NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells

Int J Med Sci. 2014 Jan 16;11(3):247-54. doi: 10.7150/ijms.6518. eCollection 2014.

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.

Keywords: HL-60 cell; NLS-RARα; differentiation; over-expression; proliferation; shRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / genetics
  • Cell Differentiation / drug effects*
  • Cell Differentiation / genetics
  • Cell Nucleus / genetics
  • Cell Nucleus / pathology
  • Cell Proliferation / drug effects
  • Cell Proliferation / genetics
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Expression Regulation, Neoplastic / genetics
  • HL-60 Cells
  • Humans
  • Leukemia, Promyelocytic, Acute / genetics*
  • Leukemia, Promyelocytic, Acute / pathology
  • Nuclear Localization Signals / biosynthesis
  • Nuclear Localization Signals / metabolism*
  • Oncogene Proteins, Fusion / biosynthesis*
  • Oncogene Proteins, Fusion / genetics
  • Proto-Oncogene Proteins c-myc / biosynthesis
  • RNA, Small Interfering / genetics
  • Receptors, Retinoic Acid / biosynthesis
  • Receptors, Retinoic Acid / metabolism*
  • Retinoic Acid Receptor alpha
  • Translocation, Genetic / genetics
  • Tretinoin / administration & dosage

Substances

  • MYC protein, human
  • Nuclear Localization Signals
  • Oncogene Proteins, Fusion
  • Proto-Oncogene Proteins c-myc
  • RARA protein, human
  • RNA, Small Interfering
  • Receptors, Retinoic Acid
  • Retinoic Acid Receptor alpha
  • promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein
  • Tretinoin