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Keywords:

  • glioma;
  • invasion;
  • migration;
  • phosphacan;
  • pleiotrophin;
  • proliferation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The protein tyrosine phosphatase ζ/receptor-type protein tyrosine phosphatase β (PTPζ/RPTPβ) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas. Both molecules are involved in neuronal cell migration during CNS development. In addition, PTN can induce glioma cell migration which is at least in part mediated through binding to PTPζ/RPTPβ. To study the relevance of this ligand–receptor pair for glioma growth in vitro and in vivo, we transfected the human glioblastoma cell line U251-MG with small interfering RNA (siRNA) directed against PTPζ/RPTPβ. Stable siRNA transfection resulted in strong down-regulation of PTPζ/RPTPβ expression. When injected subcutaneously into nude mice, clones that expressed normal levels of PTPζ/RPTPβ (PTPζ + clones) formed exponentially growing tumours, whereas tumour growth was almost completely abrogated for clones that expressed reduced PTPζ/RPTPβ levels (PTPζ – clones). Similar results were obtained using an orthotopic intracerebral model. Proliferation of PTPζ – cells in vitro was significantly reduced compared with that of control clones. Matrix-immobilized PTN stimulated the proliferation of PTPζ + cells but not of PTPζ – cells. Haptotactic migration induced by PTN was reduced for PTPζ – clones compared with control clones. Our findings suggest that antagonization of PTPζ/RPTPβ expression can inhibit glioma growth in vivo and may thus represent a potentially promising treatment strategy.

Abbreviations used
ALK

anaplastic lymphoma kinase

BSA

bovine serum albumin

con

control

DMEM

Dulbecco's modified Eagle's medium

FGF-2

fibroblast growth factor-2

FN

fibronectin

GFAP

glial fibrillary acidic protein

hpf

high-power field

i.c.

intracerebral

MK

midkine

OAS1

2′5′-oligoadenylate synthetase

PBS

phosphate-buffered saline

PTN

pleiotrophin

PTPζ/RPTPβ

protein tyrosine phosphatase ζ/receptor-type protein tyrosine phosphatase β

SAF

scaffold attachment factor-A

s.c.

subcutaneous

SCC

saline sodium citrate buffer

SDS

sodium dodecyl sulphate

siRNA

small interfering RNA

The protein tyrosine phosphatase ζ/receptor-type protein tyrosine phosphatase β (PTPζ/RPTPβ) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas (Muller et al. 2003; Ulbricht et al. 2003). PTPζ/RPTPβ is a member of a family of receptor-type transmembrane protein tyrosine phosphatases (Krueger and Saito 1992; Levy et al. 1993). Several different splice variants of human PTPζ/RPTPβ have been described, including a transmembrane long form, a truncated transmembrane short form, and a secreted form termed phosphacan. PTPζ/RPTPβ is predominantly expressed in the CNS. Interestingly, highest levels in both the developing and adult mouse brain occur in regions that have the greatest mitotic potential, such as the embryonic ventricular and subventricular zones, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle in adult brain (Levy et al. 1993). The transmembrane forms of PTPζ/RPTPβ are mainly localized on migrating neurones during brain development (Maeda and Noda 1998). In addition, radial glial cells express high levels of PTPζ/RPTPβ (Canoll et al. 1993).

Several different ligands can bind to the extracellular domain of PTPζ/RPTPβ, including the growth factors PTN and the closely related midkine (MK), the extracellular matrix molecules tenascin-C and -R, the neuronal cell adhesion molecules contactin, Nr-CAM, neural cell adhesion molecule, L1/Ng-CAM, and TAG-1/axonin-1, as well as amphoterin and fibroblast growth factor-2 (FGF-2) (Peles et al. 1998). Interactions of PTPζ/RPTPβ with neuronal adhesion molecules have been implicated in glial–neuronal interaction and neuronal migration during development. Studies on knockout mice have shown that PTPζ/RPTPβ is also important for myelinization (Harroch et al. 2000, 2002). Interactions of PTPζ/RPTPβ with PTN may be relevant for tumour progression, as PTN can stimulate tumour growth and angiogenesis (Fang et al. 1992; Chauhan et al. 1993). In addition, ribozyme targeting of PTN has been shown to inhibit the growth of gliomas in nude mice (Grzelinski et al. 2005).

Previous findings suggest that PTN can stimulate haptotactic glioma cell migration in vitro, and that up-regulated expression of PTPζ/RPTPβ and PTN in human astrocytic tumour cells may create an autocrine loop that is important for glioma cell migration (Ulbricht et al. 2003; Muller et al. 2004). In the present study we used small interfering RNA (siRNA) transfection to analyse the relevance of PTPζ/RPTPβ to glioma growth in vivo. We performed additional in vitro studies to determine the effects of down-regulated PTPζ/RPTPβ expression on cell proliferation and migration.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Selection and transfection of synthetic siRNA

Target sequences for siRNA were selected based on published optimization criteria (The siRNA user guide, http://www.rockefeller.edu/labheads/tuschl/sirna.html). Sequences were analysed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) for short nearly exact matches to avoid non-specific binding. Two different siRNA sequences were chosen for initial transient test transfections, siRNA1 (AACUGAGG,UAACACCUCAUGCUU), corresponding to nucleotides 2307–2329 of the PTPζ/RPTPβ sequence (National Center for Biotechnology Information (NCBI) GenBank NM_002851), and siRNA2 (AAUCCUAAAGC,GUUUCCUCGCUU), corresponding to nucleotides 153–175. Both siRNAs were designed to inhibit expression of all three main splice variants of PTPζ/RPTPβ. Synthetic siRNA duplexes were obtained from Dharmacon Research (Lafayette, CO, USA).

U251-MG glioblastoma cells that had been obtained from the American Type Culture Collection (Manassas, VA, USA) were a gift from Dr Wolfgang Hamel (Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany). 5 × 104 U251-MG cells were seeded into six-well plates. After 24 h, cells were transfected with 100 nm siRNA using either TKO transfection agent (Mirus Madison, WI, USA) or Effectene transfection agent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Transfection efficiency was optimized using fluorescein-labelled siRNA against luciferase (Dharmacon Research) which is not expressed in mammalian cells. After 24 and 48 h, cells were harvested for northern and western blot analyses.

Cloning and stable transfection of siRNA

The expression vector pSUPER (OligoEngine, Seattle, WA, USA) was used to express siRNA against PTPζ/RPTPβ in U251-MG cells. The siRNA2 sequence was used for stable transfection. The 19-nucleotide sequence separated by a nine-nucleotide non-complementary spacer (TTCAAGAGA) from the reverse complement of the same 19-nucleotide sequence was synthesized by MWG Biotech (Ebersberg, Germany). The sequence was inserted into the pSUPER backbone after digestion with BglII and HindIII. This vector (pSUPER-PTPζ) was transformed into XL-10 Gold ultracompetent cells (Stratagene, La Jolla, CA, USA). Several clones were obtained, and the correct vector sequence was verified by sequencing.

U251-MG cells were plated at 6 × 105 cells on to 10-cm dishes. After 24 h, cells were co-transfected with pSUPER-PTPζ and pRc/CMV (Invitrogen, Carlsbad, CA, USA) using Effectene (Qiagen). As pSUPER does not contain a resistance gene for eukaryotic cell systems, co-transfection with pRc/CMV was performed to introduce the neomycin gene for selection. Two micrograms of plasmid DNA containing pSUPER-PTPζ and co-plasmid at a ratio of 5 : 1 or 10 : 1 were transfected. The empty vector pSUPER was mock transfected as control. Twenty-four hours after transfection, selection with 400 µg/mL G418 (Invitrogen) was initiated and continued for 3 weeks. To obtain stable cell lines, clonal cells were singled out and grown separately.

Analysis of mRNA

Expression of PTPζ/RPTPβ mRNA was analysed by northern blotting. A 503-bp PCR fragment of PTPζ/RPTPβ (nucleotides 614–1117) was cloned into pGEM-T easy (Promega, Madison, WI, USA). The vector was linearized and digoxigenin (DIG)-labelled antisense RNA run-off transcripts were generated using T7 RNA polymerase and the DIG RNA Labeling kit SP6/T7 (Roche, Mannheim, Germany) according to the manufacturer's instructions. Total RNA (20 µg) derived from tumour and normal brain samples was electrophoresed on a 1% agarose gel containing 6% formaldehyde (w/v). RNA was blotted on to a nylon membrane (Roche) by transfer for 16 h, and fixed by baking for 30 min at 120°C. Hybridization with DIG-labelled antisense RNA probes (100 ng/mL) against PTPζ/RPTPβ and β-actin was carried out for 16 h at 50°C in a buffer containing 50% formamide. The membrane was washed twice at room temperature in a buffer containing 2 × saline sodium citrate buffer (SSC), 0.1% sodium dodecyl sulphate (SDS) and twice at 50°C with 0.2 × SSC, 0.1% SDS. The blot was incubated with alkaline phosphatase-labelled anti-digoxigenin antibodies (Roche), and signals were detected on X-ray film by incubation with disodium 3-(4-methoxyspiro {1,2-dioxotane-3,2′-(5′chloro)tricyclo[3.3.1.1.3,7]decan}-4-yl) phenyl phosphate (CSPD) (Roche) according to the manufacturer's protocol. To exclude non-specific activation of the interferon system by siRNA transfection, expression of the mRNA for 2′5′-oligoadenylate synthetase (OAS1), a main interferon target gene, was analysed using semiquantitative RT–PCR. Primers for OAS1 amplification have been described previously (Bridge et al. 2003). RT–PCR was performed at an annealing temperature of 60°C and PCR products were analysed by agarose gel electrophoresis after 20, 25, 30, and 35 cycles.

