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Promotion of cell migration by neural cell adhesion molecule (NCAM) is enhanced by PSA in a polysialyltransferase-specific manner.

Guan F, Wang X, He F - PLoS ONE (2015)

Bottom Line: We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7.The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied.We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

ABSTRACT
Neural cell adhesion molecule 140 (NCAM-140) is a glycoprotein and always highly polysialylated in cancer. Functions of polysialic acid (PSA) that binds to N-glycan termini on NCAM remain unclear. ldlD-14 cells, a CHO cell mutant deficient in UDP-Gal 4-epimerase, are useful for structural and functional studies of Gal-containing glycoproteins because their abnormal glycosylation can be converted to normal status by exogenous addition of galactose (Gal). We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7. The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied. We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. In addition, PSA catalyzed by different polysialyltransferases affected the adhesion of NCAM to different extracellular matrix (ECM) components.

No MeSH data available.


Related in: MedlinePlus

Effects of NCAM-140 on ldlD-14 cell behavior.(A) Morphological effects. ldlD/N140 cells were cultured as described in Materials and Methods. Magnification: 200 ×. (B) Motility assays. ldlD-14 and ldlD/N140 cells were cultured in serum-free medium with ITS or ITS+Gal, and motility assays were performed as described in Materials and Methods. Cleared areas on gold sol were measured as square pixels using the ToupView Image program and are shown as mean ± SD from three independent experiments. *, p<0.05; **, p = 0.01–0.05; NS = not significant. (C) and (D) Proliferation (MTT) assays. ldlD-14, ldlD/N140, and transfected (ldlD-14/vector) cells were seeded in equal numbers (2x103) and cultured in serum-free medium with ITS or ITS+Gal. MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Migration assays. ldlD-14, ldlD/N140, and ldlD-14/vector cells were cultured for 48 hr as described above. Migration assays were performed as described in Materials and Methods. The upper transwell insert contained ITS or ITS+Gal, and the lower chamber was supplemented with 5% FBS. Migrating cells were quantified, and values are shown as mean ± SD. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.
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pone.0124237.g003: Effects of NCAM-140 on ldlD-14 cell behavior.(A) Morphological effects. ldlD/N140 cells were cultured as described in Materials and Methods. Magnification: 200 ×. (B) Motility assays. ldlD-14 and ldlD/N140 cells were cultured in serum-free medium with ITS or ITS+Gal, and motility assays were performed as described in Materials and Methods. Cleared areas on gold sol were measured as square pixels using the ToupView Image program and are shown as mean ± SD from three independent experiments. *, p<0.05; **, p = 0.01–0.05; NS = not significant. (C) and (D) Proliferation (MTT) assays. ldlD-14, ldlD/N140, and transfected (ldlD-14/vector) cells were seeded in equal numbers (2x103) and cultured in serum-free medium with ITS or ITS+Gal. MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Migration assays. ldlD-14, ldlD/N140, and ldlD-14/vector cells were cultured for 48 hr as described above. Migration assays were performed as described in Materials and Methods. The upper transwell insert contained ITS or ITS+Gal, and the lower chamber was supplemented with 5% FBS. Migrating cells were quantified, and values are shown as mean ± SD. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.

Mentions: ldlD/N140 cells, in comparison with ldlD-14 cells, were larger and lost the spindle-like morphology (Fig 3A). They displayed significantly enhanced cell motility (Fig 3B) and proliferation under both ITS and ITS+Gal culture (Fig 3C and 3D). Up-regulation of NCAM-140 resulted in increased migration of both ITS-cultured and ITS+Gal-cultured cells (Fig 3E), suggesting a role of NCAM-140 in metastasis. However, no significant difference of cell motility and migration in ITS-cultured vs. ITS+Gal-cultured cells was observed (Fig 3E, right panel). The proposed reason was that the expression of PSA was not significant enough to make the difference (Fig 1B–1l and Fig 2D–2Dc).


Promotion of cell migration by neural cell adhesion molecule (NCAM) is enhanced by PSA in a polysialyltransferase-specific manner.

