Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 5392-5397, October 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Manfredi, M. G.
Right arrow Articles by Seyfried, T. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Manfredi, M. G.
Right arrow Articles by Seyfried, T. N.
[Cancer Research 59, 5392-5397, October 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Gangliosides Influence Angiogenesis in an Experimental Mouse Brain Tumor1

Mark G. Manfredi, Sangmook Lim, Kevin P. Claffey and Thomas N. Seyfried2

Department of Biology, Boston College, Chestnut Hill, Massachusetts 02467 [M. G. M., S. L., T. N. S.], and Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030 [K. P. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides are sialated glycosphingolipids present on the plasma membranes of all vertebrate cells. Tumors shed gangliosides into the extracellular microenvironment, which may influence tumor-host cell interactions. We have investigated the role of gangliosides on the growth and angiogenesis of the EPEN experimental mouse brain tumor. EPEN cells express only ganglioside GM3, and the solid tumors formed in vivo are sparsely vascularized with extensive necrosis. We stably transfected the EPEN cells with the cDNA for N-acetylgalactosaminyl transferase, a key enzyme for the synthesis of complex gangliosides. In addition to GM3, the transfected cell line (EPEN-GNT) expressed complex gangliosides GM2, GM1, and GD1a. The EPEN-GNT tumor was more densely vascularized with less necrosis and grew more rapidly than the nontransfected EPEN or mock-transfected (EPEN-V) control tumors. Also, VEGF gene expression was higher in the EPEN-GNT tumor than in the control tumors. The synthesis of complex gangliosides in the EPEN-GNT tumor cells also stimulated vascularization in an in vivo Matrigel assay for angiogenesis. These results indicate that the ratio of GM3 to complex gangliosides can influence the growth and angiogenic properties of the EPEN experimental brain tumor and are consistent with previous findings in other systems. We conclude that gangliosides may be important modulators of brain tumor angiogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis involves the sprouting of new microvessels from existing vessels and is a prerequisite for tumor progression (1, 2, 3) . Microvessel density is positively correlated with tumor malignancy and ultimately influences patient prognosis (4 , 5) . Brain tumors, especially glioblastomas, are highly angiogenic and are refractory to most therapies. Consequently, antiangiogenesis therapy may be an attractive treatment for brain tumors (6) .

The recruitment of blood vessels into a developing tumor is initiated by factors intrinsic to the tumor cells (7 , 8) . The migration and proliferation of endothelial cells in the tumor microenvironment are two hallmark features of angiogenesis (9) . Many factors in the tumor environment, including growth factors and extracellular matrix molecules, modulate the angiogenic response (10 , 11) . These factors are secreted by both the tumor cells and host cells (e.g., macrophages; Ref. 12 ). VEGF3 is an endothelial-specific mitogen that is important for endothelial cell proliferation and migration (13 , 14) . Experimental alterations of tumor VEGF levels can influence vessel density and tumor growth rate (15 , 16) . Antibodies that block endothelial cell adhesion to the extracellular matrix can also inhibit tumor angiogenesis and growth (17 , 18) .

Gangliosides are good candidates for modulators of tumor angiogenesis because their negatively charged oligosaccharide head group extends into the extracellular environment (19 , 20) . These molecules can also be shed into the extracellular environment, where they can influence tumor-host cell interactions (21) . Gangliosides can also modulate receptors, such as those for growth factors and cell adhesion (22) . The structurally simple ganglioside GM3 regulates the function of the epithelial growth factor and fibroblast growth factor receptors (23 , 24) . In addition, gangliosides modulate the function of the integrin receptors including {alpha}5{beta}1, {alpha}2{beta}1, and {alpha}v{beta}3 (25, 26, 27) . Hence, gangliosides influence the activity of several molecules important in angiogenesis.

In the rabbit cornea model of angiogenesis, GM3 inhibits basic fibroblast growth factor-induced angiogenesis in a dose-dependent manner (28) . However, the antiangiogenic effect of GM3 is counteracted by complex gangliosides (GM2, GM1, GD3, GD1b, and GT1b; Ref. 29 ). Complex gangliosides enhance the action of angiogenesis inducers. For example, ganglioside GD3 stimulates the release of VEGF from human glioma cell lines and also enhances the migration of cytokine-stimulated endothelial cells (28 , 30) . It is suggested that the ratio of GM3 to complex gangliosides can significantly influence angiogenesis (29) .

To directly test the hypothesis that the ratio of GM3 to complex gangliosides influence brain tumor angiogenesis, we genetically altered ganglioside biosynthesis in a well-characterized mouse brain tumor cell line and analyzed the growth and angiogenic properties of these tumors in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Experimental Brain Tumors.
The C57BL/6J (B6) strain was obtained from The Jackson Laboratory (Bar Harbor, ME). All B6 mice used in this study were propagated in the animal facility of the Department of Biology, Boston College, using animal husbandry conditions described previously (31) . Male B6 mice, ~ 6 weeks of age, were used as tumor recipients. The EPEN experimental mouse brain tumors and transfected tumor lines were maintained by serial transplantations s.c. in the flank or intracranially in the brain, as described previously (32) . Female athymic nude mice (NCr-nu/nu), 6–8 weeks of age, were obtained through the NIH, National Research Resources. All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institution Animal Care Committee.

Cell Culture and Transfections.
The EPEN cell line was established from a murine ependymoblastoma tumor as described previously (33) . The cells were maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Sigma) and 0.5% penicillin/streptomycin (Life Technologies). The cells were cultured in a humidified atmosphere with 95% air and 5% CO2 at 37°C. The EPEN cells were stably transfected with the expression vector pCMV containing a 1.6-Kb GalNAc-T cDNA insert (a gift from Dr. Richard Proia, NIH) using the Lipofectamine protocol (Life Technologies). The cells were cotransfected with the pcDNA3 vector (Invitrogen, San Diego, CA), which contained a neomycin resistance gene for selection. Cells that survived 400 µg/ml G418 (Life Technologies) treatment for 2 weeks were expanded and analyzed for the expression of GalNAc-T using RT-PCR with primers that were described previously (34) . Positive cells were subcloned to ensure a single clonal population. To confirm that GalNAc-T was expressed in the tumors grown in vivo, RT-PCR was used with primers (forward, 5'-CGGTTGACCTCACTAAAGCC-3'; reverse, 5'-TGACACTATAGAATACTCAAGC-3') that amplified a 1.3-kb portion of the transfected GalNAc-T mRNA only. RT-PCR was done as described previously (34) .

Radiolabeling of Cells.
The untransfected or stably transfected EPEN cell lines were seeded into 75-cm2 culture flasks and cultured until ~50% confluent. Cells were then labeled with 0.15 µCi/ml of [14C] galactose (New England Nuclear, Boston, MA) as described previously (35) . After 48 h, the culture medium was collected. The cells were then washed with 10 ml of PBS, which was added to the collected medium. The cells were removed from the flask with 0.25% trypsin/1 mM EDTA and harvested by centrifugation.

Ganglioside Isolation.
For tumors grown in vivo, the tissues were collected, frozen, and lyophilized. Total lipids were extracted from the radiolabeled cells or solid tissues by adding 5 ml of 1:1 chloroform:methanol (v/v) with 0.5 ml of distilled H2O and stirring for 12 h at room temperature. Samples were vortexed and centrifuged at 800 x g for 10 min. The supernatant was transferred to another tube, and the pellets were washed with 2 ml of 1:1 chloroform:methanol (v/v) and centrifuged for 10 min at 800 x g. The resulting supernatant was added to the previous supernatant. Polar and nonpolar lipids were separated by Folch partitioning (36 , 37) . Gangliosides were isolated and purified by modification of methods described previously (37 , 38) . Briefly, the Folch "upper phase" was converted to a chloroform:methanol:water ratio of 30:60:8 (v/v/v). The samples were added to a DEAE-Sephadex A-25 (Pharmacia Biotech, Uppsala, Sweden) ion exchange column that was equilibrated in 30:60:8 chloroform:methanol:water (v/v/v). Nonacidic molecules were eluted from the column with 20 ml of the same solvent. For in vitro samples, the gangliosides were eluted from the column with 20 ml of 30:60:8 chloroform:methanol:0.8 ammonium acetate (v/v/v). For in vivo samples, the gangliosides were eluted from the column with 30 ml of 30:60:8 chloroform:methanol:0.8 sodium acetate (v/v/v). The solvents were evaporated under vacuum, and then the samples were treated with base 0.5 N NaOH at 37°C for 1.5 h and desalted as described previously (32 , 37) .

Ganglioside Shedding.
To detect gangliosides that were shed from the cultured cells, the medium from radiolabeled cells was collected as described above and passed through a 0.2 µm filter (Corning) to remove dead or fragmented cells. The medium was then frozen at -80°C and lyophilized to remove water. The dried medium was then used for the isolation of gangliosides as described above. The percentage of gangliosides shed into the culture medium over a 48-h period was estimated by dividing the dpm of supernatant gangliosides by the total dpm of gangliosides synthesized (cells plus supernatant) x 100.

Ganglioside Analysis.
For radiolabeled cells, the amount of radioactive incorporation into gangliosides was determined by placing 50 µl of sample into 6 ml of Ecoscint A (National Diagnostics) scintillation solution and counting dpm on a 1219 Rackbeta Counter (LKB Wallac). The concentration of ganglioside sialic acid from solid tumors grown in vivo was quantified using a resorcinol colorimetric assay and was expressed as µg of sialic acid per 100 mg dry weight, as we described previously (39) . Ganglioside distribution was analyzed using HPTLC and quantified as described previously (37 , 40) . Cholera toxin B immunostaining and Clostridium perfringens neuraminidase (Sigma) digestion were conducted for preliminary structural studies of gangliosides, as described previously (41) .

Immunohistochemistry and Microvessel Density.
Blood vessel immunostaining and H&E staining was done on tumors grown intracranially. Briefly, tumors were fixed in 4% formaldehyde and processed for frozen sectioning. The sections (7 µm) were postfixed in 1:1 methanol:acetone (v/v) at -20°C for 10 min, and then endogenous peroxidase was inactivated by incubation in methanol containing 0.3% hydrogen peroxide for 20 min at -20°C. The sections were then blocked with goat serum and incubated with anti-type IV collagen antibody (1/400; Biodesign, Kennebunk, ME) for 2 h at room temperature to highlight blood vessel basement membrane. The primary antibody was detected using the Vectastain avidin-biotin peroxidase kit (Vector Laboratories, Burlingame, CA), according to the manufacturer’s instruction, and was visualized using 3'-diaminobenzidine with metal enhancement (Pierce, Rockford, IL). Sections were counterstained with aqueous hematoxylin. Microvessel density was quantified by examining areas of vascular hot spots, as described previously by Weidner et al. (4) with some modifications. Type IV collagen-positive microvessels appeared brown/black in color. Sections were scanned at low magnification (x4 and x10) for the localization of vascular hot spots. The 10 most vascular areas were determined and then counted for vessel number at high magnification (x40). Only darkly stained vessels, which were clearly separated from each other, were counted. For each tumor, at least two independent sections were stained with H&E and immunostained for blood vessels.

Northern Blot.
RNA was isolated from tumors that were grown in the brain to approximately equal size or grown in culture to approximately equal densities as described previously (34) . Total RNA (15 or 20 µg) was separated on a denaturing formaldehyde agarose gel, hybridized to a nylon membrane (Gene Screen Plus; DuPont NEN) by capillary action, and then fixed using a UV cross-linker (Stratagene, La Jolla, CA). The blot was hybridized with a 32P-labeled 654-bp probe that recognizes all mouse VEGF splice variants. The probe was a PCR fragment amplified from a cloned VEGF cDNA (by K. P. C.) using the primers (forward, 5' -TGGATCCATGAACTTTCTGCT-3'; reverse, 5' -GAATTCACCGCCTCGGCTTGTC-3'). The blot was stripped and rehybridized with a 32P-labeled actin probe (Stratagene). Bands were visualized and quantified using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).

In Vitro Growth Curves and Measurement of in Vivo Tumor Volumes.
For in vitro growth, approximately 1 x 104 tumor cells were seeded in wells of a 24-well plate and cultured for 8 days as described above. Cells were counted on days 2, 4, 6, and 8 using a Coulter counter. For in vivo growth, tumors of similar passage grown in the flank were enzymatically dissociated into single-cell suspension. Briefly, tumors were minced into 1-mm cubes and then incubated in RPMI 1640 containing 500 µg/ml of type II collagenase (Worthington Biochemical, Freehold, NJ) and 0.5 µg/ml of DNase I (Sigma) with gentle stirring at 37°C for 1.5 h. The single-cell suspension was then filtered through a 70 µm filter and counted using a hemocytometer. Cells were then washed with RPMI 1640 (Life Technologies) and resuspended at 1 x 107 cells/ml in PBS. One million cells were injected s.c. in the dorsal midline of B6 mice. Tumor growth was measured as described previously (42) .

In Vivo Matrigel Model of Angiogenesis.
EPEN-V and EPEN-GNT cells were grown in culture to equal densities and harvested with 0.25% trypsin containing 1 mM EDTA. Cells were washed twice, resuspended in DMEM, and then mixed thoroughly with Matrigel (Collaborative Biomedical) 1:2 (v/v) at 4°C. Nude mice were anesthetized with Avertin (0.1 ml/10 g body weight) and then injected with 1 x 106 cells in 300 µl of Matrigel s.c. in the dorsal midline using a prechilled tuberculin syringe (27-gauge needle). After 7 days, the mice were euthanized by CO2 asphyxiation, and the Matrigel plugs with the surrounding skin were removed. Vascularity was photographed at the time of removal using a dissecting photomicroscope (Leica, WILD macroskop).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stable Transfection of GalNAc-T cDNA Induced Neosynthesis of Complex Gangliosides.
The EPEN cells do not express the gene for GalNAc-T, a key enzyme for the synthesis of complex gangliosides, and thus synthesize only ganglioside GM3 (Fig. 1Citation ; Ref. 34 ). The EPEN cells stably transfected with the mouse GalNAc-T cDNA synthesized complex gangliosides (GM2, GM1, and GD1a) in addition to GM3 (Fig. 1B)Citation . The identity of these gangliosides was confirmed using neuraminidase digestion and cholera toxin B immunostaining (data not shown). The ganglioside bands in the region of GM1 and GD1a migrated slightly faster than the standards, possibly due to longer fatty acyl chains in the tumor gangliosides. The trace levels of GM2 found likely result from its rapid use as a precursor for the synthesis of GM1. The synthesized gangliosides migrated on the HPTLC as double bands because of differences in ceramide structure. Although GM3 comprised 100% of the total radiolabeled gangliosides in the control EPEN and EPEN-V cells, it was reduced to 55% of the total in the EPEN-GNT cells. In addition, the complex gangliosides, GM1 and GD1a, comprised approximately 16 and 26%, respectively, of the total radiolabeled gangliosides in the EPEN-GNT cells (Fig. 1B)Citation . These findings indicate that the stable transfection of EPEN cells with the GalNAc-T gene induced the expression of complex gangliosides.



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1. Biosynthetic pathway and distribution of gangliosides isolated from cultured EPEN, EPEN-V, and EPEN-GNT cells and conditioned medium. A, ganglioside biosynthetic pathway showing that GalNAc-T is necessary for the synthesis of complex gangliosides. B, HPTLC of 14C-labeled gangliosides from cultured cells and gangliosides shed into the cultured medium. Approximately 3000 and 1000 dpm of radiolabeled gangliosides were spotted for cells and medium, respectively. The plate was developed by one ascending run with chloroform:methanol:water containing 0.02% CaCl2 (55:45:10, v/v/v). After visualizing radiolabeled lanes with a Phosphorimager, the plates were sprayed with resorcinol reagent and heated to 100°C to visualize the standards (STD).

 
To determine whether the cultured EPEN-V and EPEN-GNT tumor cells shed gangliosides into the medium, we compared the content and distribution of gangliosides in the cultured cells with that in the conditioned medium. The EPEN-V and EPEN-GNT tumor cells shed approximately 5.7 and 6.0% of their total gangliosides, respectively, into the medium after 48 h. The distribution of shed gangliosides was similar to that of the cellular gangliosides for both tumor cell lines. For example, the EPEN-V cells shed only GM3, whereas the EPEN-GNT cells shed GM3, GM1, and GD1a (Fig. 1B)Citation . The trace band migrating in the region of GD3 stained with cholera toxin B, suggesting that it was a GM1 structure. Interestingly, the shorter chain fatty acid species (lower band of doublet) were more prevalent in the culture medium, whereas the longer chain fatty acid species (upper band of doublet) were more prevalent in the intact cells (Fig. 1B)Citation . These data show that the EPEN-V and EPEN-GNT tumors shed significant amounts of gangliosides into the extracellular environment.

The control and EPEN-GNT cells were grown s.c. in syngeneic B6 mice, where all cell lines readily formed tumors. We confirmed the expression of the transfected GalNAc-T gene in the in vivo EPEN-GNT tumor using RT-PCR (Fig. 2)Citation . The ganglioside concentration (µg of sialic acid/100 mg dry weight) of the EPEN, EPEN-V, and EPEN-GNT tumors did not differ significantly and was 44 ± 9.3, 48 ± 3.3, and 42 ± 1.2, respectively (n = 3 independent samples for each tumor, and the values are expressed as means ± SE). However, the relative distribution of gangliosides in the control and EPEN-GNT tumors differed significantly (Fig. 3A)Citation . Although GM3, GM1, and GD1a were present in all three tumors, the ratio of complex gangliosides (GD1a) to GM3 was 4–6-fold greater in the EPEN-GNT tumor than in the control tumors (Fig. 3B)Citation . It is important to mention that the ganglioside distribution of tumors grown in vivo can differ significantly from that of tumor cells grown in culture because of the presence of host infiltrating cells (e.g., macrophages) in the solid tumor (37) .



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 2. Detection of GalNAc-T gene expression in vivo using RT-PCR. GalNAc-T mRNA was detected in the EPEN-GNT tumor but was not detected in the EPEN and EPEN-V tumors. The GalNAc-T expression vector and EPEN-GNT cells grown in culture were used as controls for the PCR reaction. No bands were detected when reverse transcriptase was omitted from the cDNA reaction. To ensure that cDNA was present in samples, RT-PCR was performed using N-cadherin primers, where all samples had positive bands.

 


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 3. HPTLC of gangliosides isolated from EPEN, EPEN-V, and EPEN-GNT tumors grown in vivo. A, ~1.5 µg of ganglioside sialic acid was spotted for each lane. The plate was developed as described in Fig. 1Citation . The bands were visualized by resorcinol spray. STD, standard. B, densitometry of the HPTLC plate was used to calculate the GD1a:GM3 ratio. The values are expressed as the means of three independent tumors; bars, SE.

 
Complex Ganglioside Synthesis Stimulated Brain Tumor Growth in Vivo but not in Vitro.
Growth curves were performed to determine whether neosynthesis of complex gangliosides altered the growth of the cultured tumor cells and solid tumors. Although the EPEN, EPEN-V, and EPEN-GNT cells grew at similar rates in culture (Fig. 4A)Citation , the EPEN-GNT tumor grew significantly faster than the EPEN and EPEN-V tumors in vivo (Fig. 4B)Citation . Therefore, the neosynthesis of complex gangliosides did not affect tumor cell proliferation in the culture environment but stimulated tumor growth in the in vivo host environment.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. Growth curves of the EPEN, EPEN-V, and EPEN-GNT cells grown in culture (A) and as solid tumors grown in vivo (B). A, values are expressed as the means of three independent experiments for each cell line; bars, SE. B, day 0 represents the beginning of tumor volume measurement, when the tumors were between 150 and 200 mm3. The values for each tumor (EPEN, n = 4; EPEN-V, n = 6; and EPEN-GNT, n = 5) are expressed as the means; bars, SE. *, the EPEN-GNT tumor is significantly larger than the control tumors at the P < 0.01 level (two-tailed t test).

 
Complex Ganglioside Synthesis Increased Vascular Density and Mitotic Index.
To determine whether changes in ganglioside biosynthesis influenced angiogenesis, we examined tumor morphology and blood vessel densities using H&E staining and type IV collagen immunostaining (Figs. 5Citation and 6Citation ). Although the EPEN-GNT tumor had little or no areas of necrosis, the EPEN and EPEN-V tumors had multiple necrotic regions that were identified in at least two independent sections of each tumor (Fig. 5)Citation . The vascular density was 1.7 times greater in the EPEN-GNT tumor than in the control tumors (Figs. 5Citation and 6A)Citation . In addition, the number of thrombotic vessels was less in the EPEN-GNT tumors than in the controls (data not shown). The majority of vessels in the EPEN-GNT tumors appeared to be of typical tumor microvessel morphology. Furthermore, the number of cells undergoing mitosis was significantly greater in the EPEN-GNT tumor than in controls (Fig. 6B)Citation . Taken together, these findings indicate that the expression of complex gangliosides with a concomitant decrease in GM3 enhanced both angiogenesis and proliferation of the EPEN-GNT tumor.



View larger version (82K):
[in this window]
[in a new window]
  		Fig. 5. Histology and vascularity of tumors grown intracranially. Tissue sections for the EPEN, EPEN-V, and EPEN-GNT tumors stained with H&E (x10) or immunostained for blood vessels using an anti-type IV collagen antibody (x20) are shown. Type IV collagen-positive vessels appeared brown/black in color. N, necrotic regions in the EPEN and EPEN-V H&E sections.

 


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 6. Quantification of blood vessel density (A) and mitotic figures (B) in the EPEN, EPEN-V, and EPEN-GNT tumors. The number of vessels and cells in mitosis was determined per x40 field (hpf, high-powered field; a 0.66-mm2 area) from tissue sections as shown in Fig. 5Citation . The number of microvessels (EPEN, n = 9; EPEN-V, n = 12; and EPEN-GNT, n = 10) and mitotic figures (n = 6 for each tumor) is expressed as the means; bars, SE. *, the number of vessels and mitotic figures is significantly greater in the EPEN-GNT tumor than in the EPEN and EPEN-V control tumors at the P < 0.01 level (two-tailed t test).

 
Complex Ganglioside Synthesis Enhanced VEGF Gene Expression in Vivo but not in Vitro.
Because VEGF expression is correlated with tumor angiogenesis, we measured VEGF mRNA levels in the EPEN, EPEN-V, and EPEN-GNT tumors (Fig. 7)Citation . Although the tumor cells grown in culture did not differ in VEGF gene expression (Fig. 7B)Citation , the EPEN-GNT tumor had significantly greater VEGF mRNA levels than controls when grown in vivo (Fig. 7D)Citation . These data suggest that complex ganglioside synthesis stimulated VEGF gene expression in the EPEN-GNT tumor grown in vivo.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 7. Northern blot analysis of VEGF gene expression in the EPEN, EPEN-V, and EPEN-GNT grown as cell lines in vitro (A) and as solid tumors in vivo (C). Bands were visualized and quantified using a Phosphorimager. VEGF mRNA levels in vitro (B; EPEN, n = 3; EPEN-V, n = 6; and EPEN-GNT, n = 6) and in vivo (D; n = 6 for each tumor) were standardized to actin levels and expressed as the means; bars, SE. *, VEGF mRNA expression is significantly greater in the EPEN-GNT tumor grown in vivo than in the control EPEN and EPEN-V tumors at the P < 0.01 level (two-tailed t test).

 
Complex Ganglioside Biosynthesis Enhanced Vascularity in the in Vivo Matrigel Model of Angiogenesis.
The Matrigel angiogenesis model is highly dependent upon host stromal cell infiltration and activation, including monocytes, macrophages, and endothelial precursors. Also, when tumor cells are implanted within Matrigel, their effect on the developing stroma is more selective for soluble factors such as tumor-derived gangliosides. In addition, this model is representative of the early events initiated when tumors are formed, and angiogenic factors and inhibitor balance are critical to the overall angiogenic response. In this case, we determined whether implanting the EPEN-V control vector or EPEN-GNT cell lines would have selective effects upon initial angiogenic events as opposed to alterations in the vascularity of the tumors observed over a period of several weeks. Although both the EPEN-V and EPEN-GNT tumor cells induced angiogenesis, the number and dilation of the vessels was greater in the Matrigel plugs containing EPEN-GNT tumor cells than in plugs containing EPEN-V tumor cells (Fig. 8)Citation . Taken together, these findings indicate that EPEN-GNT tumor cells were more angiogenic than the control tumor cells in multiple assays for tumor angiogenesis.



View larger version (51K):
[in this window]
[in a new window]
  		Fig. 8. Tumor cell-induced vascularity in the in vivo Matrigel plug assay. The EPEN-V and EPEN-GNT cells in Matrigel were injected s.c. in nude mice as described in "Materials and Methods." The Matrigel plugs, with the surrounding skin, were removed on day 7 after implantation. The plugs were photographed at the same magnification (x 12.5).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that a gene-linked change in ganglioside expression significantly influenced the angiogenic response and growth of an experimental mouse brain tumor. Specifically, the neosynthesis of complex gangliosides GM2, GM1, and GD1a with a concurrent decrease in GM3 enhanced the angiogenic phenotype and growth rate of the EPEN brain tumor. This is the first report, to our knowledge, of a ganglioside influence on brain tumor angiogenesis in vivo arising through the genetic manipulation of ganglioside synthesis in the tumor cells. Interestingly, the change in ganglioside biosynthesis did not influence endogenous tumor cell proliferation because the growth rate in vitro was similar in the EPEN-GNT tumor cell line stably transfected with the GalNAc-T gene and in the untransfected or mock-transfected EPEN lines. These findings suggest that gangliosides influence angiogenesis by modulating the response of the host to the tumor.

Support for this hypothesis came from our findings that both the number and quality of blood vessels were greater in the EPEN-GNT tumor than in the control tumors. The EPEN-GNT tumor also had blood vessels of typical morphology and few necrotic regions, whereas the EPEN control tumors had numerous thrombotic vessels and necrotic regions. Furthermore, the EPEN-GNT tumor cells elicited more and larger blood vessels when implanted s.c. in Matrigel plugs than did the EPEN-V control tumor cells. These findings suggest that tumor cell gangliosides influence the angiogenic response via a paracrine mechanism on host-infiltrating stromal and vascular cells. We do not exclude the possibility that gangliosides may also influence tumor growth through other mechanisms, including the down-regulation of cellular immune responses as described by Ladisch and co-workers (43, 44, 45, 46) .

Ziche et al. (28) showed previously that the ratio of GM3 to complex gangliosides influences the proliferation and migration of microvascular endothelial cells in the rabbit cornea model of angiogenesis (28) . GM3 inhibited endothelial cell migration and proliferation and also reduced microvessel density (20 , 28) . The inhibitory effect of GM3 was counteracted, however, by the more complex gangliosides, which enhanced the angiogenic phenotype (20 , 47) . Although the more complex gangliosides are not angiogenic by themselves, they are proangiogenic in that they act synergistically with angiogenesis inducers, e.g., prostaglandin E1, basic fibroblast growth factor, and VEGF (47) . In light of these observations, we suggest that the enhanced vascularization and in vivo growth rate of the EPEN-GNT tumor compared with that of the control tumors arises from a shift in the distribution of GM3 to complex gangliosides.

The mechanisms by which gangliosides influence angiogenesis and tumor growth in vivo are not clear. The induction of complex ganglioside biosynthesis in the EPEN-GNT tumor enhanced VEGF gene expression only when the tumor cells were grown in vivo but not when they were grown in vitro. VEGF is an endothelial cell-specific mitogen and is one of the most potent inducers of angiogenesis (13 , 16) . Although most VEGF synthesized in solid tumors is derived from the neoplastic tumor cells, various host cells (macrophages and fibroblasts) can also synthesize VEGF (12 , 48 , 49) . It is therefore possible that gangliosides significantly influence VEGF gene expression in tumor-associated host cells. Alternatively, gangliosides may influence the number of tumor-infiltrating host cells. Indeed, there is a strong positive correlation between the number of tumor-infiltrating macrophages and the degree of tumor neovascularization (48 , 50 , 51) . Further studies will be needed to determine how tumor gangliosides influence the number and angiogenic properties of tumor-associated host cells.

The shedding of gangliosides from the tumor cell surface into the extracellular milieu is believed to influence tumor-host cell interactions (21 , 52 , 53) . We have shown that the EPEN-V and EPEN-GNT cells shed significant amounts of gangliosides into the culture medium. The EPEN-V cells shed only GM3, whereas the EPEN-GNT cells shed GM3 in addition to complex gangliosides (GM1 and GD1a). The total amounts shed and the preferential shedding of shorter chain fatty acid species are consistent with previous findings in other murine tumor models and in human brain tumors (21 , 54 , 55) .

