Restoration of CCAAT enhancer binding protein α P42 induces myeloid differentiation and overcomes all-trans retinoic acid resistance in human acute promyelocytic leukemia NB4-R1 cells

Abstract

All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. We restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL.

Mathematical Analysis of Cytokine-Induced Differentiation of Granulocyte-Monocyte Progenitor Cells

“…At high concentrations of GM-CSF, C/EBP increases quickly, resulting in a swift rise in Gfi-1 and repression of PU.1, thereby inducing granulopoiesis. The result, that C/EBP is an antagonist of PU.1 in granulopoiesis, is in agreement with Wang et al ( 84 ), who showed that induction of an isoform of C/EBPα downregulates the SPI1 gene (encoding PU.1) to promote granulopoiesis. Alternatively, our model predicts that C/EBP has a positive impact on PU.1 in GM-CSF induced monopoiesis, as a result of a slower increase in C/EBP, allowing PU.1 enough time to upregulate itself and establish dominance.…”

Haploinsufficiency of NR3C1 drives glucocorticoid resistance in adult acute lymphoblastic leukemia cells by down-regulating the mitochondrial apoptosis axis, and is sensitive to Bcl-2 blockage

“…The C-terminal Myc-DDK-tagged pLenti plasmid carrying the full-length Homo sapiens NR3C1 coding sequence ( NR3C1 , 2334 bp, NM_000176; hereafter called pLenti-C-Myc-DDK-NR3C1) was purchased from Biowestern Technologies (Hangzhou, China). Detailed methods for lentivirus infection have been described previously [4]. In brief, lentivirus was produced by cotransfection of packaging plasmids (PSPAX2 and PMD2.G) and lentivirus vectors into HEK293T cells using Attractene Transfection Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.…”

“…RT-qPCR and western blotting analyses were carried out as previously described [4] with NR3C1 primers 5′-ATAGCTCTGTTCCAGACTCAACT-3′ (forward) and 5′-TCCTGAAACCTGGTATTGCCT-3′ (reverse). GAPDH primers were 5′-ACAACTTTGGTATCGTGGAAGG-3′ (forward) and 5′-GCCATCACGCCACAGTTTC-3′ (reverse).…”

Role of cofilin‑1 in arsenic trioxide‑induced apoptosis of NB4‑R1 cells

“…Therefore, further studies are required to illustrate the molecular mechanisms in APL and identify novel factors that regulate differentiation and apoptosis. NB4-R1 cells are naturally resistant to ATRA but sensitive to As 2 o 3 , thus this cell line is a useful model for the study of drug resistance and apoptosis (28,29). Through protein spectrum analysis, the present study identified that the expression of a number of proteins was significantly increased in NB4-R1 cells, a retinoid-resistant cell line, compared with that in NB4 cells, a retinoid-sensitive cell line.…”