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S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells

Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21(cip1) in glioblastoma and other cancer cell lines. MGMT protein was show...

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Autores principales: Mostofa, AGM, Punganuru, Surendra R., Madala, Hanumantha Rao, Srivenugopal, Kalkunte S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Neoplasia Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909491/
https://www.ncbi.nlm.nih.gov/pubmed/29510343
http://dx.doi.org/10.1016/j.neo.2018.01.010
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author Mostofa, AGM
Punganuru, Surendra R.
Madala, Hanumantha Rao
Srivenugopal, Kalkunte S.
author_facet Mostofa, AGM
Punganuru, Surendra R.
Madala, Hanumantha Rao
Srivenugopal, Kalkunte S.
author_sort Mostofa, AGM
collection PubMed
description Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21(cip1) in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4(Cdt2) ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.
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spelling pubmed-59094912018-04-23 S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells Mostofa, AGM Punganuru, Surendra R. Madala, Hanumantha Rao Srivenugopal, Kalkunte S. Neoplasia Original article Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21(cip1) in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4(Cdt2) ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process. Neoplasia Press 2018-03-03 /pmc/articles/PMC5909491/ /pubmed/29510343 http://dx.doi.org/10.1016/j.neo.2018.01.010 Text en © 2018 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. 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
Mostofa, AGM
Punganuru, Surendra R.
Madala, Hanumantha Rao
Srivenugopal, Kalkunte S.
S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells
title S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells
title_full S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells
title_fullStr S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells
title_full_unstemmed S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells
title_short S-phase Specific Downregulation of Human O(6)-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21(cip1) Proteins in Glioma Cells
title_sort s-phase specific downregulation of human o(6)-methylguanine dna methyltransferase (mgmt) and its serendipitous interactions with pcna and p21(cip1) proteins in glioma cells
topic Original article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909491/
https://www.ncbi.nlm.nih.gov/pubmed/29510343
http://dx.doi.org/10.1016/j.neo.2018.01.010
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