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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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PRMT5 knockdown induces apoptosis in the NGP cell-line, but not in the SH-SY5Y cell-line. (A) Cell death triggered by PRMT5 in NGP cells, together with knockdown verification by immunoblotting. No phenotypic changes were observed in SH-SY5Y cells. (B) Quantification of NGP cell death by cell counting, the asterisk signifying significance (P < 0.05). (C) Confirmation of increased cleaved PARP (cPARP) by immunoblotting following PRMT5 knockdown.
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fig2: PRMT5 knockdown induces apoptosis in the NGP cell-line, but not in the SH-SY5Y cell-line. (A) Cell death triggered by PRMT5 in NGP cells, together with knockdown verification by immunoblotting. No phenotypic changes were observed in SH-SY5Y cells. (B) Quantification of NGP cell death by cell counting, the asterisk signifying significance (P < 0.05). (C) Confirmation of increased cleaved PARP (cPARP) by immunoblotting following PRMT5 knockdown.

Mentions: We evaluated the effect of short-interfering RNA (siRNA) – mediated PRMT5 depletion on the SK-N-BE(2)C cell-line, representative of the poor prognosis subset of NB, together with two other HMTs known to be involved in cancer. Knockdown of CARM1 (PRMT4), EZH2 and PRMT5 all induced some cell death in SK-N-BE(2)C cells, but PRMT5 depletion triggered a significantly greater effect with approximately double the cell death compared to the negative control siRNA, and substantially greater death than either CARM1 or EZH2 knockdowns. Treatment of knockdowns with the caspase inhibitor Q-VD-OPh rescued cell death, and apoptosis was further confirmed by immunoblotting for poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (Figure 1A–C). PRMT5 depletion also induced cell death in NGP cells, but not in SH-SY5Y NB cells or normal fibroblasts, despite efficient knockdowns (Figure 2 and Figure S1). PRMT5 depletion also had little effect on SHEP cells without high MYCN (see below). Increased cell death and PARP cleavage consistent with apoptosis was also apparent in NGP cells transfected with PRMT5 siRNA (Figure 2B and C).


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)

PRMT5 knockdown induces apoptosis in the NGP cell-line, but not in the SH-SY5Y cell-line. (A) Cell death triggered by PRMT5 in NGP cells, together with knockdown verification by immunoblotting. No phenotypic changes were observed in SH-SY5Y cells. (B) Quantification of NGP cell death by cell counting, the asterisk signifying significance (P < 0.05). (C) Confirmation of increased cleaved PARP (cPARP) by immunoblotting following PRMT5 knockdown.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
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fig2: PRMT5 knockdown induces apoptosis in the NGP cell-line, but not in the SH-SY5Y cell-line. (A) Cell death triggered by PRMT5 in NGP cells, together with knockdown verification by immunoblotting. No phenotypic changes were observed in SH-SY5Y cells. (B) Quantification of NGP cell death by cell counting, the asterisk signifying significance (P < 0.05). (C) Confirmation of increased cleaved PARP (cPARP) by immunoblotting following PRMT5 knockdown.
Mentions: We evaluated the effect of short-interfering RNA (siRNA) – mediated PRMT5 depletion on the SK-N-BE(2)C cell-line, representative of the poor prognosis subset of NB, together with two other HMTs known to be involved in cancer. Knockdown of CARM1 (PRMT4), EZH2 and PRMT5 all induced some cell death in SK-N-BE(2)C cells, but PRMT5 depletion triggered a significantly greater effect with approximately double the cell death compared to the negative control siRNA, and substantially greater death than either CARM1 or EZH2 knockdowns. Treatment of knockdowns with the caspase inhibitor Q-VD-OPh rescued cell death, and apoptosis was further confirmed by immunoblotting for poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (Figure 1A–C). PRMT5 depletion also induced cell death in NGP cells, but not in SH-SY5Y NB cells or normal fibroblasts, despite efficient knockdowns (Figure 2 and Figure S1). PRMT5 depletion also had little effect on SHEP cells without high MYCN (see below). Increased cell death and PARP cleavage consistent with apoptosis was also apparent in NGP cells transfected with PRMT5 siRNA (Figure 2B and C).

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