Clinical Cancer Research Grants Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 2178, April 1, 2007. doi: 10.1158/1078-0432.CCR-06-1711
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, L.
Right arrow Articles by Rao, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, L.
Right arrow Articles by Rao, J. S.

Cancer Therapy: Preclinical

Transfection with Anti-p65 Intrabody Suppresses Invasion and Angiogenesis in Glioma Cells by Blocking Nuclear Factor-{kappa}B Transcriptional Activity

Liang Li1, Christopher S. Gondi1, Dzung H. Dinh2, William C. Olivero2, Meena Gujrati3 and Jasti S. Rao1,2

Authors' Affiliations: 1 Program of Cancer Biology, Department of Cancer Biology and Pharmacology, 2 Department of Neurosurgery, and 3 Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois

Requests for reprints: Jasti S. Rao, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605. Phone: 309-671-3445; E-mail: jsrao{at}uic.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The strategy of intracellular antibodies to neutralize the function of target proteins has been widely developed for cancer research. This study used an intrabody against p65 subunit to prevent nuclear factor {kappa}B (NF-{kappa}B) transcriptional activity in glioma cells and to inhibit the expression of its target genes involved in the invasion and angiogenesis of human gliomas.

Experimental Design: A single-chain fragment of antibody variable region (scFv) against p65 was prepared using phage display technique. We then prepared an anti-p65 intrabody construct (pFv/nu) by cloning the scFv-encoding sequence into the mammalian nuclear-targeting vector, pCMV/myc/nuc.

Results: p65 expression in human glioma cells (U251 and] U87) transfected with pFv/nu was significantly decreased. We showed that NF-{kappa}B nuclear translocation and its DNA binding activity were blocked via intrabody transfection in electrophoretic mobility shift assays and the inhibition of NF-{kappa}B activity in nucleus resulted in the decreasing expression and bioactivity of matrix metalloproteinase-9, urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator, and vascular endothelial growth factor. The intrabody transfected glioma cells showed a markedly lower level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also repressed by coculture with the intrabody transfected glioma cells or exposure to their conditional medium. Intrabody transfection neither induced apoptosis nor altered cell proliferation in U251 and U87 cells as compared with the control vector pCMV/nu. After the injection of pFv/nu-transfected glioma cells, preestablished tumors were almost completely regressed when compared with mock, pCMV/nu, and pGFP/nu.

Conclusion: Blocking NF-{kappa}B activity via the nuclear intrabody expression might be a potential approach for cancer therapy.

Malignant gliomas are the most common primary tumors arising in the human brain, accounting for >60% of all primary brain tumors (1). Glioblastoma multiforme (WHO grade 4 glioma) is the most frequent and refractory malignant brain tumor to affect adults, with a median survival rate of <1 year and a 5-year survival rate of ≤5%, despite surgery, chemotherapy, and radiotherapy (2). Therefore, gene therapy against the characteristic invasive behavior of glioblastoma might offer novel treatment strategies when combined with conventional therapeutic agents.

Nuclear factor {kappa}B (NF-{kappa}B) is a sequence-specific transcription factor that belongs to the reticuloendotheliosis (Rel) family proteins (3, 4). Constitutive activation of NF-{kappa}B plays an important role in the tumorigenesis of diffuse gliomas and in promoting the growth of high-grade gliomas, as observed by in vitro and in vivo studies (5, 6). The autocrine activity of tumor necrosis factor-{alpha} seems likely to sustain NF-{kappa}B activation in human gliomas. Once activated by tumor necrosis factor-{alpha}, NF-{kappa}B initiates a cascade of signaling events that trigger phosphorylation, ubiquitination, and subsequent proteasomal degradation of I{kappa}B. Consequently, NF-{kappa}B is released to translocate into the nucleus and activate the transcription of a variety of genes, including a gene encoding one of its cytosolic inhibitors, I{kappa}B{alpha}, as well as other genes that encode cytokines, cytokine receptors, adhesion molecules, and proteins involved in tumorigenesis and progress. NF-{kappa}B subunits, including RelA (p65), RelB (p50), and RelC, contain two essential domains involved in NF-{kappa}B activity. One is a conserved region in their NH2 terminus, known as the Rel homology domain, which is responsible for dimerization, nuclear localization, and DNA binding; the other serves as transactivation domain within their COOH terminus, of which the activated nuclear NF-{kappa}B is phosphorylated after its translocation from cytoplasm and modulates NF-{kappa}B transcriptional activity. Growing evidence has shown that the transcriptional activity of NF-{kappa}B can be regulated both by cytosolic sequestration of I{kappa}B and by inducible phosphorylation of p65 (7–9). The molecular mechanisms and biological consequences of p65 phosphorylation are currently another crucially important determinant for NF-{kappa}B mechanism–based therapies against cancer.

Due to the presence of a strong transcriptional activation domain, RelA (p65) is responsible for most of the transcriptional activity of NF-{kappa}B (10). However, intrabody strategies based on p65 phosphorylation during NF-{kappa}B transcriptional activation have not yet been done. Therefore, we prepared a single-chain antibody against human p65 (RelA subunit of NF-{kappa}B), which recognizes a COOH-terminal epitope in the transactivation region using human single-fold single-chain variable fragment (scFv) libraries (Tomlinson I + J). We then constructed a pFv/nu vector, designed to prevent phosphorylation of transactivated p65, which could express anti-p65 intrabodies in the nucleus of human glioma cells, resulting in inhibitory expression of NF-{kappa}B–dependent genes, such as matrix metalloproteinase (MMP-9), urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and vascular endothelial growth factor (VEGF).

Our findings indicate that the anti-p65 nuclear-targeted intrabodies significantly decreased the expression of phospho-p65, p65, p50, and I{kappa}B{alpha} proteins in U251 and U87 glioma cells, whereas the nuclear translocation and sequence-specific DNA binding activities of NF-{kappa}B were blocked. In addition, the blockade of NF-{kappa}B transcriptional activity in nucleus led to the down-regulation of MMP-9, uPAR, uPA, and VEGF at both mRNA and protein levels. After intrabody transfection, the ability of in vitro migration and angiogenesis of glioma cells was inhibited. However, the NF-{kappa}B functions interrupted by intrabody therapy did not alter cell proliferation or induce apoptosis in U251 or U87 cells. In addition, the growth of the intracranial tumor established by the injection of pFv/nu-transfected U251 and U87 cells was almost completely inhibited. This finding suggests that the blockade of transcriptional activity of NF-{kappa}B by intrabody expression in the nuclei of human glioblastoma cells could contribute to the inhibition of invasion, angiogenesis, and tumor growth through down-regulation of NF-{kappa}B target proteins, uPA, uPAR, MMP-9, and VEGF, and serve as a promising therapeutic strategy for the treatment of human glioblastomas.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Selection of scFv from phage display library. ScFv selection was carried out using the human single-fold scFv libraries (Tomlinson I + J), which are being distributed by Medical Research Council HGMP Resource Centre. In the library, >100 million different scFv fragments are cloned into an ampicillin-resistant phagemid vector pIT2 under pelB-leader control and transformed into TG1 E. coli cells. All scFv fragments are composed of a single polypeptide with the VH and VL domains attached to one another by a flexible glycine-serine linker, fused with a myc tag and a His tag at the COOH terminus.

