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Ganglioside G_M3 Overexpression Induces Apoptosis and Reduces Malignant Potential in Murine Bladder Cancer

Department of Urology, Tohoku University School of Medicine Sendai 980-8574 [R. W., C. O., T. T., H. A., M. Sat., S. S., S. H., Y. A.]; and Department of Virology and Glycobiology, National Cancer Center, Research Institute, Tokyo 104-0045 [A. I., M. Sai.], Japan

	ABSTRACT

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We demonstrated previously (S. Kawamura et al., Int. J. Cancer, 94: 343–347, 2001) that large amounts of ganglioside G_M3 accumulate in superficial bladder tumor, compared with invasive bladder tumors and that exogenous G_M3 inhibits the invasive potential of bladder tumor cells. To apply the G_M3 overexpression system to bladder tumor therapy, direct evidence for the important role of G_M3 in bladder tumor invasion must be obtained through transfer of the gene responsible for G_M3 overexpression. To determine the most appropriate cancer cell line for elucidating the antitumor effect of ganglioside G_M3 overexpression, the present study examined glycolipid composition, enzyme activity, and mRNA expression of the glycosyltransferases responsible for G_M3 synthesis in the bladder tumor cell lines KK-47, J82, MGH-UI, YTS-1, and MBT-2. A murine bladder carcinoma cell line (MBT-2) was transfected with a G_M3 synthase [(lactosylceramide 2,3-N-acetyl sialic acid transferase); sialyltransferase-I; SAT-I] cDNA, because this line does not naturally express G_M3. Stable transfectants (MBT-2-SAT-I) that overexpressed G_M3 were characterized by a reduced potential for cell proliferation, motility, invasion, and xenograft tumor growth, and an increase in the number of apoptotic cells. In the proportion of synthetic S phase, cells did not differ between MBT-2-SAT-I and mock-transfectant cells. These results suggest that the decreased proliferative potential related to G_M3 overexpression was attributable to the increased number of apoptotic cells. Although details of the mechanism of apoptosis remain unclear, the overexpression of G_M3 by gene transfer of SAT-I may present a novel therapeutic modality.

	INTRODUCTION

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The bladder is the most common site of urinary tract cancer. An estimated 50,500 new patients were diagnosed with bladder cancer in the United States during 1995 (1) . Bladder cancers tend to occur in two principal forms: low-grade superficial tumors and high-grade invasive cancer. Most superficial tumors are papillary, often multifocal, and occasionally progress to invasive disease but have a good prognosis. Most invasive tumors are nodular, metastasize during the early phase, and have a poor prognosis (2) . Although recent advances in chemotherapy of invasive bladder tumors have improved the survival of patients with bladder tumor, the prognosis of invasive bladder tumor is still poor, and the therapy causes side effects. A novel therapeutic modality that could overcome these concerns is required.

Carbohydrates are major components of the cell membrane that undergo significant fluctuations in quantity and quality during differentiation and malignant transformation (3, 4, 5) . Several glycosyltransferase genes have been cloned and gene-targeting technology has demonstrated the in vivo function of products of individual glycosyltransferases (6) . The remodeling of cell surface carbohydrate structures by the gene transfer of specific glycosyltransferases has also demonstrated the important roles of carbohydrates in tumor metastasis (7 , 8) .

Among the various species of carbohydrates, gangliosides that are in general of low toxicity often have pronounced antiproliferative and differentiation-inducing properties (9) . Ganglioside G_M3 shows promise as a novel antitumor agent against human brain tumors (10) .

We indicated that a quantitative change of G_M3 expression in bladder tumors is a biochemical parameter associated with growth and invasiveness (11) . Moreover, exogenous G_M3 inhibits bladder cancer cell invasion (12) . Locally injected G_M3 also inhibits murine MBT-2 bladder tumor invasion and growth (11) .

To apply the G_M3 overexpression system as a bladder cancer therapy, direct evidence supporting the notion that G_M3 overexpression has an antitumor effect is required, such as from the gene transfer of a cDNA of the glycosyltransferase responsible for G_M3 synthesis.

Here, we present evidence showing that G_M3 synthase gene transfer has an antitumor effect on the murine bladder cancer MBT-2.

