The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

Santana-Lemos, Bárbara A.; Lange, Ana Paula Alencar de Lima; Benício, Mariana Tereza de Lira; José, Thiago Donizete da Silva; Lucena-Araújo, Antonio R.; Krause, Alexandre; Thomé, Carolina Hassibe; Rego, Eduardo Magalhães

doi:10.3324/haematol.2010.028365

Cited by 14 publications

(12 citation statements)

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“…The impact of CEBPA methylation on its expression remains controversial in AML samples. Fasan et al [24] observed a weak inverse correlation between decreased CEBPA level with CEBPA methylation in cytogenetically normal patients, whereas Santana-Lemos et al [21] did not find this association in acute promyelocytic leukemia. In this study, we disclosed the lower level of CEBPA transcript in patients with high methylation in spite of the statistically insignificant difference possibly due to the small number of patients analyzed.…”

Section: Discussionmentioning

confidence: 97%

“…CEBPA down-regulation has been identified in AML especially in patients with t(8;21) [21,31,32]. The impact of CEBPA methylation on its expression remains controversial in AML samples.…”

Section: Discussionmentioning

confidence: 98%

“…So far, single CEBPA mutations (alone or in combination) are not enough to transform normal human hematopoietic stem/progenitors (HSC/ HPCs) into leukemic cells [15]. Additionally, hypermethylation of CEBPA promoter has also been identified in AML [16][17][18][19][20][21][22][23][24]. CEBPA promoter methylation and mutation were mutually exclusive in AML [18,24].…”

Section: Introductionmentioning

confidence: 99%

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CEBPA methylation and mutation in myelodysplastic syndrome

Wen

Yang

et al. 2015

Med Oncol

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Aberrant methylation of CCAAT/enhancer-binding protein alpha (CEBPA) promoter has been observed in acute myeloid leukemia. However, little is known about CEBPA promoter in myelodysplastic syndrome (MDS). The purpose of this study was to investigate the alteration of CEBPA promoter in MDS patients and further determine the association with CEBPA expression and mutation. CEBPA promoter was significantly methylated in 105 MDS patients compared to 22 controls (median 0.016 vs. 0.000) (P < 0.0001). Receiver operating characteristic curve analysis discriminated all patients or cytogenetically normal patients from normal controls. Three cases (3 %) were identified with single-mutated CEBPA and one (1 %) with double-mutated CEBPA. CEBPA methylation and mutation occurred mutually exclusive. No significant correlation was found between CEBPA expression and methylation (P = 0.586). Our findings indicate that CEBPA methylation is a common event in MDS, but could not act as a prognostic biomarker for MDS patients.

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Section: Discussionmentioning

confidence: 97%

“…CEBPA down-regulation has been identified in AML especially in patients with t(8;21) [21,31,32]. The impact of CEBPA methylation on its expression remains controversial in AML samples.…”

Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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CEBPA methylation and mutation in myelodysplastic syndrome

Wen

Yang

et al. 2015

Med Oncol

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“…16 Further, the more relaxed DNA binding conformation of the homodimerized fusion protein allows targeting of an extended number of sites, 17 potentially contributing to gene deregulation at numerous sites in the genome. In leukemic cells, repression of two important regulators of myeloid cell development, C/EBPA and PU.1 has been described, [18][19][20] including repression of PU.1 targets, 21 effects which have been suggested to contribute to leukemogenesis. However, the transcriptional repression model of PML/RARA was built mostly on studies of fully transformed cells and cell lines, making it difficult to distinguish the contribution of PML/RARA from that of secondary events.…”

Section: Introductionmentioning

confidence: 99%

Transcription and methylation analysis of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

et al. 2015

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© F e r r a t a S t o r t i F o u n d a t i o nPML/RARA preleukemic myeloid precursors eventually give rise to leukemic promyelocytes. Methods Murine model and bone marrow harvestMRP8-PML/RARA transgenic mice have been described elsewhere. 3 Mice were bred and maintained at the University of California, San Francisco under standard conditions. For each individual sample, bones from one male and one female were pooled, thoroughly cleaned, ground and filtered. Cells were collected after centrifugation on Histopaque-1119 (Sigma #11191) and CKIT enrichment . Staining and flow cytometryCells were stained with a lymphocyte/erythrocyte/stem cell cocktail followed by secondary antibody. After blocking (Sigma #I4131), the suspension was stained with the specific cocktail. Live cells were double-sorted and purity after sorting was assessed. Compensation tubes were prepared by staining FVB/n spleen with fluorophore-conjugated CD45R/B220 antibodies. RNA isolation and microarray analysisCells (25,000-50,000) were double-sorted into Trizol (Life Technologies #15596). Following phenol/chloroform extraction, RNA was purified on PicoPure columns (Life Technologies #KIT0204). Sample preparation, labeling, and array hybridizations were performed according to standard protocols from the University of California, San Francisco Shared Microarray Core Facilities (http://www.arrays.ucsf.edu/protocols/) and Agilent Technologies. The microarray was run on a murine Agilent platform comprising 41,174 probes, including 28,283 targeting protein-coding genes (27,285 known + 998 predicted), and 1,484 targeting non-coding RNA (950 known + 534 predicted). The dataset was quantile normalized 22 without background subtraction. Differential expression analysis using moderated t statistic with Benjamini-Hochberg false discovery rate (adjusted P value) correction was performed using the limma R/Bioconductor package. 23 Below, biological replicates refer to samples harvested from different litters, over the course of several months, while a technical replicate refers to the same biological sample run twice on the array. For the preleukemic versus normal study, data are representative of three or four biological replicates/population and are available on GEO (GSE54474). For the leukemic versus preleukemic study, data are representative of two biological replicates for the preleukemic group (plus 1 technical duplicate) and three biological replicates for the leukemic group (plus 1 biological and 1 technical duplicate) and are also available on GEO (GSE59431).Genomic DNA isolation and genome-wide methylation analysis by enhanced reduced representation bisulfite sequencing Cells (25,000) were double-sorted and genomic DNA was extracted using the PureGene kit (Qiagen). DNA (25 ng) was used to perform the enhanced reduced representation bisulfite sequencing (ERRBS) assay as previously described 24 and sequenced on a HiSeq2000 Illumina sequencer. Reads were aligned against a bisulfite-converted mm9 genome using Bowtie. 25 Downstream analysis was pe...

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“…Outras alterações moleculares, tais como em C/EBPα e em MYC concomitantes a ocorrência de PML-RARα, já foram demonstradas na LPA e associadas a leucemogênese desse tipo de leucemia (Santana-Lemos et al, 2011;Jones et al, 2010).…”

Section: Leucemiaunclassified

Análise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico

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Análise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico UNIVERSIDADE DE SÃO PAULO FACULDADE DE CIÊNCIAS FARMACÊUTICAS DE RIBEIRÃO PRETOAnálise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico Heterogeneous nuclear ribonucleoprotein K (hnRNP K) and endogenous inhibitor of phosphatase 2A (SET) are overexpressed and proposed as prognostic markers in acute and chronic myeloid leukemia. The study aim was to characterize the hnRNP K and SET proteins involvement in acute promyelocytic leukemia leukemia (APL) leukemogenesis as well as all-trans retinoic acid (ATRA) induced cell differentiation. Initial qRT-PCR results demonstrate that HNRNPK and SET mRNA levels are increased in patients diagnosed with APL compare to samples from healthy donors and decrease after induction and during maintenance of treatment. The results were validated by Western blot, suggesting hnRNP K and SET as diagnostic and response to treatment markers. The knockdown of hnRNP K and SET was performed on sensitive, NB4, and ATRA-resistant, NB4-R2, LPA cells using interfering RNA. Both proteins were also tested as a therapeutic target with a use of hnRNP K (U0126) and SET inhibitors (OP449 and FTY720). The decrease of hnRNP K in cells led to increased granulocyte cell differentiation in both cells, especially in the presence of ATRA, and thus was able to reverse the NB4-R2 cells resistance to ATRA phenotype. The hnRNP K knockdown, as well as the treatment with U0126, had a loss of cell viability by induction of apoptosis accompanied by cleavage of the SET protein. The SET knockdown in APL cells confirmed that an induction of apoptosis in cells with hnRNP K knockdown occurred by cleavage and not by the SET protein decrease in the cells. Furthermore, it has also shown that SET impairs the ATRA's performance in the cellular differentiation process. The in vivo model using NB4-R2 transplant in nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has a greater potential against tumor progression compared to the treatment isolated with ATO. FTY720 and OP449 have significant anti-leukemic effect reducing cell viability. When in combination, FTY720 and OP449, they had a significant synergistic effect on NB4-R2 (CDI <0.7). We conclude that overexpression of hnRNP K and SET contributes to block cell differentiation in promyelocytes and impair the performance of ATRA in the treatment of APL and therefore hnRNPK in association with a SET protein represent a potential therapeutic target for anti-leukemic therapy of APL, mainly for patients resistant to ATRA.

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The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

Abstract: ABSTRACTinformed consent under a protocol approved by the Medical School of Ribeirão Preto, University of São Paulo.

Cited by 14 publications

References 18 publications

CEBPA methylation and mutation in myelodysplastic syndrome

CEBPA methylation and mutation in myelodysplastic syndrome

Transcription and methylation analysis of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

Análise da proteína hnRNP K nas linhagens celulares NB4 e NB4-R2 de leucemia promielocítica aguda com ênfase na patogênese e na diferenciação celular pelo ácido all-trans retinoico

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