Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov 16;13(11):15177-92.
doi: 10.3390/ijms131115177.

Detection of glycomic alterations induced by overexpression of p-glycoprotein on the surfaces of L1210 cells using sialic acid binding lectins

Affiliations

Detection of glycomic alterations induced by overexpression of p-glycoprotein on the surfaces of L1210 cells using sialic acid binding lectins

Tatiana Bubencíkova et al. Int J Mol Sci. .

Abstract

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Detection of protein bands in the crude membrane fraction of S, R and T cells by Commassie blue staining of polyacrylamide gels. Wheat germ agglutinin (WGA), Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) were detected using lectin blots and western blotting with the c219 anti-P-gp antibody. Polyacrylamide gels were stained with Commassie blue as a control for the accuracy of protein loading. These data are representative of three independent experiments.
Figure 2
Figure 2
Monitoring of S, R and T cell agglutination by WGA, MAA and SNA. After incubation with lectins (see materials and methods), cells were studied by light microscopy and counted using the CASY Model TT Cell Counter. Microscopic images are representative of three independent experiments. Data are presented as the mean ± SD of three independent experiments.
Figure 3
Figure 3
WGA, MAA and SNA induced cell death in S, R and T cells. Cell death was assessed after incubation with lectins (see materials and methods) by spectrophotometric MTT test. Control experiment in the absence of lectins was arbitrarily chosen as 1. Data represent mean ± SD from three independent experiments. Cell death effects induced by WGA and SNA in S cells vs. R or T cells were significantly different (0.002 < p < 0.02) for all concentrations tested. In R and T cells, differences in cell death in response to these two lectins were not significant. No significant effects on cell death were observed with MAA in all three L1210 cell variants.
Figure 4
Figure 4
SNA, WGA and MAA binding to the cell surface of S, R and T cells. Cells were studied by confocal microscopy after incubation with Fluorescein Isothiocyanate (FITC) linked lectins (see materials and methods) and were quantified using FACS. The red histograms represent controls in which cells were incubated in the absence of FITC labeled lectins prior to measurement. These data are representative of three independent measurements.
Figure 5
Figure 5
Sialidase induced liberation of sialic acid from S, R and T cells to the external medium. Sialic acid contents were quantified in the external medium after the incubation of cells with sialidase in phosphate buffered saline (PBS) for 10 h (E). As a control, external medium was added after incubation of the cells in the absence of sialidase (C). The net amount of sialic acid liberated by the sialidase reaction represents the difference (E-C). Data represent the mean ± SD of three independent experiments.
Figure 6
Figure 6
Effects of sialidase treatment on WGA, MAA and SNA binding to S, R and T cells. Sialidase-induced removal of external sialic acid is schematically shown in the upper left panel. Cells incubated in the absence (C) or presence (D) of sialidase were left to interact with FITC-WGA, FITC-MAA and FITC-SNA (CL and DL). Cells incubated in the absence of FITC labeled lectins (C, D) were used as control. FACS histograms are representative of three independent measurements. The median fluorescence intensity data represent the mean ± SD of three independent experiments. Differences between the median fluorescent intensity were significant when comparing CL and DL with the following probabilities for WGA binding to: S cells—p < 0.02, R cells—p < 0.01 and T cells—p <0.01; MAA binding to S cells—p < 0.02, R cells—p < 0.01 and T cells— p< 0.01; and SNA binding to S cells—p < 0.005, R cells—p < 0.01 and T cells—p < 0.01.

Similar articles

Cited by

References

    1. Perez-Tomas R. Multidrug resistance: Retrospect and prospects in anti-cancer drug treatment. Curr. Med. Chem. 2006;13:1859–1876. - PubMed
    1. Breier A., Barancik M., Sulova Z., Uhrik B. P-glycoprotein--implications of metabolism of neoplastic cells and cancer therapy. Curr. Cancer Drug Targets. 2005;5:457–468. - PubMed
    1. Breier A., Gibalova L., Seres M., Barancik M., Sulova Z. New Insight into P-Glycoprotein as a Drug Target. Anticancer Agents Med. Chem. 2012 in press. - PubMed
    1. Sulova Z., Brtko J., Macejova D., Breier A. Are Nuclear Receptors for Retinoids Involved in the Control of the Expression and Activity of P-Glycoprotein? In: Cheng L.-H., Ito Y., editors. Retinoic Acid: Structure, Mechanisms and Roles in Disease. NOVA Publisher; New York, NY, USA: 2012. pp. 29–52.
    1. Cerveny L., Svecova L., Anzenbacherova E., Vrzal R., Staud F., Dvorak Z., Ulrichova J., Anzenbacher P., Pavek P. Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways. Drug Metab. Dispos. 2007;35:1032–1041. - PubMed

Publication types

MeSH terms

Substances