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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

Detection of CD133 in the nuclei of rhabdomyosarcoma cells using transmission electron microscopy and immunoblot analysis. (A) Presence of CD133 in cell nuclei and nucleoli; more detailed images of the positive signal for CD133 in the highlighted square areas are presented on the bottom row. (B) The presence of CD133 in the cytoplasm near the nucleus (dashed square) and very close to the nuclear envelope (solid square). More detailed images of the positive signal for CD133 in the highlighted square areas, as indicated above, are shown on the right side. Representative labeling of CD133 in NSTS-28 cells is shown; CD133 was detected using 20 nm gold particles. Scale bars: (A) 250 nm and (B) 500 nm. (C) Nuclear/cytoplasmic fractionation followed by immunoblotting was performed using a rabbit polyclonal anti-CD133 primary antibody; α-tubulin and lamin B2 were used to confirm the purity of the fractions.
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f4-ijmm-36-01-0065: Detection of CD133 in the nuclei of rhabdomyosarcoma cells using transmission electron microscopy and immunoblot analysis. (A) Presence of CD133 in cell nuclei and nucleoli; more detailed images of the positive signal for CD133 in the highlighted square areas are presented on the bottom row. (B) The presence of CD133 in the cytoplasm near the nucleus (dashed square) and very close to the nuclear envelope (solid square). More detailed images of the positive signal for CD133 in the highlighted square areas, as indicated above, are shown on the right side. Representative labeling of CD133 in NSTS-28 cells is shown; CD133 was detected using 20 nm gold particles. Scale bars: (A) 250 nm and (B) 500 nm. (C) Nuclear/cytoplasmic fractionation followed by immunoblotting was performed using a rabbit polyclonal anti-CD133 primary antibody; α-tubulin and lamin B2 were used to confirm the purity of the fractions.

Mentions: Furthermore, we also used immunogold labeling with TEM to verify the localization of CD133 in the nuclei of the RMS cells. To avoid any artifacts associated with this methodological approach, the accumulation of three or more gold particles together was considered to indicate a positive signal. The results clearly indicated the presence of CD133 in both the nuclei and nucleoli (Fig. 4A and B).


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)

Detection of CD133 in the nuclei of rhabdomyosarcoma cells using transmission electron microscopy and immunoblot analysis. (A) Presence of CD133 in cell nuclei and nucleoli; more detailed images of the positive signal for CD133 in the highlighted square areas are presented on the bottom row. (B) The presence of CD133 in the cytoplasm near the nucleus (dashed square) and very close to the nuclear envelope (solid square). More detailed images of the positive signal for CD133 in the highlighted square areas, as indicated above, are shown on the right side. Representative labeling of CD133 in NSTS-28 cells is shown; CD133 was detected using 20 nm gold particles. Scale bars: (A) 250 nm and (B) 500 nm. (C) Nuclear/cytoplasmic fractionation followed by immunoblotting was performed using a rabbit polyclonal anti-CD133 primary antibody; α-tubulin and lamin B2 were used to confirm the purity of the fractions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijmm-36-01-0065: Detection of CD133 in the nuclei of rhabdomyosarcoma cells using transmission electron microscopy and immunoblot analysis. (A) Presence of CD133 in cell nuclei and nucleoli; more detailed images of the positive signal for CD133 in the highlighted square areas are presented on the bottom row. (B) The presence of CD133 in the cytoplasm near the nucleus (dashed square) and very close to the nuclear envelope (solid square). More detailed images of the positive signal for CD133 in the highlighted square areas, as indicated above, are shown on the right side. Representative labeling of CD133 in NSTS-28 cells is shown; CD133 was detected using 20 nm gold particles. Scale bars: (A) 250 nm and (B) 500 nm. (C) Nuclear/cytoplasmic fractionation followed by immunoblotting was performed using a rabbit polyclonal anti-CD133 primary antibody; α-tubulin and lamin B2 were used to confirm the purity of the fractions.
Mentions: Furthermore, we also used immunogold labeling with TEM to verify the localization of CD133 in the nuclei of the RMS cells. To avoid any artifacts associated with this methodological approach, the accumulation of three or more gold particles together was considered to indicate a positive signal. The results clearly indicated the presence of CD133 in both the nuclei and nucleoli (Fig. 4A and B).

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