Article |
Address correspondence to Erkki Ruoslahti, The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 646-3125. Fax: (858) 646-3198. email: ruoslahti{at}burnham.org
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Abstract |
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Key Words: angiogenesis; bone marrow; cell-penetrating peptides; nuclear proteins
Abbreviation used in this paper: HUVEC, human umbilical vein endothelial cell.
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Introduction |
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The molecular markers that distinguish tumor vasculature from that of normal tissues are important in a number of ways. Many of the molecules that are selectively expressed in tumor blood vessels play a functional role in development and maintenance of new blood vessels. Examples include endothelial cell growth factor receptors and integrins (Eliceiri and Cheresh, 1999; Ferrara and Alitalo, 1999; Hynes, 2002), matrix metalloproteases (Brooks et al., 1998; Bergers et al., 2000), and aminopeptidase N (Pasqualini et al., 2000). Blocking the function of these proteins inhibits angiogenesis. Furthermore, these and other molecules selectively expressed in tumor vasculature can be made use of in targeting diagnostic and therapeutic agents in tumors (Arap et al., 1998; Nilsson et al., 2001; El-Sheikh et al., 2002; Hood et al., 2002).
The identification of additional tumor blood vessel markers helps in the understanding of angiogenesis and could be useful for tumor targeting. We set out to identify the molecule, "receptor," that is recognized by a tumor-homing peptide recently identified by our laboratory. This peptide, F3, was discovered in a screening procedure that used a phage-displayed cDNA library and combined ex vivo screening on cell suspensions prepared from mouse bone marrow and in vivo screening for tumor homing. F3 is a 34amino acid fragment of a high mobility group protein, HMG2N (Porkka et al., 2002). F3 homes to the vasculature of various types of tumors by binding to the endothelial cells. F3 also binds to a subpopulation of bone marrow cells that may be precursors for endothelial cells. In some tumors, the F3 peptide also recognizes the tumor cells. A striking property of the F3 peptide is that it is internalized by its specific target cells and transported to the nucleus.
We have now identified cell surfaceexpressed nucleolin as the receptor for F3 on tumor cells and angiogenic endothelial cells. Cell surface nucleolin expression is a novel angiogenesis marker. It provides a tool for studying tumor angiogenesis, including the contribution of precursor cells to this process, and for targeting drugs into tumors.
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Results |
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We prepared antisera against nucleolin by immunizing rabbits with two different synthetic peptides from the human nucleolin sequence. Affinity-purified antibodies recognized a band that aligned with the 110-kD nucleolin band defined by the MS-3 antibody (Fig. 1 B, b). Like MS-3, the rabbit antibodies also detected smaller molecular mass bands that presumably represent nucleolin fragments. As expected, based on the fact that the immunizing peptides came from different regions of the nucleolin molecule, the sets of minor bands detected by each antibody did not overlap. Immunoblotting showed that the NCL3 antibody also recognizes a 110-kD protein in extracts of mouse cells. (Fig. 1 B, c).
Nucleolin is expressed at the cell surface
To serve as an F3 receptor, nucleolin would have to be present at the cell surface. Nucleolin is primarily known as a nuclear and cytoplasmic protein, but recent studies have shown that a cell surface form of nucleolin also exists (Said et al., 2002; Sinclair and O'Brien, 2002). To determine if F3-binding nucleolin in the MDA-MB-435 cells is expressed at the cell surface, exponentially growing cells were biotinylated with a cell-impermeable biotin reagent, and cell extracts were subjected to affinity chromatography on immobilized F3. Two biotinylated bands at 110 and 75 kD specifically bound to F3 (Fig. 2 A, a). The surface biotinylated 75-kD band was stronger than the 110-kD band, whereas the opposite was true in the affinity chromatography, possibly because the cell surface expression or accessibility to biotinylation may be different for the two forms. Notably, the histones that bound to the F3 matrix from the cell extract did not become biotin-labeled in intact cells, but were the most prominent F3-binding bands from cell surface-biotinylated serum-starved cultures, which contain many dead cells (Fig. 2 A, b). No nucleolin band was detectable in the serum-starved cells, suggesting a lack of cell surface nucleolin expression.
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To determine whether antinucleolin would similarly accumulate in tumor vessels and/or tumor cells, we intravenously injected the NCL3 antibody into mice bearing MDA-MB-435 tumors. Tissues collected 60 min after injection showed selective accumulation of the antibody in tumor blood vessels (Fig. 6, a and b). No antibody was detected in association with the tumor cells. About 70% of the tumor vessels were positive for the antibody, whereas no positive vessels were seen in the blood vessels of the normal tissues tested (skin and lung; shown for skin subcutaneous tissue in Fig. 6, c). Purified rabbit IgG, injected as a control, was not detected in tumor blood vessels (Fig. 6, d).
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Discussion |
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Several approaches were used to identify the binding molecule for the F3 peptide as nucleolin. First, nucleolin and histones were identified as the main cellular proteins that specifically bound to immobilized F3 peptide. Cell surface labeling indicated that the bound nucleolin was derived from the surface of intact cells, whereas the histones were not labeled and, therefore, likely originated from dead cells. Second, inhibition of F3 uptake into cultured cells by an antinucleolin antibody that is internalized into the nucleus provides additional evidence for the specificity of the F3nucleolin interaction and its occurrence in intact cells. Third, the specific binding of injected antinucleolin antibodies to tumor blood vessels extends the association of F3 binding and cell surface nucleolin expression to an in vivo animal model.
The nucleolin polypeptide consists of a negatively charged NH2-terminal domain, an RNA-binding domain, and a COOH-terminal domain rich in RGG motifs. The main functions of nucleolin relate to rRNA maturation and ribosome assembly (Ginisty et al., 1999; Srivastava and Pollard, 1999). Although nucleolin was originally described as a nuclear and cytoplasmic protein, a number of studies show that it can also be expressed at the cell surface (Deng et al., 1996; Larrucea et al., 1998; Said et al., 2002; Sinclair and O'Brien, 2002). Recent results also ascribe additional functions to nucleolin as a shuttle protein between the cytoplasm and the nucleus (Borer et al., 1989; Yu et al., 1998), and between the cell surface and the nucleus (Schmidt-Zachmann and Nigg, 1993; Said et al., 2002; Shibata et al., 2002). The localization of nucleolin within the cell may be regulated by phosphorylation of its NH2 terminus (Schwab and Dreyer, 1997). Our results provide additional evidence for the cell surface localization and shuttle function of nucleolin.
The expression of nucleolin at the cell surface seems to correlate with growth and metabolic activity of cells. Both the uptake of the F3 peptide and the staining of intact cells with antinucleolin antibodies were suppressed in serum-starved cells. This may be a proliferation-related effect. An association of cell surface nucleolin expression with cell proliferation in vitro has been described previously (Hovanessian et al., 2000). Other factors besides proliferation may contribute to the regulation of cell surface nucleolin expression. We found only modest levels of cell surface nucleolin on actively proliferating endothelial cells in vitro, whereas antinucleolin binding to angiogenic endothelium was readily detectable in vivo. The differentiation state of the cells may be a factor contributing to nucleolin regulation, as cultured human leukemia-60 cells induced to differentiate into nonproliferating macrophages lose their ability to bind F3 (unpublished data). The restricted expression of cell surface nucleolin and the cell-type specificity of the expression may explain why some investigators have not been able to document the presence of nucleolin at the cell surface (Yu et al., 1998). A similar explanation may apply to the heterogeneity of the cell surface nucleolin expression in the vasculature of tumors and matrigel plugs; local variation in endothelial cell proliferation is likely to occur in angiogenic lesions in vivo.
F3-displaying phage selectively homes to tumor vasculature in vivo, and fluorescein-tagged F3 also binds to and is taken up by endothelial cells in tumor vasculature. However, the peptide also spreads to tumor cells, and it appears in a few individual nonvascular cells in the skin and the gut (Porkka et al., 2002). Intravenously injected antinucleolin antibody was only detected in angiogenic vessels of tumors as well as of matrigel plugs. The restricted distribution of the antibody resembles that of the phage, probably because the size of phage and antibody limit their access to tissues, whereas the relatively small molecular mass of the peptide conjugate (5 kD) may permit wider distribution. Nonetheless, each of these reagents demonstrates the specificity of cell surface nucleolin for angiogenic vessels within the vasculature.
F3 is rich in basic amino acids and binds to cell surface heparan sulfate. However, our demonstration that CHO cells lacking heparan sulfate (and other glycosaminoglycans) internalize F3 excludes a direct role of heparan sulfate as the internalizing molecule. Indeed, binding and antibody inhibition studies show that F3 internalization is mediated by cell surface nucleolin. El-Sheikh et al. (2002) have described a peptide from the heparin-binding domain of vascular endothelial growth factor that selectively homes to tumor vasculature. The authors attributed the tumor homing to affinity of the peptide for heparan sulfate. It will be interesting to see whether this peptide might also bind to nucleolin.
The internalization of the F3 peptide and NCL3 antibody may reflect a physiological function of cell surface nucleolin. Midkine is a 13-kD cytokine that, like F3, contains a high proportion of basic amino acids (Said et al., 2002). It plays a role in neurite outgrowth and neuronal differentiation, and its mRNA is up-regulated in several human carcinomas (Tsutsui et al., 1993). The internalization of midkine by cells has been reported to be nucleolin dependent (Said et al., 2002), although lipoprotein receptor-related protein can also serve as the internalizing receptor for midkine (Shibata et al., 2002). The binding site for midkine in nucleolin has been localized to the RGG domain of nucleolin (Said et al., 2002), whereas our antibody inhibition results implicate the domain rich in acidic amino acids as the binding site for F3. Cell surface nucleolin may also be involved in the activities of basic FGF, which has been shown to bind to nucleolin in nuclear extracts (Bonnet et al., 1996). Thus, F3, midkine, and possibly basic FGF, might be internalized by a nucleolin-dependent mechanism, but distinct binding sites on nucleolin may exist to mediate the uptake.
A highly basic peptide derived from the HIV Tat protein also binds to cells and is internalized by them. The Tat peptide allows internalization of conjugated proteins and is commonly used as a cell-penetrating agent (Fawell et al., 1994; Langel, 2002). It is unlikely that the Tat peptide would use nucleolin for its internalization and nuclear transport. First, the internalization of Tat is independent of the cell type, even in vivo, whereas our results show that cell surface nucleolin is limited, it is expressed in angiogenic endothelium but not in the blood vessels in normal tissues. Second, treatment of cells with heparinase to remove heparan sulfates inhibits internalization of the Tat peptide (Suzuki et al., 2002), whereas we found that lack of heparan sulfates did not affect F3 uptake. Third, Tat internalization is independent of temperature and does not require energy, and several other cell-penetrating peptides are similar to Tat in this regard (Langel, 2002). In contrast, F3 uptake is blocked at 4°C (Porkka et al., 2002). Finally, our antibody inhibition data also suggest that Tat peptide internalization is independent of nucleolin because an antinucleolin antibody inhibited the uptake of F3 but not of the Tat peptide.
Our laboratory has recently described yet another type of a cell-penetrating peptide, LyP-1, which is also rich in basic amino acids (Laakkonen et al., 2002). This peptide specifically homes to the endothelium of tumor lymphatics and the tumor cells in certain, but not all, tumors. The internalization of this peptide is not affected by antinucleolin antibodies (unpublished data). Thus, several different internalization mechanisms for basic peptides appear to exist, both universal and cell-type specific.
Cell-penetrating peptides rich in basic amino acids are transported into the nucleus after internalization (Langel, 2002). This is also the case with F3 and LyP-1 (Laakkonen et al., 2002; Porkka et al., 2002). Nucleolin is thought to be responsible for the nuclear transport of midkine (Shibata et al., 2002), and the same may be the case with F3. It is also possible that the multiple basic amino acids in F3 form one or more independent nuclear localization signals.
The selective in vivo homing of the two nucleolin-binding reagents, the F3 peptide and the NCL3 antibody, to angiogenic blood vessels establishes cell surface nucleolin as a new angiogenesis marker. Tumor blood vessels undergo angiogenesis (Hanahan and Folkman, 1996) and have specific markers in common with other angiogenic vessels (Ruoslahti, 2002). Future studies will determine whether cell surface nucleolin might play a role in angiogenesis, possibly by binding and internalizing growth factors such as midkine and bFGF. The restricted expression of cell surface nucleolin in angiogenic vessels and in tumor cells in vivo, and its ability to internalize molecules bound to it, make nucleolin an attractive potential target for the development of agents for vascular therapy of tumors.
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Materials and methods |
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F3 affinity chromatography and mass spectroscopy
Affinity purification of nucleolin from MDA-MB-435 detergent extracts was performed as described previously (Christian et al., 2001b). In brief, 6 x 108 cells were pelleted and lysed in 60 ml of RIPA buffer (1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 1% protease inhibitor cocktail for mammalian cells; Sigma-Aldrich). The lysate was incubated with 20 µl F3 (AKVKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) affinity matrix (2 mg of peptide covalently coupled to 1 ml of affigel 10). Control fractionation was performed on a 34amino acid peptide that represents a scrambled version of F3. The matrix beads were washed three times with 0.025% Triton X-100, 50 mM Tris-HCl, pH 8.4, 150 mM NaCl, 1 mM CaCl2, and 0.02% azide; washed twice with 25 mM Tris-HCl, pH 8.4, and 250 mM NaCl; and the bound proteins were eluted with 30 µl SDS gel sample buffer. The affinity-purified proteins were reduced with 50 mM DTT and separated on an 820% polyacrylamide gel and visualized by colloidal blue staining (Invitrogen). The molecular masses of the gel bands were determined by comparing to the standards in an Alphamanager instrument (Alpha Innotech Corp.). Bands that appeared in the F3 eluate, but not in the control, were cut out, digested with trypsin, and analyzed by mass spectroscopy using a matrix-assisted laser desorption ionization, time of flight instrument (model Voyager DE-PRO; Applied Biosystems) using an -cyano-4-hydroxycinnamic acid/nitrocellulose matrix.
Immunoblot analysis
Cell extracts or affinity-purified samples were separated on an SDS-PAGE and transferred onto nitrocellulose membranes for 1 h at 100 V. The membranes were blocked overnight at 4°C with 5% milk powder in TBS-T (140 mM NaCl, 10 mM Tris-HCl, pH 7.4, and 0.05% Tween) and incubated with mouse monoclonal or rabbit polyclonal antinucleolin antibody (10 µg/ml in TBS-T) for 1 h at RT. After extensive washing, the membranes were incubated with peroxidase-coupled rabbit antimouse or goat antirabbit antibody, and bound antibody was detected with ECL (Amersham Biosciences) and exposure to Biomax MR (Kodak).
Cell surface biotinylation
For cell surface expression analysis, MDA-MB-435 cells (5 x 106 cells) were washed three times with cold PBS on a cell culture plate and incubated with biotinylation buffer (20 mM HEPES, pH 7.45, 5 mM KCl, 130 mM NaCl, 0.8 mM MgCl2, 1 mM CaCl2, and 0.5 mg/ml EZ link Sulfo-NHS-Biotin; Pierce Chemical Co.) for 1 h at 4°C. After the removal of the reagent, the cells were washed three times with wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2) and lysed in 1% Triton X-100 lysis buffer for 1 h. The lysates were centrifuged for 15 min at 15,000 g. F3 binding proteins were isolated by affinity chromatography as described above, separated on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose membranes were incubated after blocking with ExtrAvidinperoxidase conjugates diluted at 1:5,000 (Sigma-Aldrich) for 1 h at RT. Bands were detected after incubation with ECL reagent and exposure to Biomax MR.
FACS analysis of cell surface nucleolin
For FACS® analysis, MDA-MB-435 or HUVEC were detached with EDTA and 106 cells/sample were incubated with either polyclonal rabbit antinucleolin antibody NCL3 (10 µg/ml) or monoclonal antinucleolin antibody MS-3 (Santa Cruz Biotechnology, Inc.; 10 µg/ml) for 45 min on ice. Cells were washed with ice-cold PBS and incubated with Alexa-488 (Molecular Probes) secondary antibody (1:50 in PBS). As a negative control, the cells were incubated with 10 µg/ml of rabbit IgG or with 10 µg/ml monoclonal antic-myc antibody (Santa Cruz Biotechnology, Inc.), followed by the secondary antibody. As a positive control, the cells were incubated with 10 µg/ml of mouse monoclonal antiß3-integrin (CBL 479; Cymbus Biotechnology Ltd.). The antibody-treated cells were washed and resuspended in 50 µl PBS containing 2 µg/ml propidium iodide to distinguish between live and dead cells, and 10,000 cells per sample were analyzed using a FACSCalibur flow cytometer.
Detection of peptides and antibodies in cells and tissues
For internalization experiments, cells were incubated with 1 µM of fluorescein-conjugated peptide for 2 h at 37°C. The cells were washed with PBS, fixed with 4% PFA in PBS, and analyzed by confocal microscopy. The cell-penetrating basic peptide from the human immunodeficiency virus Tat protein (GRKKRRQRRR; Fawell et al., 1994) was used as a positive control in the internalization experiments. To detect nucleolin, cells were fixed with 4% PFA in PBS and stained with 10 µg/ml antinucleolin antibodies either directly or after permeabilization with Triton X-100. Bound antibodies were detected with Alexa-594labeled antirabbit antibody (Molecular Probes) and visualized by fluorescence microscopy.
In vivo distribution of circulation-accessible cell surface nucleolin was examined in mice bearing xenograft tumors or basement membrane (matrigel) plugs. Xenograft tumors were generated by subcutaneously injecting exponentially growing MDA-MB-435 human breast cancer cells (106 cells in 200 µl of culture media) into the mammary fat pad area of 2-mo-old Balb/c nu/nu mice (Animal Technologies). The animals were used for experiments 8 wk after injection. Nontumor angiogenesis was studied in matrigel plugs (Fulgham et al., 1999; Ngo et al., 2000). 2-mo-old Balb/c nu/nu mice were subcutaneously injected with 100 µl of Matrigel (Becton Dickinson) at two or three locations in the abdominal area. Each 100-µl plug contained 100 ng of recombinant human bFGF as an angiogenesis stimulant (R&D Systems). The animals were used for antibody injection experiments 8 d after the implantation.
In vivo distribution of antibodies was studied by intravenously injecting mice with 200 µg of polyclonal rabbit antinucleolin antibody or rabbit IgG. 1 h after the injection, the mice were anesthetized, perfused through the heart with 10 ml PBS, and killed by infusing 10 ml of 4% PFA in PBS. Tumors or matrigel plugs, along with various control tissues were removed, fixed in 4% PFA, and frozen in OCT embedding medium (Tissue-Tek). All procedures were performed under anesthesia induced by intraperitoneal injection of 2,2,2-tribromoethanol (Avertin) at a dosage of 0.40.75 mg/gram of body weight (500700 µl/mouse). All animal experiments were approved by the Animal Review Committee of the Burnham Institute.
For histological analyses, 5-µm sections were cut. The injected rabbit antinucleolin antibody and anti-CD31 antibody (10 µg/ml; BD Biosciences) applied on the tissue sections were detected with Alexa-594 and Alexa-488conjugated secondary antibodies, respectively. The sections were examined under an inverted fluorescent microscope (Nikon) or a confocal microscope (Bio-Rad Laboratories). Nuclei were counterstained using DAPI (Vector Laboratories).
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Acknowledgments |
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This work was supported by grants from the National Cancer Institute (CA82713), the Department of Defense (DAMD 17-02-1-0315; given to E. Ruoslahti), and the Cancer Center (CA30199). S. Christian is supported by a fellowship from the Deutsche Forschungsgemeinschaft.
Submitted: 24 April 2003
Accepted: 7 October 2003
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References |
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Akerman, M.E., W.C. Chan, P. Laakkonen, S.N. Bhatia, and E. Ruoslahti. 2002. Nanocrystal targeting in vivo. Proc. Natl. Acad. Sci. USA. 99:1261712621.
Arap, W., R. Pasqualini, and E. Ruoslahti. 1998. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 279:377380.
Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2:737744.[CrossRef][Medline]
Bonnet, H., O. Filhol, I. Truchet, P. Brethenou, C. Cochet, F. Amalric, and G. Bouche. 1996. Fibroblast growth factor-2 binds to the regulatory beta subunit of CK2 and directly stimulates CK2 activity toward nucleolin. J. Biol. Chem. 271:2478124787.
Borer, R.A., C.F. Lehner, H.M. Eppenberger, and E.A. Nigg. 1989. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell. 56:379390.[Medline]
Brooks, P.C., S. Silletti, T.L. von Schalscha, M. Friedlander, and D.A. Cheresh. 1998. Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity. Cell. 92:391400.[Medline]
Christian, S., H. Ahorn, A. Koehler, F. Eisenhaber, H.P. Rodi, P. Garin-Chesa, J.E. Park, W.J. Rettig, and M.C. Lenter. 2001a. Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium. J. Biol. Chem. 276:74087414.
Christian, S., H. Ahorn, M. Novatchkova, P. Garin-Chesa, J.E. Park, G. Weber, F. Eisenhaber, W.J. Rettig, and M.C. Lenter. 2001b. Molecular cloning and characterization of EndoGlyx-1, an EMILIN-like multisubunit glycoprotein of vascular endothelium. J. Biol. Chem. 276:4858848595.
Deng, J.S., B. Ballou, and J.K. Hofmeister. 1996. Internalization of anti-nucleolin antibody into viable HEp-2 cells. Mol. Biol. Rep. 23:191195.[Medline]
El-Sheikh, A., C. Liu, H. Huang, and T.S. Edgington. 2002. A novel vascular endothelial growth factor heparin-binding domain substructure binds to glycosaminoglycans in vivo and localizes to tumor microvascular endothelium. Cancer Res. 62:71187123.
Eliceiri, B.P., and D.A. Cheresh. 1999. The role of alphav integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J. Clin. Invest. 103:12271230.
Esko, J.D., T.E. Stewart, and W.H. Taylor. 1985. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA. 82:31973201.[Abstract]
Fawell, S., J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, and J. Barsoum. 1994. Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. USA. 91:664668.[Abstract]
Ferrara, N., and K. Alitalo. 1999. Clinical applications of angiogenic growth factors and their inhibitors. Nat. Med. 5:13591364.[CrossRef][Medline]
Fulgham, D.L., S.R. Widhalm, S. Martin, and J.D. Coffin. 1999. FGF-2 dependent angiogenesis is a latent phenotype in basic fibroblast growth factor transgenic mice. Endothelium. 6:185195.[Medline]
Ginisty, H., H. Sicard, B. Roger, and P. Bouvet. 1999. Structure and functions of nucleolin. J. Cell Sci. 112:761772.
Hanahan, D., and J. Folkman. 1996. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 86:353364.[Medline]
Harms, G., R. Kraft, G. Grelle, B. Volz, J. Dernedde, and R. Tauber. 2001. Identification of nucleolin as a new L-selectin ligand. Biochem. J. 360:531538.[CrossRef][Medline]
Hood, J.D., M. Bednarski, R. Frausto, S. Guccione, R.A. Reisfeld, R. Xiang, and D.A. Cheresh. 2002. Tumor regression by targeted gene delivery to the neovasculature. Science. 296:24042407.
Hovanessian, A.G., F. Puvion-Dutilleul, S. Nisole, J. Svab, E. Perret, J.S. Deng, and B. Krust. 2000. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Exp. Cell Res. 261:312328.[CrossRef][Medline]
Hynes, R.O. 2002. A reevaluation of integrins as regulators of angiogenesis. Nat. Med. 8:918921.[CrossRef][Medline]
Joyce, J.A., P. Laakkonen, M. Bernasconi, G. Bergers, E. Ruoslahti, and D. Hanahan. 2003. Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Cancer Cell. In press.
Laakkonen, P., K. Porkka, J.A. Hoffman, and E. Ruoslahti. 2002. A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nat. Med. 8:751755.[Medline]
Langel, Ü. 2002. Cell-Penetrating Peptides: Processes and Applications. CRC Press, Boca Raton, FL. 406 pp.
Larrucea, S., C. Gonzalez-Rubio, R. Cambronero, B. Ballou, P. Bonay, E. Lopez-Granados, P. Bouvet, G. Fontan, M. Fresno, and M. Lopez-Trascasa. 1998. Cellular adhesion mediated by factor J, a complement inhibitor. Evidence for nucleolin involvement. J. Biol. Chem. 273:3171831725.
Ngo, C.V., M. Gee, N. Akhtar, D. Yu, O. Volpert, R. Auerbach, and A. Thomas-Tikhonenko. 2000. An in vivo function for the transforming Myc protein: elicitation of the angiogenic phenotype. Cell Growth Differ. 11:201210.
Nilsson, F., H. Kosmehl, L. Zardi, and D. Neri. 2001. Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice. Cancer Res. 61:711716.
Pasqualini, R., E. Koivunen, R. Kain, J. Lahdenranta, M. Sakamoto, A. Stryhn, R.A. Ashmun, L.H. Shapiro, W. Arap, and E. Ruoslahti. 2000. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. 60:722727.
Porkka, K., P. Laakkonen, J.A. Hoffman, M. Bernasconi, and E. Ruoslahti. 2002. A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo. Proc. Natl. Acad. Sci. USA. 99:74447449.
Roghani, M., and D. Moscatelli. 1992. Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms. J. Biol. Chem. 267:2215622162.
Ruoslahti, E. 2002. Specialization of tumour vasculature. Nat. Rev. Cancer. 2:8390.[CrossRef][Medline]
Said, E.A., B. Krust, S. Nisole, J. Svab, J.P. Briand, and A.G. Hovanessian. 2002. The anti-HIV cytokine midkine binds the cell surface-expressed nucleolin as a low affinity receptor. J. Biol. Chem. 277:3749237502.
Schmidt-Zachmann, M.S., and E.A. Nigg. 1993. Protein localization to the nucleolus: a search for targeting domains in nucleolin. J. Cell Sci. 105:799806.
Schwab, M.S., and C. Dreyer. 1997. Protein phosphorylation sites regulate the function of the bipartite NLS of nucleolin. Eur. J. Cell Biol. 73:287297.[Medline]
Shibata, Y., T. Muramatsu, M. Hirai, T. Inui, T. Kimura, H. Saito, L.M. McCormick, G. Bu, and K. Kadomatsu. 2002. Nuclear targeting by the growth factor midkine. Mol. Cell. Biol. 22:67886796.
Sinclair, J.F., and A.D. O'Brien. 2002. Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7. J. Biol. Chem. 277:28762885.
Srivastava, M., and H.B. Pollard. 1999. Molecular dissection of nucleolin's role in growth and cell proliferation: new insights. FASEB J. 13:19111922.
St Croix, B., C. Rago, V. Velculescu, G. Traverso, K.E. Romans, E. Montgomery, A. Lal, G.J. Riggins, C. Lengauer, B. Vogelstein, and K.W. Kinzler. 2000. Genes expressed in human tumor endothelium. Science. 289:11971202.
Suzuki, T., S. Futaki, M. Niwa, S. Tanaka, K. Ueda, and Y. Sugiura. 2002. Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem. 277:24372443.
Tsutsui, J., K. Kadomatsu, S. Matsubara, A. Nakagawara, M. Hamanoue, S. Takao, H. Shimazu, Y. Ohi, and T. Muramatsu. 1993. A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas. Cancer Res. 53:12811285.[Abstract]
Yu, D., M.Z. Schwartz, and R. Petryshyn. 1998. Effect of laminin on the nuclear localization of nucleolin in rat intestinal epithelial IEC-6 cells. Biochem. Biophys. Res. Commun. 247:186192.[CrossRef][Medline]