	MATERIALS AND METHODS

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Cell Lines.
KK-47, J82, MGH-UI, and YTS-1 are human bladder tumor (transitional cell carcinoma) cell lines. KK-47 was provided by Dr. Takashi Masuko (Department of Hygienic Chemistry, Department of Pharmacology, Tohoku University, Sendai, Japan). YTS-1 was a gift from Dr. Hiroshi Kakizaki (Department of Urology, Yamagata University, Yamagata, Japan). J82 and MGH-UI were obtained from the American Type Culture Collection. MBT-2 is mouse transitional cell carcinoma cell line induced by the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT; Ref. 13 ). MBT-2 cells resemble their human counterpart both grossly and histologically and have been used in an animal model to evaluate the efficacy of antitumor drugs in bladder cancer. MBT-2 was a gift by Dr. Katsunori Uchida (Department of Urology, Tsukuba University, Tsukuba, Japan). These cell lines were maintained in RPMI 1640 containing 10% FCS in a humidified 5% CO2 atmosphere at 37°C.

Glycolipid Extraction and TLC.²
Glycolipids were extracted as described previously (11 , 12) . Briefly, total glycolipids were extracted from 300 µl of packed cells in chloroform/methanol [2:1, 1:1, and 1:2 (v/v)] followed by isopropanol:hexane:water [55:25:20 (v/v/v)]. After partition, the upper phase was dialyzed and separated into ganglioside and upper neutral fractions by DEAE Sephadex A 25 column chromatography. The lower neutral fraction was purified by acetylation and Florisil column chromatography. Samples of equivalent wet weight were applied to high-performance TLC plates (Baker, Philipsburg, NJ). The upper neutral and ganglioside phases were separated by chromatography in a solvent system of chloroform:methanol: 0.5% aqueous CaCl2 [60:40:9 (v/v/v)], and the lower phase in chloroform:methanol:water [60:25:4 (v/v/v)]. The coloring agent was orcinol-sulfuric acid.

Assays for Glycosyltransferase Activity.
G_M3 and G_D3 synthase activities were assayed with modifications as described previously (11) . Briefly, samples were homogenized with cell suspension solution [15 mM sodium cacodylate (pH 6.5), 5% glycerol, 1x Complete (Boehringer Mannheim), and 0.1% Lubrol], and the supernatants served as enzymatic source. For the G_M3 and G_D3 assay, lactosylceramide and G_M3 were used as acceptor, respectively, and [¹⁴C]CMP-N-acetyl neuraminic acid was used as donor. SepPak C18 cartridge (Millipore) was used for the separation of radiolabeled reaction products. For the measurement of radioactivity of the radiolabeled product, the eluates were dried up, developed on TLC with chloroform/methanol/0.5% CaCl₂ (60:40:9, by volume), and visualized with Fuji FLA-2000 Bio-imaging analyzer. All of the assays were carried out in triplicate.

Northern Blotting.
A 1086-bp open reading frame fragment of the human G_M3 synthase (SAT-I; Ref. 14 ) cDNA was gel-purified, labeled with [-³²P]dCTP, and used as a probe. Total RNA from cultured cells was isolated following a standard protocol.

Establishment of Stable Transfectant.
A cDNA encoding human SAT-I was subcloned to pMIKHygB vector containing hygromycin B-resistant gene, resulting in pMIK-hSAT-I. MBT-2 cells, which do not express G_M3, were transfected with pMIK-hSAT-I using LipofectAMINE (Life Technologies, Inc.) as described in the protocol provided by the supplier. Transfected cells were cultured in RPMI 1640 containing 10% FCS and hygromycin B (300 µg/ml) for 2 weeks; then 20 individual clones were immunocytochemically screened for G_M3 expression using the anti-G_M3 monoclonal antibody, M2590 (mouse IgM; Japan Biotest Research Institute, Tokyo, Japan). Monodispersed cells were incubated with 10 µg/ml M2590 followed by FITC-conjugated goat-affinity-purified F(ab') fragment specific to mouse IgM (Cappel); and the G_M3-positive stable transfectants, MBT-2-SAT-I-1, -2, and -3 were established.

Flow Cytometry.
MBT-2-SAT-I and mock-transfectant cells were assessed by fluorescence-activated cell sorting (FACS) analysis after incubations with M2590 followed by incubations with FITC-conjugated secondary antibody. Labeled cells were analyzed by FACSort flow cytometry using the CellQuest program (Becton-Dickinson).

In Vitro Cell Proliferation Assay.
Cells were seeded in 96-well plates at a density of 10⁵ cells/ml in RPMI 1640 containing 10% FCS and 300 µg/ml of hygromycin B, and cultured for various periods. Cell proliferation was measured daily in triplicate cultures using a cell-counting kit (Wako Pure Chemical Industries, Tokyo, Japan).

Tumor Formation in Mice.
Female C3H/He mice (6–8 weeks old) were s.c. inoculated with MBT-2-SAT-I and mock transfectants. Cell viability was assessed by trypan blue staining; then 2 x 10⁵ cells exhibiting >95% viability were suspended in 0.1 ml of serum-free RPMI 1640 and s.c. injected into the right hind limb. Tumor size was measured every other day for 10 days, and tumor volume was estimated as:

In Vitro Motility Assay and Invasion Assay.
Motility was assayed in vitro using Transwell cell culture chambers (Costar, Cambridge, MA; Refs. 11 , 15 ) with some modifications. The bottom of the upper chamber was sealed with a polyvinylpyrrolidone-free polycarbonate filter, pore size 8 mm. The lower face was covered with 50 µg/ml fibronectin (Biomedical Technologies, Stoughton, MA). Cells (1 x 10⁵) were plated into the upper chamber and incubated for 2 h, and the lower chamber was filled with serum-free RPMI 1640. Cells that did not migrate through the membrane were removed; then cells on the lower face of the membrane were fixed with methanol and visualized by Giemsa staining. Cells on the lower face were counted under a microscope, and the mean numbers of 10 fields were plotted. Motility assays were performed in triplicate, and the coefficient of variation values were always within 5%.

The upper face of the filter was covered with 1 mg/ml Matrigel (Collaborative Research, Bedford, MA) for invasion assay.

Quantitation of Synthetic S-Phase Cell Fraction.
We evaluated the influence of G_M3overexpression on the cell cycle in S phase using Cell Proliferation ELISA BrdUrd kits (Roche Diagnostics, Mannheim, Germany). This assay is based on a colorimetric immunoassay of cells labeled with BrdUrd (16) and closely correlates with [³H]thymidine assays.

Detection of Apoptotic Cells.
Apoptotic cells cultured on cover slips were detected by TUNEL assays using the Tumor TACS in Situ Apoptosis Detection kit (R&D Systems, Minneapolis, MN). One thousand cancer cells were counted in five fields and the ratio (%) of positive cells (apoptotic cells) was determined.

	RESULTS

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TLC Profile of Bladder Tumor Cell Lines.
A trace level of G_M3 was expressed in all of bladder tumor cell lines examined except in KK-47 (data not shown). All of these human cell lines except KK-47 originated from invasive bladder tumors.

Glycosyltransferase Activities of Bladder Tumor Cell Lines.
Despite the low amount of G_M3 expression on TLC in J82, YTS-1, and MGH-UI, G_M3 synthase activities in these cell lines were quite high. This is because the expression amount of G_M3 can be regulated not only by SAT-I but also by G_M2 and G_D3 synthase (11) . In contrast, MBT-2 cells were deficient in both G_M3 and G_D3 synthase activities (data not shown)

Northern Blots.
Although the amount of G_M3 synthase (SAT-I) mRNA expression in bladder tumor cell lines was not always consistent with that of G_M3 on TLC, the SAT-I mRNA in MBT-2 was not detected (data not shown). These results indicated that the optimal cell line for elucidating phenotypic alteration caused by SAT-I gene transfer would be MBT-2.

Changes in Phenotypes of MBT-2-SAT-I Cells.
The parent MBT-2 cells as well as mock transfectants were negative, whereas MBT-2-SAT-I cells were positive for anti-G_M3 (M2590 staining; Fig. 1, A and B ). Changes in the amount of G_M3 expression were also confirmed by TLC (Fig. 1C) . Morphologically, MBT-2 cells, as well as mock transfectants, are spindle shaped with long spicular protrusions, but MBT-2-SAT-I cells became cuboid (Fig. 2) . The growth of MBT-2-SAT-I cells was remarkably slower than that of mock transfectants in vitro (Fig. 3) . Motility and invasion potential were significantly suppressed in MBT-2-SAT-I cells (Figs. 4 and 5 ).

