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Clinical Cancer Research Vol. 5, 3689-3694, November 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Simplified Production of a Recombinant Human Angiostatin Derivative That Suppresses Intracerebral Glial Tumor Growth1

Patricio I. Meneses, Lauren E. Abrey, Katherine A. Hajjar, S. Humayun Gultekin, Robert M. Duvoisin, Kenneth I. Berns and Myrna R. Rosenfeld2

Division of Neurosurgery [M. R. R.], and Departments of Neurosciences [P. I. M., R. M. D., M. R. R.] and Pediatrics and Medicine [K. A. H.], Weill Medical College of Cornell University, New York, New York 10021; Departments of Neurology [L. E. A.] and Pathology [S. H. G.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; and Department of Molecular Genetics and Microbiology [K. I. B.], University of Florida College of Medicine, Gainesville, Florida 32610


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiostatin is an endogenous inhibitor of tumor neovascularization that inhibits the proliferation of endothelial cells. Production of sufficient quantities of biologically active angiostatin by the enzymatic cleavage of plasminogen has proven difficult in that it has delayed clinical testing. We have cloned, expressed, and purified a recombinant human angiostatin derivative (K1-3) using a mammalian expression system. Through the addition of a secretory signal and polyhistidine sequence tag, K1-3 can be purified from postculture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability to bind to lysine-Sepharose. In vitro, recombinant K1-3 significantly suppressed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-competent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011). Growth suppression was accompanied by a 32% decrease (P = 0.01) in tumor neovascularization.

This study demonstrates a simple method to produce a biologically active recombinant angiostatin derivative. The ability to suppress intracerebral tumor growth after systemic administration suggests that K1-3 is likely to have therapeutic value in the treatment of malignant glial tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiostatin has been recently described as an endogenous inhibitor of angiogenesis (1) . Angiostatin has been isolated by a laborious and low yield process requiring limited proteolytic digestion of plasminogen purified from urine or serum (2) . The difficulties in manufacturing sufficient quantities of biologically active angiostatin have hampered bringing this agent to clinical use (3) , and alternative production methods have been sought. Several studies suggest that recombinant angiostatin can be produced by viral or yeast expression systems (4, 5, 6) . In addition to facilitating production, the use of recombinant angiostatin derivatives avoids the risks of using human plasma, which has the potential for contamination with transmissible agents.

We have made a recombinant human angiostatin molecule consisting of the first three kringle domains (K1-3) of plasminogen (7) . Expressed and secreted by a mammalian cell line, recombinant K1-3 protein is easily purified. Recombinant K1-3 maintains antiangiogenic activity in vitro. Furthermore, we demonstrate that K1-3 is active in suppressing the growth of an intracranial glial tumor after systemic administration.

Angiogenesis plays a role in the growth of many tumors. Because malignant gliomas are among the most highly vascularized of all tumors (8) , they are likely to be affected by antiangiogenic agents such as angiostatin. In glial tumors, an increase in microvascular density is associated with higher grade and poorer prognosis (9) . Both glioma cell lines and glial tumors have been shown to express several growth factors and growth factor receptors that are capable of stimulating endothelial cell proliferation. These include bFGF3 and vascular endothelial growth factor (10, 11, 12) . In some glial tumors, the level of bFGF expression correlates with the degree of malignancy and vascularity (13) . In vitro, the growth of transformed astrocytes and glial tumor cell lines are suppressed when bFGF expression is inhibited (14 , 15) and increased when bFGF is overexpressed or blocked (16 , 17) . These studies suggest that both autocrine and paracrine angiogenic signaling pathways exist in some glioblastomas. These features make glial neoplasms reasonable targets for antiangiogenic therapy. In contrast to studies that use s.c. glioma xenografts in nude mice to test the therapeutic efficacy of novel agents (18 , 19) , we tested K1-3 against an intracerebral brain tumor model in immunocompetent rats to more closely approximate the clinical situation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Animals.
HEK 293 cells were obtained from American Type Culture Collection (Rockville, MD) and were grown in DMEM supplemented with 10% FCS and 5,000 units/ml penicillin/streptomycin. HUVE cells, which were obtained at delivery as described previously (20) , were grown in Medium 199 containing 20% pooled human serum, 2.0 mML-glutamine, 90 µg/ml porcine intestinal mucosal heparin (170 USP units/mg), 20 µg/ml endothelial cell growth factor, 2.5 µg/ml fungizone, and 50 units/ml penicillin/streptomycin. The 9L rat glioma cell line was grown in DMEM supplemented with 10% heat-inactivated FCS, 2 mML-glutamine, and 5,000 units/ml penicillin/streptomycin. Inbred female Fisher 344 rats (200 g) were purchased from Charles River Laboratories (Wilmington, MA). Animal studies were done in accordance with guidelines of the Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center.

Plasmid Construction.
A cDNA encoding kringle domains 1, 2, and 3 of human plasminogen (Val-79 to Ser-337) was amplified by PCR using human plasminogen cDNA as the template (courtesy of F. J. Castellino, University of Notre Dame, Notre Dame, IN) and oligonucleotide primers (5'-CCCAAGCTTGGTGTATCTCTCAGGG-3' and 5'-GCCTCT-AGAGGAGGAGTCACAGGA-3') containing linkers with HindIII and XbaI restriction sites, respectively. The 789-bp fragment was cloned into pSecTagA, (Invitrogen, San Diego, CA) downstream from the cytomegalovirus promoter and the murine immunoglobulin {kappa}-chain leader peptide and upstream of the myc epitope and polyhistidine tag. The integrity of the resulting plasmid, pSecK1-3, was confirmed by automated sequencing.

Purification of K1-3 Protein.
pSecK1-3 was transfected into HEK 293 cells using the calcium phosphate method (21) . Stable transfectants were obtained by selection in Zeocin (250 µg/ml). Stable transfectants were grown in 30 ml of media in T-175-cm2 flasks to 80–90% confluency. The media from each of the eight flasks was collected and passed over a Ni2+ affinity column as per the manufacturer’s instructions (Promega, Madison, WI). K1-3 protein was eluted with 1 M imidazole, and the fractions with maximum absorbance pooled and extensively dialyzed against 1000 volumes of PBS (pH 7.4) at 4°C for 16 h. K1-3 protein was also purified by passage of media over a lysine-Sepharose column, followed by elution with 200 mM {epsilon}-amino-N-caproic acid and dialysis as noted above for 24 h. The K1-3 amino terminal protein sequence was confirmed by automated Edman degradation (Rockefeller University Protein/DNA Technology Center, New York, NY). Protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA). LPS levels were determined using the Limulus Amebocyte Lysate Kinetic-QCL kit (BioWhittaker Inc., Walkersville, MD).

Western Blot.
Purified protein or 15 µl of media from transfected HEK 293 cells were resolved by 12% SDS-PAGE. To determine the purity of the protein preparations, gels were stained with Coomassie Brilliant Blue. For Western blotting, the resolved proteins were transferred to nitrocellulose. After overnight blocking in 5% Carnation nonfat milk, the nitrocellulose was sequentially incubated with mouse anti-myc IgG diluted 1:5000 (Invitrogen; 2 h) and with rabbit antimouse IgG conjugated to alkaline phosphatase diluted 1:10,000 (1 h). The blot was then immersed in an enhanced chemiluminescence solution (Amersham, Arlington, IL) for 60 s and exposed to Kodak XAR5 film (Sigma, St. Louis, MO). Between steps, the nitrocellulose was washed with 0.1% Tween Tris-buffered saline. All incubations were done at room temperature.

Cell Proliferation Assay.
HUVE cells were plated on 24 well plates at 30,000 cells/well. After 24 h, purified K1-3 protein was added to the media at increasing concentrations and then incubated for 72 h at 37°C. Cell number was quantified with a cell proliferation assay kit using 4.5 dimethylthiazol-3-carboxymethoxy-phenyl-4-sulfophenyl-tetrazolium (MTS; Promega) or by trypan blue exclusion.

Animal Model.
Rats were anesthetized by i.m. injection of chlorpromazine (1.5 mg/kg) and ketamine (100 mg/kg; Fort Dodge Laboratories, Fort Dodge, IN). After making a midline sagittal incision, a burr hole 2 mm in diameter was drilled, and a 3–4-µl suspension containing 5 x 104 or 1 x 105 9L cells in sterile saline was injected into the right basal ganglia using a 25-gauge Hamilton syringe and a small animal stereotactic frame (Kopf, Tujunga, CA). The coordinates used for injection were 4 mm lateral to the midline at the bregma and 5 mm below the dural surface. Animals were given s.c. injections of 0.5 mg/kg purified K1-3 protein in PBS every 12 h starting 24 h after 9L implantation for a total of 10 treatment days. Control animals were treated with column fractions obtained from media from nontransfected HEK 293 cells.

Tumor Size.
Following 10 days of K1-3 treatment, animals were euthanized. The brains were embedded in Optimal Cutting Temperature medium (Miles, Elkhart, IN) and snap frozen in isopentane chilled by liquid nitrogen. Consecutive 10-µm sections were cut through the extent of the tumor determined by examining hematoxylin-stained sections under light microscopy. Tumor volume was calculated by multiplying the area of the tumor by the slice thickness and the number of slides containing the tumor.

Immunohistochemical Analysis and Microvessel Quantification.
Frozen sections were fixed in 10% buffered formalin then sequentially incubated with 0.1% hydrogen peroxide to destroy endogenous peroxidase activity, 10% normal horse serum as a blocking agent, mouse antirat CD31 (1:1000; Serotec, Raleigh, NC), biotinylated rat adsorbed horse antimouse (1:1000; Vector, Burlingame, CA), and peroxidase-conjugated streptavidin (1:1000; Boehringer, Indianapolis, IN). Diaminobenzidine (0.05%) was used as the chromogenic substrate, and hematoxylin was used as the counterstain. All incubations were performed at room temperature. Quantitation of anti-CD31 staining (microvessels per field) was performed by two investigators who were blinded to the experimental status of the section. After screening the tumors at low power (x40 and x100), microvessels in the areas with the highest number of discrete microvessels were counted in the x400 field. A vessel lumen was not required for the identification of a microvessel; CD31-positive single endothelial cells or cell clusters were counted (9 , 22) . The average vessel counts of all fields studied by both investigators were recorded.

Statistical Methods.
Numerical values are expressed as the mean ± SE. Statistical significance was assessed using the Student’s t test. Ps <0.05 were considered to be statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene Construction and Expression.
The cDNA fragment corresponding to kringles 1–3 of human plasminogen was cloned into pSecTagA to produce pSecK1-3 (Fig. 1)Citation . The murine immunoglobulin {kappa}-chain leader peptide was included to serve as the secretory signal, whereas an myc epitope and a polyhistidine chain were included to allow for antibody tagging and protein purification, respectively. Using this construct, stable transfectants of HEK 293 cells were derived by antibiotic selection. Resolution of the protein preparations isolated from the media bathing stable transfectants after Ni2+ column purification on SDS-polyacrylamide gels revealed the presence of a single protein band at 32 kDa (Fig. 2A)Citation . Western blot analysis using a monoclonal anti-myc epitope antibody confirmed the identity of the protein to be the product of pSecK1-3 (Fig. 2B)Citation . A similar protein was not identified in the media of cells transfected with the pSecTagA plasmid without the K1-3 insert. Based upon the average yield of several protein preparations, 1 mg of purified K1-3 protein was obtained from the media bathing 1.2 x 108 transfected cells for 3 days. LPS levels in Ni2+ column-purified protein solutions, which were <1.2 ng/mg K1-3, were markedly below growth inhibitory levels (data not shown; 23 ).



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  		Fig. 1. Schematic representation of the pSecK1-3 plasmid used to express the K1-3 protein. The K1-3 cDNA was subcloned in frame with the immunoglobulin {kappa}-chain leader sequence and the myc epitope and polyhistidine tags.

 


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  		Fig. 2. Purification of the recombinant K1-3 protein after secretion from the HEK 293 cells. Protein from media conditioned by stable pSecK1-3 transfectants was resolved by SDS-PAGE. A, gel stained with Coomassie Brilliant Blue. B, Western analysis performed using a monoclonal anti-myc epitope antibody. Lanes 1, protein obtained after affinity chromatography over lysine-Sepharose. Lanes 2, protein obtained after Ni2+ column purification. Both gels demonstrated a single protein species. Media from cells expressing the parental pSecTagA plasmid without the K1-3 insert and subjected to Ni2+ column purification served as a negative control (Lane 3).

 
Appropriate folding of the kringle domains is essential for the antiangiogenic activity of angiostatin (7) . In the active conformation, the kringle 1 domain binds lysine (24 , 25) . To test for functional lysine binding, medium from transfected HEK 293 cells was subjected to affinity chromatography over lysine-Sepharose columns. After extensive washing, bound protein was eluted from the column by {epsilon}-amino-N-caproic acid and analyzed by Western blot, revealing a 32-kDa protein (Fig. 2, A and B)Citation .

The stability of purified K1-3 was tested by adding purified protein (1.6 µg) to the media of HUVE cells. Western blot analysis demonstrated that K1-3 was not significantly degraded for up to 72 h (data not shown).

Suppression of HUVE Cell Proliferation in Vitro.
To determine whether recombinant K1-3 protein had antiangiogenic activity, cultured HUVE cells were incubated with increasing concentrations of Ni2+ affinity column-purified K1-3 protein. After 72 h, control-treated HUVE cells had doubled in number from 32 x 103 cells to 63 x 103 cells (Fig. 3)Citation . In contrast, in the presence of purified K1-3, the HUVE cell number was reduced in a dose-dependent manner with an IC50 of 50 nM. A significant 37% reduction in cell number was seen at 47 nM K1-3 protein (P = 0.02), with a maximal reduction of 87% obtained at 125 nM K1-3 (P = 0.004). Suppression of HUVE proliferation was not seen if the recombinant K1-3 protein was boiled for 10 min prior to use (data not shown).



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  		Fig. 3. Suppression of HUVE cell proliferation by recombinant K1-3. HUVE cells were incubated with increasing concentrations of Ni2+ column-purified K1-3 for 3 days. For each concentration of K1-3, a minimum of six experiments performed in triplicate were evaluated. Cell growth curves were evaluated for the determination of IC50 by the MTS assay. The black bar represents the number of cells at the start of the experiment; bars, SE. *, P < 0.02.

 
Suppression of Intracerebral Glioma Growth.
One day after implantation of 9L glioma cells into the right basal ganglia, rats were treated with s.c. injections of 100 µg of purified K1-3 protein. Animals were dosed every 12 h and were treated for 10 consecutive days.

Analysis of tumor size demonstrated that K1-3-treated animals had tumors that were significantly smaller than control-treated animals (Table 1)Citation . The suppression of tumor growth by K1-3 was not markedly dependent upon tumor size. Animals implanted with 0.5 x 105 9L cells and treated with K1-3 had tumors that were 85% smaller than matched controls. Animals implanted with 1 x 105 9L cells had tumors that were 74% smaller than controls.


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  		Table 1 Subcutaneous treatment with K1-3 suppresses the growth of intracerebral 9L tumors

 
Tumor Vascularization.
To study the effect of K1-3 on the tumor microvasculature, representative sections of all tumors were examined for expression of CD31, a platelet endothelial cell adhesion molecule found on the surface of small and large blood vessels in pathological and normal tissue (26) . After CD31 immunoreactive hot-spots were identified at low power, microvessels were then counted at x400. The average number of blood vessels observed in tumors from K1-3-treated animals was significantly less [15 ± 2 (n = 12) per high power field] than in tumors from control-treated animals [22 ± 2 (n = 10) per high power field (P = 0.01; Fig. 4Citation )]. There were no obvious differences in the morphology of the microvasculature between the treated and control animals or in the localization of the microvessels within the tumor.



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  		Fig. 4. Immunohistochemical detection of CD31 in control- (A) and K1-3-treated (B) intracerebral 9L tumors. No counterstain (x400).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During tumorigenesis, there is disruption of the normal physiological balance between angiogenic inhibitors and stimulators leading to the rapid proliferation of endothelial cells (27 , 28) . Since the discovery of the angiogenic growth factors and the endogenous angiogenic inhibitor, angiostatin, many studies have focused on manipulating these pathways with the goal of inhibiting tumor angiogenesis (17 , 29 , 30) .

Angiostatin is derived from an internal fragment of plasminogen and contains four kringle domains (looped disulfide-linked structures; Ref. 1 ). Most of the antiproliferative activity of angiostatin resides in kringle domains 1, 2, and 3 (7) . To produce human angiostatin, plasminogen has been purified from plasma and enzymatically digested. The fragment comprising angiostatin is isolated and purified by affinity chromatography on lysine-Sepharose. This process, when successful, results in low yields of active compound making it inadequate to produce sufficient quantities for clinical testing (3) .

In the present study, we demonstrated that a recombinant protein, K1-3, containing plasminogen kringles 1–3 can be produced and secreted by mammalian cells. Upon secretion, the recombinant K1-3 protein folds appropriately, as demonstrated by its ability to bind to lysine-Sepharose, a property of native angiostatin (24 , 25) . In culture, a single dose of recombinant K1-3 protein significantly suppressed the proliferation of HUVE cells, confirming that the recombinant K1-3 protein is expressed in a conformation that is similar to native angiostatin.

To test the antiangiogenic activity of the K1-3 protein in vivo, we used a rat model of intracranial brain tumors formed by syngeneic 9L glioma cells (31) . These cells were originally derived from a nitrosourea-induced rat glioma, and in vitro are not affected by incubation with K1-3 (data not shown). Tumors of 9L cells develop a distinct neovasculature with an endothelium that expresses bFGF, a mitogen for endothelial cells (22) . Although the exact mechanism(s) by which angiostatin inhibits vascular endothelial cell growth are unknown, angiogenesis induced by bFGF is inhibited by angiostatin (1) . In addition, 9L cells express a mutant form of the p53 tumor suppressor gene (32) . Mutations with loss of p53 function are found in {approx}40% of all grades of gliomas (33) . Glioblastoma cells with loss of p53 function express strong angiogenic activity, whereas expression of wild-type p53 results in the secretion of an antiangiogenic factor (34) . These properties would be expected to render 9L cells relatively resistant to antiangiogenic therapy. However, systemic injections of recombinant K1-3 were very effective in suppressing the growth of intracranial 9L tumors. This was associated with a decrease in tumor vascularity measured by CD31 immunostaining of microvessels, which has been used to quantitate blood vessels in many tumor types (26) . Based upon the levels of LPS measured in the K1-3 preparations, it is unlikely that contaminating LPS was responsible for the tumor growth suppression that was seen (23) . These data are consistent with angiogenesis inhibition as the mechanism of tumor growth suppression rather than as an effect on tumor cell proliferation.

Although physiologically active recombinant angiostatin derivatives have therapeutic potential, there are limitations to the isolated use of angiostatin. One problem is the need for chronic treatment; studies in a variety of animal tumor models have demonstrated that if angiostatin treatment is discontinued, tumor growth resumes (35) . One approach to achieving sustained drug delivery is the injection of viral vectors directly into the tumor bed. However, if the tumor cells are transduced and secreting the active agent, there could be significant loss of drug expression as the transduced cells die. In addition, the length of time that viral vectors express their transgene is transient and variable (36, 37, 38) . Both of these issues make it likely that a patient would require repeat intracranial administration of vector, which could substantially add to morbidity. Another drawback is that the most commonly used retroviral vectors are relatively inefficient at transducing tumors in vivo. Although adenoviral vectors are very efficient at transducing target cells in vivo (39) , their use in immune-competent animals has been limited due to the development of an inflammatory response and the production of antiadenoviral antibodies (38 , 40) . The long-term use of an antiangiogenic peptide is clinically feasible. Prefilled injectors can simplify the process and slow release formulations, or the use of pumps could avoid the need for daily injections (41 , 42) . As an outpatient procedure, this form of drug delivery is economically practical.