Guan F, Wang X, He F - PLoS ONE (2015)

Effects of NCAM-140 on ldlD-14 cell behavior.(A) Morphological effects. ldlD/N140 cells were cultured as described in Materials and Methods. Magnification: 200 ×. (B) Motility assays. ldlD-14 and ldlD/N140 cells were cultured in serum-free medium with ITS or ITS+Gal, and motility assays were performed as described in Materials and Methods. Cleared areas on gold sol were measured as square pixels using the ToupView Image program and are shown as mean ± SD from three independent experiments. *, p<0.05; **, p = 0.01–0.05; NS = not significant. (C) and (D) Proliferation (MTT) assays. ldlD-14, ldlD/N140, and transfected (ldlD-14/vector) cells were seeded in equal numbers (2x103) and cultured in serum-free medium with ITS or ITS+Gal. MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Migration assays. ldlD-14, ldlD/N140, and ldlD-14/vector cells were cultured for 48 hr as described above. Migration assays were performed as described in Materials and Methods. The upper transwell insert contained ITS or ITS+Gal, and the lower chamber was supplemented with 5% FBS. Migrating cells were quantified, and values are shown as mean ± SD. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.
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pone.0124237.g003: Effects of NCAM-140 on ldlD-14 cell behavior.(A) Morphological effects. ldlD/N140 cells were cultured as described in Materials and Methods. Magnification: 200 ×. (B) Motility assays. ldlD-14 and ldlD/N140 cells were cultured in serum-free medium with ITS or ITS+Gal, and motility assays were performed as described in Materials and Methods. Cleared areas on gold sol were measured as square pixels using the ToupView Image program and are shown as mean ± SD from three independent experiments. *, p<0.05; **, p = 0.01–0.05; NS = not significant. (C) and (D) Proliferation (MTT) assays. ldlD-14, ldlD/N140, and transfected (ldlD-14/vector) cells were seeded in equal numbers (2x103) and cultured in serum-free medium with ITS or ITS+Gal. MTT assays were performed as described in Materials and Methods. Values shown are mean ± SD from three independent experiments. (E) Migration assays. ldlD-14, ldlD/N140, and ldlD-14/vector cells were cultured for 48 hr as described above. Migration assays were performed as described in Materials and Methods. The upper transwell insert contained ITS or ITS+Gal, and the lower chamber was supplemented with 5% FBS. Migrating cells were quantified, and values are shown as mean ± SD. Two independent experiments gave similar results. Magnification: 200 ×. *, p<0.05; NS = not significant.
Mentions: ldlD/N140 cells, in comparison with ldlD-14 cells, were larger and lost the spindle-like morphology (Fig 3A). They displayed significantly enhanced cell motility (Fig 3B) and proliferation under both ITS and ITS+Gal culture (Fig 3C and 3D). Up-regulation of NCAM-140 resulted in increased migration of both ITS-cultured and ITS+Gal-cultured cells (Fig 3E), suggesting a role of NCAM-140 in metastasis. However, no significant difference of cell motility and migration in ITS-cultured vs. ITS+Gal-cultured cells was observed (Fig 3E, right panel). The proposed reason was that the expression of PSA was not significant enough to make the difference (Fig 1B–1l and Fig 2D–2Dc).

Bottom Line: We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7.The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied.We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

ABSTRACT
Neural cell adhesion molecule 140 (NCAM-140) is a glycoprotein and always highly polysialylated in cancer. Functions of polysialic acid (PSA) that binds to N-glycan termini on NCAM remain unclear. ldlD-14 cells, a CHO cell mutant deficient in UDP-Gal 4-epimerase, are useful for structural and functional studies of Gal-containing glycoproteins because their abnormal glycosylation can be converted to normal status by exogenous addition of galactose (Gal). We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7. The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied. We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. In addition, PSA catalyzed by different polysialyltransferases affected the adhesion of NCAM to different extracellular matrix (ECM) components.

No MeSH data available.


Related in: MedlinePlus