Because shed tumor gangliosides can be incorporated into the membranes of neighboring host cells, it is possible that shed gangliosides modulate endothelial cell-specific growth factor receptors, such as the VEGF receptor (56) . Previous studies showed that gangliosides modify the function of tyrosine kinase receptors similar to the VEGF receptor (22) . It is also possible that shed gangliosides alter the extracellular matrix, which could influence endothelial cell/matrix adhesion, thereby altering endothelial cell proliferation and migration (20) . Taken together, these findings suggest that tumor angiogenesis may be significantly influenced by both the content and composition of shed gangliosides.

The most malignant human brain tumors (anaplastic astrocytoma and glioblastoma multiforme) are highly vascular and generally contain GM3 and GD3 as the predominant gangliosides (57, 58, 59, 60, 61, 62) . Because GD3 stimulates the release of VEGF in glioblastoma cell lines and colocalizes with areas of vascular hot spots, it is suggested that GD3 enhances malignancy by stimulating angiogenesis (28 , 30) . Although GM3 is also elevated in these tumors, its concentration is generally less than that of GD3 and may, therefore, be unable to counter the proangiogenic effects of GD3. According to our findings and those of Ziche et al. (28) , it is the balance of GM3 to complex gangliosides, including GD3, that is most critical for determining angiogenesis. We suggest that increasing the ratio of GM3 to complex gangliosides may be effective in reducing angiogenesis and growth in human glioblastomas.

Our findings suggest that the composition of brain tumor gangliosides can significantly influence the angiogenic response of the host and the growth of the tumor. Because gangliosides are synthesized and shed by all tumors examined to date, it will be important to define the mechanism(s) by which gangliosides modulate tumor angiogenesis.


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffrey A. Ecsedy, Dr. Rachael Neve, and Martin P. Keough for technical assistance and helpful discussions.


   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 NIH Grants NSCA33640 (to T. N. S.) and CA64436 (to K. P. C.) and the Boston College Research Expense Fund. Back

2 To whom requests for reprints should be addressed, at Department of Biology, Boston College, Higgins Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02467. Phone: (617) 552-3563; Fax: (617) 552-2011; E-mail: thomas.seyfried{at}bc.edu Back

3 The abbreviations used are: VEGF, vascular endothelial growth factor; GalNAc-T, N-acetylgalactosaminyl transferase; RT-PCR, reverse transcription-PCR; HPTLC, high-performance TLC. Back

