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1 Vascular and Tumor Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel,
2 ImClone Systems Inc., New York, New York
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of available clinical data, we hypothesized that heparanase functions to accelerate tumor development, possibly by releasing HS-bound angiogenic growth factors. To examine this hypothesis, we overexpressed heparanase in the U87 glioma cell line, and selected cell pools were characterized. As expected, a significant increase in cell invasion was noted upon heparanase overexpression. Interestingly, however, cells expressing high heparanase levels exhibited a low proliferative rate that correlated with increased cell spreading and ß1-integrin activation. In a xenograft tumor model, heparanase expression in U87 glioma cells accelerated tumor development that correlated with increased tumor angiogenesis. However, U87 glioma cells that exhibited the highest heparanase expression levels and were less proliferative in vitro maintained this phenotype in vivo and developed smaller tumors. These results provide a direct linkage between heparanase expression and tumor development and indicate, for the first time, that heparanase expression at high levels may activate signal transduction pathways that elicit adhesive characteristics, which may halt tumor progression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Transfection.
The U87-MG glioma cells were purchased from the American Type Culture Collection. The cells were grown in DMEM supplemented with glutamine, pyruvate, antibiotics, and 10% FCS in a humidified atmosphere containing 8% CO2 at 37°C. For stable transfection, U87 cells were transfected with the chimeric-hepa gene construct (the human heparanase cDNA fused to the chicken heparanase signal peptide; Ref. 16
) and with control pcDNA3 vector, using the FuGene reagent according to the manufacturer instructions (Roche), selected with G418 (1000 µg/ml) for 3 weeks, expanded, and pooled. This parental transfected cell population was further selected for high heparanase-expressing cells by generating pools of 
50 colonies/100-mm culture dishes and evaluating heparanase expression by immunoblot analysis. The pool with the highest expression level was further expanded and designated as "Hi" throughout the manuscript, whereas the parental transfected cells are referred to as "Low."
HEK 293 cells, stably transfected with the human heparanase gene construct in the mammalian pSecTag vector (Invitrogen), were kindly provided by ImClone Ltd. The cells were grown in DMEM supplemented with 10% FCS, glutamine, pyruvate, and antibiotics. For heparanase purification, the cells were grown overnight in serum free-DMEM, and the conditioned medium (1 liter) was applied onto a Fractogel EMD SO3- (MERCK) column. The bound material was eluted with 1 M NaCl and was further purified by affinity chromatography on an anti-c-Myc (Santa Cruz Biotechnology) column. We obtained at least 95% pure heparanase preparation by this two-step procedure.
Heparanase Activity Assay.
Preparation of ECM-coated dishes and determination of heparanase activity were performed as described in details elsewhere (16
, 17) . Briefly, cell extracts (0.5 x 106 cells/ml) were incubated (24 h, 37°C, pH 6.6) with 35S-labeled ECM. The incubation medium containing sulfate-labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS, and their radioactivity was counted in a beta scintillation counter. Degradation fragments of heparan sulfate side chains were eluted at 0.5 < Kav < 0.8 (peak II, fractions 15–30). Nearly intact HSPG were eluted just after the Vo (Kav < 0.2, peak I, fractions 3–15) (16
, 18) .
Cell Lysates, Immunoprecipitation, and Protein Blotting.
Cell cultures were pretreated with 1 mM orththovanadate for 10 min at 37°C, washed twice with ice-cold PBS containing 1 mM orththovanadate, and scraped into lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM orththovanadate, and 1 mM phenylmethylsulfonyl fluoride) containing a mixture of proteinase inhibitors (Roche). Total cellular protein concentration was determined by the bicinchoninic acid method according to the manufacturer’s instruction assay (Pierce, Rockford, IL). Forty µg of cellular protein were fractionated on SDS-PAGE, and immunoblotting was performed, as described (19)
.
Immunocytochemistry.
Cells were grown on glass coverslips for 1 h, fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.5% Triton X-100 in PBS for 1 min. Cells were then washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 h at room temperature, followed by 2-h incubation with the indicated primary antibody. Cells were then extensively washed with PBS and incubated with the relevant (Cy2/Cy3-conjugated) secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h, washed, and mounted (Vectashield, Vector, Burlingame, CA). For actin staining, cells were fixed and permeabilized as above, and after being washed three times with PBS, incubated with rhodamine-conjugated phalloidin (Sigma) for 30 min. Staining was observed under a fluorescent microscope, and photographs were taken with a digital camera, both from Nikon.
Matrigel Invasion Assay.
Invasion assay was performed using modified Boyden chambers with a polycarbonate Nucleopore membrane (Corning, Corning, NY). Filters (6.5 mm in diameter, 8-µm pore size) were coated with Matrigel (30 µl) and dried overnight at room temperature. Cells (2 x 105) in 100 µl of serum-free medium were seeded in triplicate on the upper part of each chamber, and the lower compartment was filled with 600 µl of medium conditioned by 3T3 fibroblasts. After incubation for 5 and 24 h at 37°C in a 5% CO2 incubator, non-invading cells on the upper surface of the filter were wiped with a cotton swab, and migrated cells on the lower surface of the filter were fixed, stained with 0.5% crystal violet (Sigma), and counted by examination of at least five microscopic fields (14)
.
Cell Migration Assay.
Vo, Low, and Hi cells were allowed to grow for 2 days on tissue culture plates and were then scraped with the wide end of a 1-ml tip (time 0). Plates were washed twice with PBS to remove detached cells and incubated with complete growth medium, and cell migration into the wounded empty space was followed over 2 days.
Zymographic Analysis of MMPs.
Samples of conditioned medium were mixed with non-reducing electrophoresis loading buffer and subjected to electrophoresis, carried out on a 10% SDS-PAGE copolymerized with gelatin (2 mg/ml). After electrophoresis, gels were washed with 2.5% Triton X-100 for 1 h (3 times, 20 min each) and incubated for 24 h in enzyme assay buffer (25 mM Tris, pH 7.5, 5 mM CaCl2, 0.9% NaCl, and 0.05% Na3N) for the development of enzyme activity bands. After incubation, the gels were stained with 0.25% Coomassie brilliant blue G-250 and destained in 10% methanol with 5% acetic acid. The gelatinolytic activities were detected as transparent bands against the blue background of the Coomassie brilliant blue-stained gelatin.
Cell Proliferation.
For growth curves, 5 x 104 cells were cultured in 6-cm dishes in duplicates. Cells were counted every day for 4 days after trypsinization using a Coulter Counter, and cell numbers were further confirmed by counting with a hemacytometer. Additionally, cell proliferation was analyzed by BrdUrd incorporation using a cell proliferation labeling reagent (1:1000; Amersham). Briefly, subconfluent cells grown on glass coverslips in complete growth medium were incubated for 2 h in the presence of BrdUrd, fixed with cold methanol for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 1 min, washed with PBS, and then incubated for 1 h with 2 N HCl. Next, cells were washed three times with 0.1 M borate buffer, pH 8.5, for 10 min each, at room temperature. The cells were then incubated with horseradish peroxidase-conjugated anti-BrdUrd antibody (1:20; Roche) for 2 h, washed, and visualized using an 3-amino-9-ethylcarbazole staining kit (Sigma). After hematoxylin counterstaining and mounting, the mitotic index was calculated by counting BrdUrd-positive nuclei as a percentage of total cells in at least eight different fields. At least 1000 cells were counted for each cell type.
Colony Formation in Soft Agar.
Three ml of DMEM containing 0.5% Low Melt Agarose (Bio-Rad, Hercules, CA) and 10% FCS was poured into a 6-cm Petri dish. The layer was covered with cell suspension (1 x 104 cells) in 1.5 ml of DMEM containing 0.3% Low Melt Agarose and 10% FCS, and the dish was covered with 2 ml of DMEM containing 10% FCS. Medium was exchanged every 3 days. Colonies were visualized and counted under a microscope after 3 weeks.
Flow Cytometry.
Cells were detached with trypsin, centrifuged at 1000 rpm for 4 min, washed with PBS, and counted. Cells (2 x 105) were centrifuged, and the pellet was then resuspended in PBS with 1% FCS and incubated with FITC conjugated anti-ß1-integrin antibody for 20 min on ice. Cells were then washed twice with PBS and analyzed using a FACSCalibur fluorescent activated cell sorter and CellQuest software (Becton Dickinson, Mountain View, CA).
Tumorigenicity Studies.
