From the Institute of Brain Research, University of
Tuebingen, Calwer Strasse 3, D-72076 Tuebingen, Germany, the
¶ Institute for Cell Biology, Department of Immunology, University
of Tuebingen, Auf der Morgenstelle 15, D-72076 Tuebingen, Germany, and
the
Institute for Cell Biology, Department of Molecular Biology,
University of Tuebingen, Auf der Morgenstelle 15, D-72076 Tuebingen, Germany
Received for publication, January 16, 2001
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ABSTRACT |
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Tumor microvessels differ in structure and
metabolic function from normal vasculature, and neoangiogenesis is
associated with quantitative and qualitative changes in expression of
endothelial proteins. Such molecules could serve as molecular addresses
differentiating the tumor vasculature from those of the normal brain.
We have applied Systematic Evolution of
Ligands by EXponential enrichment (SELEX)
against transformed endothelial cells as a complex target to select
single-stranded DNA-ligands (aptamers) that function as
histological markers to detect microvessels of rat experimental glioma,
a fatal brain tumor that is highly vascularized. Both the SELEX
selection procedure as well as subsequent deconvolution-SELEX were
analyzed by fluorescence based methods (flow cytometry and fluorescence
microscopy). Of 25 aptamers analyzed, one aptamer was selected that
selectively bound microvessels of rat brain glioblastoma but not the
vasculature of the normal rat brain including peritumoral areas. The
molecular target protein of aptamer III.1 was isolated from endothelial
cells by ligand-mediated magnetic DNA affinity purification. This
protein was identified by mass spectrometry as rat homologue of mouse
pigpen, a not widely known endothelial protein the expression of which
parallels the transition from quiescent to angiogenic phenotypes
in vitro. Because neoangiogenesis, the formation of new
blood vessels, is a key feature of tumor development, the presented
aptamer can be used as a probe to analyze pathological angiogenesis of
glioblastoma. The presented data show that pigpen is highly expressed
in tumor microvessels of experimental rat brain glioblastoma and may
play an important role in warranting blood supply, thus growth of brain tumors.
Neoangiogenesis, the new formation of blood vessels is associated
with endothelial cell (EC)1
proliferation, migration, and formation of new capillaries as a
response to the increased demand of tumor tissue for oxygen and
nutrients (1). In normal physiological processes, such as
wound-healing, angiogenesis is tightly balanced by positive and
negative regulators. In several disease states, such as tumor growth,
overactive angiogenesis contributes to advancement of disease (2, 3).
Positive regulators of angiogenesis are recruited by the tumor to
dominate negative regulators to ensure proliferation and organization
of microvessel-forming EC. Thus, neoangiogenic EC, associated with
tumor development, differ from preexisting, quiescent EC by qualitative
or quantitative changes of molecular addresses, which could serve as
potential targets for tumor homing diagnostic and therapeutic agents
(4-6). A series of targets and corresponding antibodies to address the
activated endothelium of pathological blood vessels are known (2). But it must been supposed that there are other regulators, so far unknown,
to be involved in the complex process of angiogenesis. To define novel
molecular addresses of the tumor vasculature, combinatorial chemistry
approaches, the libraries of which are not biased by natural mechanisms
of selection such as immune tolerance (7), have been successfully
applied to develop antibody fragments by phage display that bind to
tumor blood vessels (8-12). However, peptides require circularization,
dimerization, or presentation in the context of a larger protein for
binding to a target (13). These structural requirements limit the
number of potential ligands that can be sampled in an experiment
directed against complex targets. Systematic
Evolution of Ligands by EXponential
enrichment (SELEX) is a nucleic acid based combinatorial chemistry
procedure that has been used to isolate relatively short high affinity
ssDNA or RNA ligands, termed aptamers, to a wide variety of protein or
low molecular weight compounds (14, 15). Single-stranded nucleic acids,
which can fold into very small and complex three-dimensional shapes
with a great diversity of binding specificities, are isolated from a
large pool of random sequence molecules
(1014-1015 sequences) by reiterative rounds of
selection and amplification (reviewed in 16).
Here we describe a fluorescence-based SELEX procedure. Using
transformed EC as a complex target allowed systematic evolution of
fluorescence-labeled oligonucleotides and subsequent in situ deconvolution-SELEX by flow cytometry and fluorescence microscopy on
cryostat tissue sections (Fig. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Selection procedure for the generation
of DNA aptamers binding to angiogenic microvessels of rat experimental
glioma. (i), counterselection against N9 microglial
cells (to reduce co-selection of aptamers binding to other cell types).