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Expression of GM3 is increased in MBT-2-SAT-I cells. A, immunocytochemical detection of cell surface GM3. Left, mock transfectants were entirely negative for anti-GM3 monoclonal antibody (M2590). Right, MBT-2-SAT-I-1 cells were positive for M2590. MBT-2-SAT-I-2 and –3 cells were also positive for M2590 (data not shown). B, flow cytometry results. Cell surface expression of GM3 was analyzed using M2590. MBT-2-SAT-I-1 cells (shaded peaks) showed higher intensity than mock transfectants(solid lines). MBT-2-SAT-I-2 and MBT-2-SAT-I-3 cells also showed the same intensity as MBT-2-SAT-I-1 (data not shown). C, TLC profile. Lane 1, mock transfectants; Lane 2, MBT-2-SAT-I-1; Lane 3, MBT-2-SAT-I-2; Lane 4, MBT-2-SAT-I-3.

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Morphological changes of MBT-2-SAT-I cells. A, mock transfectants as well as parent MBT-2 cell are characterized by spindle shape with long spicular protrusions. B, MBT-2-SAT-I cells are cuboid.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Growth kinetics in vitro. In vitro proliferative potential is remarkably inhibited in MBT-2-SAT-I cells. *, statistical significance against mock transfectants (P < 0.01, Mann-Whitney’s t test).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Inhibition of motility by MBT-2-SAT-I cells. Motility is significantly inhibited in MBT-2-SAT-I cells. *, statistical significance against mock transfectants (P < 0.01, Mann-Whitney’s t test).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Inhibited invasion potential in MBT-2-SAT-I cells. Invasion potential was also significantly inhibited in MBT-2-SAT-I cells. *, statistical significance against mock transfectants (P < 0.05, Mann-Whitney’s t test).

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Tumor growth of s.c. inoculated MBT-2-SAT-I cells. A, MBT-2-SAT-I cells s.c. inoculated into syngeneic mice, significantly suppressed tumor formation compared with mock transfectant. *, statistical significance against mock transfectants (P < 0.001, Mann-Whitney’s t test). B, representative mock-transfected MBT-2 tumor on day 14. C, representative MBT-2-SAT-I-1 tumor on day 14.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. S-phase population in MBT-2-SAT-I cells. The S-phase cells were labeled with BrdUrd followed by immunostain with anti-BrdUrd monoclonal antibody. There were no significant changes in S-phase cell population between the mock transfectant and MBT-2-SAT-I cells.

View larger version (79K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Apoptotic cells in mock transfectants and MBT-2-SAT-I cells. Apoptotic cell were visualized by TUNEL assay. A, few TUNEL-positive cells were found in mock transfectant. B, a number of apoptotic cells were noted in cells. C, percentages of apoptotic cells. *, statistical significance against mock transfectants (P < 0.05, Mann-Whitney’s t test).

	DISCUSSION

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We determined the glycolipid composition of human bladder cancer cells and found a massive and specific accumulation of G_M3 in superficial papillary tumors (11) . Exogenous ganglioside modulates the growth of many cell types (18) ; we found also that when G_M3 was added to the culture medium of the human bladder cancer cell lines KK-47 and T-24, invasive activity decreased (11) . This antitumor effect of exogenous G_M3 has also been demonstrated in syngeneic xenografts of the murine bladder cancer, MBT-2 (12) . We, therefore, hypothesized that the G_M3 overexpression could be a novel bladder tumor therapy.

Such application of GM3 overexpression should be supported by direct evidence of its antitumor effect in a reconstituted system using gene transfer of glycosyltransferase responsible for G_M3 synthesis. MBT-2 is an excellent model of human invasive bladder tumors because of its aggressive properties (13) and glycolipid profile (12) . As we demonstrated previously, the expression level of G_M3 can be regulated not only by SAT-I but also by G_M2, G_D3, and CTH (G_b3) synthases (11) . In the present study, the enzymatic background of G_M3 and G_D3 synthases and the mRNA expression also supported the notion that MBT-2 is an excellent model for evaluating functional and phenotypic alterations caused by G_M3 overexpression. If MBT-2 has G_D3 synthase activity, G_M3 accumulation by gene transfer could be interfered with by the conversion of G_M3 to G_D3.