Western blot analysis

Total cell lysates were prepared using lysis buffer (50 mm HEPES pH 7.4, 1% Triton X-100, 10% glycerol, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA supplemented with protease inhibitors). Lysates were cleared from cellular debris by centrifugation, and protein concentrations were determined using the bicinchoninic assay (Pierce, Rockford, IL, USA). Proteins were separated by SDS–polyacrylamide electrophoresis on 5% gels, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Loading of equal protein amounts was verified by Coomassie staining. Membranes were blocked with 1 × Rotiblock (Roth, Karlsruhe, Germany) for 1 h at room temperature, and probed overnight at 4°C with 1 µg/mL human PTPζ/RPTPβ monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA), which detects the intracellular domain of human PTPζ/RPTPβ. Binding was detected using secondary horseradish peroxidase-conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany). Immunoreactive bands were visualized using chemiluminescent SuperSignal substrate (Pierce). As control, blots were stripped and re-probed with a polyclonal antibody against the nuclear protein scaffold attachment factor-A (SAF-A) (kindly provided by Dr Frank O. Fackelmayer).

Tumour growth in vivo

Clones were grown to subconfluence in 150-cm2 tissue culture flasks. For subcutaneous (s.c.) experiments, cells were detached with trypsin, washed twice with serum-free medium, and resuspended at 1 × 108 cells/mL in serum-free Dulbecco's modified Eagle's medium (DMEM). The cell suspension was mixed with an equal volume of matrigel (BD Biosciences), which enhances tumorigenicity in vivo and results in a 100% tumour take rate for otherwise moderately tumorigenic U251-MG cells (Akbasak et al. 1996). Cells were injected subcutaneously into the flanks of 4–7-week-old nude mice (Naval Medical Research Institute (NMRI)-nu/) at 200 µL cell suspension per injection. Each clone was injected into 10 animals. Tumour growth was measured at weekly intervals using a caliper. Tumour volume was calculated in mm3 according to the formula V = (1/6)π(d1d2d3). Animals were killed after 12 weeks when the largest tumours reached a maximal diameter of approximately 20 mm. Tumours were embedded in paraffin for histological analyses.

For intracranial experiments, a cell suspension containing 2 × 108 cells was mixed with an equal volume of matrigel. Nude mice were anaesthetized by intraperitoneal administration of ketamine (100 mg/kg bodyweight) and xylazine (5 mg/kg bodyweight). A burrhole was drilled into the skull 3.5 mm lateral to the bregma. Five microlitres of the cell suspension were slowly injected over 5 min into the basal ganglia using a 30-G needle attached to a 25-µL Hamilton syringe. All clones as well as the parental cells were injected into 10 mice each. All animals were killed when the first mice in the control groups developed symptoms (weight loss). Brains were embedded in paraffin, and serial sections (5 µm thick) were stained with haematoxylin and eosin. The maximum cross-sectional area of the glioblastoma xenografts was determined by computer-assisted image analysis using Leica IM50-software (Leica, Hamburg, Germany). Tumour volumes were estimated using the formula: volume = (square root of maximal tumour cross-sectional area)3.

Histology and immunohistochemistry

Paraffin sections (5 µm thick) were stained with haematoxylin and eosin to study tumour morphology. For immunohistochemistry, paraffin sections were dewaxed using standard histological procedures. To detect PTPζ/RPTPβ, sections were stained with the anti-human PTPζ/RPTPβ monoclonal antibody (dilution 1 : 50; BD Biosciences). To detect glial fibrillary acidic protein (GFAP), tumours were stained with a polyclonal rabbit GFAP antibody (Dako, Hamburg, Germany). To analyse the proliferative activity of the tumour cells, sections were stained with the mouse monoclonal antibody Mib-1 against the Ki-67 antigen (dilution 1 : 50; Dako) using the Envision System (Dako) according to the manufacturer's instructions and Novared (Vector, Burlingame, CA, USA) as substrate. The percentage of Mib-1-positive nuclei was determined by counting immunoreactive tumour cell nuclei in three adjacent high-power fields (hpfs) (1 hpf = 0.031 mm2) that were selected at the lowest magnification (2.5 × objective, Zeiss (Oberkochen, Germany) Axioskop microscope) within tumour areas that appeared to be most actively proliferating. To detect apoptotic cells, paraffin sections were stained using a rabbit polyclonal antibody against cleaved caspase 3 (dilution 1 : 200; Cell Signaling Technology, Beverly, MA, USA). Bound antibody was detected using the Vectastain kit (Vector). The percentage of cells expressing caspase 3 was determined by counting immunoreactive cells in five adjacent hpfs in the centre of the tumour, but avoiding necrotic tumour areas. All histological analyses were performed without knowledge of the cell type injected.

Cell proliferation assay

Transfected or control clones were seeded into 96-well tissue culture plates at 2000 cells/well in medium containing 10% fetal calf serum and cultured overnight. In some assays wells were precoated with 20 µg/mL recombinant human PTN (Sigma, St Louis, MO, USA) for 1 h at 37°C. On day 1, cells were washed once with phosphate-buffered saline (PBS) and the medium was replaced with serum-free medium which was renewed on day 4. Quadruplicate or sextuplicate wells were fixed at daily intervals using 1% glutaraldehyde. After 7 days, fixed cells were stained with crystal violet, washed with PBS, solubilized in 10% SDS, and the optical density of the lysate was quantified by reading the absorbance at 540 nm (Bio-Tek Instruments, Winooski, VT, USA).

Cell migration assay

Haptotactic migration of glioblastoma cells was analysed using a modified 96-well Boyden chamber (Neuroprobe, Cabin John, MD, USA) as described previously (Ulbricht et al. 2003). The underside of 8-µm Nucleopore filters was coated with recombinant human PTN, collagen (Vitrogen 100; Cohesion Technologies, Inc., Palo Alto, CA, USA) or human fibronectin (Chemicon, Temecula, CA, USA) at 20 µg/mL for 1 h at 37°C. Filters were washed, blocked with 1% bovine serum albumin (BSA) in PBS for 30 min, and dried. Assay medium (serum-free DMEM with 0.1% BSA) was added to the lower wells, and glioma cells (1.5 × 104 cells in 50 µL of assay medium) were added to the upper wells. After incubation for 5 h at 37°C, non-migrated cells were scraped off the upper side of the filter, and filters were stained with Diff Quick (Dade, Unterschleissheim, Germany). Nuclei of migrated cells were counted in 10 hpfs using a 40 × objective with a calibrated ocular grid. Values are mean ± SD of triplicate determinations.

Statistical analysis

Differences between staining intensities as well as differences in functional assays were analysed using the unpaired t-test or the Mann–Whitney rank-sum test. Type I error was controlled for by setting α = 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Down-regulation of PTPζ/RPTPβ expression in glioma cells

Two different siRNA oligonucleotides were designed to down-regulate PTPζ/RPTPβ expression in glioma cells. Both sequences targeted the 5′ region of the PTPζ/RPTPβ mRNA, upstream of the first splice site so that expression of all three main splice variants was antagonized. This region corresponds to the extracellular N-terminal part of the PTPζ/RPTPβ protein. The siRNA oligonucleotide duplexes were transiently transfected into U251-MG glioma cells, and PTPζ/RPTPβ expression was analysed by northern blotting 24 and 48 h following transfection.

Transfection with siRNA1 strongly down-regulated the expression of all three PTPζ/RPTPβ splice variants (Fig. 1a). Down-regulation lasted for at least 48 h. We next compared siRNA1 with siRNA2. Down-regulation of PTPζ/RPTPβ expression was stronger with siRNA2 than with siRNA1 (Fig. 1b), and TKO transfection reagent was superior to Effectene (data not shown). We performed modified Boyden chamber haptotaxis assays with the transiently transfected cells and recombinant human PTN. However, both transfection reagents by themselves strongly inhibited cell migration, and control transfections with fluorescein-labelled siRNA against luciferase showed that bright fluorescence was associated with the cell surface (not shown), suggesting that the siRNA and transfection reagent partly stuck to the cell surface which by itself impaired the migratory capacity of the cells. We therefore decided to perform stable siRNA transfections.

image

Figure 1.  Inhibition of PTPζ/RPTPβ mRNA expression by transient siRNA transfection. (a) U251-MG glioblastoma cells were transfected with siRNA1 using TKO transfection reagent. After 24 and 48 h total RNA was analysed by northern blotting. Expression of all three PTPζ/RPTPβ splice variants was strongly inhibited by siRNA transfection (+, lanes 2 and 4) compared with basal levels (lane 1) as well as with mock transfected cells (–, lanes 3 and 5). The effects lasted for at least 48 h. The blot was re-hybridized for β-actin as control (lower panel). (b) Comparison of siRNA1 with siRNA2. Down-regulation of PTPζ/RPTPβ mRNA expression was stronger for siRNA2 than for siRNA1. Controls were untransfected U251-MG cells (con, lanes 1, 2, 5 and 6). The blot was re-hybridized for β-actin (lower panel). (c) Western blot analysis showed strongly reduced PTPζ/RPTPβ expression in clones #39 and #11 (arrows) compared with wild-type and control-transfected clones. The blot was re-probed with an antibody against human SAF-A as control (lower panel).