Received 5/19/99. Accepted 8/20/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Boehm T., Folkman J., Browder T., O’Reilly M. S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature (Lond.), 390: 404-407, 1997.[Medline]
  2. Millauer B., Shawver L. K., Plate K. H., Risau W., Ullrich A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature (Lond.), 367: 576-579, 1994.[Medline]
  3. 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]
  4. Weidner N., Semple J. P., Welch W. R., Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N. Engl. J. Med., 324: 1-8, 1991.[Abstract]
  5. Martin L., Green B., Renshaw C., Lowe D., Rudland P., Leinster S. J., Winstanley J. Examining the technique of angiogenesis assessment in invasive breast cancer. Br. J. Cancer, 76: 1046-1054, 1997.[Medline]
  6. 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]
  7. Cockerill G. W., Gamble J. R., Vadas M. A. Angiogenesis: models and modulators. Int. Rev. Cytol., 159: 113-160, 1995.[Medline]
  8. Hanahan D., Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86: 353-364, 1996.[Medline]
  9. O’Reilly M. S., Boehm T., Shing Y., Fukai N., Vasios G., Lane W. S., Flynn E., Birkhead J. R., Olsen B. R., Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 88: 277-285, 1997.[Medline]
  10. Dvorak H. F., Nagy J. A., Feng D., Brown L. F., Dvorak A. M. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr. Top. Microbiol. Immunol., 237: 97-132, 1999.[Medline]
  11. Senger D. R., Claffey K. P., Benes J. E., Perruzzi C. A., Sergiou A. P., Detmar M. Angiogenesis promoted by vascular endothelial growth factor: regulation through {alpha}1{beta}1 and {alpha}2{beta}1 integrins. Proc. Natl. Acad. Sci. USA, 94: 13612-13617, 1997.[Abstract/Free Full Text]
  12. Lewis C. E., Leek R., Harris A., McGee J. O. Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages. J. Leukocyte Biol., 57: 747-751, 1995.[Abstract]
  13. Ferrara N. Vascular endothelial growth factor. Eur. J. Cancer, 14: 2413-2422, 1996.
  14. Neufeld G., Cohen T., Gengrinovitch S., Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J., 13: 9-22, 1999.[Abstract/Free Full Text]
  15. Kim K. J., Li B., Winer J., Armanini M., Gillett N., Phillips H. S., Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature (Lond.), 362: 841-844, 1993.[Medline]
  16. Cheng S. Y., Huang H. J., Nagane M., Ji X. D., Wang D., Shih C. C., Arap W., Huang C. M., Cavenee W. K. Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor. Proc. Natl. Acad. Sci. USA, 93: 8502-8507, 1996.[Abstract/Free Full Text]
  17. Brooks P. C., Stromblad S., Klemke R., Visscher D., Sarkar F. H., Cheresh D. A. Antiintegrin {alpha}v{beta}3 blocks human breast cancer growth and angiogenesis in human skin [see comments]. J. Clin. Invest., 96: 1815-1822, 1995.
  18. Friedlander M., Brooks P. C., Shaffer R. W., Kincaid C. M., Varner J. A., Cheresh D. A. Definition of two angiogenic pathways by distinct {alpha}v integrins. Science (Washington DC), 270: 1500-1502, 1995.[Abstract/Free Full Text]
  19. Hakomori S. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res., 56: 5309-5318, 1996.[Abstract/Free Full Text]
  20. Alessandri G., Cornaglia-Ferraris P., Gullino P. M. Angiogenic and angiostatic microenvironment in tumors-role of gangliosides. Acta Oncol., 36: 383-387, 1997.[Medline]
  21. Chang F., Li R., Ladisch S. Shedding of gangliosides by human medulloblastoma cells. Exp. Cell Res., 234: 341-346, 1997.[Medline]
  22. Yates A. J., Rampersaud A. Sphingolipids as receptor modulators. An overview. Ann. NY Acad. Sci., 845: 57-71, 1998.[Abstract/Free Full Text]
  23. Meuillet E., Cremel G., Dreyfus H., Hicks D. Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Muller glia. Glia, 17: 206-216, 1996.[Medline]
  24. Bremer E. G., Schlessinger J., Hakomori S. Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor. J. Biol. Chem., 261: 2434-2440, 1986.[Abstract/Free Full Text]
  25. Zheng M., Fang H., Tsuruoka T., Tsuji T., Sasaki T., Hakomori S. Regulatory role of GM3 ganglioside in {alpha}5{beta}1 integrin receptor for fibronectin-mediated adhesion of FUA169 cells. J. Biol. Chem., 268: 2217-2222, 1993.[Abstract/Free Full Text]
  26. Fang L. H., Lucero M., Kazarian T., Wei Q., Luo F. Y., Valentino L. A. Effects of neuroblastoma tumor gangliosides on platelet adhesion to collagen. Clin. Exp. Metastasis, 15: 33-40, 1997.[Medline]
  27. Cheresh D. A. Structure, function and biological properties of integrin {alpha} v{beta}3 on human melanoma cells. Cancer Metastasis Rev., 10: 3-10, 1991.[Medline]
  28. Ziche M., Morbidelli L., Alessandri G., Gullino P. M. Angiogenesis can be stimulated or repressed in vivo by a change in GM3:GD3 ganglioside ratio. Lab. Investig., 67: 711-715, 1992.[Medline]
  29. Alessandri G., De Cristan G., Ziche M., Cappa A. P., Gullino P. M. Growth and motility of microvascular endothelium are modulated by the relative concentration of gangliosides in the medium. J. Cell. Physiol., 151: 23-28, 1992.[Medline]
  30. Koochekpour S., Merzak A., Pilkington G. J. Vascular endothelial growth factor production is stimulated by gangliosides and TGF-{beta} isoforms in human glioma cells in vitro. Cancer Lett., 102: 209-215, 1996.[Medline]
  31. Flavin H. J., Wieraszako A., Seyfried T. N. Enhanced aspartate release from hippocampal slices of epileptic (El) mice. J. Neurochem., 56: 1007-1011, 1991.[Medline]
  32. Seyfried T. N., Yu R. K., Saito M., Albert M. Ganglioside composition of an experimental mouse brain tumor. Cancer Res., 47: 3538-3542, 1987.[Abstract/Free Full Text]
  33. Seyfried T. N., el-Abbadi M., Roy M. L. Ganglioside distribution in murine neural tumors. Mol. Chem. Neuropathol., 17: 147-167, 1992.[Medline]
  34. Ecsedy J. A., Manfredi M. G., Yohe H. C., Seyfried T. N. Ganglioside biosynthetic gene expression in experimental mouse brain tumors. Cancer Res., 57: 1580-1583, 1997.[Abstract/Free Full Text]
  35. Bai H., Seyfried T. Influence of ganglioside GM3 and high density lipoprotein on the cohesion of mouse brain tumor cells. J. Lipid Res., 38: 160-172, 1997.[Abstract]
  36. Folch J., Lees M., Sloane-Stanley G. H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509, 1957.[Free Full Text]
  37. Ecsedy J. A., Yohe H. C., Bergeron A. J., Seyfried T. N. Tumor-infiltrating macrophages influence the glycosphingolipid composition of murine brain tumors. J. Lipid Res., 39: 2218-2227, 1998.[Abstract/Free Full Text]
  38. Seyfried T. N., El-Abbadi M., Ecsedy J. A., Bai H. W., Yohe H. C. Influence of host cell infiltration on the glycolipid content of mouse brain tumors. J. Neurochem., 66: 2026-2033, 1996.[Medline]
  39. Seyfried T. N., Glaser G. H., Yu R. K. Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures. J. Neurochem., 31: 21-27, 1978.[Medline]
  40. el-Abbadi M., Seyfried T. N. Influence of growth environment on the ganglioside composition of an experimental mouse brain tumor. Mol. Chem. Neuropathol., 21: 273-285, 1994.[Medline]
  41. Seyfried T. N., Novikov A. M., Irvine R. A., Brigande J. V. Ganglioside biosynthesis in mouse embryos: sialyltransferase IV and the asialo pathway. J. Lipid Res., 35: 993-1001, 1994.[Abstract]
  42. Looney W. B., Mayo A. A., Allen P. M., Morrow J. Y., Morris H. P. A mathematical evaluation of tumour growth curves in rapid, intermediate and slow growing rat hepatomata. Br. J. Cancer, 27: 341-344, 1973.[Medline]
  43. Olshefski R., Ladisch S. Intercellular transfer of shed tumor cell gangliosides. FEBS Lett., 386: 11-14, 1996.[Medline]
  44. Li R., Gage D., McKallip R., Ladisch S. Structural characterization and in vivo immunosuppressive activity of neuroblastoma GD2. Glycoconj. J., 13: 385-389, 1996.[Medline]
  45. Heitger A., Ladisch S. Gangliosides block antigen presentation by human monocytes. Biochim. Biophys. Acta, 1303: 161-168, 1996.[Medline]
  46. Ladisch S., Li R., Olson E. Ceramide structure predicts tumor ganglioside immunosuppressive activity. Proc. Natl. Acad. Sci. USA, 91: 1974-1978, 1994.[Abstract/Free Full Text]
  47. Ziche M., Alessandri G., Gullino P. M. Gangliosides promote the angiogenic response. Lab. Investig., 61: 629-634, 1989.[Medline]
  48. Leek R. D., Lewis C. E., Whitehouse R., Greenall M., Clarke J., Harris A. L. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res., 56: 4625-4629, 1996.[Abstract/Free Full Text]
  49. Fukumura D., Xavier R., Sugiura T., Chen Y., Park E. C., Lu N., Selig M., Nielsen G., Taksir T., Jain R. K., Seed B. Tumor induction of VEGF promoter activity in stromal cells. Cell, 94: 715-725, 1998.[Medline]
  50. Orre M., Rogers P. A. Macrophages, and microvessel density in tumors of the ovary. Gynecol. Oncol., 73: 47-50, 1999.[Medline]
  51. Sunderkotter C., Steinbrink K., Goebeler M., Bhardwaj R., Sorg C. Macrophages and angiogenesis. J. Leukocyte Biol., 55: 410-422, 1994.[Abstract]
  52. Valentino L., Moss T., Olson E., Wang H. J., Elashoff R., Ladisch S. Shed tumor gangliosides and progression of human neuroblastoma. Blood, 75: 1564-1567, 1990.[Abstract/Free Full Text]
  53. Ladisch S., Kitada S., Hays E. F. Gangliosides shed by tumor cells enhance tumor formation in mice. J. Clin. Investig., 79: 1879-1882, 1987.
  54. Li R. X., Ladisch S. Shedding of human neuroblastoma gangliosides. Biochim. Biophys. Acta, 1083: 57-64, 1991.[Medline]
  55. Young W., Jr., Borgman C. A., Wolock D. M. Modes of shedding of glycosphingolipids from mouse lymphoma cells. J. Biol. Chem., 261: 2279-2283, 1986.[Abstract/Free Full Text]
  56. Olshefski R., Ladisch S. Synthesis, shedding, and intercellular transfer of human medulloblastoma gangliosides: abrogation by a new inhibitor of glucosylceramide synthase. J. Neurochem., 70: 467-472, 1998.[Medline]
  57. Jennemann R., Rodden A., Bauer B. L., Mennel H. D., Wiegandt H. Glycosphingolipids of human gliomas. Cancer Res., 50: 7444-7449, 1990.[Abstract/Free Full Text]
  58. Yates A. J. Glycolipids and gliomas. A review. Neurochem. Pathol., 8: 157-180, 1988.
  59. Koochekpour S., Pilkington G. J. Vascular and perivascular GD3 expression in human glioma. Cancer Lett., 104: 97-102, 1996.[Medline]
  60. Nakamura O., Iwamori M., Matsutani M., Takakura K. Ganglioside GD3 shedding by human gliomas. Acta Neurochir., 109: 34-36, 1991.[Medline]
  61. Berra B., Gaini S. M., Riboni L. Correlation between ganglioside distribution and histological grading of human astrocytomas. Int. J. Cancer, 36: 363-366, 1985.[Medline]
  62. Traylor T. D., Hogan E. L. Gangliosides of human cerebral astrocytomas. J. Neurochem., 34: 126-131, 1980.[Medline]