In summary, the clinical use of angiostatin has been limited by difficulties in producing large quantities of active compound. In the current study, we demonstrate that a recombinant angiostatin derivative with antiangiogenic activity in vitro and in vivo can be easily produced and purified. The ability of K1-3 to suppress intracerebral glial tumor growth is particularly attractive. The large majority of glial tumors are malignant and, to date, remain incurable. Except in unusual cases or for particular subtypes of brain tumors, current systemic or local chemotherapeutic regimens do not significantly affect survival (43 , 44) . These studies should encourage the development of similar antiangiogenic agents for clinical trials.


   FOOTNOTES
 
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.

1 Supported by Grant NS07384 (to P. I. M.), Grants HL42493 and HL46403 (to K. A. H.), and Grant EY09534 (to R. M. D.) from NIH, and a grant from the Brain Tumor Society (to M. R. R). Back

2 Present address and to whom requests for reprints should be addressed, at Department of Neurology, University of Arkansas for Medical Sciences Medical Center, 4301 West Markham, Slot 500, Little Rock, AR 72205. Phone: (501) 686-5135; Fax: (501) 686-7850; E-mail: rosenfeldmyrnar{at}exchange.uams.edu Back

3 The abbreviations used are: bFGF, basic fibroblast growth factor; HUVE, human umbilical vein endothelial; HEK, human embryonal kidney; LPS, lipopolysaccharide. Back

Received 4/16/99; revised 7/20/99; accepted 7/20/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. O’Reilly M. S., Holmgren L., Shing Y., Chen C., Rosenthal R. A., Moses M., Lane W. S., Cao Y., Sage E. H., Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell, 79: 315-328, 1994.[Medline]
  2. Gately S., Twardowski P., Stack M. S., Cundiff D. L., Grella D., Castellino F. J., Enghild J., Kwaan H. C., Lee F., Kramer R. A., Soff G. A. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor, angiostatin. Proc. Natl. Acad. Sci. USA, 94: 10868-10872, 1997.[Abstract/Free Full Text]
  3. Pezzella M. Drug giant turns back on cancer phenom. Biotechnology Newswatch, 2: 13-14, 1999.
  4. Griscelli F., Li H., Bennaceur-Griscelli A., Soria J., Opolon P., Soria C., Perricaudet M., Yeh P., Lu H. Angiostatin gene transfer: inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc. Natl. Acad. Sci. USA, 95: 6367-6372, 1998.[Abstract/Free Full Text]
  5. Tanaka T., Cao Y., Folkman J., Fine H. A. Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA. Cancer Res., 58: 3362-3369, 1998.[Abstract/Free Full Text]
  6. Sim B. K. L., O’Reilly M. S., Liang H., Fortier A. H., He W., Madsen J. W., Lapcevich R., Nacy C. A. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res., 57: 1329-1334, 1997.[Abstract/Free Full Text]
  7. Cao Y., Ji R. W., Davidson D., Schaller J., Marti D., Sohndel S., McCance S. G., O’Reilly M. S., Llinas M., Folkman J. Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells. J. Biol. Chem., 271: 29461-29467, 1996.[Abstract/Free Full Text]
  8. Brem S., Cotran R., Folkman J. Tumor angiogenesis: a quantitative method for histologic grading. J. Natl. Cancer Inst., 48: 347-356, 1972.
  9. Leon S. P., Folkerth R. D., Black P. M. Microvessel density is a prognostic indicator for patients with astroglial brain tumors. Cancer (Phila.), 77: 362-372, 1996.[Medline]
  10. Maxwell M., Naber S. P., Wolfe H. J., Hedley-Whyte E. T., Galanopoulos T., Neville-Golden J., Antoniades H. N. Expression of angiogenic growth factor genes in primary human astrocytomas may contribute to their growth and progression. Cancer Res., 51: 1345-1351, 1991.[Abstract/Free Full Text]
  11. Paulus W., Grothe C., Sensenbrenner M., Janet T., Baur I., Graf M., Roggendorf W. Localization of basic fibroblast growth factor, a mitogen and angiogenic factor, in human brain tumors. Acta Neuropathol., 79: 418-423, 1990.[Medline]
  12. Plate K. H., Breier G., Weich H. A., Mennel H. D., Risau W. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int. J. Cancer, 59: 520-529, 1994.[Medline]
  13. Takahashi J. A., Fukumoto M., Igarashi K., Oda Y., Kikuchi H., Hatanaka M. Correlation of basic fibroblast growth factor expression levels with the degree of malignancy and vascularity in human gliomas. J. Neurosurg., 76: 792-798, 1992.[Medline]
  14. Murphy P. R., Sato Y., Knee R. S. Phosphorothioate antisense oligonucleotides against basic fibroblast growth factor inhibit anchorage-dependent and anchorage-independent growth of a malignant glioblastoma cell line. Mol. Endocrinol., 6: 877-884, 1992.[Abstract]
  15. Morrison R. S. Suppression of basic fibroblast growth factor expression by antisense oligodeoxynucleotides inhibits the growth of transformed human astrocytes. J. Biol. Chem., 266: 728-734, 1991.[Abstract/Free Full Text]
  16. Redekop G. J., Naus C. C. Transfection with bFGF sense and antisense cDNA resulting in modification of malignant glioma growth. J. Neurosurg., 82: 83-90, 1995.[Medline]
  17. Stan A. C., Nemati M. N., Pietsch T., Walter G. F., Dietz H. In vivo inhibition of angiogenesis and growth of the human U-87 malignant glial tumor by treatment with an antibody against basic fibroblast growth factor. J. Neurosurg., 82: 1044-1052, 1995.[Medline]
  18. Begemann M., Kashimawo S. A., Choi Y. A., Kim S., Christiansen K. M., Duigou G., Mueller M., Schieren I., Ghosh S., Fabbro D., Lampen N. M., Heitjan D. F., Schiff P. B., Bruce J. N., Weinstein I. B. Inhibition of the growth of glioblastomas by CGP 41251, an inhibitor of protein kinase C, and by a phorbol ester tumor promoter. Clin. Cancer Res., 2: 1017-1030, 1996.[Abstract]
  19. Pinski J., Schally A. V., Halmos G., Szepeshazi K., Groot K. Somatostatin analogues and bombesin/gastrin-releasing peptide antagonist RC-3095 inhibit the growth of human glioblastomas in vitro and in vivo. Cancer Res., 54: 5895-5901, 1994.[Abstract/Free Full Text]
  20. Hajjar K. A., Hamel N. M., Harpel P. C., Nachman R. L. Binding of tissue plasminogen activator to cultured human endothelial cells. J. Clin. Invest., 80: 1712-1719, 1987.
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology, 52: 456-467, 1973.[Medline]
  22. Kirsch M., Strasser J., Allende R., Bello L., Zhang J., Black P. M. Angiostatin suppresses malignant glioma growth in vivo. Cancer Res., 58: 4654-4659, 1998.[Abstract/Free Full Text]
  23. Lucas R., Holmgren L., Garcia I., Jimenez B., Mandriota S. J., Boriat F., Sim B. K., Wu Z., Grau G. E., Shing Y., Soff G. A., Bouck N., Pepper M. S. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood, 92: 4730-4741, 1998.[Abstract/Free Full Text]
  24. Wiman B., Wallen P. The specific interaction between plasminogen and fibrin. A physiological role of the lysine binding site in plasminogen. Thromb. Res., 10: 213-222, 1977.[Medline]
  25. Lerch P. G., Rickli E. E., Lergier W., Gillessen D. Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming properties. Eur. J. Biochem., 107: 7-13, 1980.[Medline]
  26. Horak E., Leek R., Klenk N., LeJeune S., Smith K., Stuart N., Greenall M., Stepniewska K., Harris A. L. Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as an indicator of node metastasis and survival in breast cancer. Lancet, 340: 1120-1124, 1992.[Medline]
  27. Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue?. Cancer Res., 46: 467-473, 1986.[Free Full Text]
  28. Bicknell R., Harris A. L. Mechanisms and therapeutic implications of angiogenesis. Curr. Opin. Oncol., 8: 60-65, 1996.[Medline]
  29. Hsu S. C., Volpert O. V., Steck P. A., Mikkelsen T., Polverini P. J., Rao S., Chou P., Bouck N. P. Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res., 56: 5684-5691, 1996.[Abstract/Free Full Text]
  30. Joseph I. B., Vukanovic J., Isaacs J. T. Antiangiogenic treatment with linomide as chemoprevention for prostate, seminal vesicle, and breast carcinogenesis in rodents. Cancer Res., 56: 3404-3408, 1996.[Abstract/Free Full Text]
  31. Schmidek H. H., Nielsen S. L., Schiller A. L., Messer J. Morphological studies of rat brain tumors induced by N-nitrosomethylurea. J. Neurosurg., 34: 335-340, 1971.[Medline]
  32. Asai A., Miyagi Y., Sugiyama A., Gamanuma M., Hong S. H., Takamoto S., Nomura K., Matsutani M., Takakura K., Kuchino Y. Negative effects of wild-type p53 and s-Myc on cellular growth and tumorigenicity of glioma cells. Implication of the tumor suppressor genes for gene therapy. J. Neuro-oncol., 19: 259-268, 1994.[Medline]
  33. von Deimling A., Eibl R. H., Ohgaki H., Louis D. N., von Ammon K., Petersen I., Kleihues P., Chung R. Y., Wiestler O. D., Seizinger B. R. p53 mutations are associated with 17p allelic loss in grade II and grade III astrocytoma. Cancer Res., 52: 2987-2990, 1992.[Abstract/Free Full Text]
  34. Van Meir E. G., Polverini P. J., Chazin V. R., Su Huang H-J., De Tribolet N., Cavenee W. K. Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. Nat. Genet., 8: 171-176, 1994.[Medline]
  35. O’Reilly M. S., Holmgren L., Chen C., Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat. Med., 2: 689-692, 1996.[Medline]
  36. Ghazizadeh S., Carroll J. M., Taichman L. B. Repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy. J. Virol., 71: 9163-9169, 1997.[Abstract]
  37. Rother R. P., Fodor W. L., Springhorn J. P., Birks C. W., Setter E., Sandrin M. S., Squinto S. P., Rollins S. A. A novel mechanism of retrovirus inactivation in human serum mediated by anti-{alpha}-galactosyl natural antibody. J. Exp. Med., 182: 1345-1355, 1995.[Abstract/Free Full Text]
  38. Sparer T. E., Wynn S. G., Clark D. J., Kaplan J. M., Cardoza L. M., Wadsworth S. C., Smith A. E., Gooding L. R. Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype. J. Virol., 71: 2277-2284, 1997.[Abstract]
  39. Le Gal La Salle G., Robert J. J., Berrard S., Ridoux V., Stratford-Perricaudet L. D., Perricaudet M., Mallet J. An adenovirus vector for gene transfer into neurons and glia in the brain. Science (Washington DC), 259: 988-990, 1993.[Abstract]
  40. Yang Y., Wilson J. M. Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo. J. Immunol., 155: 2564-2570, 1995.[Abstract]
  41. Udelsman R., Chen H., Loman K., Pitt H. A., Saudek C. D. Implanted programmable insulin pumps: one hundred fifty-three patient years of surgical experience. Surgery, 122: 1005-1011, 1997.[Medline]
  42. Saudek C. D. Novel forms of insulin delivery. Endocrinol. Metab. Clin. North Am., 26: 599-610, 1997.[Medline]
  43. Fine H. A., Dear K. B., Loeffler J. S., Black P. M., Canellos G. P. Meta-analysis of radiation therapy with and without adjuvant chemotherapy for malignant gliomas in adults. Cancer (Phila.), 71: 2585-2597, 1993.[Medline]
  44. Prados M. D., Russo C. Chemotherapy of brain tumors. Semin. Surg. Oncol., 14: 88-95, 1998.[Medline]




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