Various biological activities are associated with G_M3. The growth of HL-60 and U937 was significantly inhibited by G_M3, and they morphologically matured along monocytic lineage (19) . The mechanism of this morphological change in MBT-2-SAT-I cells caused by G_M3 overexpression is unclear, but it may be the same as that in HL-60 and U937.

To understand the mechanism of inhibited motility and invasion potential associated with G_M3 overexpression, functional support of G_M3 in fibronectin-integrin interaction should be noted (11 , 12 , 20) . The adhesion of FUA169 cells (a mouse mammary carcinoma mutant cell line) to fibronectin requires the presence of G_M3, which supports the function of the 5ß1 integrin receptor (20) . Within an optimal concentration of G_M3, liposome adhesion to fibronectin-coated plates was significantly enhanced, whereas G_M3 concentrations above or below the optimal range decreased adhesion (20) . The G_M3 concentration of mock transfectants in the present study may have been optimal or excessive in MBT-2-SAT-I cells for fibronectin-mediated cell attachment. Secondly, G_M3 has been identified as a cofactor of CD9 in regulating tumor cell motility in that CD9, complexed with G_M3, inhibits motility (21) . CD9 was originally discovered as a motility-regulatory membrane receptor (22) . Our previous data also indicated that G_M3 expression is significantly reduced in the invasive bladder cancer cell lines YTS-1and J82, whereas CD9 is equally expressed in invasive and noninvasive cell lines (23) . We assume that reduced G_M3 expression causes the loss of a cofactor required for the CD9-dependent inhibition of motility.

Apoptosis is also induced by G_M3 in thymocytes (24) and in proliferating neuronal cells (25) . The cellular mechanisms through which G_D3 induces apoptosis may be Fas mediated (27) . G_D3 induces the mitochondrial permeability transition, and this event precedes apoptosis (26) . Similar effects may be induced by G_M3 ganglioside.

Our studies showed that neither exogenous (11) nor endogenous G_M3 overexpression caused by gene transfer affected the S-phase cell population in cultured bladder tumor cell lines. The major focus of cisplatin activity is at the growth (G₁) and S phases. Because cisplatin-based chemotherapy is a major therapeutic modality for invasive bladder tumors (28) , the conserved S-phase cell fraction with G_M3 overexpression is beneficial for combination therapy with conventional chemotherapy using cisplatin.

	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 To whom requests for reprints should be addressed, at Department of Urology, Tohoku University School of Medicine, 1-1 Seiryo-machi Aoba-ku, Sendai 980-8574, Japan. Phone: 81-22-717-7278; Fax: 81-22-717-7283; E-mail: coyama{at}uro.med.tohoku.ac.jp

² The abbreviations used are: TLC, thin-layer chromatography; SAT-I, lactosylceramide (2,3-N-acetyl sialic acid transferase, sialyltransferase-I); BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.

Received 12/26/01. Accepted 4/22/02.

