Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA

Tokuyama, Satoru; Moriya, Setsuko; Taniguchi, Shun’ichiro; Yasui, Akira; Miyazaki, Jun‐ichi; Orikasa, Seiichi; Miyagi, Taeko

doi:10.1002/(sici)1097-0215(19971104)73:3<410::aid-ijc16>3.0.co;2-g

Cited by 53 publications

(41 citation statements)

References 22 publications

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“…It is possible that this Cse has an alternative role in such cells or that it is left over from the earlier phase of red cell development. Even though the molecular mechanisms and topology are not easy to explain, it is noteworthy that expression of a cytosolic sialidase resulted in a reduced amount of intercellular G M3 expression and increased amounts of its hydrolyzed product lactosyl ceramide in stably transfected B16 melanoma cells (51) and epidermoid carcinoma cells (52). If a similar situation applies for O-acetylated gangliosides, it is possible that this cytosolic enzyme can still regulate O-acetylated ganglioside expression in certain cell types.…”

Section: Discussionmentioning

confidence: 99%

Lysosomal and Cytosolic Sialic Acid 9-O-Acetylesterase Activities Can Be Encoded by One Gene via Differential Usage of a Signal Peptide-encoding Exon at the N Terminus

Takematsu¹,

Díaz²,

Stoddart³

et al. 1999

Journal of Biological Chemistry

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9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acidmediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5 region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acidspecific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters.

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Section: Discussionmentioning

confidence: 99%

Lysosomal and Cytosolic Sialic Acid 9-O-Acetylesterase Activities Can Be Encoded by One Gene via Differential Usage of a Signal Peptide-encoding Exon at the N Terminus

Takematsu¹,

Díaz²,

Stoddart³

et al. 1999

Journal of Biological Chemistry

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“…11 Cell motility was determined by measuring phagokinetic tracks of the cells on colloidal gold-coated coverslips; for the assay of invasive potential, Biocoat Matrigel Invason Chambers (Becton Dickinson Labware, Braintree, MA) was used. Cell attachment assays were carried out using 3Ј-O-acetyl-2Ј,7Ј-bis(carboxyethyl)-4 or 5-carboxyfluorescein diacethoxymethyl ester (BCECF-AM; Wako, Osaka, Japan).…”

Section: In Vitro Migration Invasion and Cell Attachmentmentioning

confidence: 99%

“…Lectin blot analysis was conducted as described previously. 11 Briefly, cell homogenates were resolved by 10% SDS-PAGE and transferred to Hybond C membranes (Amersham Pharmacia, Buckinghamshire, UK). Blots were washed in TBST solution [10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween 20] and incubated with TBST containing biotinylated lectins.…”

Section: Lectin Blottingmentioning

confidence: 99%

“…The result was decreased pulmonary metastasis, invasiveness and cell motility with an associated decrease in GM3 content. 11 As we have now been able to obtain a rat lysosomal sialidase cDNA and stable transfectants by introduction of this gene into B16-BL6 cell lines, the effects of sialidase expression on malignant phenotype, including metastasis and tumor growth, could be shown to be due to completely different mechanisms from the cytosolic sialidase case. It is generally known that the major role of lysosomal sialidase is in glycoconjugate catabolism because of its subcellular location and because of sialo-oligosaccharide storage in sialidosis cases lacking this sialidase.…”

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confidence: 99%

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Overexpression of lysosomal-type sialidase leads to suppression of metastasis associated with reversion of malignant phenotype in murine B16 melanoma cells

et al. 2001

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Increased sialylation in cell surface glycoproteins is one characteristic feature of cancer cells, particularly related to their metastatic potential and invasiveness. Expression of lysosomal-type sialidase, which plays a major role in hydrolysis of such sialo-glycoproteins, is therefore considered to have a great influence on malignant properties of cancer cells. To investigate whether the sialidase expression level is linked to the malignant phenotype, we transfected B16-BL6 murine melanoma cells, a highly invasive and metastatic line, with an expression vector harboring a rat lysosomal sialidase cDNA; then clones were isolated and examined for changes in biological character. Sialidase-overexpressing cells showed suppression of experimental pulmonary metastasis and tumor progression. The transfectants exhibited diminished cell growth, anchorage-independent growth and increased sensitivity to apoptosis induced by suspension culture or serum depletion in vitro, but no significant alterations in invasiveness, cell motility and cell attachment to fibronectin, collagen IV and laminin. Flow cytometric analysis with either peanut agglutinin (PNA) or Ricinus communis agglutinin (RCA) lectin revealed that desialylated forms of glycoproteins on the cell surfaces were increased. In particular, a desialylated form of a cell surface glycoprotein of 83 kDa was prominent in the transfectants, as determined by galactose oxidase labeling. These observations indicate that sialidase expression is inversely associated with metastatic potential and tumor growth in cancer cells, probably through a regulation mechanism that suppresses cell growth and anchorage-independent growth and promotes apoptosis with deprivation of cell anchorage. © 2001 Wiley-Liss, Inc. Key words: sialidase; metastasis; anchorage-independent growth; sialic acidsSialidase (EC 3.2.1.18) is a key enzyme in catabolism of glycoproteins and glycolipids. Desialylation of these glycoconjugates is a crucial event leading to modulation of cellular functions in numerous physiological and pathological processes. 1,2 Alterations in sialylation during malignant transformation have been observed to be closely associated with the malignant phenotype in terms of metastatic potential and invasiveness. [3][4][5][6][7] To understand how sialidases are involved in this aberrant sialylation, our study has focused on mammalian forms in cancer cells.Mammalian sialidases are classified into 3 to 4 forms based on their subcellular localization and substrate preference. Cytosolic, lysosomal and membrane forms of the sialidases have been cloned; their primary sequences revealed that they are proteins encoded by distinct genes with different major subcellular locations and enzymatic properties. 8 We previously demonstrated that metastatic potential is inversely correlated with lysosomal-type sialidase activity in transformed rat 3Y1 cells 9 and in murine B16 melanoma cells, 10 whereas it did not have a significant relation to sialic acid levels. We also investigated the role of sialidas...

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“…Changes in ganglioside expression in cancer cells are strongly related to their metastatic potential. [1][2][3][4][5] Although increases in complex ganglioside expression in cancer cells have been considered to be associated with malignancy, in our previous study we found that the poorly metastatic FBJ-S1 cells highly expressed the complex ganglioside GD1a, whereas the highly metastatic FBJ-LL cells only slightly expressed this ganglioside. 6 GD1a inhibited the serum-induced motility of FBJ-LL cells and the metastatic potential of FBJ-LL cells was completely suppressed by enforced GD1a expression.…”

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confidence: 99%

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met

Hyuga¹,

Kawasaki²,

Hyuga³

et al. 2001

Int. J. Cancer

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We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, inhibits the serum-induced motility of FBJ-LL cells and that the metastatic potential of FBJ-LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999; 83:685-91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ-LL cell motility. In the present study, the HGF-induced motility of FBJ-S1 cells was found to be one-thirtieth that of FBJ-LL cells. This motility of GD1a-expressing transfectants, which were produced by transfection of FBJ-LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a-pretreated FBJ-LL cells, indicating that GD1a inhibits the HGF-induced motility of GD1a; HGF; motility; scattering Gangliosides are sialylated glycosphingolipids found on the outer layer of the plasma membrane. Changes in ganglioside expression in cancer cells are strongly related to their metastatic potential. 1-5 Although increases in complex ganglioside expression in cancer cells have been considered to be associated with malignancy, in our previous study we found that the poorly metastatic FBJ-S1 cells highly expressed the complex ganglioside GD1a, whereas the highly metastatic FBJ-LL cells only slightly expressed this ganglioside. 6 GD1a inhibited the serum-induced motility of FBJ-LL cells and the metastatic potential of FBJ-LL cells was completely suppressed by enforced GD1a expression. 7 It is possible that GD1a suppresses metastasis of FBJ-LL cells by inhibiting cell migration. The target molecules of GD1a need to be identified to clarify the mechanism of inhibition of metastasis by GD1a. Key words:We recently found that HGF induces FBJ-LL cell motility 8 and that the serum-induced motility of FBJ-LL cells was decreased by treatment of the serum with anti-HGF antibody (unpublished data), suggesting that HGF is a migration-stimulating factor in serum. Since FBJ-LL cells metastasize to the liver and HGF is secreted by nonparenchymal liver cells, HGF may promote liver metastasis of FBJ-LL cells. We suspect that HGF may play a key role in FBJ-LL cell metastasis. HGF is a multipotential modulator of biologic activities in a variety of cell types and it influences the growth, motility, differentiation and morphogenesis of its target cells through the c-Met tyrosine kinase receptor. 9 HGF has been described as a potent chemotactic factor 10 and has been associated with the progression of carcinoma cells. 11 Overexpression of the HGF receptor, c-Met, in malignant cells has been reported in various tissues. 12 Hence HGF may play a crucial role in cancer metastasis.In the present study, we attempted to identify the target molecules of GD1a by analyzing the molecular mechanism of the inhibition of cell migration by GD1a. We found that GD1a inhibited the HGFinduced motility, morphologic change and formation of actin stress fibers in FBJ-LL cells through suppression of tyrosine phosphorylation ...

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Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA

Cited by 53 publications

References 22 publications

Lysosomal and Cytosolic Sialic Acid 9-O-Acetylesterase Activities Can Be Encoded by One Gene via Differential Usage of a Signal Peptide-encoding Exon at the N Terminus

Lysosomal and Cytosolic Sialic Acid 9-O-Acetylesterase Activities Can Be Encoded by One Gene via Differential Usage of a Signal Peptide-encoding Exon at the N Terminus

Overexpression of lysosomal-type sialidase leads to suppression of metastasis associated with reversion of malignant phenotype in murine B16 melanoma cells

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met

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