title: NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.
abstract: A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The expression of C-MYC increased strikingly in HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. Down-regulation of NLS-RARα expression inhibited the proliferation and induced the differentiation of HL-60 cells. On the contrary, over-expression of NLS-RARα promoted proliferation and reduced the ATRA-induced differentiation of HL-60 cells.
authors: 1. Central Laboratory of Yong-chuan hospital, Chongqing Medical University, Chongqing 402160, China. ; 2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors: Xiu-Xiu
authors: XX
authors: Hu
authors: 2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors: Liang
authors: L
authors: Zhong
authors: 2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors: Xi
authors: X
authors: Zhang
authors: 2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors: Yuan-Mei
authors: YM
authors: Gao
authors: 1. Central Laboratory of Yong-chuan hospital, Chongqing Medical University, Chongqing 402160, China. ; 2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors: Bei-Zhong
authors: BZ
authors: Liu
mesh_terms: D017209:Apoptosis / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics* / Q000473:pathology; D019913:Nuclear Localization Signals / Q000096:biosynthesis / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis* / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D014178:Translocation, Genetic / Q000235:genetics; D014212:Tretinoin / Q000008:administration & dosage
chemical_list: C489427:MYC protein, human; D019913:Nuclear Localization Signals; D015514:Oncogene Proteins, Fusion; D016271:Proto-Oncogene Proteins c-myc; C000607071:RARA protein, human; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D014212:Tretinoin
keywords: HL-60 cell; NLS-RARα; differentiation; over-expression; proliferation; shRNA