	REFERENCES

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1. Messing E. M., Catalona W. Urothelial tumors of the urinary tract Walsh P. C. Retic A. B. Vaughan E. D. Wein A. J. eds. . Campbell’s Urology, 2327-2410, W. B. Saunders Company Philadelphia 1998.
2. Kakizoe T., Tobisu K., Takai K., Tanaka Y., Kishi K., Teshima S. Relationship between papillary and nodular transitional cell carcinoma in the human urinary bladder. Cancer Res., 48: 2293-2303, 1988.
3. Hakomori S., Kannagi R. Glycosphingolipids as tumor-associated and differentiation markers-a guest editorial. J. Natl. Cancer Inst. (Bethesda), 71: 231-251, 1983.
4. Hakomori S. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res., 56: 5309-5318, 1996.[Abstract/Free Full Text]
5. Fukuda M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res., 56: 2237-2244, 1996.[Abstract/Free Full Text]
6. Yeh J. C., Hiraoka N., Petryniak B., Nakayama J., Ellies L. G., Rabuka D., Hindsgaul O., Marth J. D., Lowe J. B., Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension ß1,3-N-acetylglucosaminyltransferase. Cell, 105: 957-969, 2001.[Medline]
7. Yoshimura M., Nishikawa A., Ihara Y., Taniguchi S., Taniguchi N. Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. Proc. Natl. Acad. Sci. USA, 92: 8754-8758, 1995.[Abstract/Free Full Text]
8. Aubert M., Panicot L., Crotte C., Gibier P., Lombardo D., Sadoulet M-O., Mas E. Restoration of (1, 2) fucosyltransferase activity decreases adhesive and metastatic properties of human pancreatic cancer cells. Cancer Res., 60: 1449-1456, 2000.[Abstract/Free Full Text]
9. Svennerholm L. Gangliosides—a new therapeutic agent against stroke and Alzheimer’s disease. Life Sci., 55: 2125-2134, 1994.[Medline]
10. Noll E. N., Lin J., Nakatsuji Y., Miller R. H., Black P. M. GM3 as a novel growth regulator for human gliomas. Exp. Neurol., 168: 300-309, 2000.
11. Kawamura S., Ohyama C., Watanabe R., Satoh M., Saito S., Hoshi S., Gasa S., Orikasa S. Glycolipid composition in bladder tumor: a crucial role of GM3 ganglioside in tumor invasion. Int. J. Cancer, 94: 343-347, 2001.[Medline]
12. Ohyama C., Kawamura S., Suzuki K., Numahata K., Tokuyama S., Ito A., Satoh M., Saito S., Yoshikawa K., Hoshi S., Orikasa S. GM3 inhibits murine MBT-2 tumor invasion and growth. Int. J. Oncol., 8: 809-813, 1996.
13. Soloway M. S., DeKernion J. B., Rose D., Persky L. Effect of chemotherapeutic agents on bladder cancer: a new animal model. Surg. Forum, 13: 542-544, 1973.
14. Ishii A., Ohta M., Watanabe Y., Matsuda K., Ishiyama K., Sakoe K., Nakamura M., Inokuchi J., Sanai Y., Saito M. Expression cloning and functional characterization of human cDNA for ganglioside GM3 synthase. J. Biol. Chem., 48: 31652-31655, 1998.
15. Albini A., Iwamoto Y., Kleinman H. K., Martin G. R., Aaronson S. A., Kazlowski J. M., McEwan R. M. A rapid in vitro assay for quantitating the invasive potential of cancer cells. Cancer Res., 47: 3239-3245, 1987.[Abstract/Free Full Text]
16. Thomton J. G., Wells M., Hume W. L. Measurement of the S-phase duration in normal and abnormal human endometrium by in vitro double labelling with bromodeoxyuridine and tritiated thymidine. J. Pathol., 157: 109-115, 1989.[Medline]
17. Lunec J., Mellon J. K. Molecular biology and bladder cancer Neal D. E. eds. . Tumours in Urology, 19-45, Springer-Verlag London 1994.
18. 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]
19. Nojiri H., Takaku F., Terui Y., Miura Y., Saito M. Ganglioside GM3: an acidic membrane component that increases during macrophage-like cell differentiation can induce monocytic differentiaton of human myeloid leukemic cell lines HL-60 and U937. Proc. Natl. Acad. Sci. USA, 83: 782-786, 1986.[Abstract/Free Full Text]
20. Zheng M., Fang H., Tsuruoka T., Tsuji T., Sasaki T., Hakomori S. Regulatory role of GM3 ganglioside in 5ß1 integrin receptor for fibronectin-mediated adhesion of FUA169 cells. J. Biol. Chem., 268: 2217-2222, 1993.[Abstract/Free Full Text]
21. Ono M., Handa K., Sonnino S., Wither D. A., Nagai H., Hakomori S. GM3 ganglioside inhibits CD9-facilitated haptotactic cell motility: coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy. Biochemistry, 40: 6414-6421, 2001.[Medline]
22. Miyake M., Koyama M., Seno M., Ikeyama S. Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31–15, which inhibits cell motility. J. Exp. Med., 174: 1347-1354, 1991.[Abstract/Free Full Text]
23. Satoh M., Itoh A., Nojiri H., Handa K., Numahata K., Ohyama C., Saito S., Hoshi S., Hokomori S. Enhanced GM3 expression, associated with decreased invasiveness, is induced by brefeldin A in bladder cancer cells. Int. J. Oncol., 19: 723-731, 2001.[Medline]
24. Zhou J., Shao H., Cox N. R., Baker H. J., Ewald S. J. Gangliosides enhance apoptosis of thymocytes. Cell Immunol., 183: 90-98, 1998.[Medline]
25. Nakatsuji Y., Miller R. H. Selective cell-cycle arrest and induction of apoptosis in proliferating neural cells by ganglioside GM3. Exp. Neurol., 168: 290-299, 2001.[Medline]
26. Kristal B. S., Brown A. M. Apoptogenic ganglioside GD3 directly induces the mitochondrial permeability transition. J. Biol. Chem., 274: 23169-23175, 1999.[Abstract/Free Full Text]
27. Okada M., Itoh M., Haraguchi M., Okajima T., Inoue M., Ohishi H., Matsuda Y., Iwamoto T, Kawano T., Fukumoto S., Miyazaki H., Furukawa K., Aizawa S., Furukawa K. b-series ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis, but impairs regeneration of the lesioned hypoglossal nerve. J. Biol. Chem., 277: 1633-1636, 2002.[Abstract/Free Full Text]
28. Scher H. I., Yogoda A., Herr H. W., Sternberg C. N., Morse M. J., Sogani P. C., Watson R. C., Reuter V., Whitmore W. F., Fair W. R. Neoadjuvant M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin) for extravesical urinary tract tumors. J. Urol., 139: 475-477, 1988.[Medline]

