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Merkel cell polyomavirus small T antigen mediates microtubule destabilization to promote cell motility and migration.

Knight LM, Stakaityte G, Wood JJ, Abdul-Sada H, Griffiths DA, Howell GJ, Wheat R, Blair GE, Steven NM, Macdonald A, Blackbourn DJ, Whitehouse A - J. Virol. (2014)

Bottom Line: These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin by its known association with the cellular phosphatase catalytic subunit PP4C.These findings highlight stathmin as a possible biomarker of MCC and as a target for novel antitumoral therapies.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.

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MCPyV ST expression leads to the differential expression of proteins involved in microtubule-associated cytoskeletal organization and dynamics. (A) (i) i293-ST cells were grown in DMEM with R0K0 and induced (IN) for 24 h or grown in DMEM with R6K4 and remained uninduced (UN). Cell lysates were analyzed by immunoblotting with a FLAG-specific antibody. (ii) To confirm that induced levels of MCPyV ST in i293-ST cells are representative of ST expression in the MCPyV-positive MCC cell lines, immunoblotting was performed using an MCPyV T-specific antibody comparing cell lysates from 1 × 105 cells of uninduced and induced i293-ST, MCC13, and MKL-1 cells. (B) i293-ST cells remained uninduced or were incubated for either 24 or 48 h in the presence of doxycycline hyclate. After induction, cell lysates were analyzed by immunoblotting using a FLAG-specific antibody and a range of microtubule-associated-specific antibodies highlighted by quantitative proteomic analysis. GAPDH was used as a measure of equal loading. (C) FFPE sections of a primary MCC tumor were stained with stathmin- and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was performed with bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (D) FFPE sections of two additional primary MCC tumors were stained with stathmin-, MCPyV LT-, and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was labeled using bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope.
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Figure 1: MCPyV ST expression leads to the differential expression of proteins involved in microtubule-associated cytoskeletal organization and dynamics. (A) (i) i293-ST cells were grown in DMEM with R0K0 and induced (IN) for 24 h or grown in DMEM with R6K4 and remained uninduced (UN). Cell lysates were analyzed by immunoblotting with a FLAG-specific antibody. (ii) To confirm that induced levels of MCPyV ST in i293-ST cells are representative of ST expression in the MCPyV-positive MCC cell lines, immunoblotting was performed using an MCPyV T-specific antibody comparing cell lysates from 1 × 105 cells of uninduced and induced i293-ST, MCC13, and MKL-1 cells. (B) i293-ST cells remained uninduced or were incubated for either 24 or 48 h in the presence of doxycycline hyclate. After induction, cell lysates were analyzed by immunoblotting using a FLAG-specific antibody and a range of microtubule-associated-specific antibodies highlighted by quantitative proteomic analysis. GAPDH was used as a measure of equal loading. (C) FFPE sections of a primary MCC tumor were stained with stathmin- and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was performed with bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (D) FFPE sections of two additional primary MCC tumors were stained with stathmin-, MCPyV LT-, and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was labeled using bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope.

Mentions: To analyze the effect of MCPyV ST expression on the cellular proteome, SILAC-based quantitative proteomics was performed utilizing a 293 Flp-In cell line capable of inducible MCPyV ST expression, termed i293-ST (15). The i293-ST line is a robust model for this analysis, as inducible levels of MCPyV ST are representative of ST expression in an MCPyV-positive MCC cell line, MKL-1 (Fig. 1A). Bioinformatic analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (51) highlighted that a significant proportion of the highly differentially expressed proteins were implicated in gene ontology groupings involving microtubule-associated cytoskeletal organization and dynamics (see Fig. S1 in the supplemental material), which is the focus of this study. Figure S2 in the supplemental material summarizes the differential expression of proteins associated with other gene ontology groupings.


Merkel cell polyomavirus small T antigen mediates microtubule destabilization to promote cell motility and migration.

Knight LM, Stakaityte G, Wood JJ, Abdul-Sada H, Griffiths DA, Howell GJ, Wheat R, Blair GE, Steven NM, Macdonald A, Blackbourn DJ, Whitehouse A - J. Virol. (2014)

MCPyV ST expression leads to the differential expression of proteins involved in microtubule-associated cytoskeletal organization and dynamics. (A) (i) i293-ST cells were grown in DMEM with R0K0 and induced (IN) for 24 h or grown in DMEM with R6K4 and remained uninduced (UN). Cell lysates were analyzed by immunoblotting with a FLAG-specific antibody. (ii) To confirm that induced levels of MCPyV ST in i293-ST cells are representative of ST expression in the MCPyV-positive MCC cell lines, immunoblotting was performed using an MCPyV T-specific antibody comparing cell lysates from 1 × 105 cells of uninduced and induced i293-ST, MCC13, and MKL-1 cells. (B) i293-ST cells remained uninduced or were incubated for either 24 or 48 h in the presence of doxycycline hyclate. After induction, cell lysates were analyzed by immunoblotting using a FLAG-specific antibody and a range of microtubule-associated-specific antibodies highlighted by quantitative proteomic analysis. GAPDH was used as a measure of equal loading. (C) FFPE sections of a primary MCC tumor were stained with stathmin- and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was performed with bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (D) FFPE sections of two additional primary MCC tumors were stained with stathmin-, MCPyV LT-, and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was labeled using bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4301106&req=5

Figure 1: MCPyV ST expression leads to the differential expression of proteins involved in microtubule-associated cytoskeletal organization and dynamics. (A) (i) i293-ST cells were grown in DMEM with R0K0 and induced (IN) for 24 h or grown in DMEM with R6K4 and remained uninduced (UN). Cell lysates were analyzed by immunoblotting with a FLAG-specific antibody. (ii) To confirm that induced levels of MCPyV ST in i293-ST cells are representative of ST expression in the MCPyV-positive MCC cell lines, immunoblotting was performed using an MCPyV T-specific antibody comparing cell lysates from 1 × 105 cells of uninduced and induced i293-ST, MCC13, and MKL-1 cells. (B) i293-ST cells remained uninduced or were incubated for either 24 or 48 h in the presence of doxycycline hyclate. After induction, cell lysates were analyzed by immunoblotting using a FLAG-specific antibody and a range of microtubule-associated-specific antibodies highlighted by quantitative proteomic analysis. GAPDH was used as a measure of equal loading. (C) FFPE sections of a primary MCC tumor were stained with stathmin- and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was performed with bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (D) FFPE sections of two additional primary MCC tumors were stained with stathmin-, MCPyV LT-, and CK20-specific antibodies or an isotype negative control. After washing, sections were incubated with Alexa Fluor-labeled secondary antibodies. Nuclear staining was labeled using bis-benzimide. Slides were then analyzed using a Zeiss LSM 510 confocal laser scanning microscope.
Mentions: To analyze the effect of MCPyV ST expression on the cellular proteome, SILAC-based quantitative proteomics was performed utilizing a 293 Flp-In cell line capable of inducible MCPyV ST expression, termed i293-ST (15). The i293-ST line is a robust model for this analysis, as inducible levels of MCPyV ST are representative of ST expression in an MCPyV-positive MCC cell line, MKL-1 (Fig. 1A). Bioinformatic analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (51) highlighted that a significant proportion of the highly differentially expressed proteins were implicated in gene ontology groupings involving microtubule-associated cytoskeletal organization and dynamics (see Fig. S1 in the supplemental material), which is the focus of this study. Figure S2 in the supplemental material summarizes the differential expression of proteins associated with other gene ontology groupings.

Bottom Line: These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin by its known association with the cellular phosphatase catalytic subunit PP4C.These findings highlight stathmin as a possible biomarker of MCC and as a target for novel antitumoral therapies.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.

Show MeSH
Related in: MedlinePlus