Glycobiology Program, Center for Cancer and Transplantation Biology, Childrens Research Institute and Department of Pediatrics and Biochemistry/Molecular Biology, George Washington University School of Medicine, Washington, DC 20010, USA
Received on April 13, 2001; revised on November 26, 2001; accepted on November 27, 2001.
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Abstract |
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Key words: antisense transfection/glucosylceramide synthase/glycosphingolipids/mouse melanoma/tumorigenesis
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Introduction |
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Gangliosides have a range of biological properties. They enhance the growth factormediated proliferation of fibroblasts (Li et al., 2000) and the proliferation and migration of human vascular endothelial cells (Lang et al., 2001
), and they exert potent immunosuppressive activity (Ladisch et al., 1983
; Floutsis et al., 1989
; Li et al., 1996
; Lu and Sharom, 1996
). By multiple mechanisms, gangliosides inhibit normal human lymphoproliferative responses to mitogens and antigens (Gonwa et al., 1984
), interleukin 2dependent cell proliferation (Robb, 1986
), antigen presenting cell function (Ladisch et al., 1984
; Heitger and Ladisch, 1996
), helper T cell proliferation (Chu and Sharom, 1995
), and NK cell activity (Bergelson et al., 1989
), and they enhance apoptosis of thymocytes (Zhou et al., 1998
). Because of their cell surface location, gangliosides are likely to be involved in various membrane-triggered cellular functions, including signal transduction, regulation of cell proliferation and differentiation, and cell death (De Maria et al., 1997
; Iwabuchi et al., 1998
; Kopitz et al., 1998
). Evidence implicating tumor gangliosides in modulating tumor formation in vivo includes (1) a correlation between tumor cell ganglioside content and tumor forming ability and (2) direct experiments showing that addition of tumor gangliosides to the tumor cell inoculum enhances tumor formation in vivo (Ladisch et al., 1987
).
MEB4 mouse melanoma is characterized by high expression of NeuNAc2-3Galß1-4Glcß1-1Cer (GM3) ganglioside; this high expression is associated with enhanced tumor formation in mice (Ichikawa et al., 1994). Recently, we found that the pharmacologic inhibitor of glucosylceramide synthase, 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) (Lee et al., 1999
), was effective in reducing ganglioside content and in inhibiting tumor formation and metastasis by MEB4 cells (Deng et al., 2000
). However, because the inhibition of ganglioside synthesis by PPPP can only be achieved with the constant presence of this inhibitor in the cell culture, and because cellular recovery from inhibition is relatively rapid (4872 h after the removal of the inhibitor), we sought to develop a more permanent approach to the alteration of ganglioside metabolism in tumor cells.
Here we transfected MEB4 melanoma cells with an antisense sequence to the gene encoding glucosylceramide synthase. The highly specific inhibition of glucosylceramide synthase, a key enzyme in the pathway of ganglioside synthesis, resulted in substantially reduced ganglioside content and markedly reduced tumorigenicity of these murine melanoma cells.
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Results |
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In a second approach we used 3H-serine to metabolically radiolabel cellular ceramide and glucosylceramide. Here we observed a 60% reduction in glucosylceramide content by HPTLC autoradiography (Figure 3B). Following autoradiography, the HPTLC bands were visualized with iodine vapor, scraped off the plate, and quantified by scintillation counting. In MA173 cells the radiolabeled glucosylceramide concentration was decreased by 65%, to 4.2 x 103 dpm or 0.96 nmol/107 cells from 12.2 x 103 dpm or 1.8 nmol/107 MEB4 cells. The level of ceramide was somewhat increased, to 1863 dpm or 2.15 nmol/107 cells from 860 dpm or 0.98 nmol/107 MEB4 cells (Figure 3B). These combined findings clearly demonstrate that antisense transfection was highly efficient in inhibiting glucosylceramide synthase activity.
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Discussion |
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In earlier studies, pharmacologic inhibition of ganglioside synthesis were used to block endogenous cellular ganglioside production. The first such molecule was D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase (Inokuchi et al., 1990; Radin, 1994
; Li and Ladisch, 1996
). Recently we used the improved, more potent inhibitor PPPP (Lee et al., 1999
) to inhibit ganglioside synthesis of MEB4 cells. Treatment of MEB4 cells with 0.5 µM PPPP for 4 days resulted in a significant reduction of ganglioside content of MEB4 cells (Deng et al., 2000
). However, a rapid reversal of the inhibition of ganglioside synthesis was seen when PPPP was removed from the culture medium, and ganglioside content recovered to the original level within 3 days. The requirement of continuing presence of an exogenous inhibitor for efficient inhibition is a disadvantage of this approach.