Cells from exponential cultures of control (Vo), Low, and Hi cell transfectants were detached with trypsin, washed with PBS, and brought to a concentration of 5 x 107cells/ml. Cell suspension (5 x 106/0.1 ml) was inoculated s.c. at the right flank of 5-week-old female BALB/c athymic nude mice (Harlan, Jerusalem, Israel). Xenograft sizes were determined weekly by externally measuring tumors in two dimensions using a caliper. Tumor volume (V) was determined by the equation 
, where L is the length and W is the width of the xenograft. At the end of the experiment, mice were sacrificed by cervical dislocation, xenografts were resected, weighted, and fixed in formalin. Paraffin-embedded 5-µm sections were stained with H&E or immunostained with anti-heparanase, anti-VEGF, and anti-PECAM-1 antibodies, using the Envision kit, according to the manufacturer’s instructions (Dako, Glostrup, Denmark). Apoptotic cell death was evaluated by the in situ cell death detection kit (terminal deoxynucleotidyl transferase-mediated nick end labeling), according to the manufacturer’s instructions (Roche).
Statistics.
Data are presented as mean ± SE. Statistical significance was analyzed by two-tailed Student’s t test. The value of P < 0.05 is considered significant.
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RESULTS |
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, Vo). A significant increase in heparanase activity (release of sulfate-labeled HS degradation fragments eluted from Sepharose 6B column in fractions 15–26) was observed in U87 cells that survived G418 selection after heparanase transfection (Fig. 1A
, Low), and heparanase activity was further amplified in one of the selected cell pools (Fig. 1A
, Hi). The heparanase activity results were further confirmed by immunoblot analysis (Fig. 1B)
, revealing a 4–5-fold increase in expression of both the latent (65-kDa) and active (50-kDa) heparanase forms in the Hi cells compared with Low, as determined by densitometry analysis. Heparanase activity was traditionally correlated with the invasive properties of tumor-derived cell types (14
, 18)
, and this correlation is supported by more recent clinical studies (4
, 7
, 8
, 10)
. We, therefore, evaluated the invasive capacity of the U87 cells in a Boyden chamber assay. Matrigel, a reconstituted basement membrane containing HSPG, was used as a relevant barrier. Heparanase-transfected Low and Hi cell populations exhibited 2.5- and 3.5-fold increase, respectively, in their invasive capacity compared with the Vo control cells (Fig. 1, C and D)
, as might have been expected. Interestingly, no difference or a slight decrease in MMP2 secretion was detected by zymography analysis of Vo, Low, and Hi cells conditioned medium (Fig. 1E)
, suggesting that the observed increase in cell invasion is attributable primarily to heparanase elevation.
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, Vo). In striking contrast, the heparanase-transfected Hi cells adopted apparent anchorage-dependent growth, failed to form such cell aggregates, and grew as a tight monolayer (Fig. 2A
, Hi), whereas the Low cells developed an intermediate phenotype (Fig. 2A
, Low). Because cell clumps produced by the control Vo cells continued to develop, while heparanase transfectants assumed a monolayer morphology, we suspected that changes in cell proliferation may be involved. To better correlate phenotypic appearance with proliferative behavior, cell proliferation was measured during 4 days in culture (Fig. 2B)
. Initially plated at a density of 5 x 104 cells/dish, 5.8 ± 0.28 x 105 of control Vo cells were counted 4 days later, as compared to 2.7 ± 0.14 x 105 and 4.2 ± 0.56 x 105 cells/dish of the heparanase-transfected Hi and Low cells, respectively, with no apparent change in apoptosis (data not shown). Differences were statistically significant (P < 0.04). These results were further confirmed by direct measurements of DNA synthesis by staining cells for incorporated BrdUrd. Positively stained nuclei represented 5.5% of the total control Vo cells, compared with 0.9 and 2.7% in the Hi and Low cells, respectively (Fig. 2C)
, further supporting significant attenuation of cell proliferation in cells with high heparanase expression levels. Moreover, whereas the Vo control cells produced large colonies in soft agar (115 ± 23 µM; Fig. 4D
), both the Hi and Low cells developed significantly smaller colonies (11 ± 4 and 14 ± 7 µM, respectively).
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7) conditions that are unfavorable for its enzymatic activity (20)
. Given the observed phenotypic appearance of our heparanase-transfected U87 cells, we considered changes in cell adhesion properties as a possible explanation for this behavior. Careful examination yielded no differences between control and heparanase-transfected cell attachment rates to fibronectin-coated or uncoated surfaces (data not shown). Interestingly, however, significant differences in cell spreading were noted. One h after plating, control Vo cells appeared rounded or slightly spread (Fig. 3A
, left panel). In contrast, heparanase-transfected U87 Low and Hi cells appeared to be significantly better spread. This increase in cell spreading was confirmed by staining for several characteristic components of focal adhesion complexes, i.e., phospho-tyrosine (second panel), phospho-FAK (third panel), paxillin (fourth panel), and filamentous actin (fifth panel). The staining pattern of all of the above markers strongly argues for an increased cell spreading and focal contact formation in heparanase-transfected cells. Moreover, a marked increase in FAK (Fig. 3B
, upper panel) and Akt (second panel) phosphorylation levels was observed in heparanase-transfected Low and Hi cell extracts prepared 1 h after plating, whereas other signaling components such as phospho-p38 (third panel) and phospho-JNK (fourth panel) were unchanged or even reduced (phospho-ERK, fifth panel) upon heparanase overexpression, at the time point examined. In addition, an increase in Rac activation was noted in the heparanase transfectants (Fig. 3B
, bottom panel), whereas Rho activation was not detected (data not shown). Furthermore, heparanase-transfected Low and Hi cells exhibited an increase in cell migration (Fig. 3C)
, further supporting an elevation of cell motility upon heparanase overexpression (Figs. 1D
and 3C
) mediated, possibly, by Akt activation (21, 22, 23)
.
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4ß1 integrin) have been implicated in heparanase-mediated T-cell adhesion, because blocking antibodies were able to partially inhibit this phenomenon (20)
. We therefore, analyzed the 4 and ß1-integrin profile in the U87 cell system by FACS analysis. The 4 integrin was not detected in the U87 cells. In contrast, ß1-integrin surface localization was noted to be significantly elevated in the Low, and even more so in the Hi, heparanase-transfected cells (Fig. 4A)
. The increase in ß1-integrin reactivity observed by FACS was not attributable to changes in expression levels, as evident by immunoblot analysis (Fig. 4A
, inset). ß1-Integrin activation was further confirmed by FACS analysis with a monoclonal antibody (12G10) that specifically recognizes the activated state of ß1-integrin (29
, 30)
, revealing a similar increase in reactivity with the heparanase-transfected U87 Hi and Low cells (Fig. 4B)
. We considered two basic possible alternatives for the heparanase-mediated integrin localization and activation: an inside-out versus outside-in mechanism. The latter possibility is based upon the secreted nature of heparanase and its ability to interact with cell surface molecules, presumably HSPG such as syndecan-4 (31, 32, 33, 34)
. We could not, however, demonstrate stable interaction of heparanase with syndecan-4 by coimmunoprecipitation experiments. Moreover, we could not demonstrate heparanase localization to the plasma membrane by FACS analysis or by surface biotinylation (data not shown). Exposure of U87 cells to exogenously added heparanase (0.2 µg/ml) was able to modestly activate ß1-integrin (Fig. 4C)
, supporting the ß1-integrin activation observed in stably transfected cells (Fig. 4B)
. However, heparanase was noted to interact rapidly, within 15–30 min, with cell surface molecules of a number of cell types (31)
, including U87 cells.4
We, therefore, expected an outside-in mechanism to initiate signaling short after heparanase application. Nevertheless, heparanase-mediated ß1-integrin activation was not observed at early time points (30 min or 1 h) but only at late time points (24 h, Fig. 4C
) after its addition. These observations may suggest that heparanase functions from inside the cell.
Heparanase Overexpression Affects Tumorigenic Potential of U87 Cells in Nude Mice.