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Based on the findings obtained with transiently transfected cells, stable transfections were performed using siRNA2. The sequence was ligated into the siRNA expression vector pSUPER and transfected into U251-MG cells. After 3 weeks of selection with neomycin, 31 clones were screened for down-regulation of PTPζ/RPTPβ expression by immunoblot analysis. Strong down-regulation of PTPζ/RPTPβ expression was observed in clones #11 and #39 (PTPζ – clones), whereas only weak down-regulation was found in clone #6 (Fig. 1c). No down-regulation of the nuclear protein SAF-A was observed in these cells. Clones #11 and #39 as well as two control clones (PTPζ + clones #8 and #12) were therefore selected for all further experiments. Expression of the interferon target gene OAS1 was detected in all clones by RT–PCR. Densitometric analysis showed that band intensities for PTPζ – clones after 20–35 PCR cycles were slightly lower than those for PTPζ + clones, excluding non-specific activation of the interferon system in PTPζ – clones (data not shown).

Tumour growth in vivo

To analyse growth of siRNA-transfected clones in vivo, cells were first injected subcutaneously into nude mice and measured at weekly intervals starting 2 weeks after injection. After 12 weeks all animals injected with control clones #8 and #12 had developed tumours, and these tumours displayed exponential growth (Fig. 2a). In contrast, after 12 weeks measurable tumours were only present in six of 10 mice injected with clone #11 and in four of 10 mice injected with clone #39; the other tumours derived from siRNA-transfected clones had regressed over time. Those tumours that were still measurable did not show any significant growth compared with their initial sizes 2 weeks after injection.

image

Figure 2.  Growth of PTPζ siRNA-transfected clones in vivo. Cells were injected subcutaneously or intracerebrally. (a) The size of s.c. tumours was measured over 12 weeks, after which tumours were analysed histologically; s.c.PTPζ + clones (#8 and #12) grew at an exponential rate, whereas PTPζ – clones (#11 and #39) did not exceed their initial size as measured 2 weeks after injection. (b) After 7 weeks of growth, i.c. tumours derived from PTPζ – clones (#11 and #39) were significantly smaller than tumours derived from PTPζ + clones (#8 and #12) or from the parental U251-MG cells (p = 0.001, power 0.944–0.998 with α = 0.05 for all individual comparisons between PTPζ – clones with PTPζ + clones or U251-MG cells, unpaired t-tests). (c) The fraction of cells expressing the Ki-67 antigen (Mib-1 labelling index) in s.c. tumours derived from PTPζ + clones was significantly larger than that in PTPζ – tumours (for all comparisons between clone #11 or #39 with clone #8 or #12: p = 0.01, power 0.767–1.000 with α = 0.05, unpaired t-tests). (d) Similarly, the percentage of Mib-1-labelled cells was smaller in i.c. PTPζ – tumours than in i.c. PTPζ + tumours (for all comparisons of PTPζ – clones with PTPζ + clones or parental cells: p < 0.01, power 0.846–0.999, unpaired t-tests). (e) No significant differences in the fraction of apoptotic cells immunoreactive for cleaved caspase 3 were detected between s.c. tumours derived from PTPζ + and PTPζ – clones. (f) No differences in apoptosis were observed between i.c. PTPζ – and PTPζ + tumours. Values in (a) are mean ± SEM; values in (b–f) are mean ± SD. Asterisks indicate significance, but have been omitted from (a) for clarity.

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Orthotopic tumour growth was analysed using an intracerebral (i.c.) nude mouse model. PTPζ – and PTPζ + clones as well as parental U251-MG cells were engrafted stereotactically into the caudate–putamen of nude mice. Animals were killed 7 weeks later when animals in the controls groups developed clinical symptoms (weight loss). Histological analysis of the brains showed that tumours had formed in all mice that had received clones #8, #12, #39 or parental U251-MG cells. In two of 10 mice that received clone #11 no tumours were found. Tumours derived from PTPζ – clones were more than 98% smaller than tumours from PTPζ + clones or parental U251-MG cells (Fig. 2b, and Figs 3a and b).

