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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 inhibits growth of ARMS cells.A) ARMS cells (Rh30 and Rh41) and ERMS cells (RD and JR1) were treated with 0.69 µM fascaplysin for indicated times before cell viability was determined. Fascaplysin at 0.69 µM started to inhibit cell viability of Rh30 cells as shown in Figure 1B. B) Cdk4 levels in various cell lines.
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pone-0058193-g006: Fascaplysin inhibits growth of ARMS cells.A) ARMS cells (Rh30 and Rh41) and ERMS cells (RD and JR1) were treated with 0.69 µM fascaplysin for indicated times before cell viability was determined. Fascaplysin at 0.69 µM started to inhibit cell viability of Rh30 cells as shown in Figure 1B. B) Cdk4 levels in various cell lines.

Mentions: Downregulating PAX3-FOXO1 by using RNA interference (RNAi) markedly decreased anchorage-independent growth and cell motility of Rh30 cells [7], [13]. Although fascaplysin is toxic to both ARMS (Rh41 and Rh30; PAX3-FOXO1–positive) and ERMS cells (RD and JR-1; PAX3-FOXO1–negative), ARMS cells were more sensitive to fascaplysin than ERMS cells in a cell viability assay (Fig. 6A). All cell lines tested expressed Cdk4 (Fig. 6B), which is in agreement with previously reported results [19]. Interestingly, Rh41, in which the level of endogenous PAX3-FOXO1 is much higher than in Rh30 [20], was most sensitive to fascaplysin. The level of Cdk4 is not highest in Rh41, suggesting that the sensitivity to fascaplysin might be determined by the levels of both Cdk4 and PAX3-FOXO1 in ARMS cells, and the inhibitory effect of fascaplysin on ARMS cells is PAX3-FOXO1–dependent.


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 inhibits growth of ARMS cells.A) ARMS cells (Rh30 and Rh41) and ERMS cells (RD and JR1) were treated with 0.69 µM fascaplysin for indicated times before cell viability was determined. Fascaplysin at 0.69 µM started to inhibit cell viability of Rh30 cells as shown in Figure 1B. B) Cdk4 levels in various cell lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0058193-g006: Fascaplysin inhibits growth of ARMS cells.A) ARMS cells (Rh30 and Rh41) and ERMS cells (RD and JR1) were treated with 0.69 µM fascaplysin for indicated times before cell viability was determined. Fascaplysin at 0.69 µM started to inhibit cell viability of Rh30 cells as shown in Figure 1B. B) Cdk4 levels in various cell lines.
Mentions: Downregulating PAX3-FOXO1 by using RNA interference (RNAi) markedly decreased anchorage-independent growth and cell motility of Rh30 cells [7], [13]. Although fascaplysin is toxic to both ARMS (Rh41 and Rh30; PAX3-FOXO1–positive) and ERMS cells (RD and JR-1; PAX3-FOXO1–negative), ARMS cells were more sensitive to fascaplysin than ERMS cells in a cell viability assay (Fig. 6A). All cell lines tested expressed Cdk4 (Fig. 6B), which is in agreement with previously reported results [19]. Interestingly, Rh41, in which the level of endogenous PAX3-FOXO1 is much higher than in Rh30 [20], was most sensitive to fascaplysin. The level of Cdk4 is not highest in Rh41, suggesting that the sensitivity to fascaplysin might be determined by the levels of both Cdk4 and PAX3-FOXO1 in ARMS cells, and the inhibitory effect of fascaplysin on ARMS cells is PAX3-FOXO1–dependent.

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