To obtain anti-p65 scFv fragments from the two libraries, 10 µg of a bioactive peptide from the COOH-terminal region of the human p65 subunit of transcription factor NF-{kappa}B (New England Peptide Co.; p65 bioactive peptide: amino acids 463-472, located within transactivation domain 2) were coated overnight in Maxisorb tubes (Nunc Corp., Naperville, IL) and four rounds of selections were undertaken according to a standard protocol. Briefly, E. coli TG-1 was infected with the eluted phages, rescued by the helper phage, and finally the antigen-bound phages were prepared for the further selection after enrichment, amplification, and purification. After four rounds, E. coli TG-1 was infected with the final phage preparations and individual ampicillin-resistant colonies (phage clones) were selected for further analysis.

ELISA. To measure antigen-binding specificity of scFvs, polyclonal and monoclonal phage ELISA were used to screen phage populations produced during each round of selection and phages from single colonies, respectively. The antigen, p65 bioactive peptide coated in 96-well plates was incubated with either polyethylene glycol–precipitated phage from the end of each round of selection or single-phage suspension for 3 h, with bovine serum albumin or PBS as negative controls. The amount of bound phages was determined using horseradish peroxidase/anti-M13 monoclonal conjugate in polyclonal ELISA, and the incorporation of a small peptide epitope (myc-tag) at the COOH-terminal end of scFv allowed detection with the primary antibody [anti–c-myc monoclonal antibody (mAb)] to determine its specific affinity to p65 in monoclonal ELISA. After visualization with the 3,3',5,5'-tetramethylbenzidine substrate (GE Healthcare, Waukesha, WI), the colorimetric evaluation of binding activity was measured in an ELISA reader (Benchmark, Bio-Rad, Hercules, CA) at 450 nm, with a reference wavelength of 650 nm.

To determine the affinity of scFv to p65 bioactive peptide, competitive ELISA was done. In brief, the supernatants of total lysates from HB2151 bacteria culture were extracted to isolate soluble scFv and measure the concentration using a BCA assay. The different scFvs were incubated with p65 bioactive peptide (competitor) at serial concentrations ranging from 10–11 to 10–5 for 2 h. Then, ELISA was done as described above. PBS and scFvs without preincubation with competitor were also used as negative and positive controls, respectively. The affinity constant was defined as the reciprocal of the competitor concentration required for 50% inhibition of maximal scFv binding (determined by the positive control, non-preincubated antibody) and was expressed as per molar concentration.

PCR screening and sequence alignments of p65-specific phage clones. To check individual clones for the presence of full-length VH and VL inserts, the selected clones were screened using PCR with the following conditions: 35 cycles of 94°C for 1 min, 55°C for 1 min (annealing), and 72°C for 2 min (extension) with a terminal 72°C, 10 min final extension cycle. The positive selected phage clones were sequenced using the primers LMB3 (5'-CAGGAAACAGCTATGAC) and pHEN Seq (5'-CTATGCGGCCCCATTCA) anti-p65 scFv DNA fragment. Sequence alignments to National Center for Biotechnology Information database were carried out using Immunoglobulin BLAST.

Construction of the nuclear-targeted anti-p65 intrabody. ScFv coding sequence was isolated from pIT2 by NcoI/NotI restriction and subcloned into the vector pCMV/myc/nuc (pCMV/nu) digested with the same enzymes. The anti-p65 intrabody construct (named pFv/nu) was cloned in-frame with the nuclear location signal and c-myc epitope at the COOH terminus and expressed as a fusion protein, subsequently confirmed by DNA sequencing.

Cell culture and transfection. Human glioma cell lines, U251 and U87, were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum in a humidified incubator with 5% CO2 at 37°C. For transfection with pFv/nu, pCMV/nu, and pGFP/nu, Lipofectamine reagent (Invitrogen) was used according to the manufacturer's instructions. After 48 h posttransfection, cells were harvested for isolation of total RNA and/or whole-cell lysate for reverse transcription-PCR (RT-PCR) or Western blotting. For zymography and in vitro angiogenic assays, the complete medium was replaced with serum-free medium for 12-h incubation after transfection, and conditioned medium was collected. DMEM containing G418 (800 µg/mL) was used for selection of stable clones of transfection, and DMEM with 400 µg/mL G418 was used for maintenance of the stable transfectants.

Western blotting assay. Western blotting was done. Equal amounts of protein from cell lysates were mixed with 2x SDS loading buffer and were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with primary antibodies. The enhanced chemiluminescence system was used for detection of immunoreactive proteins with horseradish peroxidase–conjugated immunoglobulin G (IgG) as secondary antibodies. The following antibodies were used: anti–c-myc mAb clone 9E10 (Sigma, St. Louis, MO), anti-p65, anti–phospho-p65, anti-p50, anti-I{kappa}B{alpha}, anti–phospho-I{kappa}B{alpha}, anti–I{kappa}B kinase {alpha}, anti–MMP-9, anti-uPA, anti-uPAR, anti-VEGF, and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All antibodies were obtained from (Biomeda, Foster City, CA) and Santa Cruz Biotechnology (Santa Cruz, CA).

Immunofluorescence assay. Cells were fixed with 10% buffered formalin for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and incubated with primary antibodies followed by fluorescent secondary antibody. A Leitz fluorescence microscope was used to acquire the images for further analysis.

Cell proliferation assay. Colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT; Chemicon, Temecula, CA) assay was used for the evaluation of glioma cells proliferation according to the manufacturer's instructions. After seeding cells into 96-well plates, MTT was added into the plates for additional incubation for 3 to 4 h in a humidified incubator with 5% CO2 at 37°C. Following addition of detergent reagent, the absorbance at 570 nm was measured on a microplate reader (Benchmark, Bio-Rad) with a reference wavelength of 630 nm.

Flow cytometry. Samples were incubated in the presence of anti-p65 antibody at 4°C overnight at various time points posttransfection. FITC-conjugated IgG (final concentration, 50 nmol/L) was added and incubated for 1 h at room temperature. Finally, the cells were analyzed with a FACScan cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon ion laser emitting at 488 nm.

Electrophoretic mobility shift assay. To detect the DNA binding activity of NF-{kappa}B, electrophoretic mobility shift assay was done according to the manufacturer's instructions (Panomics, Inc., Redwood City, CA). Briefly, nuclear proteins were prepared using a nuclear extraction kit and their concentrations determined by protein estimation procedure. A biotin-labeled NF-{kappa}B probe with a 5'-AGTTGAGGGGACTTTCCCAGGC-3' sequence or an unlabeled cold probe was used to bind nuclear proteins at 15°C to 20°C for 30 min. Products were run on a 6% nondenaturing polyacrylamide gel in 0.5x Tris-borate EDTA at 120 V for 60 min at 4°C; the shifted bands corresponding to the protein/DNA complexes were separated relative to the unbound dsDNA. The gel was then transferred onto a presoaked membrane (Pall Biodyne B membrane) at 300 mA for 30 min at 4°C. Following the immobilization of bound oligonucleotides in the membrane by a UV-cross-linking oven for 5 min, the shifted bands were visualized after exposure to film.