image

Figure 3.  Immunohistochemical analysis of tumours grown intracerebrally (a, b) or subcutanously (c–i) in nude mice. (a) Only small tumours formed from PTPζ – clones 7 weeks after i.c. injection. (b) Large tumours formed in mice that received PTPζ + clones or parental U251-MG cells. (c) Strong PTPζ/RPTPβ immunoreactivity was detected in tumours derived from PTPζ + clones. (d) Only faint PTPζ/RPTPβ immunoreactivity was found in PTPζ – tumours. (e) Tumour cells expressed GFAP (PTPζ – tumour). (f) Tumour cells in PTPζ + tumours showed high proliferative activity. (g) The fraction of proliferating tumour cells was low in PTPζ – tumours. (h) Few apoptotic tumour cells were detectable in PTPζ + tumours. (i) Apoptotic cells were infrequent in PTPζ – tumours. Magnifications for (a, b) are 5.6-fold; for (c, d, h, i) are 116-fold; and for (e, f, g) are 58-fold.

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Histological analysis of tumours grown in vivo

Tumours derived from the four clones as well as from the parental U251-MG cells were composed of pleomorphic, poorly differentiated cells with nuclear atypia and occasional necrosis. Immunohistochemistry for PTPζ/RPTPβ showed that the strong down-regulation of PTPζ/RPTPβ protein expression persisted in tumours derived from PTPζ – clones (Figs 3c and d). Tumours derived from all clones as well as those from the parental cell line expressed GFAP (Fig. 3e and data not shown).

To determine the proliferative activity of the tumour cells, sections were immunostained with the Mib-1 antibody against the Ki-67 antigen. High proliferative activity was detected in tumours derived from PTPζ + clones (Fig. 3f), whereas low proliferation was found in PTPζ – tumours (Fig. 3g). The mean proliferative activity in s.c. PTPζ – tumours was reduced by at least 94% compared with that in PTPζ + tumours (Fig. 2c). Similarly, proliferation in i.c. PTPζ – tumours was reduced by more than 97% compared with that in PTPζ + tumours or parental cells (Fig. 2d).

To analyse tumour cell apoptosis, sections were immunostained for cleaved caspase 3. The fraction of apoptotic cells was generally low in both PTPζ – and PTPζ + tumours. No significant differences were found between the percentages of apoptotic tumour cells in PTPζ – tumours compared with PTPζ + tumours or parental cells in either the s.c. or the i.c. model (Figs 2e and f, and Figs 3h and i).

Cell proliferation in vitro

Cell proliferation was analysed over 9 days using a colorimetric assay. Both PTPζ – clones (#11 and #39) displayed strong inhibition of proliferation compared with PTPζ + clones (Fig. 4a). On day 9 inhibition was 78.8% and 89.6% for clone #11 compared with clones #12 and #8 respectively. Inhibition for clone #39 was 55.7% and 78.2% compared with clones #12 and #8 respectively (p < 0.001 for all clone comparisons, power 1.000 with α = 0.05, unpaired t-test). In contrast, clone #19, in which no down-regulation of PTPζ was observed (Fig. 1c), showed a similar proliferation rate to control clone #12. Clone #6, in which only weak down-regulation of PTPζ was obtained, showed only a moderate reduction in proliferation compared with clone #12 (Fig. 4a).

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Figure 4. In vitro proliferation of U251-MG cells transfected with siRNA against PTPζ/RPTPβ. (a) Cells were seeded into 96-well tissue culture plates and allowed to proliferate for 9 days. Cell numbers were quantified using a colorimetric assay. PTPζ – clones (#11 and #39) proliferated at a significantly slower rate than PTPζ + clones (#8 and #12) and parental U251-MG cells (on day 9 of the assay all comparisons between clones #11 or #39 and #8 or #12 or U251-MG cells: p < 0.001, power 1.000 with α = 0.05, unpaired t-test). Values are mean ± SD of quadruplicate determinations. (b) Proliferation rates on coated PTN and tissue culture plastic were compared. Both PTPζ + clones proliferated significantly faster on PTN than on plastic (clone #8: p < 0.001, power 1.000 with α = 0.05; clone #12: p < 0.001, power 0.998 with α = 0.05, unpaired t-tests), whereas PTPζ – clones proliferated at similar rates on PTN and plastic. Values are mean ± SD of sextuplicate determinations. Asterisks in (b) indicate significance; Asterisks have been omitted from (a) for clarity.

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We previously showed that the U251-MG cell line secretes PTN (Ulbricht et al. 2003), so that autocrine stimulation via PTPζ/RPTPβ is likely. However, PTN in solution had no significant effect on the proliferation of glioma cell lines (Mentlein and Held-Feindt 2002; Muller et al. 2003; Ulbricht et al. 2003). PTN in solution also had almost no effect on the chemotactic migration of glioma cells but, when presented as an immobilized coating substrate, it strongly stimulated haptotactic migration (Ulbricht et al. 2003). The observation that down-regulation of PTPζ/RPTPβ expression strongly decreased cell proliferation prompted us to analyse whether immobilized PTN would also have a different effect on proliferation than PTN in solution. Indeed, both PTPζ + clones proliferated at a significantly faster rate on a PTN coating than on tissue culture plastic (Fig. 4b). In contrast, no different growth rates on PTN versus plastic were observed for clones #11 and #39.

