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CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells.

Barrantes-Freer A, Renovanz M, Eich M, Braukmann A, Sprang B, Spirin P, Pardo LA, Giese A, Kim EL - PLoS ONE (2015)

Bottom Line: CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133.A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein.Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of Neuronal Signals, Max-Planck-Institute of Experimental Medicine, Göttingen, Germany; Institute of Neuropathology, University Medical Centre, Göttingen, Germany.

ABSTRACT
A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.

No MeSH data available.


Related in: MedlinePlus

Divergent patterns detected by anti-AC133 and anti-CD133CT Abs in CaCo-2 cells.A. Representative flow cytometry histograms of CaCo-2 cells immunolabeled with anti-AC133 Ab (left panel) or anti-CD133CT Ab (right panel). B. Quantification of AC133- and CD133CT-positive cells from three independent experiments. Bars represent mean percentage of positive cells ± SEM. C. Immunofluorescence microscopy of CaCo-2 cells co-stained with anti-AC133 Ab (green) and anti-CD133CT Ab (red). Nuclei were counterstained using DAPI (blue). Confocal microscopy images (inset) show the subcellular distribution of the staining. Merged image shows overlapping signals generated by anti-AC133 Ab (green) and anti-CD133CT Ab (red) antibodies. Scale bars correspond to 50 μm (main fields) and 7 μm (insets).
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pone.0130519.g001: Divergent patterns detected by anti-AC133 and anti-CD133CT Abs in CaCo-2 cells.A. Representative flow cytometry histograms of CaCo-2 cells immunolabeled with anti-AC133 Ab (left panel) or anti-CD133CT Ab (right panel). B. Quantification of AC133- and CD133CT-positive cells from three independent experiments. Bars represent mean percentage of positive cells ± SEM. C. Immunofluorescence microscopy of CaCo-2 cells co-stained with anti-AC133 Ab (green) and anti-CD133CT Ab (red). Nuclei were counterstained using DAPI (blue). Confocal microscopy images (inset) show the subcellular distribution of the staining. Merged image shows overlapping signals generated by anti-AC133 Ab (green) and anti-CD133CT Ab (red) antibodies. Scale bars correspond to 50 μm (main fields) and 7 μm (insets).

Mentions: To assess surface CD133 by flow cytometry, we have used anti-AC1331 (CD133/1, Miltenyi Biotec) and anti-CD133CT antibodies binding to different regions in the CD133 protein. Anti-AC1331 Ab binds to the externally located epitope AC133 whereas anti-CD133CT Ab recognizes the C-terminal domain of CD133 (CD133CT). We first analyzed CD133 binding patterns in the human colon carcinoma cell line CaCo-2, which is well characterized with respect to CD133/AC133 [43]. In agreement with previous reports on CaCo-2 cells, our assessments confirmed the expression of membrane-associated CD133 in CaCo-2 cells detected by both anti-AC133 and anti-CD133CT Abs (Fig 1A).


CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells.

Barrantes-Freer A, Renovanz M, Eich M, Braukmann A, Sprang B, Spirin P, Pardo LA, Giese A, Kim EL - PLoS ONE (2015)

Divergent patterns detected by anti-AC133 and anti-CD133CT Abs in CaCo-2 cells.A. Representative flow cytometry histograms of CaCo-2 cells immunolabeled with anti-AC133 Ab (left panel) or anti-CD133CT Ab (right panel). B. Quantification of AC133- and CD133CT-positive cells from three independent experiments. Bars represent mean percentage of positive cells ± SEM. C. Immunofluorescence microscopy of CaCo-2 cells co-stained with anti-AC133 Ab (green) and anti-CD133CT Ab (red). Nuclei were counterstained using DAPI (blue). Confocal microscopy images (inset) show the subcellular distribution of the staining. Merged image shows overlapping signals generated by anti-AC133 Ab (green) and anti-CD133CT Ab (red) antibodies. Scale bars correspond to 50 μm (main fields) and 7 μm (insets).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4472699&req=5

pone.0130519.g001: Divergent patterns detected by anti-AC133 and anti-CD133CT Abs in CaCo-2 cells.A. Representative flow cytometry histograms of CaCo-2 cells immunolabeled with anti-AC133 Ab (left panel) or anti-CD133CT Ab (right panel). B. Quantification of AC133- and CD133CT-positive cells from three independent experiments. Bars represent mean percentage of positive cells ± SEM. C. Immunofluorescence microscopy of CaCo-2 cells co-stained with anti-AC133 Ab (green) and anti-CD133CT Ab (red). Nuclei were counterstained using DAPI (blue). Confocal microscopy images (inset) show the subcellular distribution of the staining. Merged image shows overlapping signals generated by anti-AC133 Ab (green) and anti-CD133CT Ab (red) antibodies. Scale bars correspond to 50 μm (main fields) and 7 μm (insets).
Mentions: To assess surface CD133 by flow cytometry, we have used anti-AC1331 (CD133/1, Miltenyi Biotec) and anti-CD133CT antibodies binding to different regions in the CD133 protein. Anti-AC1331 Ab binds to the externally located epitope AC133 whereas anti-CD133CT Ab recognizes the C-terminal domain of CD133 (CD133CT). We first analyzed CD133 binding patterns in the human colon carcinoma cell line CaCo-2, which is well characterized with respect to CD133/AC133 [43]. In agreement with previous reports on CaCo-2 cells, our assessments confirmed the expression of membrane-associated CD133 in CaCo-2 cells detected by both anti-AC133 and anti-CD133CT Abs (Fig 1A).

Bottom Line: CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133.A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein.Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of Neuronal Signals, Max-Planck-Institute of Experimental Medicine, Göttingen, Germany; Institute of Neuropathology, University Medical Centre, Göttingen, Germany.

ABSTRACT
A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.

No MeSH data available.


Related in: MedlinePlus