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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (4) : 420-429     DOI: 10.1007/s11684-016-0478-3
RESEARCH ARTICLE |
Repression of CDKN2C caused by PML/RARα binding promotes the proliferation and differentiation block in acute promyelocytic leukemia
Xiaoling Wang1,Yun Tan1,Yizhen Li1,Jingming Li1,Wen Jin1,2,3(),Kankan Wang1,2,3()
1. State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China
3. Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Abstract  

Inappropriate cell proliferation during oncogenesis is often accompanied by inactivation of components involved in the cell cycle machinery. Here, we report that cyclin-dependent kinase inhibitor 2C (CDKN2C) as a member of the cyclin-dependent kinase inhibitors is a target of the PML/RARα oncofusion protein in leukemogenesis of acute promyelocytic leukemia (APL). We found that CDKN2C was markedly downregulated in APL blasts compared with normal promyelocytes. Chromatin immunoprecipitation combined with quantitative polymerase chain reaction demonstrated that PML/RARα directly bound to the CDKN2C promoter in the APL patient-derived cell line NB4. Luciferase assays indicated that PML/RARα inhibited the CDKN2C promoter activity in a dose-dependent manner. Furthermore, all-trans retinoic acid treatment induced CDKN2C expression by releasing the PML/RARα binding on chromatin in NB4 cells. Functional studies showed that ectopic expression of CDKN2C induced a cell cycle arrest at the G0/G1 phase and a partial differentiation in NB4 cells. Finally, the transcriptional regulation of CDKN2C was validated in primary APL patient samples. Collectively, this study highlights the importance of CDKN2C inactivation in the abnormal cell cycle progression and differentiation block of APL cells and may provide new insights into the study of pathogenesis and targeted therapy of APL.

Keywords CDKN2C      acute promyelocytic leukemia      cell cycle arrest      differentiation     
Corresponding Authors: Wen Jin,Kankan Wang   
Just Accepted Date: 25 October 2016   Online First Date: 23 November 2016    Issue Date: 01 December 2016
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0478-3     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I4/420
Fig.1  Repression of CDKN2C by PML/RARα in APL cells. (A) CDKN2C was significantly lower expressed in APL patient cells compared with the normal promyelocytes. We analyzed the mRNA expression levels of CDKN2C in APL patient samples (n = 14) and normal promyelocytes (n = 5) and compared their absolute intensities of mRNA expression values after log transformation. ** P<0.01. (B) The expression level of CDKN2C was repressed by PML/RARα. The mRNA expression levels of PML/RARα and CDKN2C were examined five days after transfection of the expression plasmid of PML/RARα or the empty vector into U937 cells.
Fig.2  Direct binding of PML/RARα on the CDKN2C promoter in APL cells. (A) Overview of PML/RARα binding sites on the CDKN2C promoter in NB4 cells. ChIP assays were carried out in NB4 cells using anti-PML and anti-RARα antibodies. The arrow denotes the transcription direction of CDKN2C. The peaks represent the enriched regions ChIPed by anti-PML and anti-RARα antibodies. (B) PML/RARα was significantly enriched on the CDKN2C promoter in NB4 cells. The DNA binding of PML/RARα on the CDKN2C promoter was analyzed by ChIP-qPCR assays. NC was amplified by a pair of negative primers for an irrelevant region. ChIP with the normal rabbit immunoglobulin G (IgG) was used as a negative control. The fold change of PML/RARa enrichment was calculated relative to normal IgG. Data represent the average of three replicates±SD.
Fig.3  PML/RARα repressing the promoter activity of CDKN2C. (A) Schematic diagram showing the promoter region of the CDKN2C constructed into the luciferase report vector (pGL3-CDKN2C). The gray rectangles represent the peak regions bound by PML/RARα. (B) PML/RARα represses the activity of CDKN2C promoter in a dose-dependent manner. Increasing quantities of the PML/RARα expression plasmid (pSG5-PML/RARα) were cotransfected into 293T cells with the CDKN2C luciferase reporter plasmid (pGL3-CDKN2C). The relative luciferase activity was standard with the activity of pGL3-CDKN2C cotransfected with the pSG5 empty expression vector. Data represent the average of three replicates±SD.
Fig.4  Induction of CDKN2C expression by ATRA in APL cells. (A, B) The relative mRNA and protein levels of CDKN2C were increased after ATRA treatment of NB4 cells for the indicated times. The mRNA levels of CDKN2C were detected by qRT-PCR assays and normalized to GAPDH expression. The relative mRNA expressions were all compared with the values in ATRA-untreated NB4 cells (Ctrl). (C) ATRA severely decreased the binding capacity of PML/RARα on the CDKN2C promoter after ATRA treatment of NB4 cells for 24 h. ChIP-qPCR assays were carried out using anti-PML and anti-RARα antibodies before and after ATRA treatment of NB4 cells for 24 h. ChIP using normal IgG was used as a negative control. The fold change of PML/RARα enrichment by anti-PML and anti-RARα antibodies is calculated relative to normal IgG. (D) The repression of CDKN2C was only conferred by PML/RARα. NB4 cells were pretreated with or without CHX at a final concentration of 10 mg/ml for 30 min and then treated with 1 mmol/L ATRA for 8 h. Data represent the mean of three replicates±SD. * P<0.05, ** P<0.01, *** P<0.001. ns, non-significant.
Fig.5  Role of CDKN2C in cell cycle arrest and differentiation of APL cells. (A) The mRNA and protein levels of CDKN2C after overexpression. (B) Restored expression of CDKN2C induced the G0/G1 phase arrest. GFP-positive NB4 cells were sorted by FACS three days after infection. Then, the distribution of cell cycle phases was analyzed by flow cytometry analysis. Data represent the mean of three replicates±SD. (C) Ectopic expression of CDKN2D induced a partial differentiation. Flow cytometry analysis of cell surface marker CD11b in GFP-positive NB4 cells is shown on the left. Wright’s staining of GFP-positive NB4 cells is shown on the right. Data represent the mean of three replicates±SD. *** P<0.001.
Fig.6  Validation of the PML/RARα binding to the CDKN2C locus and the increase of CDKN2C expression by ATRA in primary APL patient cells. (A) Overview of the PML/RARα binding sites on CDKN2C in APL patient blast cells. Blue represents the PML ChIP-seq signals retrieved from the ChIP-seq profiling performed by Martens et al. (GSE18886) [6]. (B) ATRA increased the mRNA level of CDKN2C expression in primary APL patient cells. The gene expression data for CDKN2C were retrieved from microarray gene expression profiling performed by Stegmaier et al. (GSE976) using U133A Affymetrix microarrays [26]. (C) A schematic illustration of CDKN2C expression in an APL patient sample before and after ATRA treatment. The peak represents the RNA-seq signals for CDKN2C. The RefSeq annotation for CDKN2C is shown at the bottom. The expression values are the absolute intensities.
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