RT-PCR. Total RNA was isolated from the transfected cells using the Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol, and OneStep RT-PCR kit (Qiagen) was used for detecting mRNA expression of MMP-9, uPA, and VEGF. First-strand cDNA was prepared using Omniscript and Sensiscript reverse transcriptases at 50°C for 30 min. PCR amplification was then carried out under the following conditions: 95°C for 15 min, followed by 35 cycles at 94°C for 1 min, at either 48°C (for amplification of uPAR) or 56°C (for MMP-9, VEGF, uPA, and GAPDH as an internal control) for 1 min, and at 72°C for 1 min. The final extension was completed at 72°C for 10 min. The primers used are shown in Table 1 .


View this table:
[in this window]
[in a new window]

  		Table 1. Oligonucleotide primers of human uPAR, uPA, MMP-9, VEGF, and GAPDH used in RT-PCR assay

 
Zymography. Gelatin and fibrin zymography were done to detect MMPs and uPA, respectively, as previously described (11). For gelatin zymography, the cell conditioned medium was collected following protein estimation and equal amounts of protein were subjected to SDS-PAGE under nonreducing conditions in 10% SDS-PAGE gel containing 0.1% gelatin. Gels were washed in 2.5% Triton X-100 and incubated in Tris-CaCl2 buffer at 37°C for 18 to 22 h. The gels were then stained with 0.2% Coomassie blue for 30 min and destained in 20% methanol and 10% acetic acid. The clear bands represented gelatinase activity. Fibrin zymography was similarly done to detect uPA using the above-mentioned method instead of the gel-containing fibrin and plasminogen.

Matrigel invasion assay. The invasiveness of glioma cells was tested after transfection as previously described. The cells (1 x 106/mL) were added to the upper wells coated with Matrigel (1 mg/mL; Collaborative Research, Inc., Boston, MA) with serum-free medium containing 25 µg/mL fibronectin as a chemoattractive agent in the lower wells. After a 24-h incubation period, cells that migrated through the filters into the lower chamber were counted by the number of cells on the lower side of the membrane in five random fields after staining with Hema-3 kit.

In vitro angiogenesis assay. Glioma cells (2 x 104/mL) were transfected with various plasmids for 48 h and the conditioned medium was filtered off for future research as previously described (12). HMEC-1 endothelial cells (4 x 104) were seeded onto eight-well chamber slides and the aforementioned conditioned medium was added. Cells were cultured for 72 h until capillary network formation was observed. The number of branch points and total number of branches per point were counted after H&E staining to quantify the degree of angiogenesis.

Capillary-like structure formation in coculture assay. Glioma cells (2 x 104) were seeded onto eight-well chamber slides and transfected with various plasmids for 72 h. Cell medium was removed and allowed to coculture with HMEC-1 endothelial cells (4 x 104) for 72 h. Capillary networks, indicators of angiogenesis, were assessed with VIII antibody and then with FITC-conjugated secondary antibody under fluorescence microscopy.

Apoptosis assay. Cells were cultured in six-well plates (1 x 106 per well) and transfected for 72 h. The apoptotic cells were stained with Hoechst 33244 dye, which selectively stains nuclei of apoptotic cells. Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling apoptosis assay was done for the DNA fragmentation fluorescence staining under the manufacturer's instructions (Upstate Cell Signaling Solution, Charlottesville, VA). Briefly, cells in eight-well chamber slides were fixed with 4% paraformaldehyde-PBS for 15 min at room temperature and then incubated with 50 µL of terminal deoxynucleotidyl transferase end-labeling cocktail for 1 h, following washing in PBS thrice and permeabilization with 0.05% Tween 20, 0.2% bovine serum albumin in PBS for 15 min at room temperature. After the steps including immersion of cells in Tris-borate buffer to stop the reaction, washing in PBS, and blocking in blocking buffer, the slides were subsequently incubated with avidin-FITC solution in the dark for 30 min at room temperature and viewed under a fluorescent microscope, followed by washing and mounting coverslip with gel.

Intracranial tumor growth. Athymic male nude mice (nu/nu, 6-8 weeks age) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Animal handling and experimental procedures were approved by the University of Illinois College of Medicine Institutional Animal Care and Use Committee. For the intracerebral tumor model, 2 x 106 U87 (or U251) cells and stable transfected cells were counted and intracerebrally inoculated into nude mice (five mice per group). On day 45 after orthotopic tumor implantation, mouse brains were removed and fixed. Paraffin sections (3-5 µm) were prepared and observed blindly to evaluate tumor size. Tumor size was measured semiquantitatively in terms of maximum cross-section diameter as an index of intracranial tumor size. The variation between the sections in each group was <10% for tumor regression experiments.

Statistical analysis. The significance of the results was determined by the Student's t test (two-tailed). Values are expressed as mean ± SD from at least three separate experiments and differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Preparation of anti-p65 human scFv and construction of intrabody. Two large phage display libraries, human single-fold scFv libraries (Tomlinson I + J; Cambridge Antibody Technology, Cambridge, United Kingdom), were panned to obtain anti-p65 scFv fragments. After four rounds of panning by polyclonal phage ELISA and monoclonal phage ELISA, crude scFv preparations bound to the target molecule were eluted. The clones that showed a 3- to 5-fold higher specific affinity to the target (86 of 200 randomly picked) were then used for downstream applications. The specific affinity to antigen p65 and the lower affinity binding to nonrelated bovine serum albumin of 28 selected scFv clones were shown in ELISA (Fig. 1A ).


Figure 1
View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Characterization and construction of the anti-p65 scFv and intrabody. A, specificity of scFv to p65 bioactive peptide. After four rounds of panning by polyclonal ELISA, 28 scFvs from the positive clones were selected to detect their binding activity in monoclonal ELISA. Briefly, 100 µL of single-phage suspension were added into 96-well plates precoated with p65 peptide or unrelated antigen, bovine serum albumin (BSA), for 3 h of incubation, as well as PBS for negative control. The anti–c-myc mAb, recognizing myc-tag at the COOH-terminal end of scFv, was used as the primary antibody to determine its specific affinity to p65. Following visualization by 3,3',5,5'-tetramethylbenzidine substrate, colorimetric evaluation was measured at 450 nm. B, structural schema of both scFv and intrabody-encoding genes. NLS, nucleotide sequence of a nuclear localization signal from SV40; arrows, restricted enzyme sites. C, the nucleotide sequence encoding cDNA of nuclear-targeted intrabody against p65 (DNA sequence, 900 bp) was obtained from DNA sequencing of the selected scFvs. D, amino acid sequence of nuclear-targeted intrabody against p65 (protein sequence, 299AA; protein MW, 31,896 g/mol).

 
The scFv affinity constant was ~107 (mol/L)–1 according to the result from competitive ELISA. Then the eluted clones were digested with BstNI to analyze the restriction patterns of the samples in agarose gel assays. The results (data not shown) indicate that the selected positive clones were derived from a single group and possessed the same pattern of restriction sites.