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				H. Ishimura, T. Takahashi, H. Nakagawa, S.-I. Nishimura, Y. Arai, Y. Horikawa, T. Habuchi, E. Miyoshi, A. Kyan, S. Hagisawa, et al. N-Acetylglucosaminyltransferase V and {beta}1-6 Branching N-Linked Oligosaccharides Are Associated with Good Prognosis of Patients with Bladder Cancer Clin. Cancer Res., April 15, 2006; 12(8): 2506 - 2511. [Abstract] [Full Text] [PDF]

				S. Hagisawa, C. Ohyama, T. Takahashi, M. Endoh, T. Moriya, J. Nakayama, Y. Arai, and M. Fukuda Expression of core 2 {beta}1,6-N-acetylglucosaminyltransferase facilitates prostate cancer progression Glycobiology, October 1, 2005; 15(10): 1016 - 1024. [Abstract] [Full Text] [PDF]

				T.-W. Chung, H.-J. Choi, Y.-C. Lee, and C.-H. Kim Molecular mechanism for transcriptional activation of ganglioside GM3 synthase and its function in differentiation of HL-60 cells Glycobiology, March 1, 2005; 15(3): 233 - 244. [Abstract] [Full Text] [PDF]

				X. Zhang and F. L. Kiechle Glycosphingolipids in Health and Disease Ann. Clin. Lab. Sci., January 1, 2004; 34(1): 3 - 13. [Abstract] [Full Text] [PDF]

				X.-Q. Wang, P. Sun, and A. S. Paller Ganglioside GM3 Inhibits Matrix Metalloproteinase-9 Activation and Disrupts Its Association with Integrin J. Biol. Chem., July 3, 2003; 278(28): 25591 - 25599. [Abstract] [Full Text] [PDF]

				S. Uemura, K. Kabayama, M. Noguchi, Y. Igarashi, and J.-i. Inokuchi Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth, apoptosis, and gene expression in3LL Lewis lung carcinoma cells Glycobiology, March 1, 2003; 13(3): 207 - 216. [Abstract] [Full Text] [PDF]

				A. Prinetti, L. Basso, V. Appierto, M. G. Villani, M. Valsecchi, N. Loberto, S. Prioni, V. Chigorno, E. Cavadini, F. Formelli, et al. Altered Sphingolipid Metabolism in N-(4-Hydroxyphenyl)- retinamide-resistant A2780 Human Ovarian Carcinoma Cells J. Biol. Chem., February 14, 2003; 278(8): 5574 - 5583. [Abstract] [Full Text] [PDF]

				R. Paris, A. Morales, O. Coll, A. Sanchez-Reyes, C. Garcia-Ruiz, and J. C. Fernandez-Checa Ganglioside GD3 Sensitizes Human Hepatoma Cells to Cancer Therapy J. Biol. Chem., December 13, 2002; 277(51): 49870 - 49876. [Abstract] [Full Text] [PDF]

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