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The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexp...

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Autores principales: Huun, Johanna, Gansmo, Liv B., Mannsåker, Bård, Iversen, Gjertrud T., Sommerfelt-Pettersen, Jan, Øvrebø, Jan Inge, Lønning, Per E., Knappskog, Stian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Neoplasia Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576977/
https://www.ncbi.nlm.nih.gov/pubmed/28844019
http://dx.doi.org/10.1016/j.tranon.2017.07.006
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author Huun, Johanna
Gansmo, Liv B.
Mannsåker, Bård
Iversen, Gjertrud T.
Sommerfelt-Pettersen, Jan
Øvrebø, Jan Inge
Lønning, Per E.
Knappskog, Stian
author_facet Huun, Johanna
Gansmo, Liv B.
Mannsåker, Bård
Iversen, Gjertrud T.
Sommerfelt-Pettersen, Jan
Øvrebø, Jan Inge
Lønning, Per E.
Knappskog, Stian
author_sort Huun, Johanna
collection PubMed
description BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P = 4.3 × 10(−4) and P = 7.1 × 10(−4), respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA (P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.
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spelling pubmed-55769772017-09-06 The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells Huun, Johanna Gansmo, Liv B. Mannsåker, Bård Iversen, Gjertrud T. Sommerfelt-Pettersen, Jan Øvrebø, Jan Inge Lønning, Per E. Knappskog, Stian Transl Oncol Original article BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P = 4.3 × 10(−4) and P = 7.1 × 10(−4), respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA (P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene. Neoplasia Press 2017-08-29 /pmc/articles/PMC5576977/ /pubmed/28844019 http://dx.doi.org/10.1016/j.tranon.2017.07.006 Text en © 2017 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original article
Huun, Johanna
Gansmo, Liv B.
Mannsåker, Bård
Iversen, Gjertrud T.
Sommerfelt-Pettersen, Jan
Øvrebø, Jan Inge
Lønning, Per E.
Knappskog, Stian
The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells
title The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells
title_full The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells
title_fullStr The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells
title_full_unstemmed The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells
title_short The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells
title_sort functional roles of the mdm2 splice variants p2-mdm2-10 and mdm2-∆5 in breast cancer cells
topic Original article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576977/
https://www.ncbi.nlm.nih.gov/pubmed/28844019
http://dx.doi.org/10.1016/j.tranon.2017.07.006
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