This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
P. Mukherjee, A. C. Faber, L. M. Shelton, R. C. Baek, T. C. Chiles, and T. N. Seyfried
Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a
J. Lipid Res., May 1, 2008; 49(5): 929 - 938.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Biswas, A. Richmond, P. Rayman, S. Biswas, M. Thornton, G. Sa, T. Das, R. Zhang, A. Chahlavi, C. S. Tannenbaum, et al.
GM2 Expression in Renal Cell Carcinoma: Potential Role in Tumor-Induced T-Cell Dysfunction.
Cancer Res., July 1, 2006; 66(13): 6816 - 6825.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. Ogushi, S. Takahashi, T. Takeuchi, T. Urano, K. Horie-Inoue, J. Kumagai, T. Kitamura, Y. Ouchi, M. Muramatsu, and S. Inoue
Estrogen Receptor-Binding Fragment-Associated Antigen 9 Is a Tumor-Promoting and Prognostic Factor for Renal Cell Carcinoma
Cancer Res., May 1, 2005; 65(9): 3700 - 3706.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Liu, R. Li, and S. Ladisch
Exogenous Ganglioside GD1a Enhances Epidermal Growth Factor Receptor Binding and Dimerization
J. Biol. Chem., August 27, 2004; 279(35): 36481 - 36489.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Weiss, S. Hettmer, P. Smith, and S. Ladisch
Inhibition of Melanoma Tumor Growth by a Novel Inhibitor of Glucosylceramide Synthase
Cancer Res., July 1, 2003; 63(13): 3654 - 3658.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
G. Chatterjee, A. Rosner, Y. Han, E. T. Zelazny, B. Li, R. D. Cardiff, and A. S. Perkins
Acceleration of Mouse Mammary Tumor Virus-Induced Murine Mammary Tumorigenesis by a p53172H Transgene: Influence of FVB Background on Tumor Latency and Identification of Novel Sites of Proviral Insertion
Am. J. Pathol., December 1, 2002; 161(6): 2241 - 2253.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Hakomori
Glycosylation defining cancer malignancy: New wine in an old bottle
PNAS, August 6, 2002; 99(16): 10231 - 10233.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
G. Zeng, L. Gao, S. Birklé, and R. K. Yu
Suppression of Ganglioside GD3 Expression in a Rat F-11 Tumor Cell Line Reduces Tumor Growth, Angiogenesis, and Vascular Endothelial Growth Factor Production
Cancer Res., December 1, 2000; 60(23): 6670 - 6676.
[Abstract] [Full Text]

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

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS