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Tumor-specific expression of the RGD-3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice

Toru Miyoshi^*,1, Satoshi Hirohata^*,,1, Hiroko Ogawa^*,, Masayuki Doi^*, Masanari Obika^*,, Tomoko Yonezawa, Yoshikazu Sado, Shozo Kusachi, Satoru Kyo^||, Seiji Kondo^¶, Yasushi Shiratori^*, Billy G. Hudson and Yoshifumi Ninomiya^,2

^* Department of Medicine and Medical Science,

Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama;

Division of Immunology, Shigei Medical Research Institute, Okayama;

Department of Medical Technology, Faculty of Health Science, Okayama University Medical School, Okayama;

^|| Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Ishikawa, Japan;

^¶ Department of Neurosurgery, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA; and

Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

²Correspondence: Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2–5-1, Shikata-cho, Okayama 700-8558 Japan. E-mail: yoshinin{at}cc.okayama-u.ac.jp

ABSTRACT

Angiogenesis plays an essential role in tumor growth. This study investigated expression of the noncollagenous domain of 3(IV) collagen (3(IV)NC1) transduced into tumors and its inhibition of tumor growth. We hypothesized that if a human telomerase reverse transcriptase (hTERT) promoter-driven RGD motif containing 3(IV)NC1 (hTERT/RGD-3(IV)NC1) were expressed in telomerase-expressing tumor cells, it would inhibit tumor growth by its anti-angiogenic property. Adenoviral transduction of hTERT/RGD-3(IV)NC1 expressed RGD-3(IV)NC1 in hTERT-positive tumor cell lines. However, hTERT/RGD-3(IV)NC1 did not express RGD-3(IV)NC1 in hTERT-negative cells such as keratinocytes and fibroblasts. The secreted RGD-3(IV)NC1 in the conditioned medium from tumor cells inhibited cell proliferation as well as tube formation in cultured endothelial cells, but had no effect on other types of cells. In an in vivo model, adenoviral hTERT/RGD-3(IV)NC1 gene therapy showed limited expression of RGD-3(IV)NC1 in tumors and resulted in a significant decrease of vessel density in tumors. The growth of subcutaneous (s.c.) tumors in nude mice was significantly suppressed by treatment with hTERT/RGD-3(IV)NC1. In addition, long-term inhibition of tumor growth was achieved by intermittent administration of hTERT/RGD-3(IV)NC1. In conclusion, our findings demonstrate that tumor-specific anti-angiogenic gene therapy utilizing RGD-3(IV)NC1 under the hTERT promoter inhibited angiogenesis in tumors, resulting in an antitumor effect.—Miyoshi, T., Hirohata, S., Ogawa, H., Doi, M., Obika, M., Yonezawa, T., Sado, Y., Kusachi, S., Kyo, S., Kondo, S., Shiratori, Y., Hudson, B. G., Ninomiya, Y. Tumor-specific expression of the RGD-3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice.

Key Words: adenovirus • angiogenesis • basement membrane • cancer dormancy • hTERT

ANGIOGENESIS IS A COMPLEX process consisting of endothelial cell proliferation, migration, basement membrane degeneration, and new lumen formation. It is required for a variety of physiological processes, such as development and tissue regeneration. It also contributes to the development and progression of a number of pathological conditions, including tumor growth and tumor metastasis (1 , 2) . Since angiogenesis plays an essential role in tumor growth and invasion, anti-angiogenesis may have therapeutic potential in cancer therapy. To date, many anti-angiogenic therapies have been designed to inhibit tumor growth as well as cancer cell dissemination (3 , 4) . In the last few years a number of proteins or molecules with anti-angiogenic activity have been identified, some of which are protein domains, such as angiostatin of plasminogen or endostatin of collagen XVIII (5 6 7) .

The noncollagenous (NC1) domain of certain -chains of the collagen IV family also displays activity as an inhibitor of angiogenesis and tumor growth. The capacity of the exogenous 1(IV)NC1 domain of the 1 chain and 2(IV)NC1 domain of the 2 chain to disrupt basement membrane assembly, blocking tissue development in vivo, was first described for Hydra vulgaris (8) . Subsequent to these observations, we demonstrated that the recombinant RGD motif-containing NC1 domain of the human 3 chain, as denoted RGD-3(IV)NC1 in this article, potently inhibited angiogenesis and tumor growth, and endothelial cells adhered to these domains in an integrin vß3-dependent manner (9) . The anti-angiogenic antitumor activity of recombinant human 3(IV)NC1 protein (also referred as tumstatin) was confirmed (10) . The action mechanism of the 3(IV)NC1 domain is attributed to its interaction with integrins vß3 and vß5 (11 12 13) , and it is mediated by 1ß1 integrin in the case of 1(IV)NC1 (14) . We recently reported that the RGD motif of RGD-3(IV)NC1 is involved in endothelial adhesion and binding of vß3 and vß5 integrins, while another group demonstrated that the action of tumstatin is not RGD-dependent (15 , 16) . A potential problem with the clinical application of the 3(IV)NC1 domain as an inhibitor of tumor growth is that extratumoral overexpression of this domain might lead to the production of autoantibodies. Notably, autoantibodies to the 3(IV)NC1 domain cause severe and rapidly progressive glomerulonephritis and lung bleeding in patients with Goodpasture’s syndrome (17 18 19 20) . To avoid the production of autoantibodies against 3(IV)NC1, the utilization of a tissue- or tumor-specific promoter is a candidate strategy to express the 3(IV)NC1 domain only at the tumor site.

Telomerase is a particularly attractive target for tumor-specific therapy because human telomerase is highly active in 85% of primary cancers but is inactive in most normal somatic cells (21) . Telomerase is an RNP enzyme that plays an important role in the replication of chromosomal ends or telomeres. The enzyme is composed of a functional or template RNA component (hTER) and a telomerase catalytic protein subunit or human telomerase reverse transcriptase (hTERT) (22 , 23) . Although hTER and hTERT are both necessary for telomerase activity, hTERT is specifically expressed in tumor cells whereas hTER is expressed in both normal and tumor cells (24) . Recently, the hTERT gene was cloned by several groups, including ours (25 , 26) , and found to have a key role in telomerase activity. The hTERT promoter is highly tumor-selective, indicating its potential application in tumor-specific gene therapy. The hTERT promoter has been tested for use in targeted cancer gene therapy by several groups, including our own (27 , 28) . In previous studies it was shown that target genes such as lacZ were successfully driven by the hTERT promoter dependent on the hTERT promoter activity in various organs/cells (27) . Thus far, proapoptotic, cytotoxic, and prodrug-activating genes have been tried for targeted cancer gene therapy (28 , 29) . However, there have been no reports of studies employing this strategy for anti-angiogenic molecules such as tumstatin and endostatin.

We hypothesized that the transfer of the RGD-3(IV)NC1 domain gene under the hTERT promoter would restrict its expression to telomerase-expressing tumor cells. To test our hypothesis, in the present study we constructed an hTERT-driven adenovirus containing RGD-3(IV)NC1 domain cDNA and investigated its effects in vitro and in vivo.

MATERIALS AND METHODS

Cell lines
Human prostate cancer DU145 and PC3 cells, human fibrosarcoma HT1080 cells, and human lung cancer H1299 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). DU145, PC3, HT1080 cells, and H1299 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) or RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS (JRH Bioscience, Lenexa, KS, USA), 100 U/ml penicillin, and 100 U/ml streptomycin. Human umbilical vein endothelial cells (HUVEC; Clonetics, San Diego, CA, USA) were grown in EGM-2 medium according to the protocols provided by the supplier. Human foreskin keratinocytes (HFK; Cascade Biologics, OR, USA) were cultured in K110-TypeII medium (Kyokuto Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. Human skin fibroblasts (HSF) were derived from human adult volunteers and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin as previously reported (30) . Cells at passage 3–6 were used for all experiments.

Quantitative real-time RT-polymerase chain reaction (RT-PCR) analysis
To measure the expression levels of hTERT mRNA in the cells used in this study, real-time reverse transcription polymerase chain reaction (PCR) (RT-PCR) was performed. Cells were grown to 90% confluence in a 6-cm dish and washed twice with PBS. Total RNA was extracted from the cultured cells using RNA-Stat60 (Tel-Test, Friendswood, TX, USA) according to the manufacturer’s instructions. Quantitative detection of hTERT mRNA was performed with a LightCycler TeloTAGGG hTERT Quantification Kit (Roche Diagnostics, Mannheim, Germany) using a LightCycler instrument (Roche Diagnostics). The hTERT mRNA signals were normalized by the porphobilinogen deaminase (PBGD) mRNA levels.

Plasmid construction and preparation of recombinant adenovirus
To construct a human RGD-3(IV)NC1 domain expression plasmid vector under the hTERT promoter (phTERT/RGD-3(IV)NC1), an hTERT promoter expression vector containing luciferase cDNA (pGL3–378) (31) and a cytomegalovirus (CMV) promoter expression vector containing the cDNA for the FLAG-tagged RGD-3(IV)NC1 domain (pCMV/RGD-3(IV)NC1) (32) were ligated. The RGD-3(IV)NC1 domain has a 12-residue collagenous extension at the N terminus that contains a RGD motif, as described previously (9) . This domain was originally produced with this extension by collagenase digestion of native basement membranes to ensure the preservation of epitopes for Goodpasture autoantibodies (32 ,33) . This recombinant protein is equivalent to tumstatin (NCBI accession number AAF72632) in other reports (10) . The hTERT promoter region was cut out from pGL3–378 at the Mlu I/Hind III site. The fragment was used to replace the CMV promoter region of pCMV/RGD-3(IV)NC1 at the Mlu I/Hind III site. Reconstruction of the adenovirus was entrusted to Oriental Yeast Co. (Tokyo, Japan). Briefly, a recombinant adenovirus carrying RGD-3(IV)NC1 domain under the control of the hTERT promoter (hTERT/RGD-3(IV)NC1) was constructed by blunt insertion of the fragment obtained by Mlu I and XhoI cleavage of phTERT/RGD-3(IV)NC1. This adenovirus vector (pAFC3) is replication-deficient, since it lacks the E1A and E3 regions. Adenovirus were then amplified in HEK 293 cells and purified by cesium chloride density gradient centrifugation. Adenovirus carrying the lacZ reporter gene under the control of the CAG promoter (CAG/lacZ, clone; AxCALacZ, RIKEN, Ibaraki, Japan) was also amplified as described above.

Transient transfection and adenoviral infection in vitro
Transfection was performed as we described (34) . Briefly, cells were plated in 24-well plates at densities of 5 x 10⁴ – 1 x 10⁵ cells/well for transient transfection. After 24 h, the cells were transfected with 1 µg of pCMV/RGD-3(IV)NC1 plasmid using lipofectamine 2000 (Invitrogen, CA, USA). Adenoviral infection was performed at a multiplicity of infection (MOI) from 0 to 100 for 1 h in a minimal volume of medium, then the medium was replaced with 2% FBS-containing medium. Incubation was continued until 48 h and conditioned medium was collected.

Western blot analysis
Western blot was performed following the protocol we have described (35 , 36) . Conditioned medium (15 µl) was subjected to 4% t o12% sodium dodecyl sulfate/PAGE (SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes using transfer buffer that contained 25 mM Tris-HCl and 200 mM glycine, then blocked for 1 h in 5% nonfat dried milk in PBS containing 0.1% Tween 20 (PBS-T) and washed with PBS-T. The membranes were hybridized at room temperature for 2 h with mouse anti-FLAG M2 monoclonal antibody (mAb) (Sigma) (at 1:500 dilution) or rat mAb against human 3 (IV) collagen (H31) (at 1:100 dilution) (37) . After stringent washing with PBS-T three times for 5 min each at room temperature, the membranes were incubated with the appropriate secondary antibodies (ICN Pharmaceuticals, Aurora, OH, USA) for 1 h. After five successive washes with PBS-T, immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Bioscience, Piscataway, NJ, USA).

Preparation of conditioned medium
Conditioned medium from DU145 cells that had been transfected with pCMV/RGD-3(IV)NC1 or infected with hTERT/RGD-3(IV)NC1 (conditioned medium-3(IV)NC1) or CAG/lacZ (conditioned medium-lacZ) at an MOI of 100 was electrophoresed in 4% to 20% SDS-polyacrylamide gels. Alternatively, RGD-3(IV)NC1 was depleted from conditioned medium-3(IV)NC1 by immunoprecipitation of RGD-3(IV)NC1 with anti-FLAG M2 affinity gel (Sigma). Briefly, conditioned medium was incubated with anti-FLAG M2 affinity gel for 2 h at 4°C, then centrifuged at 4000 rpm for 2 min at 4°C. The presence of RGD-3(IV)NC1 in the immunoprecipitate or conditioned medium was confirmed by Western blot analysis with H31 (data not shown). The supernatant after centrifugation, in which RGD-3(IV)NC1 was no longer detectable, was used as "conditioned medium-RGD-3(IV)NC1FLAG" in further experiments.

Cell proliferation assay and cell viability assay
Cell proliferation was assessed by the bromodeoxyuridine (BrdU) assay (BrdU Cell Proliferation Assay, Oncogene, San Diego, CA, USA). The BrdU assay was performed according to the supplier’s protocol. Briefly, cells were plated in 96-well plates at 7000–10,000 cells/well for 24 h. The medium was then replaced with a mixture of fresh medium and conditioned medium-RGD-3(IV)NC1. Control cells were incubated with a mixture of fresh medium and conditioned medium-lacZ or conditioned medium-RGD-3(IV)NC1FLAG. Cell culturing was continued for 48 h after labeling with BrdU. The cells were then fixed to the wells, reacted with anti-BrdU primary antibody (Ab) and secondary antibodies, then developed using a colorimetric reaction. The absorbance was read at a wavelength of 450 nm using a plate reader (Bio-Rad, Hercules, CA, USA).

Cell viability was assessed by the MTT (3-(4, 5-dimethlthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay according to the manufacturer’s instructions (Chemicon, Temecula, CA, USA). Briefly, HUVEC or DU145 cells were seeded in 96-well plates in DMEM with 10% FBS. The medium was replaced the next day with a mixture containing conditioned medium-lacZ, conditioned medium-RGD-3(IV)NC1, or conditioned medium-RGD-3(IV)NC1FLAG. Then 10 µl of 5 mg/ml MTT solution was added to each well and cells were incubated for 4 h at 37°C. The medium was removed from each well, and 200 µl of DMSO and 25 µl of Sorensen’s glycine buffer were added to each well. The absorbance was read at a wavelength of 570 nm using a plate reader.

Flow cytometric analysis
The expression of vß3 integrin was evaluated by flow cytometric analysis using FACScalibur (Becton Dickinson, San Jose, CA, USA). Cells were washed twice with PBS containing 2% BSA; for Ab incubation, 2 x 10⁵ cells were incubated with anti-vß3 (LM609; Chemicon) for 30 min on ice. Isotype-matched normal mouse IgG1 conjugated to FITC (PharMingen, San Diego, CA, USA) was used as a negative control. The cells were then washed twice with PBS containing 2% FCS and incubated with FITC-labeled rabbit antimouse IgG (PharMingen) for 30 min on ice. The cells were again washed twice with PBS containing 2% BSA and resuspended in 1 ml of PBS containing 2% BSA. They were analyzed immediately by flow cytometry.

Tube formation
HUVEC (5000/well) were seeded in 96-well plates coated with Matrigel (Biosciences Discovery Labware, Bedford, MA, USA), then incubated in conditioned medium for 24 h at 37°C. Control cells were incubated with conditioned medium-lacZ. Three wells were examined, and the tube formation was quantified by counting the number of connecting branches between two discrete endothelial cells as reported earlier (11) .

Animal study
All experiments involving animals were carried out in accordance with the Guidelines for Animal Experiments at Okayama University Medical School, which are comparable with the "Guide for the Care and Use of Laboratory Animals" published by the Institute for Laboratory Animal Research (National Institutes of Health publication No. 85–23, revised 1996). First, HT1080 cells were inoculated s.c. into the right flank (1x10⁶ cells) of 6- to 8-wk-old female BALB/c nude mice (Charles River Japan, Yokohama, Japan). Tumor growth was monitored with Vernier calipers every 3 or 4 days. The volume was calculated according to the formula (vol=lengthxwidth²x0.52) (38) . When the tumor reached between 30 and 120 mm³ in volume, phTERT/RGD-3(IV)NC1 was transfected. 10 µg of phTERT/RGD-3(IV)NC1 plasmid and cationic lipid (DMRIE-C, 2 µg; Invitrogen) dissolved in 20 ml of sterile PBS was injected directly into the tumor every 24 h for 7 days and tumor size was monitored (three mice per group). As a negative control, plasmid encoding the lacZ gene instead of RGD-3(IV)NC1 domain (phTERT/lacZ) was used. For the adenoviral experiments, DU145 cells were inoculated s.c. into the right flank (1x10⁶ cells) of 6- to 8-wk-old female BALB/c nude mice (Charles River Japan, Yokohama, Japan) (five mice per group). When the tumor reached between 25 and 50 mm³ in volume, treatment was started. In a series of single-injection experiments, mice received an intratumor injection of hTERT/RGD-3(IV)NC1 or CAG/lacZ at a dose of 1 x 10⁹ pfu or PBS only on day 0 (five mice per group). In another series of experiments, intratumor injection was performed four times on days 0, 7, 14, and 21. After the fourth injection, tumor size was monitored without any special treatment until 60 days. When the tumor volume became >1000 mm³, the experiments were discontinued from the aspect of the protection of animals from harm.

Immunofluorescent staining
Because Goodpasture’s syndrome is clinically characterized by rapid progressive glomerulonephritis and the presence of circulating and organ-bound autoantibodies against RGD-3(IV)NC1 (20) , we examined the expression of hTERT/RGD-3(IV)NC1 in vivo. Tumors were removed on day 10 and livers, kidneys, and lungs were taken on day 28 after intratumor treatment with hTERT/RGD-3(IV)NC1 at a dose of 1 x 10⁹ pfu. Tissues were snap frozen in liquid N₂ and embedded in OCT compound (Sakura, Tokyo, Japan), and cryostat sections (7 µm) were fixed with acetone for 20 min. After blocking of nonspecific epitopes with 1% BSA for 1 h, the sections were incubated with primary Ab overnight at 4°C in a humidified chamber. For the primary Ab, anti-FLAG M2 mAb (at 1:100 dilution), rabbit anti-FLAG polyclonal antibody (pAb) (Sigma), a rat anti-CD31 Ab (at 1:100 dilution) (PharMingen), or H31 (at 1:20 dilution) was used. The sections were washed with PBS, then incubated with secondary Ab diluted in blocking solution. The following secondary antibodies were used: FITC-conjugated goat anti-rabbit IgG (Amersham Bioscience) or Cy3-conjugated donkey antirat IgG with minimal cross-reaction with other species (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The samples were examined with a BX50 microscope (Olympus, Tokyo, Japan) equipped with an AxioVision system (CarlZeiss, Germany) as previously reported (39 , 40) . All images were processed for publication using Adobe Photoshop.

Immunohistochemistry of intratumor vascularization
Intratumor vascularization was examined by immunohistochemical analysis as described previously (36 , 41) . Frozen sections of tumors on day 10 after a single viral or vehicle injection were fixed in acetone. The sections were stained with anti-CD31 Ab (at 1:100 dilution) and a second peroxidase-conjugated goat anti-rat IgG Ab. Immunoperoxidase staining was carried out using a Simplestain mouse MAX-PO Kit (Nichirei, Tokyo, Japan). The density of microvessels was quantified by first scanning the tumor at low power and identifying five areas at the tumor periphery that contained the maximum number of discrete microvessels, then counting individual microvessels.

Statistical analysis
Statistical analysis was performed using unpaired Student’s t tests. P values < 0.05 were considered statistically significant.

RESULTS

Expression of hTERT mRNA
Tumor cell lines (DU145, HT1080, PC3, and H1299) examined in this study were all hTERT mRNA-positive, although expression levels of hTERT mRNA varied considerably depending on the cell line (Fig. 1 a). Neither type of nontumor cells (HFK and HSF) examined showed detectable hTERT mRNA expression (Fig. 1a , lanes 5 and 6). Quantitative real-time RT-PCR analysis revealed that DU145 cells expressed high levels of hTERT mRNA compared to other cell lines.

View larger version (24K): [in this window] [in a new window]   Figure 1. a) Expression of human telomerase reverse transcriptase (hTERT) mRNA in tumor and normal cells. The hTERT mRNA expression level was measured by quantitative real-time RT-PCR analysis. The hTERT mRNA expression level of DU145 cells was taken as 1.0, and the hTERT mRNA expression level of other cell lines was compared with that of DU145 cells (lane 1, DU145 cells; lane 2, HT1080 cells; lane 3, PC3 cells; lane 4, H1299 cells; lane 5, HSF; lane 6, HFK). Each hTERT mRNA expression value was standardized relative to that of the PBGD mRNA level used as an internal control. Each column represents the mean value ± SD from 3 independent experiments. b) Secretion of induced RGD-3(IV)NC1 into conditioned medium (CM). DU145 cells were infected or transfected with hTERT/RGD-3(IV)NC1, pCMV/RGD-3(IV)NC1, or CAG/lacZ. CM from hTERT/RGD-3(IV)NC1-infected cells (lane 1), pCMV/RGD-3(IV)NC1-transfected cells (lane 2; positive control), or CAG/lacZ-infected cells (lane 3; negative control) was collected and subjected to Western blot analysis with an anti-FLAG M2 mAb. RGD-3(IV)NC1 was detected at the size of 28 kDa in CM from DU145 cells infected with hTERT/RGD-3(IV)NC1 at an MOI of 100 for 48 h. c) RGD-3(IV)NC1 secretion increased in a time-dependent manner in hTERT/RGD-3(IV)NC1-infected cells. DU145 cells were infected with hTERT/RGD-3(IV)NC1 and CM was collected at 0 h (lane 1), 24 h (lane 2), 48 h (lane 3), and 72 h (lane 4) and subjected to Western blot analysis with anti-FLAG M2 mAb. d) RGD-3(IV)NC1 was secreted into the CM only from hTERT-positive cells. DU145 cells (lane 1), PC3 cells (lane 2), HT1080 cells (lane 3), H1299 cells (lane 4), HSF (lane 5) and HFK (lane 6) were infected with hTERT/RGD-3(IV)NC1 at an MOI of 100. After 48 h of infection, each type of CM was collected and subjected to Western blot analysis. Note that RGD-3(IV)NC1 was not detected in CM from HSF or HFK.

Construction of adenovirus containing RGD-3(IV)NC1 domain under control of the hTERT promoter
We constructed a recombinant adenovirus containing the RGD-3(IV)NC1 domain driven by the hTERT promoter as described in Materials and Methods. As shown in Fig. 1b , a single band at 28 kDa was observed in conditioned medium from cells infected with hTERT/RGD-3(IV)NC1 (lane 1), and a band of the same size was detected in cells transfected with pCMV/RGD-3(IV)NC1 serving as a positive control (lane 2). RGD-3(IV)NC1 expression was also detected in cell lysates in addition to conditioned medium by Western blot analysis (data not shown). RGD-3(IV)NC1 was not detected in conditioned medium from cells infected with control adenovirus CAG/lacZ (Fig. 1b , lane 3). The level of RGD-3(IV)NC1 in conditioned medium from hTERT/RGD-3(IV)NC1-infected cells increased in a time-dependent manner (Fig. 1c ). To evaluate whether the hTERT promoter-driven RGD-3(IV)NC1 domain is secreted by telomerase-positive tumor cells but not by normal somatic cells, we examined conditioned medium from tumor cells, HSF and HFK infected with hTERT/RGD-3(IV)NC1 by Western blot. As shown in Fig. 1d , the secretion of RGD-3(IV)NC1 domain was detected in conditioned medium from hTERT-expressing cells such as DU145 cells (lane 1), PC3 cells (lane 2), HT1080 cells (lane 3), and H1299 cells (lane 4) infected with hTERT/RGD-3(IV)NC1. In contrast, HSF (lane 5), HFK (lane 6), and HUVEC (data not shown) infected with hTERT/RGD-3(IV)NC1 did not secrete RGD-3(IV)NC1 domain into conditioned medium. Western blot was also performed with H31 Ab and the results were the same as those obtained using anti-FLAG M2 Ab (data not shown).

Function of RGD-3(IV)NC1 produced by hTERT/RGD-3(IV)NC1 in vitro
The proliferation of tumor cells and somatic cells treated with conditioned medium-RGD-3(IV)NC1 was examined (Fig. 2 a). The proliferation of HUVEC treated with conditioned medium-RGD-3(IV)NC1 was significantly reduced to 67% of the level of proliferation of HUVEC treated with conditioned medium-lacZ (P=0.022) (Fig. 2b ). In addition, the proliferation of HUVEC was not inhibited when conditioned medium-RGD-3(IV)NC1FLAG from which RGD-3(IV)NC1 domain had been depleted by immunoprecipitation with anti-FLAG Ab, indicating that the inhibitory effect on proliferation was due to the secreted RGD-3(IV)NC1 (Fig. 2b , lane 3). This inhibitory effect on proliferation appeared to be specific to endothelial cells because conditioned medium-RGD-3(IV)NC1 did not affect the growth rate of DU145 cells when compared to conditioned medium-lacZ (Fig. 2c ). We also evaluated the effect on cell viability using the MTT assay. Conditioned medium-RGD-3(IV)NC1 significantly decreased cell viability of HUVEC by 51% compared with that of HUVEC treated with conditioned medium-lacZ (P=0.004), whereas conditioned medium-RGD-3(IV)NC1FLAG did not show a significant inhibitory effect on HUVEC (Fig. 2d ). Neither conditioned medium-RGD-3(IV)NC1 nor conditioned medium-lacZ had any effect on the viability of DU145 cells (Fig. 2e ). These results indicate that conditioned medium from cells infected with hTERT/RGD-3(IV)NC1 exerts its effects only on endothelial cells.

View larger version (21K): [in this window] [in a new window]   Figure 2. Inhibition of cell proliferation and decrease of cell viability by CM-RGD-3(IV)NC1. a) Cell proliferation was quantified by the BrdU assay. The proliferation of each type of cell treated with CM-RGD-3(IV)NC1 was examined and expressed as percentage compared with that of cells treated with CM-lacZ (lane 1, DU145 cells; lane 2, PC3 cells; lane 3, H1299 cells; lane 4, HT1080 cells; lane 5, HUVEC; lane 6, HFK; lane 7, HSF). The proliferation of HUVEC was inhibited by treatment with CM-RGD-3(IV)NC1. b) HUVEC were cultured in 96-well plates with CM-lacZ (lane 1), CM-RGD-3(IV)NC1 (lane 2), or CM-RGD-3(IV)NC1FLAG (lane 3) for 48 h. CM-RGD-3(IV)NC1 significantly inhibited the proliferation of HUVEC, while CM-RGD-3(IV)NC1FLAG caused no inhibition of proliferation. #P < 0.05 vs. CM-lacZ. c) Proliferation of DU145 cells was not inhibited by CM-RGD-3(IV)NC1 (lane 1, CM-lacZ; lane 2, CM-RGD-3(IV)NC1). d) Cell viability was assessed by the MTT assay. CM-RGD-3(IV)NC1 significantly decreased the viability of HUVEC after 48 h incubation, while CM-RGD-3(IV)NC1FLAG showed no decrease of viability (lane 1, CM-lacZ; lane 2, CM-RGD-3(IV)NC1; lane 3, CM-RGD-3(IV)NC1FLAG). *P < 0.01 vs. CM-lacZ. e) The viability of DU145 cells was not decreased by CM-RGD-3(IV)NC1 (lane 1, CM-lacZ; lane 2, CM-RGD-3(IV)NC1). Each column represents the mean ± SD of triplicate samples.

Differences of _Vß₃ integrin expression among tumor cells and somatic cells
Because RGD-3(IV)NC1 was reported to function via the _Vß₃ integrin signaling pathway, we then examined whether the inhibitory effects of RGD-3(IV)NC1 are related to the integrin expression profiles of the cell surface. The expression of vß3 integrin was measured by flow cytometric analysis. As shown in Fig. 3 , a large population of HUVEC expressed vß3 integrin compared with the populations of tumor cells and other somatic cells used in this study. Some of the other cells (e.g., HT1080) showed low levels of vß3 integrin expression.

View larger version (24K): [in this window] [in a new window]   Figure 3. Cell surface integrin vß3 expression was quantitated by flow cytometric analysis. Cells were incubated with LM609 (anti-vß3 Ab), followed by FITC-labeled secondary Ab. An isotype-matched normal mouse IgG1 conjugated to FITC was used as a control in all experiments. The gray histogram represents nonspecific fluorescence obtained with the isotype-matched IgG control, and the open black-lined histogram represents specific fluorescence. Note that a large population of HUVEC expressed integrin vß3 compared with other cell types.

Inhibitory effect of RGD-3(IV)NC1 on tube formation
The effects of RGD-3(IV)NC1 on tube formation of endothelial cells were examined by a tube formation assay performed using HUVEC on Matrigel. As shown in Fig. 4 a, HUVEC formed tubes on Matrigel-coated plates supplemented with conditioned medium-lacZ. Conditioned medium-RGD-3(IV)NC1 strongly reduced the formation of tube-like structures (Fig. 4b ). Tube formation was not reduced when HUVEC were incubated with conditioned medium-RGD-3(IV)NC1FLAG (Fig. 4c ). Quantitative analysis of the tube formation showed significant inhibition by conditioned medium-RGD-3(IV)NC1 compared with conditioned medium-lacZ or conditioned medium-RGD-3(IV)NC1FLAG (P=0.002 and 0.013, respectively) (Fig. 4d ).

View larger version (86K): [in this window] [in a new window]   Figure 4. Inhibition of endothelial tube formation by CM-RGD-3(IV)NC1 obtained from hTERT/RGD-3(IV)NC1-infected DU145 cells. HUVEC were allowed to form tubes on Matrigel-coated plates and incubated with CM-lacZ (a), CM-RGD-3(IV)NC1 (b), or CM-RGD-3(IV)NC1FLAG (c) for 24 h. d) The number of tube branches in the lower field was counted (three independent wells were counted and averaged). CM-RGD-3(IV)NC1 significantly decreased tube formation (lane 1, CM-lacZ; lane 2, CM-RGD-3(IV)NC1; lane 3, CM-RGD-3(IV)NC1FLAG). *P < 0.01 and #P < 0.05, respectively. Each column represents the mean ± SD.

In vivo expression of RGD-3(IV)NC1 directed by hTERT/3(IV)NC1
The expression of RGD-3(IV)NC1 in hTERT/RGD-3(IV)NC1-treated mice was examined by immunofluorescent staining. RGD-3(IV)NC1 was expressed restrictively in tumors (Fig. 5 a–c) but not in organs such as the kidney (Fig. 5e ). Treatment with CAG/lacZ instead of hTERT/RGD-3(IV)NC1 did not induce RGD-3(IV)NC1 signals (Fig. 5d ). Furthermore, in hTERT/RGD-3(IV)NC1-treated mice, there were no clear signs of inflammation as indicated by cell infiltration (Fig. 5f ).

View larger version (106K): [in this window] [in a new window]   Figure 5. In vivo expression of RGD-3(IV)NC1 directed by hTERT/RGD-3(IV)NC1 treatment in DU145 tumor-bearing BALB/C nude mice. DU145 cells were s.c. inoculated into 6- to 8-wk-old female BALB/c nude mice and tumor volumes were measured. When the tumors reached a mean volume of 35–50 mm3, the mice received an intratumor injection of 1 x 109 pfu of recombinant adenovirus or vehicle. Frozen sections of the tumors on day 10 and the kidney on day 28 after a single viral or PBS injection were fixed in acetone. a–c) Sections of the tumor in hTERT/RGD-3(IV)NC1-treated mice were doubly stained with anti-FLAG Ab (a, FITC) and anti-CD31 Ab (b, Cy3). c) Merged images obtained with anti-FLAG (FITC) and anti-CD31 (Cy3). d) Sections of tumors in PBS-injected mice were also doubly stained with anti-FLAG Ab (FITC) and anti-CD31 Ab (Cy3). Note that there were only negligible FITC signals (green) whereas Cy3 signals (red) were more abundant than in panel c. e) The kidney was doubly stained with anti-FLAG Ab (FITC) and anti-3(IV)NC1 Ab (H31, Cy3) in hTERT/RGD-3(IV)NC1-treated mice. Note that endogenous mouse 3(IV)NC1 (i.e., 3NC1 of mouse type IV collagen) cross-reacted with H31. The kidney, including glomeruli and tubulus renalis, showed H31-positive signals (red), whereas secreted FLAG-tagged-hTERT/RGD-3(IV)NC1 (green) in tumors was not observed in the kidney. f) Hematoxylin-eosin staining of sections contiguous to those in panel e. Note that there were no clear signs of inflammation such as cell infiltration in the kidney of hTERT/RGD-3(IV)NC1-treated mice. The scale bar shown in each panel represents 100 µm.

Inhibition of angiogenesis by hTERT/RGD-3(IV)NC1 in tumor-bearing mice
We then examined the effect of RGD-3(IV)NC1 on angiogenesis in vivo in tumors by immunostaining with CD31. Tumors of each group of mice were removed 10 days after the initial treatment. Tumors from animals receiving PBS (Fig. 6 a) or CAG/lacZ (Fig. 6b ) showed intense signals for CD31 staining, indicating the presence of extensive angiogenesis in the tumors. However, tumors from hTERT/RGD-3(IV)NC1-treated animals showed marked reduction in microvessel density (Fig. 6c ). Quantitative analysis demonstrated a significant reduction of the intratumor microvessel density in hTERT/RGD-3(IV)NC1-treated animals compared with that in control animals receiving PBS or CAG/lacZ (P=0.027 and 0.022, respectively) (Fig. 6d ).

View larger version (90K): [in this window] [in a new window]   Figure 6. Inhibition of tumor angiogenesis by treatment with hTERT/RGD-3(IV)NC1. Frozen tumor sections were stained with anti-CD31 Ab as described in Material and Methods. Representative light micrographs showing microvessels in tumors from animals that received PBS (a), CAG/lacZ (b), or hTERT/RGD-3(IV)NC1 (c). d) Quantitative analysis of microvessel density was performed by counting the positively stained cells in five different fields (x200). Significantly fewer blood vessels were observed in tumor sections of the hTERT/RGD-3(IV)NC1-treated group. #P < 0.05.

Inhibition of tumor growth by hTERT/RGD-3(IV)NC1 in tumor-bearing mice
Treatment with phTERT/RGD-3(IV)NC1 demonstrated considerable inhibition of tumor growth (Fig. 7 a) compared with the extent of tumor growth shown in animals transfected with the control plasmid (phTERT/lacZ). Tumor size at day 10 was significantly smaller than in control group animals (P=0.0041). Tumor growth using other cancer cell lines (PC3 and DU145) in this model was also inhibited (data not shown). In the case of adenoviral treatment, a single injection of hTERT/RGD-3(IV)NC1 resulted in a significant inhibition of tumor growth (Fig. 7b ) compared with the extent of tumor growth shown in animals treated with PBS or control adenovirus CAG/lacZ. Tumor size at day 20 was significantly smaller than that in animals from the control groups (i.e., PBS or CAG/lacZ, P=0.0002 and 0.007, respectively) (Table 1 ). However, with a single injection of hTERT/RGD-3(IV)NC1, suppression was not maintained for >10 days because tumors with and without treatment progressed at similar speeds from 10 days after the first adenovirus injection. Accordingly, we examined whether weekly intratumor treatment with hTERT/RGD-3(IV)NC1 was able to exert a long-term inhibitory effect on tumor growth (Fig. 7c ), resulting in an increased number of mice showing adequate tumor control (i.e., tumor vol<1000 mm³) (Table 2 ). Treatment with hTERT/RGD-3(IV)NC1 had a greater inhibitory effect on tumor growth than treatment with PBS or CAG/lacZ (P=0.001 and 0.002, respectively). In the case of weekly injections, mice were killed when tumor volumes exceeded 1000 mm³. As shown in Table 3 , all animals treated with vehicle were killed by day 60, and only one mouse in the CAG/lacZ group survived until day 60. In contrast, all mice in the hTERT/RGD-3(IV)NC1-treated group survived past 60 days. The maximal tumor volume in the hTERT/RGD-3(IV)NC1-treated group was <1000 mm³ at 60 days after the initial treatment (Table 3) .

View larger version (19K): [in this window] [in a new window]   Figure 7. Inhibition of tumor progression by treatment with hTERT/RGD-3(IV)NC1. a) Mice were treated with phTERT/RGD-3(IV)NC1 or phTERT/lacZ from day 1 until day 7 and the tumor volumes were measured. Tumor volumes were expressed as the percentage relative to the values on day 0 in each group and plotted. hTERT/RGD-3(IV)NC1 caused significant inhibition of HT1080 tumor growth in tumor-bearing mice. Tumor growth was inhibited by the intratumoral administration of hTERT/RGD-3(IV)NC1 in DU145 tumor-bearing BALB/c nude mice. b) Mice were treated once on day 0 with hTERT/RGD-3(IV)NC1, CAG/lacZ, or PBS only and the tumor volumes were measured. Tumor volumes were expressed as the percentage relative to the values on day 0 in each group and plotted. A single administration of hTERT/RGD-3(IV)NC1 caused significant inhibition of tumor growth in DU145 tumor-bearing mice. c) Intermittent administration of hTERT/RGD-3(IV)NC1 achieved tumor dormancy in DU145 tumor-bearing mice. hTERT/RGD-3(IV)NC1 was administered a total of four times on days 0, 7, 14, and 21. Tumor volumes were expressed as the percentage relative to the values on day 0 in each group and plotted. Open arrows indicate the time points at which treatment was given. *P < 0.01

DISCUSSION

This is the first report of the application of RGD-3(IV)NC1 for gene therapy using adenovirus vector. The expression of RGD-3(IV)NC1 domain under the control of the hTERT promoter was observed in tumor cells but not in normal tissues. Treatment with RGD-3(IV)NC1 domain significantly inhibited the growth of s.c. tumors in nude mice. These findings indicate the potential therapeutic usefulness of tumor-specific anti-angiogenic therapy using the hTERT promoter system.

We used adenovirus carrying a lacZ reporter gene under the control of the conventional CAG promoter (CAG/lacZ) as a negative control in this study. Adenovirus vector using the hTERT promoter has been used in several studies, including studies by our group, and has been extensively characterized (27 , 28 , 31) . Gu et al. reported that the hTERT promoter has high transcriptional activity in a variety of human cancer cell lines. The expression of hTERT-driven lacZ was shown to be limited to hTERT-positive cells. There is no particular toxicity related to hTERT-carrying adenovirus compared to that of the CAG-carrying adenovirus. The expression level under the control of the hTERT promoter is relatively low compared with that under the control of conventional promoters (e.g., CAG). In fact, the CMV promoter expressed lacZ at higher levels than the hTERT promoter, presumably due to differences of promoter activity, as determined by ß-gal staining in the initial plasmid transfection experiments (data not shown). To examine the effects of hTERT/RGD-3(IV)NC1, the use of CAG promoter instead of hTERT promoter for a negative control (i.e., lacZ-carrying vector) may be acceptable because of the high promoter activity of CAG promoter. Accordingly, we used CAG/lacZ instead of hTERT/lacZ for a negative control throughout this study.