(ii), incubation of successive SELEX pools with YPEN-1
endothelial cells. (iii), removal of unbound sequences.
(iv), PCR amplification of cell-bound aptamers using
modified primers. (v), FITC-ssDNA generation.
(vi), PCR amplification using unmodified primers for
(vii) sorting round 8 pool aptamers into individual clones.
(viii), deconvolution-SELEX (step 1) of cloned aptamers as a
preselection to evaluate aptamers binding to endothelial cells.
(ix), deconvolution-SELEX (step 2) to evaluate preselected
aptamers selectivity against tumor microvessels embedded in their
natural surrounding on tissue sections of rat brain glioblastoma.
Finally aptamers were generated that function as a histological marker
to selectively stain microvessels of experimental rat glioblastoma, a
brain tumor that is highly vascularized (17). We present aptamer III.1,
a ssDNA-ligand binding to pigpen, a not widely known endothelial
protein of the Ewing's sarcoma family that parallels the
transition from quiescent to angiogenic phenotypes in
vitro (18).
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EXPERIMENTAL PROCEDURES |
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Cell Lines-- Endothelial cell line YPEN-1 (CRL-222) immortalized by an Adenovirus-12 SV40 hybrid virus (19), microglial cell line N9 (20), and rat glioblastoma cell line C6 (21) were obtained from ATCC (American Type Culture Collection). YPEN-1 cells and N9 microglial cells were cultured in RPMI 1640 (Life Technologies, Inc.), supplemented by 10% fetal calf serum (Seromed) to subconfluence, and harvested by carefully dislodging the cells from tissue culture flasks.
SELEX Library and Primers-- The high pressure liquid chromatography-purified library contained a central randomized sequence of 60 nucleotides (nt) flanked by 18-nt primer hybridization sites (5'-ATA CCA GCT TAT TCA ATT- 60-nt -AGA TAG TAA GTG CAA TCT-3'; MWG-Biotech AG) (22). A fluorescein isothiocyanate (FITC)-labeled 5'-primer (5'-FITC-18C-ATA CCA GCT TAT TCA ATT-3'; 18C denotes an 18-carbon ethylene glycol spacer) and a triple biotinylated (trB) 3'-primer (5'-trB-AGA TTG CAC TTA CTA TCT-3') were used in the PCR reactions for the synthesis of double-labeled, double-stranded DNA molecules. After alkaline melting, the biotinylated ssDNA strand was separated from the FITC-conjugated ssDNA aptamer by streptavidin-coated magnetic beads (M-280 streptavidin dynabeads were obtained from Dynal) according to the supplier's instructions (23). FITC- and biotin-labeled primers were synthesized by Operon Technologies, Inc. Unmodified primers were purchased from Amersham Pharmacia Biotech. All steps with FITC-labeled DNA were performed in light-protecting (ambra) reaction tubes (Eppendorf GmbH, Hamburg, Germany). Incubation of cells and tissue sections with the FITC-aptamers was performed in the dark.
SELEX-- Selection of ssDNA against YPEN-1 endothelial cells was carried out essentially as described for pure protein targets (14, 15, 24, 25). Briefly, ssDNA pools were denatured by heating at 80 °C for 10 min in selection buffer (initial round: 1015 sequences in 1 ml; subsequent rounds: 200 pmol in 200 µl) containing 50 mM Tris·HCl (pH 7.4), 5 mM KCl, 100 mM NaCl, 1 mM MgCl2, and 0.1% NaN3 and then renatured at 0 °C for 10 min. To reduce background binding, a 5-fold molar excess of yeast tRNA (Life Technologies, Inc.) and bovine serum albumin (BSA; Merck) were added. To reduce co-selection of ssDNA binding to other cell types, in particular macrophages, counterselection was performed with 3 × 107 N9 microglial cells, a population of brain monocytes, by incubation at 37 °C for 30 min in selection buffer. Partitioning of bound and unbound ssDNA sequences was done by centrifugation. To ensure elimination of aptamers against the polypropylene-partitioning matrix of the reaction tubes, tubes were replaced against polyethylene ones after the third round of selection. The counterselected SELEX library was incubated with endothelial cells (106 in the initial round, 105 in subsequent rounds) at 37 °C for 30 min. After centrifugation and three washes with 1 ml selection buffer (with 0.2% BSA), cell-bound aptamers were amplified by PCR (Taq-polymerase and dNTP's were obtained from Promega). FITC- and biotin-labeled primers were used in PCR amplification (28 cycles of 1 min at 94 °C, 1 min at 46 °C, and 1 min at 72 °C, followed by 10 min at 72 °C). For cytofluorometric analysis FITC-labeled ssDNA was prepared as described above.
