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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

Nuclear localization of CD133 in rhabdomyosarcoma cells. (A) Example of the frequency of cells with CD133 nuclear positivity in the NSTS-11 cell line, as detected using three independent primary antibodies. Cells with exclusive nuclear positivity for CD133 are indicated by arrows; cells with the typical membrane positivity are indicated by arrowheads. (B) Details of cells that exhibited exclusive nuclear positivity for CD133 in all five rhabdomyosarcoma cell lines. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bars, (A) 50 µm and (B) 10 µm.
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f2-ijmm-36-01-0065: Nuclear localization of CD133 in rhabdomyosarcoma cells. (A) Example of the frequency of cells with CD133 nuclear positivity in the NSTS-11 cell line, as detected using three independent primary antibodies. Cells with exclusive nuclear positivity for CD133 are indicated by arrows; cells with the typical membrane positivity are indicated by arrowheads. (B) Details of cells that exhibited exclusive nuclear positivity for CD133 in all five rhabdomyosarcoma cell lines. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bars, (A) 50 µm and (B) 10 µm.

Mentions: For all five RMS cell lines examined in this study, we performed a detailed analysis of the presence of cells with nuclear CD133 positivity using indirect immunofluorescence with three independent anti-CD133 antibodies (Fig. 1). A subset of cells showed only nuclear CD133 positivity, i.e., no detectable membrane or cytoplasmic positive signal. The results were markedly similar in all five cell lines analyzed, regardless of the primary antibody utilized, and the proportion ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies (Fig. 2A and Table I). We also performed a detailed morphological analysis of the cells that exhibited exclusive nuclear positivity for CD133 (Fig. 2B); as can be seen on these micrographs, the pattern of CD133 nuclear positivity was markedly similar in all of the cell lines.


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)

Nuclear localization of CD133 in rhabdomyosarcoma cells. (A) Example of the frequency of cells with CD133 nuclear positivity in the NSTS-11 cell line, as detected using three independent primary antibodies. Cells with exclusive nuclear positivity for CD133 are indicated by arrows; cells with the typical membrane positivity are indicated by arrowheads. (B) Details of cells that exhibited exclusive nuclear positivity for CD133 in all five rhabdomyosarcoma cell lines. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bars, (A) 50 µm and (B) 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijmm-36-01-0065: Nuclear localization of CD133 in rhabdomyosarcoma cells. (A) Example of the frequency of cells with CD133 nuclear positivity in the NSTS-11 cell line, as detected using three independent primary antibodies. Cells with exclusive nuclear positivity for CD133 are indicated by arrows; cells with the typical membrane positivity are indicated by arrowheads. (B) Details of cells that exhibited exclusive nuclear positivity for CD133 in all five rhabdomyosarcoma cell lines. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bars, (A) 50 µm and (B) 10 µm.
Mentions: For all five RMS cell lines examined in this study, we performed a detailed analysis of the presence of cells with nuclear CD133 positivity using indirect immunofluorescence with three independent anti-CD133 antibodies (Fig. 1). A subset of cells showed only nuclear CD133 positivity, i.e., no detectable membrane or cytoplasmic positive signal. The results were markedly similar in all five cell lines analyzed, regardless of the primary antibody utilized, and the proportion ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies (Fig. 2A and Table I). We also performed a detailed morphological analysis of the cells that exhibited exclusive nuclear positivity for CD133 (Fig. 2B); as can be seen on these micrographs, the pattern of CD133 nuclear positivity was markedly similar in all of the cell lines.

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