Quantitative RT-PCR analysis revealed that hTERT mRNA was expressed at considerable levels in tumor cells, but not in normal cells. It was reported that prostate cancer cell lines DU145 and PC3 had not only strong expression of hTERT mRNA, but also high activity of telomerase (28) . We showed here that normal cells (fibroblasts and keratinocytes) infected with hTERT/RGD-3(IV)NC1 did not produce RGD-3(IV)NC1, presumably because the hTERT promoter has little or no activity in these cells. Although it has been reported that some normal human cells, such as lymphocytes (42) , germ cells (43) , and hematopoietic progenitor cells (44) , express hTERT at a certain level, hTERT expression is transient in these cells, including stem cells (45) . However, telomerase activity in stem cells is stringently regulated (46) , and normal cells were reported to regulate telomerase and telomere structure in a complex manner (47 , 48) . These observations indicate that hTERT expression is not constant, but rather is restricted via a sophisticated mechanism in nontumor tissues (49) . In addition, adenovirus was reported to infect stem cells poorly (45) . Our results demonstrated that RGD-3(IV)NC1 was not detected in the kidney in hTERT/RGD-3(IV)NC1-treated mice. Taken together, our findings suggested that hTERT/RGD-3(IV)NC1 restricted the expression of RGD-3(IV)NC1 to the tumors, and the expression of RGD-3(IV)NC1 may be negligible in other cells/tissues.

The antitumor activity mechanism of the hTERT/RGD-3(IV)NC1 domain observed in hTERT/RGD-3(IV)NC1-treated mice is not yet clear, although there are several possible explanations. First, the N-terminal part of the 3(IV)NC1 domain was reported to possess anti-angiogenic activity in vitro and in vivo, possibly because of its endothelial cell-specific inhibition of protein synthesis, which requires binding to _Vß₃ integrin (11 , 13 , 16) . 3(IV)NC1 (tumstatin) has been reported to suppress the growth of various cell lines, including CT26 (colon adenocarcinoma), LLC (Lewis lung carcinoma), renal cell carcinoma (786-O), and prostate carcinoma (PC3), by inhibiting tumor angiogenesis (10 , 50) . The results of Western blot analysis in this study demonstrated that RGD-3(IV)NC1 was secreted from tumor cells, and both BrdU and MTT assays revealed that the secreted RGD-3(IV)NC1 inhibited endothelial cell proliferation and viability, indicating that RGD-3(IV)NC1 functions via a cell surface protein/receptor. We also observed that RGD-3(IV)NC1 itself did not have an antiproliferative effect on the tumor cells used in this study (i.e., DU145, PC3, H1299, and HT1080). Second, there are several reports regarding extracellular matrix (ECM) degradation by the 3(IV)NC1 domain. 3(IV)NC1 was reported to decrease the plasminogen activators u-PA and t-PA, and to increase plasminogen activator inhibitor-1 (PAI-1) expression, both leading to a decrease in plasmin generation (51) . Plasmin affects tumor growth either directly by the breakdown of the ECM or indirectly by activating promatrix metalloproteinases (MMPs). Other evidence has shown that 3(IV)NC1 decreased MMP activities (51) . Whether these mechanisms were involved in the effects observed in this study remains to be determined. However, the fact that there were few vessels in the RGD-3(IV)NC1-treated tumor-bearing mice indicates that the anti-angiogenic activity of RGD-3(IV)NC1 was associated with the inhibition of tumor growth in mice.

In this report we showed that intermittent administration of hTERT/RGD-3(IV)NC1 achieved an inhibitory effect against tumor growth for up to 2 months. Such intermittent application may minimize the amount of adenovirus used for anti-angiogenic gene therapy. A previous report using endostatin revealed that anti-angiogenic molecules are effective only if they are administered daily (52) , and there have been no reports showing the long-term effects of intermittent administration. Our finding suggests that intermittent anti-angiogenic therapy may be useful for the inhibition of tumor growth, although careful surveillance for any toxic effects or unfavorable immune response to long-term therapy will be necessary.

There were several limitations in our study. First, the hTERT promoter is probably not a perfect promoter for tumor-specific gene therapy. Recent reports showed that hTERT is expressed in stem cells, and thus it may be impossible to avoid unexpected hTERT expression in nontumor tissues when using our construct. However, the activity of the hTERT promoter is not strong compared with that of conventional promoters such as CMV; therefore, the nontumor expression by the hTERT promoter was, at least at the gross anatomy level, not detectable, although careful studies are needed to confirm this. Another limitation of this study was the immunogenic response caused by RGD-3(IV)NC1. As mice are not very immuno-responsive to 3(IV)NC1, the safety of our system should be clarified in other animal models (53) . Nevertheless, the fact that hTERT/RGD-3(IV)NC1 limited the detectable expression of 3(IV)NC1 to tumors indicates that our system succeeded in eliminating the systemic expression of 3(IV)NC1, which is essential to avoid the production of autoantibodies against 3(IV)NC1.

In conclusion, our findings demonstrated the potential therapeutic usefulness of tumor-specific anti-angiogenic therapy with RGD-3(IV)NC1 under the hTERT promoter. Our results suggest that intermittent tumor-specific anti-angiogenic gene therapy is a novel approach for cancer dormancy therapy.

ACKNOWLEDGMENTS

The authors thank Dr. Toshiyoshi Fujiwara for his outstanding help and thoughtful suggestions. We would also like to acknowledge Dr. Kadir Demircan, Dr. Toshitaka Oohashi, Dr. Ichiro Naito, Dr. Aiji Ohtsuka, and other members of our Department for stimulating discussions and comments. The authors also thank Dr. Shunji Hattori for providing HSF and HFK cell, and Dr. Hideaki Ito (Department of Neurosurgery, University of Texas M. D. Anderson Cancer Center, TX, USA) for great help with the animal experiments, and Mr. Mehmet Zeynel Cilek, Ms. Tomoko Maeda, and Ms. Kahori Tanaka for technical help. B.G.H. is cofounder and equity holder in BioStratum, Inc, a company with an interest in angiogenic inhibitors. BioStratum did not provide financial support for the present study. He is also a cofounder and equity holder in NephroGenex, Inc., a company with an interest in developing drugs to treat kidney diseases. NephroGenex did not provide support for the present study. This work was supported in part by funding from grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (grant 16591755 to S.K. and 18390416 to S.H.) and a National Institutes of Health Grant (DK 18381 to B.G.H.).

FOOTNOTES

¹ These authors contributed equally to this work.

Received for publication December 14, 2005. Accepted for publication April 24, 2006.

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