After putative binders were isolated depending on their affinity in ELISA and were analyzed for diversity by their BstNI restriction enzyme mapping pattern, scFv-encoding inserts of phage clones were amplified by PCR using the primers LMB3 (CAGGAAACAGCTATGAC) and pHEN (CTATGCGGCCCCATTCA).

The PCR products were visualized at 935 bp in agarose gel analysis, which further confirmed the expected size of the entire scFv cDNA insert (data not shown). The structure of scFv-encoding gene derived from pIT2 vector shown in the scheme (Fig. 1B) was confirmed using both DNA sequencing and restriction endonuclease digestion analysis (data not shown).

The results from DNA sequencing revealed that of 28 clones selected, only 4 clones (named clone 1, clone 2, clone 3, and clone 4) had intact, full-length cDNA without TAG stop codons; the others had internal amber (TAG) stop codons, probably as a result of the synthetic assembly process. The four intact scFv-encoding clones contained 732-bp full-length fragments, coding for 244 amino acids, without any premature stop codons, and their molecular weights were ~27 kDa.

For nuclear-targeted expression of intrabodies in mammalian cells, the scFv-encoding gene was PCR-modified and cloned into the NcoI and NotI sites of an intrabody plasmid pCMV/nu, which is a 5.0-kb expression vector designed to express recombinant protein as a fusion to a SV40 nuclear location signal and c-myc epitope. The nuclear-targeted anti-p65 intrabody was obtained after cloned in-frame with the nuclear location signal and c-myc epitope at the COOH terminus and expressed as a fusion protein in the nucleus. The scheme in Fig. 1B showed the structure of the intrabody-encoding gene, confirmed by restriction endonuclease digestion analysis (data not shown).

Because the clinical efficacy of the intrabodies depends on their stability rather than their epitope affinity, we did immunoblotting and immunofluorescence assays to analyze their longevities when expressed in glioblastoma cells with recombinant transfection. Compared with the other experimental clones, the recombinant derived from clone 1 presented the longest half-life, indicating its efficient and steady-state accumulation in the nucleus after transit through the cytosol. Therefore, we chose the recombinant, which we named pFv/nu, for further research due to its intracellular stability and activity. The construct pFv/nu was confirmed by double digestion with NcoI and NotI in agarose gel analysis (data not shown) and DNA sequencing (the sequence of its nucleic acid and amino acid was shown in Fig. 1C and D, respectively).

Expression and location of anti-p65 intrabody in glioma cells. Western blotting analysis indicated that the anti-p65 intrabody (the below band) was expressed primarily in the nuclei of U251 and U87 cells and only minutely in the cytosolic compartments, whereas samples from mock-transfected and empty vector (pCMV/nu)–transfected cells did not show any bands of the appropriate molecular weight besides the above band of endogenous c-myc protein in each lane (Fig. 2A ). Because the nuclear location signal and the c-myc epitope added ~5 kDa to scFv at its COOH terminus, the size of the total scFv-fusion protein was ~32 kDa; the bands of GFP proteins were also detected as positive control.


Figure 2
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Expression and location of anti-p65 intrabody in glioma cells. A, expression of anti-p65 intrabody in U251- and U87-transfected cells was determined by Western blotting assay. During the detection of the myc-tagged fusion protein by the anti–c-myc (9E10) mAb, the upper bands were endogenous c-myc proteins in U251 or U87 cells whereas the endogenous c-myc protein presents in both cytosolic and nuclear compartments; the lower bands showed expression of GFP or scFv fusion proteins. As described in Materials and Methods, the cytosolic and nuclear fractions from the 48-h posttransfected cells was extracted and loaded into each well of both cytosolic and nuclear lanes of the 12% SDS-PAGE gel with 30 µg of total proteins per well. Following transfer, the nitrocellulose membrane was incubated with anti–c-myc mAb (clone 9E10) and goat anti-mouse horseradish peroxidase–conjugated IgG as the primary and secondary antibodies, respectively. Consequently, the band of anti-p65 intrabody was visualized with the enhanced chemiluminescence system. B, location of the intrabody in U251 and U87 cells as detected by immunofluorescence assay. Briefly, cells with 48-h posttransfection of either pFv/nu or empty control vector, pCMV, were fixed, permeabilized, and incubated with primary antibody, anti–c-myc mAb, followed by fluorescent secondary antibody, goat anti-mouse FITC-conjugated IgG. With a fluorescence microscope, the images of the expressed intrabody in nucleus were acquired for the further analysis. C, colocalization of p65 and anti-p65 intrabody in U251 (top) and U87 (bottom) transfected cells was determined in immunofluorescence assay, as described in (B). Left, distribution of p65 (red) detected with polyclonal anti-p65 antibody, followed by Texas red–conjugated antirabbit IgG. Middle, localization of anti-p65 intrabody (green) detected with c-myc (9E10) mAb, followed by FITC-conjugated antimouse IgG. Right, merged signal by p65 (red) and intrabody (green) in yellow.

 
The nuclear location of anti-p65 intrabody was confirmed by immunofluorescence assay after transfection with both pCMV/nu and pFv/nu. The results (Fig. 2B) concur with those from the immunoblotting analysis. In addition, colocalization of p65 and anti-p65 intrabody is shown in Fig. 2C. The results suggest that anti-p65 intrabody fusion protein presented exactly in the nuclei of human glioblastoma cells. Furthermore, the subcellular localization of anti-p65 intrabody was consistent with the distribution of p65 in the nucleus.

Kinetic analysis of intrabody expression. The result from Western blotting assay (Fig. 3A ) shows that a gradual increase in the nuclear expression of the anti-p65 intrabody in U251 and U87 cells was observed on days 0, 1, 2, 3, 5, and 7 after transfection with pFv/nu. Meanwhile, the nuclear p65 expression in the intrabody-transfected cells was reduced by the maximal inhibition rate at 40% (U251) or 51.1% (U87) on day 3 or 5 compared with mock cells (Fig. 3B and C), determined by flow cytometry. p65 expression level inversely correlated with the increasing expression of the intrabody on days 1, 2, 3, and 5, which was consistent with the hypothesis that the increased expression of anti-p65 intrabody in nucleus might contribute to the reduced p65 expression in tumor cells after pFv/nu transfection.


Figure 3
View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Kinetic analysis of intrabody expression. A, increased expression of anti-p65 intrabody from nuclear fractions of U251 and U87 cells was detected on days 0, 1, 2, 3, 5, and 7 after pFv/nu transfection, as determined by Western blotting assay. To monitor the expression of the intrabody, the nuclear extract from the pFv/nu-transfected cells was collected for Western blotting assay with anti–c-myc mAb and goat anti-mouse horseradish peroxidase–conjugated IgG as the primary and secondary antibodies, respectively, whereas anti-GAPDH antibody served as an internal control. B, densitometry results of immunublots described in (A), plotted as change from control amount on day 0 after normalization to GAPDH expression level of each lane. C, depletion of p65 nuclear expression in U251 and U87 cells with anti-p65 intrabody transfection was monitored for 7 days by flow cytometry. The nuclear fractions were incubated with anti-p65 antibody as a primary antibody and then with FITC-conjugated goat anti-rabbit IgG antibody as a secondary antibody. D, quantitation of nuclear p65 expression on days 0, 1, 2, 3, 5, and 7 after transfection. The formula used for calculation of expression inhibition was percent inhibition = (p65mock – p65Fv) / p65 mock, which is shown in (C). Points, mean from three separate experiments; bars, SD (P < 0.001).

 
Effect of anti-p65 intrabody on NF-{kappa}B activity. There was no apparent reduction in both fractions of the control samples and cytosolic fraction of intrabody-transfected samples, as determined by Western blotting (Fig. 4A ), whereas a significant decrease in expression of p50, p65, and their phosphorylated forms was detected in the nuclear fraction of intrabody-transfected samples. This implicated that the translocation ability of p65/p50 dimers into the nuclei was apparently inhibited via the intrabody transfection, leading to their down-regulation mainly in the nucleus of intrabody-transfected cells. In addition, the result (Fig. 4B) from Western blotting showed that anti-p65 intrabody significantly down-regulated I{kappa}B{alpha} and phospho-I{kappa}B{alpha} by blocking nuclear translocation of NF-{kappa}B and transcription of its target genes. However, the expression of I{kappa}B kinase {alpha} as the upstream protein of NF-{kappa}B activation signal pathway did not significantly alter. These findings were further shown to be consistent with the results shown in Fig. 4A.


Figure 4
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of anti-p65 intrabody on NF-{kappa}B activity. A, reduced expression of p65, p50, and their phosphorylated forms in cell nucleus. Expression of p65/p50 heterodimer in nuclear extractions and cytosolic fractions of U251 and U87 tumor cells after transfection with anti-p65 intrabody recombinant was determined by Western blot assay, as described in Materials and Methods. GAPDH antibody was used to verify that the same amount of protein has been loaded in each lane. B, the expressions of NF-{kappa}B activity–related proteins in human glioma U251 and U87 cells after transiently transfection with recombinants were analyzed by Western blot assay, as described in Materials and Methods. GAPDH antibody was used as an internal control. Western blot results are representative of three independent preparations. A (right) and B (bottom), quantitation analysis of the respective data. Columns, mean of three observations followed by normalization with GAPDH protein amount. The relative protein expression in each lane was compared with untreated mock cells, arbitrarily as one unit (AU, arbitrary unit). C, effect of anti-p65 intrabody expression on the DNA binding activity of NF-{kappa}B in human glioma U251 (left) and U87 (right) cells after transient transfection with recombinant pFv/nu in electrophoretic mobility shift assay, detected as described in Materials and Methods. First, nuclear proteins were incubated with a biotin-labeled NF-{kappa}B probe or an unlabeled cold probe after preparation of nuclear extracts. Subsequently, the shifted bands of the protein/DNA complexes were separated from the unbound dsDNA by running on a 6% nondenaturing polyacrylamide gel. Following Pall Biodyne B membrane transfer, immobilization of bound oligonucleotides, and exposure to film, the shifted bands were visualized by enhanced chemiluminescent system. For supershift assay, anti-p65 and anti-p50 antibodies were added to the nuclear extracts before incubation with the DNA probe.

 
The results (Fig. 4C) from electrophoretic mobility shift assay shows that the ability of NF-{kappa}B to bind to probe DNA was almost completely inhibited in the nuclei of U251 and U87 cells transfected with pFv/nu as compared with mock cells and control vector–transfected cells, which showed that NF-{kappa}B transcriptional activity was significantly inhibited by the intrabody transfection.

Effect of transfection on cell proliferation and apoptosis of glioma cells. Figure 5A shows that the growth rate of all the cells of 72-h posttransfection was very similar in MTT assay, suggesting that transfection does not alter cell proliferation rate in these cells.


Figure 5
View larger version (46K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Effect of transfection on cell proliferation and apoptosis of glioma cells. A, effect of transfection on cell proliferation. In the MTT assay, U251 and U87 cells were trypsinized after 48-h posttransfection with mock, pCMV/nu, pGFP/nu, and pFv/nu, respectively, and continually cultured for 48 h. The number of metabolically active cells (5 x 103) was detected following the addition of MTT for 3-h incubation at 37°C followed by detergent reagent for additional 1-h incubation. Absorbance of living cells in 96-well plates was measured on an ELISA reader at 570 nm with a reference wavelength of 630 nm. Columns, mean from three separate experiments; bars, SD (P < 0.001). B, effect of transfection on apoptosis. U251 and U87 cells after 48-h transfection were stained with Hoechst 33244. The apoptotic cells were counted under a fluorescent microscope in five random 200-fold fields, as compared with the control, arbitrarily chosen as one unit. Right, columns, mean from three separate experiments; bars, SD (P < 0.001). C, terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling apoptosis assay was done for DNA fragmentation/fluorescence staining. Briefly, pFv/nu- or pCMV/nu-transfected cells in eight-well chamber slides were fixed, washed, permeabilized, and incubated with terminal deoxynucleotidyl transferase end-labeling cocktail. Following immersion in Tris-borate buffer to stop the reaction and blocking, the cells were subsequently incubated with avidin-FITC solution. Finally, the slides were observed to count the apoptotic cells stained in green under a fluorescent microscope by the same method as in (B). Right, columns, mean from three separate experiments; bars, SD (P < 0.001).

 
The result (Fig. 5B and C) indicates that transfection might, to a small degree, induce apoptosis in glioma cells. However, there was no significant difference between pCMV/nu (control vector) and pFv/nu (experimental construct), which implies that the intrabody does not seem to induce NF-{kappa}B–mediated apoptosis.

Effect of anti-p65 intrabody on invasion and angiogenesis via blocking NF-{kappa}B bioactivity. Western blotting assay (Fig. 6A ) and RT-PCR assay (Fig. 6B) showed reduced expression of protein and mRNA of MMP-9, uPAR, uPA, and VEGF after transfection with anti-p65 intrabody. Enzymatic activity of MMP-9 and uPA was significantly inhibited as compared with the controls by gelatin and fibrin zymography (Fig. 6C). The expression level in related target proteins and mRNAs of each cell line showed a cell line–specific difference (Fig. 6A and B), in which VEGF decreased dramatically with intrabody expression in U251 but not in U87 cells, whereas MMP-9 expression showed more reduction in U87 than in U251 cells. It implied that decreased NF-{kappa}B–mediated protein expression by intrabody expression is attributed to the cell line–specific difference and the effect of other transcriptional cofactors besides those controlled by NF-{kappa}B.


Figure 6
View larger version (59K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. The effects of mock-, pCMV/nu-, pGFP/nu-, and pFv/nu-transfected cells on the expression of MMP-9, uPAR, uPA, and VEGF at protein and mRNA levels by Western blotting assay (A) and RT-PCR assay (B), respectively, as described in Materials and Methods. The used primers for mRNA expression detection of MMP-9, uPAR, uPA, VEGF, and GAPDH were shown in Table 1. Results from Western blots and RT-PCR are representative of three independent preparations. A and B, right, quantitation of the respective data. Columns, mean of three observations and normalized to untreated mock cells, arbitrarily chosen as one unit. C, effect on enzymatic activities of MMP-9 and uPA in transfected cells. Gelatin zymography for MMP-9 and fibrin zymography for uPA in U251 and U87 cells after 48-h transfection with mock, pCMV/nu, pGFP/nu, and pFv/nu were done. Briefly, the conditioned medium from transfected cells was collected, followed by incubation with serum-free medium and protein estimation. Equal amount of protein was loaded into nondenaturing SDS-PAGE gel containing relevant substrates. Subsequently, the gel was incubated at 37°C overnight, stained, and destained; the clear bands in the gels represent enzymatic activity of MMP-9 or uPA. Representative of three separate experiments.

 
The result (Fig. 7A ) from Matrigel invasion assay indicates that intrabody transfection significantly inhibited the invasion of U251 and U87 cells by 88.6% and 84%, respectively, as compared with mock-transfected and control vector–transfected cells. Furthermore, the results (Fig. 7B) showed that HMECs treated with conditioned media from mock- and pCMV/nu-transfected glioblastoma cells were able to form capillary-like structures. It shows that HMECs treated with conditioned media from pFv/nu-transfected U251 and U87 cells showed less capillary-like networks (60.5% and 62.1%, respectively), as compared with the controls. Additionally, capillary-like structure formation was observed using immunofluorescent staining for factor VIII in cocultured assay. HMECs cultured with mock- and pCMV/nu-transfected cells exhibited capillary networks, whereas the formation of capillary-like structures in pFv/nu-transfected U251 and U87 cells was almost completely suppressed (87.8% and 78.6%, respectively; Fig. 7C).


Figure 7
View larger version (88K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Effect of anti-p65 intrabody on invasion and angiogenesis. A, invasiveness of U251 and U87 cells transfected with recombinants in Matrigel invasion assay. Cells were transfected with empty vector pCMV/nu or anti-p65 intrabody construct pFv/nu. After 2 d, 1 x 105 cells were allowed to invade through transwell inserts (8 µm) coated with Matrigel. The cells on lower surface of chambers were stained, counted, and photographed under a light microscope. The in vitro inhibition of invasiveness was calculated in five random 200-fold fields using the following formula: percent inhibition = (mock – Fv) / mock. Right, columns, mean from three separate experiments; bars, SD (P < 0.001). B, HMEC endothelial cells treated with conditional medium from U251 and U87 glioma cells after intrabody transfection by in vitro angiogenesis assay. The conditioned medium of glioma cells was collected after 48-h transfection following filtering of medium. HMEC-1 endothelial cells seeded in eight-chamber slides were cultured with the above medium for 72 h until the formation of capillary network was observed. In the end of the experiment, angiogenesis was assessed by H&E staining and photographed under a microscope. Right, columns, mean from three separate experiments; bars, SD (P < 0.001). C, formation of capillary-like structures in coculture of endothelial HMEC cells and glioma U251/U87 cells after intrabody transfection. The mock or 48-h posttransfected glioma cells were plated onto eight-chamber slides following seeding of HMEC-1 endothelial cells to coculture constitutively for 72 h until the formation of capillary network. The angiogenesis was assessed by the immunofluorescent staining of factor VIII and quantified under a fluorescent microscope. Right, columns, mean from three separate experiments; bars, SD (P < 0.001).

 
Furthermore, results from the invasion and angiogenesis assay indicated that the abilities of local invasiveness and neovascularization of glioma cells showed cell line–specific differences associated with intrabody expression. As we detected, the invasive ability of U87 cells was significantly impaired, as well as that of U251 cells in which the expression of uPA and MMP-9 showed less reduction. Although the VEGF expression in U87 cells was not inhibited as much as that in U251 cells, the formation of capillary-like structure was almost completely suppressed in U87 cells with the intrabody expression. It suggested that the proposed molecular basis for endothelial tube formation and local invasion is a very complicated process, which involved interactions of multiple molecules such as adhesive molecules, epidermal growth factor receptor family, and other transcription cofactors.

Inhibition of anti-p65 intrabody on intracranial tumor growth in nude mice. Having shown that anti-p65 intrabody decreased invasion and angiogenesis by down-regulation of MMP-9, uPA, and VEGF in U251 and U87 cells in vitro, we tested the growth of the tumors accomplished by pFv/nu-transfected cells using an orthotopic intracranial glioma model. As shown in Fig. 8A and B , the tumors intracranially injected with pFv/nu-transfected U251 or U87 cells showed a significant reduction as compared with control groups, whereas U251 (or U87) cells developed obvious intracranial tumors in nude mice.


Figure 8
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Evaluation of intracranial tumor growth in the brains of nude mice by intrabody transfection. A, intracranial tumor assay. The growth of the tumor accomplished glioma cells was tested using an orthotopic intracranial glioma model. Briefly, U251 (or U87) glioma mock cells (2 x 106 in serum-free medium), control vector (pCMV/nu and pGFP/nu), and intrabody (pFv/nu)–transfected cells were injected intracranially, respectively. After 5 wk, mice from each group (five per group) were killed and their brains removed, fixed in formaldehyde, sectioned, and stained (H&E). B, semiquantification of tumor volume in control, pCMV/nu-, pGFP/nu-, and pFv/nu-treated groups. Sections were observed blindly and scored semiquantitatively for tumor size under a microscope (x100; Olympus BX16). Columns, mean from five animals in each group; b Bars, SD (P < 0.001).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
With the advent of phage display libraries derived from the human IgG repertoire, an scFv against the target of interest can be selected in a relatively short time. Over the past decade, there has been growing interest in the use of scFv fragments aimed at intracellular targets to inhibit protein expression and trafficking as a gene therapeutic strategy against cancer, commonly referred to as intrabody. Because intrabodies can be directed to specific subcellular compartments and precise epitopes on target proteins, they have the potential to block only one of several functions of an expressed protein at the posttranslational level. This potential has been exploited by several laboratories in gene therapeutic strategies as well as in revealing the biological function of proteins in cancer, AIDS, and other research areas (13–15). Intrabodies present a potent alternative to methods of gene inactivation that allows for targeting at the level of DNA or mRNA, such as antisense oligonucleotides (16), zinc finger proteins (17), and RNA interference (18). In the present study, we succeeded in using the human single-fold scFv libraries (Tomlinson I + J) to prepare for the humanized anti-p65 intrabody precisely expressed in the nucleus of glioma cells as determined by immunoblotting and immunofluorescent results and also proved that the recombinant presented a longer half-life and more effective intracellular activity.

In the past, scFvs derived from high-affinity antibodies were thought to be most desirable for use as intrabodies due to their inherent antigen-binding properties. However, growing evidence suggests that the intracellular stability of an intrabody, not its affinity, is most critical for its efficacy. A useful description of an effective intrabody should include specificity, affinity, and intracellular stability, where stability is the determining factor for intrabody efficacy. Stability seems to be especially important when intrabodies are expressed in the strongly reducing environment of cytosol, which disrupts the formation of intrachain disulfide bridges that can be essential for the proper folding, solubility, and inherent stability of scFvs. ScFvs with extended half-lives have been shown to achieve higher steady-state expression levels, a by-product of which seems to be a more effective target molecule inactivation. Removal from the harsh cytosolic environment by directing scFvs expression to the ER or nucleus can also increase half-life, as long as the desired localization was detectable for interaction with the target protein (19–24). Our construct pFv/nu, which displayed a long half-life and accumulated at steady-state level in the nucleus even after transit through the reducing cytosolic environment, proved to be suitable for the interaction with the target molecule owing to its intracellular stability and activity. In future research, to enhance intracellular stability and activity of the intrabody in the in vivo environment, it is proposed that we could fuse the scFv with other stabilized fragments such as Fc of human IgG, or establish other additional intracellular screening steps such as ribosome display, mRNA display, and yeast two-hybrid screening after the primary phage display (25–28).

Local invasive infiltration and growth are key features in glioblastoma, which are accompanied by remodeling of the vasculature and by the destruction of the surrounding tissues. Several reports in human gliomas showed overexpression of uPA, uPAR, MMP-9, and VEGF, which are involved in the adhesion, invasion, metastasis, and angiogenesis of human glioblastomas. Numerous studies on interference with the uPA-uPAR system and the MMP and VEGF pathways have shown the successful inhibition of neovascularization and tumor growth in preclinical or clinical trials (29–40). It has been shown that the {kappa}B binding site of NF-{kappa}B was identified in the promoter regions of uPA, uPAR, MMP-9, and VEGF genes. As such, their transcriptional activation could be controlled by NF-{kappa}B in the nucleus where it recognizes and functionally binds to their promoters and further triggers their transcription (29–33). Moreover, aberrant or constitutive activated NF-{kappa}B has been detected in human high-grade gliomas (3–5). Based on the important role of NF-{kappa}B and its mediated genes in tumor growth, angiogenesis, invasion, and metastasis, blocking NF-{kappa}B activity to inhibit target gene expression could then serve as a promising approach for cancer therapy. According to reports, constitutive NF-{kappa}B activity in cancer cells can be inhibited through down-regulation of the NF-{kappa}B intrinsic inhibitor I{kappa}B; its specific upstream kinases such as phosphatidylinositol 3-kinase, protein kinase C, mitogen-activated protein kinase, and I{kappa}B kinase; and a nonspecific proteasome inhibitor, PS-341 (40–43). We therefore hypothesized that direct blocking NF-{kappa}B transcriptional activity by intrabody expression could contribute to the inhibitory expression and activity of MMP-9, uPA, uPAR, and VEGF, resulting in inhibition of tumor invasion, metastasis, and angiogenesis of human gliomas.

For the first time, we reported that the inhibition of p65 transactivation by anti-p65 intrabody expression in the nucleus might suppress the transcriptional activity of NF-{kappa}B, resulting in the inhibition of the expression and activity of its target proteins associated with human glioma invasiveness and angiogenesis. Based on the kinetic analysis (Fig. 3), we showed that nuclear p65 level decreased maximally with inhibition rates of 40% (U251) and 50% (U87) on day 3 (in U251) or day 5 (in U87), respectively. After that time point, the expression level of nuclear p65 started to be restored although the intrabody amount maintained a raised trend until day 7 after transfection, suggesting that the action mechanism of the intrabody herein, which is able to neutralize its target protein (p65), change its space conformation by forming the antigen-antibody (p65-intrabody) complex, and then inhibit the nuclear p65 expression, was a reversible and time-limited reaction based on a certain level of expression of the intrabody in nucleus. As such, the depleted nuclear p65 could be free partly once the nuclear anti-p65 intrabody was degraded to release p65 protein from the complex due to the transient expression of the intrabody after transient pFv/nu transfection into the glioma cells. In addition, the expression of the intrabody showed a cell line–specific difference that the increasing ratio of the intrabody in U87 cells was significantly more than that in U251 cells, which might explain why the depletion of nuclear p65 in U87 cells could maintain longer than in U251 cells. To overcome this transient depletion of p65, the selection of a stably transfected clone could be a helpful approach to obtain a stable nuclear expression of the intrabody. In addition, we are planning to screen an intrabody able to directly degrade its target protein (p65) after binding reaction, which could be another more effective way to completely inhibit the activity of NF-{kappa}B in nucleus. The result from intracranial tumor experiment showed remarkable inhibition of tumor growth after injection with stable clones of pFv/nu-transfected U251 or U87 cells compared with obvious intracranial tumors established by control vector–transfected U251 (or U87) cells in nude mice, which further showed that the constant inhibition of tumor growth by pFv/nu transfection could be observed after stable expression of the intrabody.

Because most of studies on NF-{kappa}B indicate that it mediates the expression of antiapoptotic and proapoptotic proteins, we detected whether apoptotic cells were induced after transfection. However, there was no apparent difference in the number of apoptotic cells (Fig. 5B and C), suggesting that anti-p65 intrabody expression did not induce the NF-{kappa}B–mediated apoptosis pathway. Although the expression of its target antiapoptotic proteins, such as FLIP and Bcl-XL, was decreased by the inhibition of NF-{kappa}B transcription activity in our experiment (data not shown), the caspase expression associated with apoptotic phenotype did not increase. This implies that there perhaps exists an alternative-signaling pathway to prevent apoptosis of glioma cells.

In conclusion, blocking the transcriptional activity of NF-{kappa}B using anti-p65 nuclear-targeting intrabody inhibited glioblastoma cell angiogenesis, invasion, and intracranial tumor growth via the down-regulation of MMP-9, uPAR, uPA, and VEGF. As such, this approach may be useful for the treatment of gliomas as well as other tumors. Furthermore, the intrabody strategy provides a novel way to interfere with NF-{kappa}B activity in the nuclear compartment and to study the biological significance of the NF-{kappa}B signaling pathway in cancer biology.


   Acknowledgments
 
We thank Shellee Abraham for manuscript preparation and Diana Meister and Sushma Jasti for manuscript review.


   Footnotes
 
Grant support: National Cancer Institute grants CA75557, CA92393, CA95058, and CA116708; National Institute of Neurological Disorders and Stroke grant NS47699; Caterpillar, Inc.; and OSF Saint Francis, Inc., Peoria, IL (J.S. Rao).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/13/06; revised 11/21/06; accepted 1/23/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cavenee WK, Furnari FB, Nagane M. Diffusely infiltrating astrocytomas. In: Kleihues P, Cavenee WK, editors. Tumors of the nervous system. Lyon: IARC Press; 2000. p. 9–51.
  2. Burger PC, Vogel FS, Green SB, Strike TA. Glioblastoma multiforme and anaplastic astrocytoma. Pathologic criteria and prognostic implications. Cancer 1985;56:1106–11.[CrossRef][Medline]
  3. Karin M, Cao Y, Greten FR, Li ZW. NF-{kappa}B in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002;2:301–10.[CrossRef][Medline]
  4. Ravi R, Bedi A. NF-{kappa}B in cancer—a friend turned foe. Drug Resist Updat 2004;7:53–67.[CrossRef][Medline]
  5. Kanzawa T, Ito H, Kondo Y, Kondo S. Current and future gene therapy for malignant gliomas. J Biomed Biotechnol 2003;2003:25–34.[CrossRef][Medline]
  6. Wang H, Wang H, Zhang W, Huang HJ, Liao WS, Fuller GN. Analysis of the activation status of Akt, NF{kappa}B, and Stat3 in human diffuse gliomas. Lab Invest 2004;84:941–51.[CrossRef][Medline]
  7. Buss H, Dorrie A, Schmitz ML, et al. Phosphorylation of serine 468 by GSK-3ß negatively regulates basal p65 NF-{kappa}B activity. J Biol Chem 2004;279:49571–4.[Abstract/Free Full Text]
  8. Egan LJ, Mays DC, Huntoon CJ, et al. Inhibition of interleukin-1-stimulated NF-{kappa}B RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity. J Biol Chem 1999;274:26448–53.[Abstract/Free Full Text]
  9. Wang D, Westerheide SD, Hanson JL, Baldwin AS, Jr. Tumor necrosis factor {alpha}-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 2000;275:32592–7.[Abstract/Free Full Text]
  10. Schmitz ML, dos Santos Silva MA, Baeuerle PA. Transactivation domain 2 (TA2) of p65 NF-{kappa}B. Similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. J Biol Chem 1995;270:15576–84.[Abstract/Free Full Text]
  11. Gondi CS, Lakka SS, Dinh D, Olivero W, Gujrati M, Rao JS. Down-regulation of uPA, uPAR and MMP-9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth. Neuron Glia Biology 2004;1:165–76.[CrossRef][Medline]
  12. Lakka SS, Gondi CS, Yanamandra N, et al. Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angiogenesis, and tumor growth. Cancer Res 2003;63:2454–61.[Abstract/Free Full Text]
  13. Heng BC, Cao T. Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (Intrabody). Med Hypotheses 2005;64:1105–8.[CrossRef][Medline]
  14. Popkov M, Jendreyko N, Gonzalez-Sapienza G, Mage RG, Rader C, Barbas CF III. Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies selected from an immune b9 allotype rabbit antibody library. J Immunol Methods 2004;288:149–64.[CrossRef][Medline]
  15. Wheeler YY, Chen SY, Sane DC. Intrabody and intrakine strategies for molecular therapy. Mol Ther 2003;8:355–66.[CrossRef][Medline]
  16. Wagner RW, Flanagan WM. Antisense technology and prospects for therapy of viral infections and cancer. Mol Med Today 1997;3:31–8.[CrossRef][Medline]
  17. Beerli RR, Barbas CF III. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 2002;20:135–41.[CrossRef][Medline]
  18. Hannon GJ. RNA interference. Nature 2002;418:244–51.[CrossRef][Medline]
  19. Caron dF, Gruel N, Venot C, et al. Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments. Oncogene 1999;18:551–7.[CrossRef][Medline]
  20. Mary MN, Venot C, Caron de Fromentel C, et al. A tumor specific single chain antibody dependent gene expression system. Oncogene 1999;18:559–64.[CrossRef][Medline]
  21. Rajpal A, Turi TG. Intracellular stability of anti-caspase-3 intrabodies determines efficacy in retargeting the antigen. J Biol Chem 2001;276:33139–46.[Abstract/Free Full Text]
  22. Ramm K, Gehrig P, Pluckthun A. Removal of the conserved disulfide bridges from the scFv fragment of an antibody: effects on folding kinetics and aggregation. J Mol Biol 1999;290:535–46.[CrossRef][Medline]
  23. Wu Y, Duan L, Zhu M, et al. Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition. J Virol 1996;70:3290–7.[Abstract]
  24. Zhu Q, Zeng C, Huhalov A, et al. Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen, caspase-7. J Immunol Methods 1999;231:207–22.[CrossRef][Medline]
  25. Amstutz P, Forrer P, Zahnd C, Pluckthun A. In vitro display technologies: novel developments and applications. Curr Opin Biotechnol 2001;12:400–5.[CrossRef][Medline]
  26. der Maur AA, Zahnd C, Fischer F, et al. Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J Biol Chem 2002;277:45075–85.[Abstract/Free Full Text]
  27. Strube RW, Chen SY. Enhanced intracellular stability of sFv-Fc fusion intrabodies. Methods 2004;34:179–83.[CrossRef][Medline]
  28. Worn A, Pluckthun A. Stability engineering of antibody single-chain Fv fragments. J Mol Biol 2001;305:989–1010.[CrossRef][Medline]
  29. Chintala SK, Tonn JC, Rao JS. Matrix metalloproteinases and their biological function in human gliomas. Int J Dev Neurosci 1999;17:495–502.[CrossRef][Medline]
  30. Choong PF, Nadesapillai AP. Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis. Clin Orthop Relat Res 2003;(415 Suppl):S46–58.[CrossRef][Medline]
  31. Machein MR, Plate KH. VEGF in brain tumors. J Neurooncol 2000;50:109–20.[CrossRef][Medline]
  32. Hasan J, Jayson GC. VEGF antagonists. Expert Opin Biol Ther 2001;1:703–18.[CrossRef][Medline]
  33. Ikeda E, Achen MG, Breier G, Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Biol Chem 1995;270:19761–6.[Abstract/Free Full Text]
  34. Kerr DJ. Targeting angiogenesis in cancer: clinical development of bevacizumab. Nat Clin Pract Oncol 2004;1:39–43.[CrossRef][Medline]
  35. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993;362:841–4.[CrossRef][Medline]
  36. Lin P, Sankar S, Shan S, et al. Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor. Cell Growth Differ 1998;9:49–58.[Abstract]
  37. Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992;359:845–8.[CrossRef][Medline]
  38. Pulukuri SM, Gondi CS, Lakka SS, et al. RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo. J Biol Chem 2005;280:36529–40.[Abstract/Free Full Text]
  39. Lakka SS, Gondi CS, Yanamandra N, et al. Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis. Oncogene 2004;23:4681–9.[CrossRef][Medline]
  40. Huang S, Pettaway CA, Uehara H, Bucana CD, Fidler IJ. Blockade of NF-{kappa}B activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene 2001;20:4188–97.[CrossRef][Medline]
  41. Sliva D, Rizzo MT, English D. Phosphatidylinositol 3-kinase and NF-{kappa}B regulate motility of invasive MDA-MB-231 human breast cancer cells by the secretion of urokinase-type plasminogen activator. J Biol Chem 2002;277:3150–7.[Abstract/Free Full Text]
  42. Sliva D, English D, Lyons D, Lloyd FP, Jr. Protein kinase C induces motility of breast cancers by up-regulating secretion of urokinase-type plasminogen activator through activation of AP-1 and NF-{kappa}B. Biochem Biophys Res Commun 2002;290:552–7.[CrossRef][Medline]
  43. Adams J. Development of the proteasome inhibitor PS-341. Oncologist 2002;7:9–16.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
D. Sarkar, E. S. Park, L. Emdad, S.-G. Lee, Z.-z. Su, and P. B. Fisher
Molecular Basis of Nuclear Factor-{kappa}B Activation by Astrocyte Elevated Gene-1
Cancer Res., March 1, 2008; 68(5): 1478 - 1484.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, L.
Right arrow Articles by Rao, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, L.
Right arrow Articles by Rao, J. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online