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Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines.

Nunukova A, Neradil J, Skoda J, Jaros J, Hampl A, Sterba J, Veselska R - Int. J. Mol. Med. (2015)

Bottom Line: In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies.Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines.The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Biology, Faculty of Science, Masaryk University, 61137 Brno, Czech Republic.

ABSTRACT
CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.

No MeSH data available.


Related in: MedlinePlus

Confocal microscopy analysis of CD133-positive cell nuclei. (A) The planes of software cross-sections through the CD133-positive nuclei are highlighted by simple yellow lines. (B) The cross-sections at these yellow lines are shown at the bottom. (C) The yellow arrows indicate lines drawn across individual CD133-positive nuclei in the confocal image; (D) matching plots reporting the fluorescence intensity according to these arrows are given below. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 5 µm.
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f3-ijmm-36-01-0065: Confocal microscopy analysis of CD133-positive cell nuclei. (A) The planes of software cross-sections through the CD133-positive nuclei are highlighted by simple yellow lines. (B) The cross-sections at these yellow lines are shown at the bottom. (C) The yellow arrows indicate lines drawn across individual CD133-positive nuclei in the confocal image; (D) matching plots reporting the fluorescence intensity according to these arrows are given below. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 5 µm.

Mentions: To confirm the presence of CD133 in the nuclei of the RMS cells visualized using indirect immunofluorescence, we employed confocal microscopy and software cross-section analysis through these CD133-positive nuclei (Fig. 3). As is apparent from the results, the localization of the fluorescence signal for CD133 was detected within the cell nuclei both on the software cross-sections (Fig. 3B) and on the plot diagrams of the fluorescence intensity (Fig. 3D).


Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines.

Nunukova A, Neradil J, Skoda J, Jaros J, Hampl A, Sterba J, Veselska R - Int. J. Mol. Med. (2015)

Confocal microscopy analysis of CD133-positive cell nuclei. (A) The planes of software cross-sections through the CD133-positive nuclei are highlighted by simple yellow lines. (B) The cross-sections at these yellow lines are shown at the bottom. (C) The yellow arrows indicate lines drawn across individual CD133-positive nuclei in the confocal image; (D) matching plots reporting the fluorescence intensity according to these arrows are given below. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494592&req=5

f3-ijmm-36-01-0065: Confocal microscopy analysis of CD133-positive cell nuclei. (A) The planes of software cross-sections through the CD133-positive nuclei are highlighted by simple yellow lines. (B) The cross-sections at these yellow lines are shown at the bottom. (C) The yellow arrows indicate lines drawn across individual CD133-positive nuclei in the confocal image; (D) matching plots reporting the fluorescence intensity according to these arrows are given below. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 5 µm.
Mentions: To confirm the presence of CD133 in the nuclei of the RMS cells visualized using indirect immunofluorescence, we employed confocal microscopy and software cross-section analysis through these CD133-positive nuclei (Fig. 3). As is apparent from the results, the localization of the fluorescence signal for CD133 was detected within the cell nuclei both on the software cross-sections (Fig. 3B) and on the plot diagrams of the fluorescence intensity (Fig. 3D).

Bottom Line: In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies.Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines.The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Biology, Faculty of Science, Masaryk University, 61137 Brno, Czech Republic.

ABSTRACT
CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.

No MeSH data available.


Related in: MedlinePlus