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Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, Catchpoole D, Deery MJ, Malik K - Mol Oncol (2014)

Bottom Line: PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells.By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein.Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.

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Cell-cycle changes accompanying PRMT5 knockdowns. Following PRMT5 knockdown, moderate increases in G1 were apparent in NGP and Kelly cell-lines, and statistically significant G1-arrest was apparent in uninduced SHEP-Tet21N cells (asterisked, P < 0.0001, Student's t-test). In induced SHEP-Tet21N cells, no G1-arrest was evident; however a significant G2/M-phase decrease was apparent (asterisked, P < 0.005). The latter analysis was done with duplicate samples.
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fig6: Cell-cycle changes accompanying PRMT5 knockdowns. Following PRMT5 knockdown, moderate increases in G1 were apparent in NGP and Kelly cell-lines, and statistically significant G1-arrest was apparent in uninduced SHEP-Tet21N cells (asterisked, P < 0.0001, Student's t-test). In induced SHEP-Tet21N cells, no G1-arrest was evident; however a significant G2/M-phase decrease was apparent (asterisked, P < 0.005). The latter analysis was done with duplicate samples.

Mentions: In neuronal progenitor cells, MYCN is proteosomally regulated during mitosis (Sjostrom et al., 2005), so we investigated whether the PRMT5-depletion effect on MYCN might be indirect and attributable to solely to G1 cell-cycle arrest. Whilst a degree of G1 arrest was apparent in NGP and Kelly cells after PRMT5 knockdown, it was below statistical significance (Figure 6A and B). Furthermore, G1 arrest was not observed after PRMT5 knockdown in SHEP-Tet21N cells overexpressing MYCN, in contrast to uninduced SHEP-Tet21N cells where G1 arrest was pronounced (Figure 6C). Interestingly however, MYCN-induced SHEP-Tet21N cells exhibited a significant decrease of cells in G2/M-phase after PRMT5 knockdown (P < 0.005), concomitant with an increase of cells in S-phase. A similar effect on S-phase has been demonstrated for c-myc in medulloblastoma cells (Zhang et al., 2006). Together these experiments strongly support PRMT5 being able to directly influence MYCN protein stability and modulate its biological activities.


Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, Catchpoole D, Deery MJ, Malik K - Mol Oncol (2014)

Cell-cycle changes accompanying PRMT5 knockdowns. Following PRMT5 knockdown, moderate increases in G1 were apparent in NGP and Kelly cell-lines, and statistically significant G1-arrest was apparent in uninduced SHEP-Tet21N cells (asterisked, P < 0.0001, Student's t-test). In induced SHEP-Tet21N cells, no G1-arrest was evident; however a significant G2/M-phase decrease was apparent (asterisked, P < 0.005). The latter analysis was done with duplicate samples.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359099&req=5

fig6: Cell-cycle changes accompanying PRMT5 knockdowns. Following PRMT5 knockdown, moderate increases in G1 were apparent in NGP and Kelly cell-lines, and statistically significant G1-arrest was apparent in uninduced SHEP-Tet21N cells (asterisked, P < 0.0001, Student's t-test). In induced SHEP-Tet21N cells, no G1-arrest was evident; however a significant G2/M-phase decrease was apparent (asterisked, P < 0.005). The latter analysis was done with duplicate samples.
Mentions: In neuronal progenitor cells, MYCN is proteosomally regulated during mitosis (Sjostrom et al., 2005), so we investigated whether the PRMT5-depletion effect on MYCN might be indirect and attributable to solely to G1 cell-cycle arrest. Whilst a degree of G1 arrest was apparent in NGP and Kelly cells after PRMT5 knockdown, it was below statistical significance (Figure 6A and B). Furthermore, G1 arrest was not observed after PRMT5 knockdown in SHEP-Tet21N cells overexpressing MYCN, in contrast to uninduced SHEP-Tet21N cells where G1 arrest was pronounced (Figure 6C). Interestingly however, MYCN-induced SHEP-Tet21N cells exhibited a significant decrease of cells in G2/M-phase after PRMT5 knockdown (P < 0.005), concomitant with an increase of cells in S-phase. A similar effect on S-phase has been demonstrated for c-myc in medulloblastoma cells (Zhang et al., 2006). Together these experiments strongly support PRMT5 being able to directly influence MYCN protein stability and modulate its biological activities.

Bottom Line: PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells.By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein.Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus