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Experimental Therapeutics |
Department of Pathology [L. Z., W. S. G., S. A. S., O. B. I., M. A. D., A. J. M.] and Greenebaum Cancer Center [S. A. S., O. B. I., M. A. D., A. J. M.], University of Maryland Baltimore, Baltimore, Maryland 21201
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNAzymes offer several significant advantages compared with ribozyme molecules, including easier synthesis and decreased sensitivity to chemical or enzymatic degradation. Indeed, after modifying the 5' and 3' ends of DNAzymes, little degradation has been observed after 24 h of exposure to serum (2 , 3) . Although DNAzymes may be further modified, it is a requirement that synthetic ribozymes are extensively modified to maintain stability in serum. Ribozymes that have phosphorothioate backbones to maintain stability have reduced binding affinity for target mRNA (4) , increased binding avidity for serum and/or cellular protein (5) , and may be cytotoxic (6) . Furthermore, DNAzymes exhibit greater substrate flexibility compared with conventional and hammerhead ribozymes (1 , 7, 8, 9) . The 10–23 DNAzymes can cleave effectively between any unpaired purine and pyrimidine of mRNA transcripts (1) . As a result, DNAzymes can be designed specifically to recognize the AU nts of the start codon. Because the translation start site and its neighboring bases have little secondary structure, DNAzymes often reduce their substrate mRNA levels without significant amounts of screening. These characteristics of DNAzymes make them promising candidates for in vivo oligonucleotide therapy.
One potential target for DNAzyme therapy is angiogenesis of solid tumors. It has been well recognized that growth and metastasis of solid tumors require persistent angiogenesis and that induction of angiogenesis is a discrete component of the tumor phenotype (10) . VEGF and its cognate receptors (VEGFR1 and 2) are critical factors in the balance between activation and inhibition of angiogenesis. Several reports demonstrating the utility of antiangiogenic gene therapy in tumor-bearing mouse models (11 , 12) have shown modest antitumor activity. At present, there are no antisense and only one therapeutic angiogenic ribozyme targeting the VEGFR in clinical trials (13 , 14) . As a result, new gene therapy and oligonucleotide strategies and carriers that target angiogenesis are essential to determining improved approaches. In this study, we explored the use of a synthetic DNAzyme targeting VEGFR2 transcripts as a potential tool to inhibit tumor angiogenesis. The VEGFR2 DNAzyme was found to cleave its substrate efficiently, to inhibit the proliferation of endothelial cells with a concomitant reduction of VEGFR2 mRNA, and to inhibit tumor growth in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of PCI-VEGFR2f
A 2.5-kb N-terminal fragment of the mouse VEGFR2 cDNA was inserted into the XbaI site of PCI expression vector (Promega). Orientation of this plasmid (PCI-VEGFR2f) was verified with restriction enzyme digest (SalI) and with sequence analysis.
Oligonucleotide Synthesis and Labeling
The DNAzyme, the disabled DNAzyme oligonucleotide (also named control ODN), and their oligoribonucleotide substrates were prepared with an oligonucleotide synthesizer (Beckman). The 5' and 3' termini of the oligonucleotides were protected from exonucleases by a phosphorothioate linkage and a CPG-C3 cap, respectively (Glen Research, Sterling, VA). The sequences of the ODNs that target the VEGFR2 are as follows: DNAzyme, 5'-tgctctccaGGCTAGCTACAACGAcctgcacct-3' Control ODN, 5'-ctctccaGGTATGTACAACGAcctgcacct-3', and substrate, 5'-GCGCGAGGUGCAGGAUGGAGAGCAAGGC-3' (GenBank accession no. X70842 for mouse VEGFR2 sequence). The capitalized nts (15 nts) within the DNAzyme sequence represent the catalytic domain, whereas the capitalized nts within the control ODN represent a disabled catalytic domain. Transversion of two nts in the control oligonucleotide are sufficient to inactivate the catalytic activity (1
, 15, 16, 17)
. The bold-faced nts within the DNAzyme and control oligonucleotides anneal to the translational start site region of VEGFR2. The oligoribonucleotide substrate of the DNAzyme was labeled at the 5' end with [
-33P]ATP (2500 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) by using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Unincorporated labeled nts were then removed by centrifuging the reaction sample on a Spin-25 minicolumn (BioMax Inc., Odenton, MD).
