The DNA Binding Property of PML/RARA but Not the Integrity of PML Nuclear Bodies Is Indispensable for Leukemic Transformation

Liu, Xi; Yuan, Hao; Pérès, Laurent; Chen, Sai‐Juan; Chen, Zhu; Zhou, Jun; Zhu, Jun

doi:10.1371/journal.pone.0104906

Cited by 4 publications

(2 citation statements)

References 33 publications

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“…HL-60 and CCRF-CEM human leukemic cell lines were extensively used as in vitro models in cancer research, for example to investigate anticancer drug action and gene/protein analysis (Fard et al, 2012;Perri et al, 2014;Wang et al, 2014). Both cell lines activated proto-oncogenes that lead to cell immortalization (Drexler et al, 2000;Liu et al, 2014). The main objective of this study was to screen the alteration of proteome profiles of HL-60 and CCRF-CEM cell due to epigenetic treatment by 5-Aza and TSA.…”

Section: Discussionmentioning

confidence: 99%

Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

Aziee

Yassim

Yusoff

et al. 2019

APJMBB

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Leukemia is classified as a malignant disease of hematopoietic stem cells (HSCs) that fails in cell differentiation but preserve their self-renewal. It is caused by genetic alterations and epigenetic modifications resulting in the activation or inactivation of particular genes for transcription. Epigenetic causes changes in gene expression without any alteration in the DNA sequence. The most common epigenetic modifications are DNA methylation and histone acetylation. 5-Azacitidine (5-Aza) is a DNA methytransferase inhibitor (DNMTi) that inhibits DNA methyltransferase enzymes resulting in hypomethylation. Trichostatin A (TSA) is a histone deacetylase inhibitor which inhibits deacetylation of both histone and non-histone proteins resulting in chromatin relaxation. This present study focused on the alteration of proteome profile on 2D gel electrophoresis (2-DE) induced by 5-Aza and TSA in HL-60 and CCRF-CEM cell lines as in vitro model to represent acute promyelocytic leukemia (APL) and T-lymphoblastic leukemia (T-ALL), respectively. Total proteins of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM cell lines were extracted using urea/thiourea buffer and stained with Coomassie Blue. Comparative analysis of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM was performed by PDQuest software. Qualitative analysis identified 190-659 protein spots detected in untreated, 5-Aza and TSA-treated HL-60 and CCRF-CEM. Quantitative comparison analysis was analyzed by over 2-fold change in 5-Aza/TSA-treated cells compared to untreated. One and eight upregulated proteins were detected in 5-Aza and TSA-treated HL-60, respectively. While five and one upregulated proteins were detected in 5-Aza and TSA-treated CCRF-CEM, respectively. These preliminary results suggested that 5-Aza and TSA induced proteome profiles alterations due to their inhibition effects in HL-60 and CCRF-CEM cell lines.

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Section: Discussionmentioning

confidence: 99%

Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

Aziee

Yassim

Yusoff

et al. 2019

APJMBB

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“…The importance of the PML-RARα gene has been confirmed in transgenic mice ( 16 ). Direct DNA-binding is indispensable for PML/RARA to transform hematopoietic cells and the disruption of PML-nuclear bodies is not sufficient for full cell leukemic transformation ( 17 ); however, disruption of PML may facilitate the acquisition of leukemia-promoting mutations by disabling oncogene-induced senescence ( 18 ).…”

Section: Discussionmentioning

confidence: 99%

Location of NLS-RARα protein in NB4 cell and nude mice

et al. 2017

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In the majority of acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RAα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. NLS-RARα promotes cell growth and inhibits differentiation in response to ATRA. However, the mechanisms by which NLS-RARα affects cell biological characteristics are yet to be fully elucidated. The present study found that the location of RARαwas altered after it was cleaved by NE. Firstly, NE was overexpressed during the preparation of recombinant plasmid NB-4/pCMV6-NE-Myc to cleave PML-RARα. The total protein expression levels of myc and NE and expression levels of NLS-RARα in nucleoprotein were detected by western blotting. Location of NLS-RARα protein was detected by immunofluorescence and confocal laser scanning. Secondly, a nude mice model was constructed and NE protein, NLS-RARα and RARα protein assays, and the location of NLS-RARα and RARα proteins were assessed as described. The present results showed that, compared with the control groups, the location of NLS-RARα protein was predominantly detected in the nucleus, whereas RARα was mainly distributed in the cytoplasm. These findings were consistent with those of the nude mice model, and these may be used as a foundation to explain the occurrence mechanism of APL.

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PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells

Katerndahl,

Rogers,

Day

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are “open” prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin “pioneering factor” GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA , Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA . These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

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The DNA Binding Property of PML/RARA but Not the Integrity of PML Nuclear Bodies Is Indispensable for Leukemic Transformation

Cited by 4 publications

References 33 publications

Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

Location of NLS-RARα protein in NB4 cell and nude mice

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells

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