Cell migration in vitro

Haptotactic cell migration was analysed using a modified Boyden chamber migration assay. PTN, collagen and fibronectin were used as coating substrates. Migration of PTPζ – clones towards PTN was significantly reduced compared with that of PTPζ + clones (Fig. 5). Reductions were 75.2% and 76.3% for clone #11 compared with clones #8 and #12, respectively, and 51.7% and 53.7% for clone #39 compared with clones #8 and #12 respectively (p < 0.001 for all clone comparisons, power 1.000 with α = 0.05, unpaired t-test). No significant inhibition of migration for PTPζ – clones was obtained when migration was stimulated by collagen or fibronectin as control substrates.

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Figure 5.  Haptotactic cell migration was analysed using a modified Boyden chamber assay. The underside of the filter was coated with collagen, PTN or fibronectin (FN). Haptotactic migration of PTPζ – clones (#11 and #39) was significantly reduced compared with that of PTPζ + clones (#8 and #12) when PTN was used as haptoattractant but not when collagen or fibronectin was used. Values are mean ± SD of triplicate determinations. Asterisks indicate significance (p < 0.001, power 1.000 with α = 0.05, unpaired t-test) for comparisons of each PTPζ + clone with each PTPζ – clone).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study has shown that RNA interference targeting PTPζ/RPTPβ can inhibit glioblastoma growth in vivo. Mechanisms of this antitumour effect appear to include inhibition of tumour cell proliferation as well as migration.

Previous in vitro studies suggested that PTPζ/RPTPβ is mainly involved in glioma cell migration. Although PTN is a secreted growth factor it has little chemotactic activity for glioma cells but stimulates haptotactic migration up to 200-fold (Ulbricht et al. 2003). PTN-induced haptotactic migration of C6 rat glioma cells could specifically be inhibited by an PTPζ/RPTPβ antibody which, however, only recognized rat PTPζ/RPTPβ and not the human form (Ulbricht et al. 2003). The present study showed that the PTN-induced migration of human glioma cells was reduced by 51.7–76.3% when PTPζ/RPTPβ expression was down-regulated. Importantly, no such inhibition of migration was observed when collagen or fibronectin was used as haptoattractant, suggesting that stimulation of migration is specifically mediated by the interaction between PTN and PTPζ/RPTPβ.

Proliferation was also inhibited in cells that expressed reduced levels of PTPζ/RPTPβ. In vivo, the fraction of proliferating cells was less than 3% in all tumours derived from s.c. or i.c. PTPζ – clones, whereas it was greater than 40% in PTPζ + tumours. In vitro, proliferation of PTPζ – cells was reduced by 55.7–89.6% after 9 days of growth. The effect of PTPζ/RPTPβ down-regulation on cell proliferation was to some extent unexpected as we and others were previously unable to detect effects of PTN on glioma cell proliferation in vitro (Mentlein and Held-Feindt 2002; Muller et al. 2003). Interestingly, Lu et al. recently reported that PTN exists in an 18-kDa form and a 15-kDa form, the latter of which is most probably generated by post-translational cleavage of the longer form (Lu et al. 2005). They further showed that the 18-kDa form stimulates haptotactic glioma cell migration by binding PTPζ/RPTPβ, whereas the 15-kDa form stimulates proliferation by binding anaplastic lymphoma kinase (ALK), another PTN receptor. However, previous studies by other groups demonstrated that also the long form of PTN can the have mitogenic and transformic activity (Zhang et al. 1999; Bernard-Pierrot et al. 2001). Differences in protein folding or modifications resulting from diverse strategies of PTN purification or recombinant production are thought to account for the different functional effects.

In the present study we analysed the effect of matrix-immobilized PTN on glioma cell proliferation. Interestingly, when PTPζ + cells were grown on a PTN-coated surface they proliferated at a significantly faster rate than on plastic. This effect is most likely mediated through PTPζ/RPTPβ because (i) the PTPζ + cells but not the PTPζ – cells proliferated at an increased rate on PTN, and (ii) U251-MG cells do not express ALK mRNA (as analysed by RT–PCR, unpublished observation, Ulbricht, U.). It is possible that matrix-immobilized PTN assumes a solid-phase conformation that is capable of triggering mitogenic signals via PTPζ/RPTPβ, whereas PTN in solution lacks this property. Alternatively, binding of U251-MG cells to PTN-coated surfaces may not directly induce mitogenic signalling, but attachment and spreading on PTN may interfere with other signalling pathways, rendering the cells less responsive to other mitogenic signals, and/or causing alterations in cytoskeletal organization that affect cell cycling.

The mitogenic effect of immobilized PTN, however, cannot explain why PTPζ – clones proliferated significantly slower on plastic than PTPζ + clones. Although it is possible that some of the PTN secreted by the U251-MG cells was adsorbed on to the culture plate during the assay and could function in a similar fashion as precoated PTN, such an effect should then also be expected in assays in which recombinant 18-kDa PTN in solution was added to the cells. It is therefore likely that other PTPζ/RPTPβ ligands also contributed to the stimulation of proliferation of U251-MG cells. Several other PTPζ/RPTPβ ligands with mitogenic effects have been described, including MK, which is closely related to PTN, FGF-2, amphoterin and tenascin-C. The MK protein is also expressed by U251-MG cells (unpublished observation, Ulbricht, U.); MK expression is known to be increased in human malignant gliomas (Mishima et al. 1997; Kato et al. 1999), and MK can stimulate the growth of various tumours (Takei et al. 2005). Amphoterin is also expressed by glioma cells (Punnonen et al. 1999) and can stimulate the growth of tumours in mice (Taguchi et al. 2000; Huttunen and Rauvala 2004). FGF-2 is expressed by glioma cells including the U251-MG cell line (unpublished observation, Ulbricht, U.); FGF-2 can stimulate the growth of various tumours, and treatment with a monoclonal FGF-2 antibody was shown to inhibit glioma growth in vivo (Stan et al. 1995). Finally, tenascin-C is strongly expressed in human malignant gliomas and can stimulate glioma cell proliferation and angiogenesis (Herold-Mende et al. 2002; Zagzag et al. 2002; Ruiz et al. 2004). However, little is known about the interactions between PTPζ/RPTPβ and these ligands, and the picture is further complicated by the fact that all four ligands have alternative receptors: MK can also bind to ALK, amphoterin can bind to the receptor for advanced glycation end-products (RAGE), FGF-2 binds to the FGF receptors 1–4, and tenascin-C binds to integrins and annexin II. Further research is required to elucidate the complex interactions between the components of this multiligand–multireceptor conglomerate.

Little is known about the intracellular binding partners of PTPζ/RPTPβ and its signalling pathways. The involvement of a phosphatase in glioma formation and/or progression may seem unexpected. Phosphorylation cascades usually have stimulatory effects on tumour cell proliferation whereas phosphatases are generally assumed to antagonize such pathways, a well known example being the pro-apoptotic phosphatase and tensin homolog (PTEN). However, phosphatases are not always tumour suppressive; for example, the phosphatase PTPα can dephosphorylate and thereby activate kinases of the src family, which leads to cell transformation (Zheng et al. 1992). Thus, depending on the dephosphorylation target and the signalling pathway, phosphatases can also have oncogenic activity. Meng et al. reported that PTPζ/RPTPβ interacts with β-catenin (Meng et al. 2000). PTN dephosphorylates the constitutively active PTPζ/RPTPβ, which upon binding PTN dimerizes and becomes inactivated, resulting in an increase in tyrosine-phosphorylated β-catenin. Tyrosine phosphorylation of β-catenin is known to cause loss of cell–cell adhesion. It further leads to increased levels of cytoplasmic β-catenin which translocates to the nucleus where it binds to the transcription factor TCF/LEF; this causes increased expression of MYC and Cyclin D1, stimulating cell cycling and proliferation (Nelson and Nusse 2004). Activation of this pathway could explain the mitogenic effects of immobilized PTN on PTPζ + glioma cells in our study. In addition, a recent report suggested that the nuclear factor-κB signalling pathway can also be activated through PTPζ/RPTPβ (Lorente et al. 2005).

To conclude, we showed that siRNA transfection targeting PTPζ/RPTPβ can inhibit glioma growth in vivo. Given the strong up-regulation of both PTPζ/RPTPβ in human malignant gliomas, antagonization of PTPζ/RPTPβ expression and/or signalling may be a promising strategy to inhibit the growth of these tumours. This approach appears particularly promising as multiple PTPζ/RPTPβ ligands with mitogenic and motogenic properties, including PTN, MK, FGF-2, amphoterin and tenascin-C, are also simultaneously expressed by glioma cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by the Deutsche Forschungsgemeinschaft (WE 928/4-1, and 928/4-2). We thank Dr Frank O. Fackelmayer (Heinrich-Pette-Institut, Hamburg) for providing the SAF-A antibody.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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