In Vitro Transcription
To prepare the target mRNA of VEGFRf for cleavage experiments, in vitro transcription was performed as described in the T7 MAXIscript kits (Ambion Inc, Austin, TX; see below for details about the cleavage experiments). In brief, the PCI-VEGFR2f plasmid was digested with SalI before transcription. With a T7 polymerase in vitro transcription kit (Ambion Inc.), the expected 572-nt VEGFR2 RNA transcript was generated. The transcript was prepared in a total volume of 20 µl containing 50 µCi of [
-33P]UTP (2500 Ci/mmol), 40 µmol of nucleotide triphosphate, 1 µg of template, and 30 units of T7 polymerase for 1 h at 37°C. Unincorporated labeled nts were removed by centrifugation on Spin-50 minicolumn.
Cleavage Experiments
Cleavage experiments were performed as described previously (18)
in 20 µl of the reaction buffer [5 mM Tris buffer (pH 7.50), 10 mM MgCl2, and 150 mM NaCl]. These cleavage reactions were allowed to proceed at 37°C and were stopped by transferring aliquots of the reaction into formamide loading buffer (Ambion).
To measure substrate cleavage as a function of DNAzyme concentration, we added increasing amounts of DNAzyme (0, 2.5, 10, 50, and 250 pmol) to 1 pmol of the synthetic oligonucleotide RNA substrate. Consequently, the DNAzyme:substrate ratio ranged from 0:1 to 250:1. A time course of DNAzyme and substrate was then done after adding 50 pmol of DNAzyme to 1 pmol of synthetic RNA substrate. The reaction was stopped by removing aliquots at times 0, 10, 20, 30, 60, 90, and 120 min. For in vitro transcript experiments, 50 pmol of DNAzyme were added to 4 or 40 pmol of the in vitro transcript substrate; the reaction was then stopped at several time points. In these cleavage experiments, the cut and uncut substrates were separated by electrophoresis on a 5% (for in vitro transcript) or a 19% (for oligonucleotide) urea denaturing polyacrylamide gel and detected by autoradiography at 4°C. Signals were then scanned by Storm 840 instrument and analyzed by ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA).
In Vitro Delivery of Oligonucleotides
Cells were first plated into 12-well plates with 3 x 104cells/well. When the cells were 40–50% confluent, ODN/polymer/liposome complexes were incubated with the cells for 4 h. The cells were then washed with PBS solution, and Opti-MEM medium with 7.5% serum was added to the cells. For preparation of the complexes, the detailed procedure is as follows: first, 2 µg of ODN were incubated with 9 µg of the HHK4b polymer for 30 min in 120 µl of Opti-MEM (Life Technologies, Inc., Rockville, MD); then, 3 µg of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes in 80 µl of Opti-MEM was gently mixed with the polymer/DNA complex and allowed to stand for an additional 30 min; finally, the complex was diluted in 400 µl of Opti-MEM +7.5% serum, and transferred to each well. HHK4b is a highly branched polymer composed of histidine and lysine, which, in combination with liposomes, enhances gene expression (19)
. Compared with liposomes only, we found that the HHK4b/liposome combination is a more effective carrier of DNAzymes (data not shown). For Northern blot analysis, the cells were harvested for purification of total RNA 24 h after the cellular uptake of ODN. For cell growth studies, the cell numbers were determined 48 h after ODN delivery.
Cell Counting
Forty-eight h after ODN uptake, cells in a 12-well plate were washed with PBS. Then 500 µl of Sytox Green (5 µg/ml; Molecular Probes) in PBS +0.1% Triton X-100 was added to the cells in each well for 15 min at room temperature; after incubation, the cells were washed with PBS, and total fluorescence per well was measured with a Cytofluor microplate reader (Excitation/Emission = 504/523; Perkin-Elmer, Woburn, MA). The cell number per well was calculated after determining the standard curve for each cell line.
Northern Blot Analysis
To prepare the VEGFR2 probe, PCI-VEGFR2f was initially digested with XbaI. The resulting 2.5-kb cDNA fragment of the VEGFR2 was then purified on a 1% agarose gel. After purification, the cDNA fragment was labeled with [32P]dCTP by random priming with the use of Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech). Total cellular RNA was isolated from BAE or MDA-MB-435 cells with the RNAwiz reagent (Ambion Inc.). Cellular RNA (30 µg) was subjected to electrophoresis on 1.0% (w/v) agarose-formaldehyde gels and blotted to nylon membrane (Bio-Rad, Hercules, CA) by capillary transfer. After UV cross-linking, blots were prehybridized in ExpressHybrid solution (Clontech Laboratories Inc., Palo Alto, CA) at 65°C for 30 min. Hybridization was performed by reacting with fresh ExpressHybrid solution containing the denatured 32P-cDNA probes at 65°C for 16 h. The blots were washed twice with 2x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate) and 0.05% SDS at room temperature for 20 min, then washed twice with 0.1x SSC and 0.1% SDS at 50°C for 20 min. Blots were exposed to film with intensifying screens at -80°C. After developing the film, the signal was scanned by Personal Densitometer SI and analyzed by ImageQuant 1.1 software (Molecular Dynamics). The housekeeping gene, ß-actin, was used as internal control to normalize the RNA loading. After the signals for VEGFR2 and ß-actin were quantified, the ratio of VEGFR2:ß-actin for each treatment group was determined. These experiments, including ODN treatment of the cells, were repeated twice.
Intratumoral Injections of Cells and Complexes into Mice
Complexes and intratumoral injections were prepared as described previously (19
, 20)
. In brief, after administering the Metofane anesthetic, female athymic nude mice received injections of 2.5 x 105 MDA-MB-435 tumor cells bilaterally into thoracic mammary fat pads with a stepper (Tridak) and a 27.5-gauge needle. Five days after the injection of cells when the tumor was visible, the mice were randomly divided into four treatment regimens: (a) saline-treated; (b) HHK4b (carrier only)-treated; (c) HHK4b+DNAzyme-treated; and (d) HHK4b+control oligonucleotide-treated. Each treatment group contained six mice, each with two tumors. The mice received four injections of 30 µl of the therapeutic complexes, and the tumors were measured with skin calipers before each injection and 5 days after the last injection. The intratumoral injections consisted of a histidine-lysine polymer (1.092 nmol) in complex with 2.9 µg of the oligonucleotide. The complexes were prepared as described above in the in vitro cellular ODN delivery studies, except that water replaced Opti-MEM.
Quantitative Measurement of Blood Vessels
Three days after the last intratumoral injection of complex, the tumor blood vessels were quantified as described previously (21)
. Four tumors (two tumors per mouse) of similar size were selected from each treatment group. One ml of carmine suspension (10% wt/vol) in 2.5% gelatin (300 bloom, Aldrich Co.) solution, warmed to 42°C, was injected into the tail vein of each mouse. The mice survived for 
20 s after this injection. After the gelatin solidified by cooling the cadaver at 4°C for a few hours, the tumor was removed and fixed in formalin overnight. The tumor sections were counterstained with hematoxylin, and the blood vessels were counted in the two areas of each tumor section with the highest blood vessel density ("neovascular hot spots"; Refs. 22, 23, 24, 25
).
Histology Analysis and Immunohistochemistry Detection of Ki67
Primary tumors were fixed in Streck tissue fixative (Streck Laboratories, Inc., Omaha, NE), placed in a paraffin block, and then sectioned. H&E staining was performed routinely. For immunohistochemistry, sections were deparaffinized and treated with 3% H2O2 followed by 0.01% trypsin. The sections were first incubated with a polyclonal rabbit anti-Ki67 (Novocastra, Burlingame, CA), diluted 1:1000, for 1 h and then with a biotinylated goat antirabbit IgG antiserum, diluted 1:400, for 1 h at room temperature. After washing, the sections were labeled with the avidin-biotin-peroxidase complex system (Vectastain ABC kit; Novocastra) followed by diaminobenzidine as a substrate for peroxidase. Positive staining was observed as a brown granular nuclear precipitate. No counterstain was used.