To further evaluate the relevance of our in vitro observations, Vo control and heparanase-transfected Low and Hi U87 cells were implanted s.c into nude mice. Implantation of 5 x 106 Vo, Low, and Hi cells yielded tumor formation in all mice (n = 6; Fig. 5A
). Xenografts from the Hi cells developed much slower, yielding a low average tumor volume (532 ± 132 mm3) and weight (0.67 ± 0.07 grams), compared with control Vo xenografts (956 ± 205 mm3 and 1.27 ± 0.39 grams, respectively), differences which are statistically highly significant (P < 0.0009). Surprisingly, however, xenografts from the Low cells developed tumors (1911 ± 606 mm3 and 2.1 ± 0.41 grams), twice as much in volume (Fig. 5B)
and 1.7-fold higher in weight (Fig. 5C)
, compared with control tumors (P < 0.005). Interestingly, differences in tumor volume were not evident on day 24 after Vo and Low cell inoculation (Fig. 5B)
but started to appear on day 31 in a pattern that was maintained until the end of the experiment on day 38. In contrast, Hi cell xenografts appeared smaller from the very beginning of the experiment. Moreover, a similar apoptotic index was calculated for xenografts produced by the Vo, Low, and Hi cells, determined by means of terminal deoxynucleotidyl transferase-mediated nick end labeling assay performed on the respective tumor tissue sections (data not shown). These results suggest that heparanase expression at relatively low levels accelerates tumor development in vivo by means others than cell proliferation, possibly by contributing to the angiogenic switch necessary for tumor growth. To evaluate this hypothesis, 5-µm sections from control Vo, Low, and Hi xenografts were immunostained for PECAM-1 (CD31), a most commonly used endothelial cell marker (Fig. 5D)
. In control Vo xenograft sections, PECAM-1 mainly stained small capillary-like vessels (Fig. 5D
, g). In contrast, heparanase-transfected Low cell xenograft sections exhibited bigger vessels (Fig. 5D
, h) that correlated with elevated VEGF expression levels noted in the Low cells (Fig. 5D
, n) and in the respective xenograft tumor section (Fig. 5D
, k). Interestingly, increased vessel diameter (Fig. 5D
, i) and VEGF expression were also observed in the heparanase-transfected Hi cells (Fig. 5D
, o) and the respective xenograft sections (Fig. 5D
, l), suggesting that coordinate cell proliferation and angiogenesis are required for tumor progression.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Surprisingly, however, this effect on tumor growth largely depends upon heparanase expression levels. Thus, although relatively low heparanase expression levels significantly enhanced tumor growth (Fig. 5
, Low), high levels exerted the opposite effect and inhibited tumor development (Fig. 5
, Hi). This inhibitory effect on tumor growth correlated with an increase in cell spreading, formation of a tight cell monolayer instead of large cell aggregates (Fig. 2A)
, and a decrease in cell proliferation (Fig. 2, B–D)
. Interestingly, not only the heparanase-transfected U87 Hi, but also the Low, cell transfectants exhibited an increased cell spreading and more adhesive phenotype in vitro, yet the Low cells seem to completely overcome this inhibitory effect in vivo and produced larger tumors. This may suggest a very high tumorigenic potential of heparanase in vivo. Thus, heparanase at relatively low levels, which more closely resembled in vivo expression levels, may affect in vivo cellular characteristics other than cell proliferation per se. The increase in tumor progression may be explained by a strong angiogenic response reported previously for heparanase-transfected Eb lymphoma (13
, 14) . Indeed, staining tumor sections with the endothelial cell marker PECAM-1 (CD31) revealed an increase in blood vessel diameter (Fig. 5D
, g–i), in agreement with the enhanced tumor vascular functionality noted in the Eb lymphoma model (14)
. Moreover, the observed increase in tumor volume at late stages (31 and 38 days after cell inoculation; Fig. 5B
) may reflect a heparanase-mediated angiogenic switch necessary for a rapid tumor growth. This effect may be mediated by the elevated levels of Akt phosphorylation observed in heparanase-transfected U87 cells (Fig. 3B
, second panel). Akt-mediated angiogenesis may result from induction of VEGF expression (44
, 45)
, a potent in vivo angiogenic factor (46)
. Indeed, VEGF was noted to be elevated in the heparanase-transfected Low and Hi cells, both in vitro (Fig. 5D
, m–o) and in vivo (Fig. 5D
, j–l), further supporting the correlation between Akt activation and induced angiogenic response.
Importantly, thus far, metastatic potential was correlated with heparanase activity, participating in breaking and penetrating the ECM barrier. The results presented here may provide additional insight into this aspect of heparanase function. Clearly, adhesion to vascular endothelial cells, and subsequently to the subendothelial ECM, plays a crucial role in the metastatic process. Thus, the observed increase in cell spreading and adhesive properties of the heparanase-transfected cells (Fig. 3)
, suggests an additional aspect in heparanase function in the metastatic cascade, which may be independent of heparanase enzymatic activity, as was demonstrated recently (47)
. Heparanase, therefore, may accelerate tumor cell dissemination by virtue of its enzymatic activity, enabling penetration of cells through the ECM barrier, and by improving tumor cell adhesion to endothelial cells and the subendothelial matrix. Previously, it has been suggested that heparanase may mediate cell adhesion physically, by binding HS molecules and thereby anchoring T cells to the ECM (20)
. The results presented here may suggest a different mechanism, which seems to operate, at least in part, from inside the cells, involving increased ß1-integrin cell membrane localization and activation (Fig. 4)
. The observed activation of ß1-integrin molecules led us to examine cell spreading and adhesive behavior. Nevertheless, it may well be that integrin activation by itself cannot account for all of the phenotypic alterations noted upon heparanase overexpression, and that other mechanisms are operating as well, all contributing to the attenuation of tumor progression exhibited by the Hi cell (Fig. 5)
. Our preliminary results point to reduced ß-catenin transcriptional activity in the Hi cells, correlating with the attenuated tumor progression. Studies examining alterations in cadherin-mediated cell-cell adhesion and catenins transactivation are currently in progress. Taken together, our results indicate that heparanase expression levels may control and dictate different outcomes in human glioma cells, making inhibitory strategies quite complex in the context of cancer treatment. Studies examining the effect of heparanase overexpression levels on additional glioma cell lines, as well as breast, colon, and prostate cells, are currently under way.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Requests for reprints: Israel Vlodavsky, Vascular and Tumor Biology Research Center, Faculty of Medicine, Technion, P. O. Box 9649, Haifa 31096, Israel. Phone: 972-4-8295410; Fax: 972-4-8523947; E-mail: Vlodavsk{at}cc.huji.ac.il
3 The abbreviations used are: HS, heparan sulfate; HSPG, heparan sulfate proteoglycans; ECM, extracellular matrix; VEGF, vascular endothelial growth factor; BrdUrd, bromodeoxyuridine; PECAM-1, platelet endothelial cell adhesion molecule-1; MMP, matrix metalloproteinase; FACS, fluorescence-activated cell sorter. 
4 A. Zetser, F. Levy-Adam, V. Kaplan, S. Gingis, Y. Bashenko, S. Schubert, M. Y. Flugelman, I. Vlodavsky, and N. Ilan. Heparanase uptake, localization and processing as evidenced by isoform-specific antibodies, submitted for publication.
Received 5/ 1/03. Revised 9/10/03. Accepted 9/12/03.
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S. Gingis-Velitski, A. Zetser, V. Kaplan, O. Ben-Zaken, E. Cohen, F. Levy-Adam, Y. Bashenko, M. Y. Flugelman, I. Vlodavsky, and N. Ilan Heparanase Uptake Is Mediated by Cell Membrane Heparan Sulfate Proteoglycans J. Biol. Chem., October 15, 2004; 279(42): 44084 - 44092. [Abstract] [Full Text] [PDF]
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 E. Edovitsky, M. Elkin, E. Zcharia, T. Peretz, and I. Vlodavsky Heparanase Gene Silencing, Tumor Invasiveness, Angiogenesis, and Metastasis J Natl Cancer Inst, August 18, 2004; 96(16): 1219 - 1230. [Abstract] [Full Text] [PDF]
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S. Gingis-Velitski, A. Zetser, M. Y. Flugelman, I. Vlodavsky, and N. Ilan Heparanase Induces Endothelial Cell Migration via Protein Kinase B/Akt Activation J. Biol. Chem., May 28, 2004; 279(22): 23536 - 23541. [Abstract] [Full Text] [PDF]
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 A. Zetser, F. Levy-Adam, V. Kaplan, S. Gingis-Velitski, Y. Bashenko, S. Schubert, M. Y. Flugelman, I. Vlodavsky, and N. Ilan Processing and activation of latent heparanase occurs in lysosomes J. Cell Sci., May 1, 2004; 117(11): 2249 - 2258. [Abstract] [Full Text] [PDF]
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