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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 PSA on MCF-7 cell behavior.(A) and (B) Western blot analysis of NCAM, STX, PST and PSA-NCAM expression, using anti-NCAM, anti-His-tag antibody respectively and β-tubulin as a loading control. (C) Immunofluorescence staining of PSA-NCAM. MCF7/N140 and MCF7/N140 cells transfected with transfection reagent (MCF7/N/R) as mock and control, respectively. Cell nuclei were visualized by Hoechst staining. Size bars: 20 μm. (D) Proliferation (MTT) assays. Transfected MCF-7 cells were cultured and MTT assays were performed as described in Materials and Methods. MCF7/3.1: MCF-7 transfected with pcDNA3.1; MCF7/N/R: MCF7/N140 cells transfected with transfection reagent. Values shown are mean ± SD from three independent experiments. (E) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. *, p<0.05.
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pone.0124237.g005: Effects of PSA on MCF-7 cell behavior.(A) and (B) Western blot analysis of NCAM, STX, PST and PSA-NCAM expression, using anti-NCAM, anti-His-tag antibody respectively and β-tubulin as a loading control. (C) Immunofluorescence staining of PSA-NCAM. MCF7/N140 and MCF7/N140 cells transfected with transfection reagent (MCF7/N/R) as mock and control, respectively. Cell nuclei were visualized by Hoechst staining. Size bars: 20 μm. (D) Proliferation (MTT) assays. Transfected MCF-7 cells were cultured and MTT assays were performed as described in Materials and Methods. MCF7/3.1: MCF-7 transfected with pcDNA3.1; MCF7/N/R: MCF7/N140 cells transfected with transfection reagent. Values shown are mean ± SD from three independent experiments. (E) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. *, p<0.05.

Mentions: In order to confirm the above result, a noninvasive breast cancer cell line, MCF-7, was used[36,37]. Stable overexpression of NCAM (MCF7/N140), and transient overexpression of STX or PST (MCF7/N/S and MCF7/N/P) in MCF-7 cells were constructed (Fig 5A and 5B). MCF7/N/S and MCF7/N/P cells showed a strong signal of PSA-NCAM, compared with control cells (Fig 5C). Overexpression of NCAM resulted in slightly increased proliferation, while STX expression, but not PST expression, increased proliferation further in comparison to MCF7/N140 cells (Fig 5D). Similarly, increased migration of MCF7/N140 cells was further enhanced by overexpression of STX, but not PST (Fig 5E). These findings suggested that PSA catalyzed by STX vs. PST has a different effect on cell proliferation and migration, presumably because of a differing degree of polymerization of PSA.


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 PSA on MCF-7 cell behavior.(A) and (B) Western blot analysis of NCAM, STX, PST and PSA-NCAM expression, using anti-NCAM, anti-His-tag antibody respectively and β-tubulin as a loading control. (C) Immunofluorescence staining of PSA-NCAM. MCF7/N140 and MCF7/N140 cells transfected with transfection reagent (MCF7/N/R) as mock and control, respectively. Cell nuclei were visualized by Hoechst staining. Size bars: 20 μm. (D) Proliferation (MTT) assays. Transfected MCF-7 cells were cultured and MTT assays were performed as described in Materials and Methods. MCF7/3.1: MCF-7 transfected with pcDNA3.1; MCF7/N/R: MCF7/N140 cells transfected with transfection reagent. Values shown are mean ± SD from three independent experiments. (E) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. *, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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pone.0124237.g005: Effects of PSA on MCF-7 cell behavior.(A) and (B) Western blot analysis of NCAM, STX, PST and PSA-NCAM expression, using anti-NCAM, anti-His-tag antibody respectively and β-tubulin as a loading control. (C) Immunofluorescence staining of PSA-NCAM. MCF7/N140 and MCF7/N140 cells transfected with transfection reagent (MCF7/N/R) as mock and control, respectively. Cell nuclei were visualized by Hoechst staining. Size bars: 20 μm. (D) Proliferation (MTT) assays. Transfected MCF-7 cells were cultured and MTT assays were performed as described in Materials and Methods. MCF7/3.1: MCF-7 transfected with pcDNA3.1; MCF7/N/R: MCF7/N140 cells transfected with transfection reagent. Values shown are mean ± SD from three independent experiments. (E) Migration assays. Cells were cultured and migratory cells were quantified as described in Fig 3E. Two independent experiments gave similar results. *, p<0.05.
Mentions: In order to confirm the above result, a noninvasive breast cancer cell line, MCF-7, was used[36,37]. Stable overexpression of NCAM (MCF7/N140), and transient overexpression of STX or PST (MCF7/N/S and MCF7/N/P) in MCF-7 cells were constructed (Fig 5A and 5B). MCF7/N/S and MCF7/N/P cells showed a strong signal of PSA-NCAM, compared with control cells (Fig 5C). Overexpression of NCAM resulted in slightly increased proliferation, while STX expression, but not PST expression, increased proliferation further in comparison to MCF7/N140 cells (Fig 5D). Similarly, increased migration of MCF7/N140 cells was further enhanced by overexpression of STX, but not PST (Fig 5E). These findings suggested that PSA catalyzed by STX vs. PST has a different effect on cell proliferation and migration, presumably because of a differing degree of polymerization of PSA.

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