Detection of Apoptosis
In Vitro.
After plating cells in a Lab-Tek chamber slide (Nunc, Rochester, NY) for 24 h, the DNAzyme or the control oligonucleotide, in complex with the HHK4b/liposome carrier, was added to the cells. Twenty-four h later, the cells were washed with PBS and then fixed with 2% paraformaldehyde for 30 min at 4°C. Twenty µl of mounting material, Mowiol, containing the nuclear DAPI stain (final concentration, 0.1 µg/ml) were added to the fixed cells, and the cells were overlaid with coverslips. After solidification, cellular apoptosis was quantified by nuclear fragmentation with a Nikon TE 200 fluorescent microscope (Beckman) by counting 10 contiguous x40 fields.
In Vivo.
Tumor tissue from mice was sectioned after fixation with Streck tissue fixative. Apoptotic nuclei were detected by the TUNEL method with an In Situ Cell Death detection kit (Roche, Indianapolis, IN). To calculate the ratio of the number of apoptotic nuclei to the total number of nuclei, the numbers of all of the nuclei and apoptosis-positive-stained nuclei were counted in three x40 fields of two different tumors.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The 5' and 3' termini of the molecule are protected from exonucleases by a phosphorothioate linkage and a CPG-C3 cap, respectively. To inactivate the DNAzyme and to generate a control oligonucleotide, two nts were changed in the catalytic domain of VEGFR2 DNAzyme (Refs. 1
and 15, 16, 17
; Fig. 1C
).
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. Cleavage products were seen within 10 min (1.86% of total) and progressively increased until the last time point at 120 min (19.6% of total). The greatest catalytic activity was seen when the DNA:substrate ratio was 250:1 (Fig. 2B)
. At this ratio, 21.2% of the substrate product was digested in 1 h at 37°C. Furthermore, the 33P-labeled 572-nt in vitro transcript was cleaved by VEGFR2 DNAzyme to produce two expected cleavage products, 326-nt and 246-nt (Fig. 1D
; Fig. 2, C and D
). With 4 pmol of the RNA transcript as the target, the DNAzyme at 10 min, 60 min, and 120 min digested 8.8, 13.1, and 20.3%, respectively, of the product (Fig. 2C)
. In contrast, the control oligonucleotide did not show any enzymatic activity. Because the in vitro catalytic activity of our DNAzyme was 1/5 that of a highly active DNAzyme (26)
, it may be beneficial to either modify the current DNAzyme or screen the VEGFR2 transcript for more effective DNAzymes.
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. Induction of apoptosis in BAECs was illustrated by marked nuclei fragmentation or condensation in the DNAzyme-treated group (Fig. 3B)
. By examining 10 contiguous x40 fields, 12.6% of cells were found to be apoptotic in the DNAzyme group, whereas only 1.9 and 0% of cells were apoptotic, respectively, in the control ODN and untreated groups. Thus, the DNAzyme demonstrates significantly greater potency at inducing apoptosis in endothelial cells than does its control oligonucleotide counterpart.
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. These findings, which were verified in a second experiment, established the ability of the DNAzyme to reduce cellular mRNA levels of VEGFR2.
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, tumors from the DNAzyme-treated group were significantly reduced compared with the control groups after the third measurement. By the fifth measurement, nearly a 75% reduction of tumor growth was found in the DNAzyme group, whereas 20 and 35% reductions were shown in the carrier-alone and control-ODN groups, respectively, compared with the saline-treated group. The difference in tumor size between the DNAzyme- and saline-treated groups was significant (P = 0.024). Similar tumor reductions on intratumoral injections with the DNAzyme have been found in four other experiments, with liposomes as the sole carrier or with the HHK4b/liposome combination. We also observed diffuse distribution of a fluorescently labeled (AlexaFluor 546; Molecular Probes) oligonucleotide within the tumor 4 h after injection of the HHK4b/oligonucleotide complex (data not shown). These results indicate that local DNAzyme ODN therapy with the polymer carrier effectively inhibits tumor growth in vivo. The effects of therapeutic DNAzyme and control ODN in reducing tumor size in vivo and endothelial cell number in vitro show similar trends, which indicates greater potency of the DNAzyme versus the control oligonucleotide.
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). In the DNAzyme-treated MDA-MB-435 nude mice, there were 1.5 ± 0.25 blood vessels per x10 field compared with 11.0 ± 1.47 in untreated cells. H&E staining showed large areas of peripheral cell death in the DNAzyme-treated tumor (Fig. 6, C and D)
; because angiogenesis occurs primarily at the leading edge of the tumor, tumor cell death at the periphery with the VEGFR2 DNAzyme therapy supports the antiangiogenic mechanism of this therapy. Although the corrected VEGFR2 mRNA levels were similar in the saline- and DNAzyme-treated groups, the VEGFR2 and ß-actin mRNA levels were markedly reduced in the DNAzyme-treated tumors, perhaps reflecting the amount of tumor cell death in this group (data not shown).
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. We found that 0.5% stained positive for Ki67 in the DNAzyme-treated tumors compared with 16% in the saline-treated tumors. Approximately 22% of the cells in the mouse tumors were apoptotic in the DNAzyme-treated group (Fig. 6H)
, whereas only 0.8% of cells were apoptotic (Fig. 6G)
in the saline-treated tumors, as demonstrated by the positive TUNEL staining. In addition, tumor histology revealed an increased number of apoptotic cells in the DNAzyme-treated group. Thus, proliferation was decreased, whereas apoptosis was increased in VEGFR2 DNAzyme-treated tumors compared with the untreated group (Fig. 6E–H)
, which indicated that retardation of tumor growth is caused by apoptosis and decreased proliferation. In tumors of mice treated with control oligonucleotides, apoptosis (7% of cells) and Ki67 expression (9% of cells) were intermediate between the saline- and DNAzyme-treated groups. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With the development of DNAzymes through PCR and in vitro evolution, ODNs have gained several intrinsic catalytic functions (1 , 29) . In addition to cleaving the phosphodiester bonds of RNA as demonstrated in this and other studies, DNAzymes are quite versatile, catalyzing many different chemical reactions including RNA cleavage (29, 30, 31, 32) , ligation (33) , phosphorylation (34) , and porphyrin metallation (35) . Similar to antisense and ribozyme technologies, DNAzymes confront many developmental challenges such as biostability and catalytic efficiency (3 , 18 , 32) .
On the basis of the above mentioned studies, we were able to develop a DNAzyme that targeted and cleaved VEGFR mRNA in vitro and markedly reduced blood vessel density and tumor growth in vivo. The successful application of VEGFR2 DNAzyme suggests a therapeutic role of DNAzymes for other important angiogenic factors with the possibility of using combination therapy in future studies. Nevertheless, we believe that the efficacy of the currently designed DNAzyme in targeting the VEGFR2 translational start site may be further improved by varying the length of its target-binding arms or by increasing the number of modified bases. For example, altering the target arms by a single base may affect the catalytic efficiency of the DNAzyme by >100-fold (1) . Alternatively, a second type of DNAzyme, the "8–17" model (whose substrate is AG), may be more effective in digesting VEGFR2; one of the 8–17 DNAzymes exhibited greater catalytic efficiency in cleaving the BCR-ABL fusion mRNA in another study (9) . With the 8–17 DNAzyme, there are several potential cleavage sites in the mouse VEGFR mRNA near the translation start site (-9, -3, +4, and +6). Because several studies suggest these modifications and strategies may increase the efficacy significantly (1 , 3 , 9) , we are currently examining these approaches to improve the ability of the DNAzyme to digest the VEGFR2 mRNA target.