Sorting into Individual Clones-- Aptamers obtained from the eighth round of selection were PCR-amplified using unmodified primers and cloned into Escherichia coli using the TA cloning kit (Invitrogen). Plasmids of individual clones were isolated by alkaline lysis (26), and inserts were amplified by PCR. For binding studies of individual aptamers by flow cytometry and fluorescence microscopy, FITC-ssDNA was prepared as described above.
Flow Cytometric Analysis of Aptamers Bound to YPEN-1 EC in Successive SELEX Rounds-- Specific EC-binding, FITC-labeled aptamers were monitored in the second and subsequent rounds. After incubation of 105 EC with 200 pmol aptamer in 200 µl of selection buffer (with a 5-fold molar excess of tRNA and BSA), cells were washed twice in 1 ml of selection buffer (with 0.2% BSA), and FITC fluorescence was monitored with a FACScan cytometer (BD Pharmingen) by counting 5000 events. Nonspecific control ssDNA (FITC-18C-96-nt) was prepared from the starting pool by PCR as described above.
Deconvolution-SELEX (Step 1): Flow Cytometric Analysis of Individual Aptamer-- 5 × 104 YPEN-1 endothelial cells and N9 microglial cells, respectively, were each preincubated with selection buffer containing 1 µg/µl tRNA for 20 min on ice and incubated with 200 µl of denatured and renatured FITC-aptamer solution (0.25 pmol/µl with 1 µg/µl tRNA) for 30 min at 37 °C. Cells were washed twice in selection buffer (with 0.2% BSA), and fluorescence was determined by flow cytometry.
Deconvolution-SELEX (Step 2): Preparation of Rat Brain Glioblastoma Tissue Sections and Histological Analysis-- 2-2.5 × 104 tumor cells (C6 glioblastoma cell line) were transplanted intracranially into Harlan Sprague-Dawley rats as described (21). Rats were perfused 23 days after transplantation with phosphate-buffered saline, and brains were removed for cryostat sectioning. Binding of individual FITC-aptamers was analyzed on rat cryostat sections of normal and glioblastoma brain. Tissue sections (10 µm) were preincubated with selection buffer (with 1 µg/µl tRNA) at 4 °C for 20 min and then incubated with FITC-aptamer solution (0.25 pmol/µl with 1 µg/µl yeast tRNA) at room temperature for 40 min. Tissue sections were washed twice in selection buffer (with 0.2% BSA), and fluorescence patterns were analyzed microscopically. For double labeling, cryostat tissue sections of rat brain glioblastoma were stained with aptamer III.1 as described above and subsequently stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (300 nM, 2-3 min; Molecular Probes) or mouse CD-31 (1 h at a dilution of 1/1000; Dako) with CyTM3 anti-mouse secondary IgG (30 min at a dilution of 1/200; Amersham Pharmacia Biotech), respectively. After two washes in selection buffer (with 0.2% BSA) fluorescence patterns were analyzed microscopically.
Wounding experiments were performed in Petri dishes. The confluent
monolayer of YPEN-1 EC was wounded by carefully dislodging cells with a
sterile pipette tip. The cells in Petri dishes were cultured for an
additional 4 h, washed twice with selection buffer, and
subsequently fixated with methanol (10 min, 20 °C; Merck). After
incubation of the cell layer with FITC-18C-aptamer III.1 (0.25 pmol/µl with 1 µg/µl tRNA) in selection buffer (30 min, 37 °C), staining patterns were analyzed by fluorescence microscopy.
