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Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Liu L, Wu J, Ong SS, Chen T - PLoS ONE (2013)

Bottom Line: Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1.In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430).Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.

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Related in: MedlinePlus

Fascaplysin treatment does not affect the levels of PAX3-FOXO1 in Rh30 cells.A) Fascaplysin reduced the mRNA levels of CPT1A but not PAX3-FOXO1 (PF) in Rh30 cells. B) Fascaplysin did not affect protein levels of PAX3-FOXO1. Top panel: Western blot using anti-FOXO1 antibody. Bottom panel: Western blot using anti-actin antibody. Cells were treated with indicated concentrations of fascaplysin (Fas) for 2 h. M, protein molecular weight marker.
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pone-0058193-g004: Fascaplysin treatment does not affect the levels of PAX3-FOXO1 in Rh30 cells.A) Fascaplysin reduced the mRNA levels of CPT1A but not PAX3-FOXO1 (PF) in Rh30 cells. B) Fascaplysin did not affect protein levels of PAX3-FOXO1. Top panel: Western blot using anti-FOXO1 antibody. Bottom panel: Western blot using anti-actin antibody. Cells were treated with indicated concentrations of fascaplysin (Fas) for 2 h. M, protein molecular weight marker.

Mentions: To determine the mechanism by which fascaplysin inhibits the transcriptional activity of PAX3-FOXO1, we first analyzed the mRNA and protein levels of PAX3-FOXO1 in response to 2 h of fascaplysin treatment in Rh30 cells. As shown in Fig. 4, fascaplysin decreased the transcriptional activity of PAX3-FOXO1, as evidenced by the decreased mRNA levels of CPT1A, without decreasing the mRNA (Fig. 4A) or protein (Fig. 4B) levels of PAX3-FOXO1, suggesting that some mechanism other than decreased protein levels is responsible for the inhibitory effect of fascaplysin on PAX3-FOXO1.


Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Liu L, Wu J, Ong SS, Chen T - PLoS ONE (2013)

Fascaplysin treatment does not affect the levels of PAX3-FOXO1 in Rh30 cells.A) Fascaplysin reduced the mRNA levels of CPT1A but not PAX3-FOXO1 (PF) in Rh30 cells. B) Fascaplysin did not affect protein levels of PAX3-FOXO1. Top panel: Western blot using anti-FOXO1 antibody. Bottom panel: Western blot using anti-actin antibody. Cells were treated with indicated concentrations of fascaplysin (Fas) for 2 h. M, protein molecular weight marker.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585270&req=5

pone-0058193-g004: Fascaplysin treatment does not affect the levels of PAX3-FOXO1 in Rh30 cells.A) Fascaplysin reduced the mRNA levels of CPT1A but not PAX3-FOXO1 (PF) in Rh30 cells. B) Fascaplysin did not affect protein levels of PAX3-FOXO1. Top panel: Western blot using anti-FOXO1 antibody. Bottom panel: Western blot using anti-actin antibody. Cells were treated with indicated concentrations of fascaplysin (Fas) for 2 h. M, protein molecular weight marker.
Mentions: To determine the mechanism by which fascaplysin inhibits the transcriptional activity of PAX3-FOXO1, we first analyzed the mRNA and protein levels of PAX3-FOXO1 in response to 2 h of fascaplysin treatment in Rh30 cells. As shown in Fig. 4, fascaplysin decreased the transcriptional activity of PAX3-FOXO1, as evidenced by the decreased mRNA levels of CPT1A, without decreasing the mRNA (Fig. 4A) or protein (Fig. 4B) levels of PAX3-FOXO1, suggesting that some mechanism other than decreased protein levels is responsible for the inhibitory effect of fascaplysin on PAX3-FOXO1.

Bottom Line: Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1.In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430).Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.

Show MeSH
Related in: MedlinePlus