One heralded advantage of DNAzymes is that they are frequently effective at cleaving and reducing the target mRNA (1) . Because DNAzymes can potentially cleave RNA at any purine-pyrimidine junction, this allows the catalytic DNA to cut at the AUG site, a region of the mRNA with decreased secondary structure. Without any prior screening, we found that our initially synthesized DNAzyme that targeted VEGFR2 cleaved its mRNA substrate and reduced tumor growth. Obviously, DNAzymes may still be ineffective if there are high levels of the target mRNA expression or if their target site is within the secondary structure of the RNA (26) . Nevertheless, the ability of local in vivo therapy with the VEGFR2 DNAzyme to inhibit tumor growth suggests that sufficient amounts of the DNAzyme are accumulating within the mitogenic endothelial cells.
Furthermore, DNAzymes offer several potential advantages compared with plasmid-based therapy. Several plasmid-based antiangiogenic gene therapies have demonstrated antitumor efficacy (11
, 12)
; unfortunately, plasmids almost always contain unmethylated palindromic immunostimulatory sequences that have cellular toxicity and interfere with expression of proteins of plasmids (36, 37, 38, 39)
. These sequences can activate a wide variety of immunological mechanisms in a dose-dependent manner, including cell proliferation and immunoglobulin secretion, monocyte/macrophage cytokine secretion (i.e., interleukin 4, interleukin 12, IFN-, tumor necrosis factor ), and activation of natural killer cell lytic activity (36)
. Although some groups have taken advantage of these immunostimulatory sequences to inhibit tumors, in general, such sequences are an unwanted side effect when a therapeutic protein is expressed. A significant advantage of DNAzymes is that they commonly lack these immunostimulatory sequences, allowing the carrier-DNAzyme combination, to be administered more frequently with less toxicity. Nevertheless, our nonviral carrier and probably the DNA appear to have a modest direct toxic effect on the tumor, independent of the DNAzyme or antisense effects.
In summary, this is the first study that demonstrates the inhibition of tumor growth in vivo with an RNA-cleaving DNAzyme. This DNAzyme, targeting VEGFR2, exhibited higher antitumor activity than its control ODN, which indicated its potential advantages as a biocatalyst in oligonucleotide therapy. To extend the efficacy of the DNAzyme to systemic therapy, one approach that we are pursuing is the development of more efficient and specific endosomolytic carriers (40 , 41) . With systemic therapy, these carriers delivering DNAzymes will have to overcome a multitude of challenges from blood-borne components (e.g., aggregation of complexes) to escaping from the endosomal/lysosomal pathway. Similar to plasmid-based therapies (42) , most carriers deliver ODNs inefficiently to their cellular target (40 , 41 , 43 , 44) , and, consequently, improved carriers of ODNs are being developed (40 , 41 , 45) . Although carriers of ODNs and plasmids injected systemically will face many of the same obstacles in reaching their target, it is likely, however, that the most effective carriers of these different molecular forms of DNA (plasmids versus ODNs) will differ (40 , 46) . Other potential systemic approaches that may achieve sufficient amounts of DNAzyme within the tumor endothelium include conjugation of the ODNs to "membrane-penetrating " peptides and nuclease-resistant modification of ODNs that are stable in serum without a carrier (40 , 41) . Despite the challenges in systemic delivery of ODNs, the selective, growth-inhibitory properties of VEGFR2 DNAzyme indicate that DNAzymes may be a valuable addition to the armamentarium of antiangiogenic therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grant CA70394. 
2 To whom requests for reprints should be addressed, at Department of Pathology, University of Maryland Baltimore, Building MSTF, Room 759, Baltimore, MD 21201. Phone: (410) 706-3223; Fax: (410) 706-8414; E-mail: amixson{at}umaryland.edu
3 The abbreviations used are: ODN, oligodeoxynucleotide; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; VEGFR2, VEGF receptor 2; BAEC, bovine aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; nt, nucleotide; DAPI, 4',6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
Received 12/26/01. Accepted 7/26/02.
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DNA enzymes with potential therapeutic application. J. Mol. Biol., 296: 937-947, 2000.[Medline]
(v)ß3: a new prognostic indicator in breast cancer. Clin. Cancer Res., 4: 2625-2634, 1998.[Abstract]
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