Two alternative approaches to modify ganglioside synthesis that have been explored by others are antisense oligodeoxynucleotide administration and antisense transfection. Treatment of the human promyelocytic leukemia cell line HL-60 with an antisense oligodeoxynucleotide to II3NeuNAcGgOse3Cer (GM2) synthase down-regulated the synthesis of GM2 and the more complex ganglioside II3NeuNAcGgOse4Cer (GM1) by ~50%. Specificity of the effect was suggested by a concomitant increase in GM3 content without a change in total cellular ganglioside content. Antisense oligodeoxynucleotides to NeuNAc2-8NeuNAc2-3Galß1-4Glcß1-1Cer (GD3) synthase were also used to interfere with ganglioside synthesis (Zeng et al., 1995) and to study the function of GD3 in cell apoptosis (De Maria et al., 1997
). The drawback of antisense oligodeoxynucleotide administration is that oligodeoxynucleotides must be constantly present in the cell culture to achieve sustained inhibition of ganglioside synthesis. This is the same disadvantage as that of standard pharmacological inhibition. Antisense transfection, on the other hand, could provide a way to alter permanently the kinetics of glycosphingolipid synthesis. In the only published study of alteration of ganglioside metabolism using this technique, the GD3 level in rat F11 hybrid neuroblastoma cells was reduced by stable transfection of an antisense vector to GD3 synthase (Zeng et al., 1999
). This was associated with reduced cell migration in vitro and reduced metastatic potential in a nude mouse model (Zeng et al., 2000
). Though both of these antisense studies targeted the synthesis of a specific ganglioside (GM2 or GD3), the reduction of total cellular gangliosides by antisense transfection has not been reported.
Here we provide a first demonstration that the ganglioside level of cells can be permanently reduced by transfection of cells with an antisense vector targeting glucosylceramide synthase. The resulting cell line, MA173, expressed the antisense RNA and showed reduction in half of both glucosylceramide and GM3 ganglioside content. Also, synthesis of metabolically radiolabeled glucosylceramide (Ichikawa et al., 1994) in MA173 cells was greatly reduced, suggesting that glucosylceramide synthase activity was efficiently inhibited.
The reduction of tumor formation associated with the inhibition of glucosylceramide synthesis by antisense transfection was striking and shows that reduction of tumor cell glycosphingolipids is associated with significantly reduced tumor incidence. These results are consistent with our previous findings using the pharmacologic inhibitor PPPP (Deng et al., 2000), in which reduction of tumor cell glycosphingolipids was also associated with significant reduction in tumor incidence. In the present study, the constitutive inhibition of ganglioside synthesis circumvented possible complications that could result from the recovery of ganglioside synthesis after the removal of an inhibitor.
The mechanism(s) underlying significant inhibition of tumor formation after inhibition of glucosylceramide synthesis remain to be elucidated. Several possible mechanisms must be considered. First, because inhibition of glucosylceramide synthase may cause accumulation of ceramide (the substrate for this enzyme), and because elevated ceramide levels have been associated with increased apoptosis, the possibility that increased apoptotic cell death could be the cause of decreased tumorigenicity was investigated. In the MA173 cell line, in which ceramide was somewhat elevated, there was neither a reduction in cell viability nor an increase in apoptosis compared with the control MEB4 cells. These findings exclude decreased intrinsic cell survival as a cause for decreased tumor formation. Second is a possible effect of decreased concentrations of the neutral glycosphingolipid products of glucosylceramide synthase, glucosylceramide, and lactosylceramide. However, to the extent studied, NGSLs (lacking sialic acid) have not been found to affect host cell responses that may influence tumor formation (such as the cellular immune response), which are affected by their sialic acidcontaining products, gangliosides (Lengle and Krishnaraj, 1979; Ladisch et al., 1992
).
Existing knowledge of these biological properties of gangliosides may give some clues to the mechanism of decreased tumor formation. One is that the inhibition of shedding caused by blockade of synthesis stops the release of immunosuppressive gangliosides (Li and Ladisch, 1996), which act to inhibit host immune responses to tumors through suppression of immune cell function at the tumor site (Ladisch et al., 1987
; Li et al., 1995
; McKallip et al., 1999
). Most recently we have obtained direct evidence that GM1b ganglioside shed by a murine lymphoma inhibits the specific immune response against this syngeneic tumor in vivo, including the primary anti-tumor immune response, the secondary response, and the generation of tumor-specific cytotoxic lymphocytes (McKallip et al., 1999
). Another property that may be operative is ganglioside enhancement of growth factormediated proliferation of normal fibroblasts (Li et al., 2000
) and of human vascular endothelial cells (Lang et al., 2001
), important in tumor-associated angiogenesis.
Although the cellular mechanisms remain to be fully elucidated, the significant reduction of tumor formation by antisense transfection of glucosylceramide synthase suggests that highly specific pharmacologic interference with glycosphingolipid metabolism warrants further study as a potential experimental therapeutic approach to cancer.
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Materials and methods |
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Vector construction and transfection
A glucosylceramide synthase cDNA fragment corresponding to the nucleotides 821128 of the published sequence of human glucosylceramide synthase (Ichikawa et al., 1996) was obtained by RT-PCR from total human RNA (human medulloblastoma DAOY cells) with two primers: AGTCCTGACGCGTCATGGCTATCATCTACACCCGA (forward primer specific to nucleotides 82102) and AGTCCTGACGCGTCTCTCCAGCTTATAGTTGGGTC (reverse primer specific to nucleotide of 11081128). Both primers contain an extra 14-nucleotide segment, which has a restriction site for Mlu-1 endonuclease. The restriction site permits the cloning of the 1075-bp-long cDNA fragment into the polylinker region of the pCI-neo mammalian expression vector at the Mlu-1 site. The orientation of inserts was identified by restriction mapping with Hind III enzyme, for which there is one restriction site on the inserts and two sites on the vector. Antisense and sense vectors were identified by different digestion patterns, with three distinguishable bands.
MEB4 cells were transfected by electroporation using the gene pulser II (Bio-Rad) at 375 µF and 500 V. Stable transfectants were selected by incubating the cells in 1 mg/ml of the antibiotic G-418 for 1014 days beginning 2 days after the electroporation. Individual surviving and proliferating colonies were then collected and cultured in fresh medium containing 500 µg/ml G418. The transfection efficiency was about 0.05%, which is the range of anticipated efficiency by electroporation (Potter, 1996).
Antisense expression by RT-PCR
Expression of the antisense was studied by RT-PCR with a pair of primers: TACGACTCACTATAGGCTAGC (the same sequence as a part of the polylinker region of the vector between the transcription starting point and the insert) and ATCAGGTGGACCAAACTACGA (the same sequence as the nucleotides from 819 to 839). Successful use of this pair of primers to amplify the cDNA fragment, which is 349 bp in length, showed that the cDNA fragment was inserted into the vector in antisense orientation.
Flow cytometry
To determine cellular GM3 expression, 2 x 105 MEB4 cells or the transfectants were detached using trypsin, which was neutralized by adding 10-fold serum-containing medium. The cells were incubated for 1 h at 37°C to repair any membrane damage due to the trypsinization, resuspended in cold Hanks buffered saline solution (HBSS)/0.5% bovine serum albumin (BSA)/0.1% NaN3, sequentially incubated with DH2, a murine anti-GM3 antibody (Dohi et al., 1988) and phycoerythrin-conjugated goat monoclonal anti-mouse antibody on ice for 30 min, and washed three times with HBSS/0.5% BSA/0.1% NaN3. To quantify apoptosis, cells were assessed (Schutte et al., 1995
; Vermes et al., 1995
) for expression of annexin V and uptake of 7-aminoactinomycin D. Apoptotic cells were identified as annexin Vpositive, 7-aminoactinomycin Dnegative. Flow cytometry was performed using a FACStar Plus flow cytometer (Becton Dickinson). Fluorescence was measured as molecules of equivalent soluble fluorochrome.
Lipid analyses of antisense-transfected cells
To assess the effect of the antisense transfection on incorporation of UDP-glucose into glucosylceramide, cells were cultured to subconfluence and then labeled with 5 µCi/ml 14C-UDP-glucose for 18 h, harvested, and lyophilized. Ten nmol unlabeled GM3 ganglioside was added to the cells as a cold carrier before extraction twice with 5 ml chloroform:methanol (1:1). The combined extracts were dried down under N2, lyophilized, and partitioned twice with 2 ml di-isopropyl ether/1-butanol (60:40, by vol) and 1 ml 0.1% aqueous NaCl (Ladisch and Gillard, 1985). The upper organic phases, which contain most of the NGSLs, were combined and dried down under N2, lyophilized, and redissolved in chloroform:methanol (1:1) for aliquoting for ß-scintillation counting and HPTLC analysis.
Cell lipids were also analyzed by metabolic radiolabeling of the cells with 1.0 µCi/ml [3H] serine (21.7 Ci/mmol, NEN). After a 24-h incubation, the cells were washed three times with phosphate buffered saline (PBS) and the cell pellet processed for sphingolipid analysis. For ceramide determination (Rani et al., 1995), the pellet was extracted twice with 4 ml chloroform:methanol (1:1), centrifuged at 1000 x g for 10 min, and the supernatants pooled. Chloroform (5 ml) and 0.9% NaCl (4 ml) were added to the supernatants, which were then vortexed and centrifuged at 1000 x g. The lower phase was retained, dried under a nitrogen stream, and resuspended in chloroform:methanol (1:1), and the radioactivity determined by scintillation counting. For glucosylceramide determination (Lavie et al., 1997
), the pellets were resuspended in 2 ml methanol with 2% acetic acid. Unlabeled glucosylceramide (5 µg) was added as a carrier to aid in recovery. The lipids were extracted with 2 ml chloroform and 2 ml water, by vortexing, sonication, and centrifugation at 1000 x g for 10 min. The lower organic phase was retained, dried under a nitrogen stream, and radioactivity quantified.
Equivalent amounts of radiolabeled lipid extracts and unlabeled ceramide or glucosylceramide standards were analyzed by HPTLC following resolution in either chloroform:acetic acid (9:1) for ceramide or chloroform:methanol:ammonium hydroxide (65:25:5) for glucosylceramide. The HPTLC plate was split to separate the unlabeled standards, which were visualized by charring (Fewster et al., 1969). The portion of the plate containing the radioactive lipids was sprayed with En3hanceTM (NEN) and analyzed by HPTLC autoradiography by exposure of the plate to XRP X-ray film (Eastman Kodak, Rochester, NY). Densitometric scanning analysis of the samples was performed using a Scan Maker 5 scanner (Microtek) and Scion Image Analysis (NIH Image 160) software. To test for ceramide, after development of the HPTLC lipids were visualized by iodine vapor and the ceramide area was scraped into 0.5 ml water. Counting fluid was added, and the radioactivity was quantified by scintillation counting (Liu et al., 2000
). Total cellular protein was quantified by a modification of the Lowry method (Markwell et al., 1981
).
Gangliosides were also isolated from the cell pellets. The gangliosides were recovered from the final lower aqueous phase of the di-isopropyl ether/1-butanol partition method as described but without the addition of cold carrier. This aqueous phase was lyophilized and redissolved in a small volume of distilled water. Salts and low-molecular-weight impurities were removed from the total ganglioside fraction by Sephadex G-50 gel filtration. Gangliosides, recovered in the void volume, were lyophilized and quantified as nanomoles of lipid-bound sialic acid by the colorimetric resorcinol assay (Ledeen and Yu, 1982). For HPTLC analysis, they were spotted on 10 x 20 cm precoated Silica Gel-60 HPTLC plates, which were then developed in chloroform:methanol, 0.25% CaCl2 2H2O (60:40:9, by vol). Gangliosides were visualized as purple bands with resorcinol-HCl reagent (Ledeen and Yu, 1982
), and quantified by densitometry.
Cell proliferation assay
To assess the effect of antisense transfection on the proliferation kinetics of MEB4 cells, the cells were cultured in 96-well plates. Triplicate cultures were harvested daily for up to 4 days. Before harvesting, cells were incubated for 4 h with [3H]thymidine at 3.3 µCi/ml and then rinsed twice with ice-cold PBS. Cells were detached in 1% trypsin/ethylenediamine tetra-acetic acid. Incorporated [3H]thymidine was quantified by ß-scintillation counting.
Tumorigenicity assay
Tumor formation was detected as the development of palpable tumors following ID injection of tumor cells. Female syngeneic C57BL/6 mice, 68 weeks old, were used. MEB4, MS2, and MA173 cells were washed in PBS and resuspended, and viability was assessed by trypan blue dye exclusion. Twenty microliters of the resuspended cells (104 or 105 cells) were injected ID on the back of each mouse. The mice were examined for tumor formation twice weekly for 10 weeks.
Statistical analysis
To test significance of differences in the absolute proportion of tumors formed in each of the three groups at the final time point, a logistic regression was performed on data for each cell number administered (104 or 105). Logistic regression allowed estimation of odds ratios (ratio of the odds of tumor production between two groups). Because 105 control MEB4 cells/mouse caused a 100% tumor incidence at 8 weeks, we used an exact logistic procedure to examine these odds ratios and test significance, using LogExact software. To examine the proportion of tumors over time a generalized estimating equation approach was used; this allows for the testing of the difference in tumor growth between the three groups over the entire study period.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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