jun-NH2-terminal kinase/c-Jun/AP-1 pathway; and (d) IL-8 is one of the major downstream effectors that are responsible for pro-migratory effects of IGF-I in melanoma cells.
IGF-I and IL-8 are both critical proteins in melanoma progression. Whereas IGF-I imparts its mitogenic, motogenic, and antiapoptotic effects through paracrine actions, IL-8 functions through autocrine mechanisms. We have observed that although melanoma cells do not secrete IGF-I, they possess functional IGF-IR, which can be triggered to subsequently activate extracellular signal-regulated kinase 1 and 2 and Akt/protein kinase B via phosphorylation (10) . The precise effects of IL-8 on tumor progression are only beginning to be understood. Melanoma cells express IL-8 receptors IL-8RA and IL-8RB, both of which are G-protein linked seven-transmembrane molecules. Melanoma cells, but not normal melanocytes, synthesize IL-8 (16 , 31) , which can be further stimulated by IGF-I. The convergence of signaling pathways of IL-8 and IGF-I may provide integrated cellular responses, such as proliferation, survival, and tumor angiogenesis. In fact, it has been shown that IGF-I and IL-8 have parallel mitogenic (5 , 19) , motogenic (9 , 20 , 22 , 32) , angiogenic (25 , 33) , and pro-proteolytic (22 , 34) functions in various cancers, including melanoma.
On the basis of the facts that IGF-I increases expression of IL-8 and that IGF-I and IL-8 have extensive parallel functions, we proposed that IL-8 is the downstream effector of IGF-I and is, in part, responsible for the biological actions of IGF-I. In this study, we provide evidence that antibody blockage of IL-8 signaling could abolish IGF-I-induced cell migration, suggesting that the motility effect of IGF-I on melanoma is mediated by subsequent events of IL-8 induction and autocrine stimulation. Thus, the present investigation adds a new and important activity to the IGF-I repertoire.
Angiogenesis has been shown to be a significant prognostic factor (24 , 35, 36, 37) and a major requirement for tumor outgrowth and metastasis in melanoma (15 , 38, 39, 40) . Whether angiogenic effect of IGF-I is mediated by IL-8 induction remains unknown. However, the facts that both IGF-I and IL-8 are angiogenic (15 , 23 , 25 , 33 , 41) and that IGF-I increases expression of IL-8 point to this possibility. Alternatively, the angiogenic activity of IGF-I could also be mediated by induction of VEGF (25, 26, 27) or by induction of both IL-8 and VEGF, as detected by our microarray profiling. The involvement of multiple angiogenic factors has been a major obstacle in antiangiogenic treatment of malignant melanoma, and efficient treatment may require identification and blockage of common functional features or common signaling cascades of multiple angiogenic factors. Our study suggests that IGF-I might be a master regulator of migration and angiogenesis in melanoma and justifies targeting IGF-I signaling pathway as a potential therapeutic approach to reverse melanocytic malignancy.
Materials and Methods
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Abstract
Introduction
Results
Discussion
Materials and Methods
References
Reagents.
MCDB153, L15, and insulin were purchased from Sigma Chemical Co. (St. Louis, MO). FBS was from Hyclone (Logan, UT). Recombinant IGF-I, anti-IGF-I, and anti-IGF-IR antibodies, PD98059, and Wortmannin were obtained from Calbiochem (San Diego, CA). IL-8 antibody was from R&D Systems, Inc. (Minneapolis, MN). IL-8RA and IL-8RB antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphospho-c-Jun (Ser 63) antibody was from New England Biolabs (Beverly, MA).
Cell Culture.
The following melanoma cell lines were used, which have been described before (42) : WM1552C, SBcl2, WM115, WM1341D, 1205Lu, WM852, WM9, and WM1158. Melanoma cells were maintained in "2% Tumor" medium composed of MCDB153:L15 (4:1) supplemented with 2% FBS and 5 µg/ml insulin, unless otherwise stated. When a specific growth factor was tested, FBS and insulin were omitted from the medium ("PF medium").
Adenoviruses.
The generation of IGF-I adenovirus has been described (10) . Control adenoviral vector containing the LacZ cDNA (LacZ/Ad5) was provided by the Vector Core at the Institute for Human Gene Therapy, University of Pennsylvania (Philadelphia, PA). Adenoviruses were purified by CsCl density centrifugation and tittered on 293 cells using agar overlay technique (43) .
ELISA.
IL-8 protein in the culture supernatants was measured with an ELISA kit (Biosource International, Camarillo, CA), according to manufacturer’s protocol. All experiments were repeated three times.
Northern Blotting.
Total RNA was isolated using Tri Reagent (MRC, Inc., Cincinnati, OH), separated on 1.2% formaldehyde-agarose gel and transferred on to nylon membranes (MSI, Westboro, MA). Radioactive probes were prepared using High Prime DNA labeling Kit (Roche Diagnostics, Indianapolis, IN) and hybridized overnight to the membranes at 65°C. The membranes were visualized by autoradiography and quantitated using NIH Image software.
Nuclear Run-on Assay.
Melanoma cells were grown to 90% confluence in 60-mm dishes. After 24-h starvation in PF medium, cells were treated with IGF-I (100 ng/ml) or left untreated. Cells were harvested at different times, and nuclei were purified from these cells using run-on lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40 in DEPC-H2O]. The purity of the nuclei was examined by microscopy. The nuclei were resuspended in 200 µl of storage buffer [50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA in DEPC- H2O] and kept at -70°C until use. For each reaction, 100 µl of thawed nuclei were mixed with 30 µl of 5 x run-on buffer [25 mM Tris-HCl (pH 8.0), 12.5 mM MgCl2, 750 mM KCl, and 1.25 mM each of ATP, GTP, and CTP], supplemented with 100 µCi of -32P-UTP and 0.06% Sarkosyl, to a final volume of 150 µl. The reaction mixtures were incubated for 30 min at 30°C, followed by the addition of 15U DNase I and incubation for another 15 min. TRIzol was used to extract total RNA from the reaction mixtures. The extracted probes were then hybridized to nylon membrane slot-blotted with IL-8 cDNA (0.5 µg/dot). After hybridization, the signals were captured by X-ray films and quantified using NIH Image software.
mRNA Stability.
Melanoma cells were grown to 90% confluence in 60-mm dishes. After a 16-h incubation in PF medium with or without recombinant IGF-I (100 ng/ml), actinomycin D (5 µg/ml) was added to block transcription. Cells were then harvested 0, 1, 2, 4, 6, and 8 h after the addition of actinomycin D, and Northern blotting was performed for IL-8 mRNA expression. To correct for differences in loading, GAPDH probe was hybridized to the same blot. The corrected densities were calculated as percentages of the value at time 0 and plotted on a logarithmic scale.
Western Blotting.
Cell lysates were prepared using radioimmunoprecipitation assay buffer. Protein estimation was performed using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Equal amounts of proteins were loaded onto 10% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membranes (MSI), and probed with primary and secondary antibodies. Signals were developed by enhanced chemiluminescence kit (Amersham, Farmington, IL).
EMSA.
Cells were starved in PF medium for 24 h and treated with 100 ng/ml IGF-I for 30 min. Nuclear and cytoplasmic fractions were isolated by hypotonic solution followed by nuclear protein extraction (44) . Oligonucleotides derived from IL-8 promoter used in this study are AP-1 (wild type) 5'-AGT GTG ATG ACT CAG GTT TG-3'; AP-1 (mutant) 5'-AGT GTG TAT CTC AGG TTT G- 3'; NF-B (wild type) 5'-ATC GTG GAA TTT CCT CTG ACA-3'; and NF-B (mutant) 5'-ATC GTT AAC TTT CCT CTG ACA-3'. Oligonucleotides were end labeled with -32P-ATP. The reaction mixture consisted of 10 mM HEPES (pH 7.6), KCl (60 mM), 1 mM DTT, protease inhibitors (1 µg/ml leupeptin, pepstatin, and aprotinin and 10 µM phenylmethylsulfonyl fluoride), and 1 µg of poly (dI-dC). The reaction mixture was incubated at room temperature for 20 min with radiolabeled oligonucleotides. The preincubation with the antibodies for supershift analysis was performed using anti-p65 and anti-p50 antibodies obtained from Santa Cruz Biotechnology. After the incubation, the reaction mixtures were separated in 0.5XTBE in a 5% nondenaturing polyacrylamide gel, dried, and exposed for autoradiography. Specificity of the complexes was examined using 40-fold higher concentrations of cold wild-type, as well as mutated, oligonucleotides.
Migration Assay.
Chamber assembly (48 wells; AC48) from NeuroProbe, Inc. (Gaithersburg, MD) was used for migration analysis. Fifty µl (106cells/ml) was placed in the upper chamber and 30 µl of PF medium containing 0.1% BSA (control), or PF medium containing recombinant IGF-I (200 ng/ml) in the lower chamber, separated by an 8-µm polycarbonate filter paper. Antibodies were included in the upper chamber where mentioned. The chamber assembly was kept in 37°C for 4 h. The filter was then removed, fixed, and stained using Diff-Quick (Baxter, Miami, FL) and mounted on slide. Numbers of cells were counted in five fields at x40 magnification.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by NIH Grants CA25874, CA47159, CA76674, and CA10815.
2 K. S. and G. L. contributed equally to this study.
3 Current address: Center for Molecular and Cellular Biology, MAHE, Manipal, India.
4 To whom requests for reprints should be addressed, at The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: (215) 898-3950; Fax: (215) 898-0980; E-mail: herlynm@wistar.upenn.edu.
5 The abbreviations used are: RGP, radial growth phase; EMSA, electromobility shift assay; IGF, insulin-like growth factor; IL, interleukin; MAP, mitogen-activated protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FBS, fetal bovine serum; VEGF, vascular endothelial growth factor; VGP, vertical growth phase; PDGF, platelet-derived growth factor; pfu, plaque-forming unit(s); PF, protein-free; IGF-IR, IGF-I receptor.
Received for publication 10/ 8/01. Revision received 11/26/01. Accepted for publication 12/ 7/01.
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Abstract
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Discussion
Materials and Methods
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