Ligand Mediated Protein Purification-- 1 mg (100 µl) of magnetic streptavidin beads were coated with 200 pmol of trB aptamer III.1 (MWG-Biotech AG) by incubation in 1 ml selection buffer (30 min, room temperature). As a control, 100 µl of magnetic beads were coated with 200 pmol of unselected FITC-ssDNA (trB-96-nt). 1.5 × 108 Ypen-1 endothelial cells were lysed in the presence of protease inhibitors as described (27). After centrifugation, the protein pellet was resuspended in 400 µl of selection buffer, treated by ultra-sonication (0 °C, 20 s), and incubated with aptamer III.1-coated magnetic beads in the presence of a 100-fold excess of tRNA (20 nmol) as an unspecific competitor in selection buffer (total volume 1.5 ml, 0 °C, 15 min). The protein-aptamer-magnetic bead-complex was removed in a magnet stand and washed four times (first wash: 1 ml of selection buffer with 150 mM NaCl; second through fifth wash: 200 µl of selection buffer with 100 mM NaCl with 2 nmol tRNA). Protein is removed from aptamer-coated beads by incubation in 30 µl of 1 M NaCl (0 °C, 30 min) and analyzed by polyacrylamide gel electrophoresis after staining with Coomassie Blue.
Protein Identification--
In gel tryptic digestion was
performed as described (28) and modified as outlined below. Briefly,
the protein band was excised from the gel, fully destained, and
digested for 3 h with porcine trypsin (sequencing grade, modified;
Promega) at a concentration of 67 ng/µl in 25 mM ammonium
bicarbonate, pH 8.1, at 37 °C. Prior to peptide mass mapping and
sequencing of tryptic fragments by tandem mass spectrometry, the
peptide mixture was extracted from the gel by two changes of 50%
trifluoroacetic acid/50% water followed by two changes of 50%
trifluoroacetic acid/50% acetonitrile. The combined extracts were
vacuum-dried. The dried peptides were redissolved in 0.1%
trifluoroacetic acid and purified using a ready-to-go pipette tip
filled with C18 spherical silica reverse phase material (ZipTipC18TM, Millipore). Peptides were eluted with 10 µl
of 50% methanol/1% formic acid, and sequencing was performed by
nanoelectrospray tandem mass spectrometry on a hybrid quadrupole
orthogonal acceleration time of flight tandem mass spectrometer (Q-Tof,
Micromass, Manchester, England) equipped with a nanoflow electrospray
ionization source. Gold-coated glass capillary nanoflow needles were
obtained from Protana (Type Medium NanoES spray capillaries for the
Micromass Q-Tof, Odense, Denmark). The needle was filled with 3 µl of
the sample and subsequently opened by breaking the tapered end of the
tip under a microscope. A stable spray was observed applying a needle
voltage of 1200-1400 V, a back pressure of 2 p.s.i., and a source
temperature of 40 °C. The estimated flow rate was 20-50 nl/min. For
nanoflow electrospray ionization tandem mass spectrometry experiments, fragmentation was achieved by
collision with argon atoms. Q1 was set to the mass of interest, and an
optimized collision energy was applied. The integration time for the
time of flight analyzer was 1 s with an interscan delay of
0.1 s. Database searches (NCBInr, non-redundant protein database)
were done using the MASCOT software from Matrix Science (29).
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RESULTS |
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Selection of ssDNA Aptamers that Bind Transformed Endothelial
Cells--
SELEX was used essentially as described (30) to generate
ssDNA aptamers against Adenovirus-12 SV40-transformed YPEN-1 rat endothelial cells. Commonly used radioactive labeling of nucleic acids
(to monitor the enrichment of aptamers in successive SELEX rounds) was
replaced by attaching FITC as a fluorescence reporter molecule. The
selection was started with a SELEX library of ~1 × 1015 sequences, each containing a central randomized region
of 60 nt flanked by two primer hybridization sites (22). To reduce co-selection of aptamers binding to other cell types, counterselection against N9 microglial cells (20), a population of brain monocytes, was
performed prior to each selection round. To monitor the enrichment of
specific cell-binding aptamers during selection, SELEX pools of the
second and following rounds were analyzed by flow cytometry after
incubation with YPEN-1 endothelial cells (Fig.
2). Analysis of fluorescence-labeled
pools in successive cycles of selection showed a pronounced shift of
second round histogram toward higher fluorescence intensity. The
histograms of SELEX pools 3-7 showed slow but steady increases in
fluorescence intensity and thus enrichment of endothelial cell-binding
aptamers.
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Deconvolution-SELEX-- As the utility of a selected pool of nucleic acids to dissect complex systems is contingent upon the ability to quickly identify which sequences are ligands for components of interest within the targeted mixture (30), individual aptamers cloned from the eighth round of selection were evaluated by a two step deconvolution-SELEX procedure: (i) each sequence affinity against the transformed YPEN-1 EC cell line was preevaluated by flow cytometry, and (ii) the selective binding of aptamers to microvessels of C6 brain tumor was analyzed by fluorescence microscopy on cryostat tissue sections of rat brain glioblastoma. Of 25 sequences (FITC-18C-96-nt) tested by flow cytometry, by comparison of endothelial cells stained with unselected ssDNA (FITC-18C-96-nt), 23 showed binding to YPEN-1 endothelial cells. Of these, 16 bound to the pathological microvasculature of C6 glioblastoma, and 7 showed no binding. Aptamer III.1 displayed the most intensive staining of the vasculature exclusively in areas of solid tumor growth and was therefore chosen as the candidate to be further characterized as outlined below.
Increased fluorescence intensity of YPEN-1 EC stained with
fluorescence-labeled aptamer III.1 (sequence and proposed secondary structure is illustrated in Fig.
3C) by comparison with
unselected ssDNA (FITC-18C-96-nt) as a negative control is shown in
fluorescence-activated cell sorter histograms of Fig. 3A. To
demonstrate also EC-specific binding by flow cytometry, binding
analyzes against N9 microglial cells was performed (Fig.
3B).
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Histological analyzes of aptamer III.1. is illustrated in Fig.
4. FITC-labeled aptamer stained the
complex architecture of the pathological microvasculature (Fig.
4A). Counterstaining of cellular nuclei with DAPI
(blue) demonstrated selective targeting of microvessels
within the cell-rich tumor region but not of vasculature in peritumoral
areas (Fig. 4B). Fluorescence microscopy of rat normal brain
after incubation with aptamer III.1 did not show any staining of EC or
vessel-associated structures (data not shown). Staining of tumor tissue
sections with mouse IgG directed against EC (CD-31, visualized with
anti-mouse IgG-CyTM3, red) and subsequent
labeling with aptamer III.1 (FITC, green) countersigned the
aptamers target as EC (Fig. 4D). Rat brain tumor tissue
sections were analyzed after incubation with unselected FITC-18C-ssDNA
and endothelial CD-31 (CyTM3, red) as a control.
Fig. 4C demonstrates negative binding.
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To get further evidence for aptamer III.1 pathological association, cultured EC were analyzed after wounding the confluent cell layer with a pipette tip. Subsequent staining with aptamer III.1 and fluorescence microscopy showed exclusive up-regulation of the molecular target of aptamer III.1 in endothelial cells next to the lesion, a region that is characterized by increased proliferation compared with areas of adjacent contact-inhibited cells of the confluent monolayer (Fig. 4E).
Thus, our histological data gave strong indication that aptamer III.1 binds an endothelial molecular target that is involved in the process of EC proliferation, a key step of angiogenesis.
Aptamer III.1-Mediated Target Identification--
The molecular
target has been isolated by magnetic DNA affinity purification.
Solubilized EC proteins were incubated with trB aptamer III.1
beforehand coupled to magnetic streptavidin beads. After thorough
washing and subsequent release of aptamer-bound protein in high salt
solution, proteins were analyzed by SDS/polyacrylamide gel
electrophoresis (Fig. 5).
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Polyacrylamide gel analyses revealed a distinct band migrating at ~70
kDa in the lane of aptamer III.1, which is absent in the lane
containing a product of the control reaction (performed with unselected
ssDNA) which revealed one single band of ~50 kDa. Peptide mass
fingerprinting and sequencing of three of the tryptic peptide fragments
of the protein by mass spectrometry and tandem mass spectrometry,
respectively, were used to identify the aptamer-specific protein as the
rat homologue of mouse pigpen protein (67 kDa), a so far,
widely unknown endothelial protein that has been considered to parallel
the transition from quiescent to angiogenic phenotypes in
vitro (31).
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DISCUSSION |
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Selection by Intact Biological Entities-- To select a brain tumor-homing ssDNA ligand we applied SELEX against Adenovirus-12 SV40-transformed YPEN-1 rat endothelial cell line, thus against a pathologic endothelial target for several reasons (32): (i) it can be supposed that in actively growing endothelial cells the balance between positive and negative angiogenic regulators has been shifted toward the side of regulators required for EC proliferation and thus the transformed endothelial cells bear a profile of molecular addresses predominantly expressed by angiogenic tumor microvessels; (ii) the selection by intact biological entities does not require a full understanding of the complex mechanism of EC proliferation, whereas the selection by an isolated protein is limited to cases in which the proteins have been identified and isolated; (iii) because of structural differences in protein conformation, the aptamers selected to bind the protein in its purified form will not automatically bind the protein embedded in its natural surrounding; (iv) the selection using intact, activated EC give promises in the identification of components that have not been known for their critical role in angiogenesis, (v) moreover, the cellular target allowed fluorescence-based selection thus to monitor the enrichment of cell-binding, fluorescence-labeled ssDNA-ligands in successive pools of selection and amplification by flow cytometry.
Deconvolution-SELEX-- The eighth round pool was deconvoluted, and single binding of individual aptamers was quantified by flow cytometry (33-36). Further, screening of these individual aptamers on cryostat brain tissue sections allowed deconvolution for their discriminatory binding to neoangiogenic tumor microvessels but not to vessels of the normal rat brain. Thus histological staining allowed the selection of those aptamer candidates recognizing the differentiation of endothelial cells under in vivo conditions of angiogenesis.
These two steps of library deconvolution result in aptamer libraries selectively addressing molecular structures, differentiating neoangiogenic endothelium of the brain. The alignment of the 16 aptamer sequences after eight cycles of systematic evolution and the two subsequent steps of deconvolution did not reveal any obvious binding motifs. This appears to be due to the complexity of the endothelial cell with its high number of potential molecular targets. However, the combination of fluorescence-based SELEX and deconvolution-SELEX allowed identification of aptamer candidates, which perform the desired task to act as a histological marker to selectively stain tumor microvessels.
Aptamers as Histological Markers-- Aptamers were used as histological markers in combination with chromosomal dyes and conventional antibodies for immunostaining. This demonstrates that aptamers are versatile tools that rival antibodies in diagnostic applications (reviewed in 7). Unlike antibodies, synthetic aptamers can easily be produced with a high degree of accuracy, reproducibility ,and purity. Therefore, little or no batch to batch variation is expected in aptamer production. They are not sensitive to temperature and undergo reversible denaturation, thus having a much longer self-life (7). Further, aptamers can easily be conjugated to a variety of reporter molecules, chemotherapeutics, or photosensibilisators at precise locations. Thus aptamers selectively binding to tumor endothelium might be of value not only in the development of tumor-homing diagnostics but also in therapeutics.
Complex-SELEX as a Tool to Dissect EC Differentiation-- The pronounced and selective staining of rat brain tumor vessels by aptamer III.1 suggested a selective addressing of a molecule involved in neoangiogenesis. We therefore used this aptamer to purify and identify its binding partner, the rat homologous of mouse endothelial pigpen protein. Previous cell culture experiments (18, 31) demonstrate pigpen to be synthesized at barely detectable levels in non-proliferating EC, whereas its expression is up-regulated under conditions where EC are actively migrating and dividing (i.e. in subconfluent or wounded cultures) (18). Our observation that the pigpen molecule is highly up-regulated in angiogenic microvessels suggests pigpen as a target for diagnostic imaging or therapeutic strategies in vivo.
The combinatorial chemistry procedure SELEX, applied to select ssDNA
ligands against transformed EC as intact biological entities followed
by deconvolution-SELEX and aptamer-mediated target identification, has
demonstrated its potential to identify compounds that are unknown or
have not been supposed in probably playing a key role in complex
mechanisms like angiogenesis (30, 32). In general, generation of
fluoresceinated, cell-binding aptamers by SELEX opens new avenues in
the study of cellular differentiation and tumor diagnostics.
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FOOTNOTES |
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* This work was supported by the Wilhelm Sander Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Institute of Brain Research, Calwer Str. 3, D-72076 Tuebingen, Germany. Tel.: 0049-7071-2984881; Fax: 0049-7071-295456; E-mail: hirnforschung@uni-tuebingen.de.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100347200
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ABBREVIATIONS |
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The abbreviations used are: EC, endothelial cell(s); ssDNA, single-stranded DNA; SELEX, Systematic Evolution of Ligands by EXponential enrichment; nt(s), nucleotide(s); FITC, fluorescein isothiocyanate; 18C, 18-carbon ethylene glycol spacer; trB, triple biotin; PCR, polymerase chain reaction; BSA, bovine serum albumin; DAPI, 4',6 diamidino-2-phenylindole.
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