The DNA Binding Property of PML/RARA but Not the Integrity of PML Nuclear Bodies Is Indispensable for Leukemic Transformation

<div><p>PML/RARA is the oncoprotein driving acute promyelocytic leukemia (APL). It suppresses genes expression by recruitment of a number of transcriptional repressors, resulting in differentiation block and malignant transformation of hematopoietic cells. Here, we found that mice primary hematopoietic progenitor cells (HPCs), transduced by DNA-binding-defective PML/RARA mutants, were deficient in colony formation. Further experiments showed that DNA-binding-defective PML/RARA mutants could not repress the transcription of retinoic acid regulated genes. Intriguingly, there were no significant differences of the micro-speckled intracellular distribution between the mutants and wild-type PML/RARA. Some retinoic acid target genes regulated by PML/RARA are involved in not only differentiation block but also hematopoietic cell self-renewal. Altogether, our data demonstrate that direct DNA-binding is essential for PML/RARA to immortalize hematopoietic cells, while disruption of PML-nuclear body does not seem to be a prerequisite for hematopoietic cell transformation.</p></div

DNA-binding defective PML/RARA mutants disrupt PML nuclear bodies.

<p>NIH-3T3 cells (A and C) or mouse HPCs (B and D) were infected with retroviral constructs expressing wild type or the indicated mutant PML/RARA respectively. Immunofluorescence analysis (A and B) was carried out with a rabbit anti-PML specific for PML/RARA to show distribution of wild type or mutant PML/RARA in nucleus, and with DAPI to visualize nuclei (top). Mouse PML was not recognized by the rabbit anti-PML. HPCs were harvested from the first round colonies. Representative of three independent experiments. Scale bar, 10 µm. Western blotting analysis of fusion proteins with anti-RARA in transduced NIH-3T3 cells (C) or mouse HPCs (D).</p

PML/RARA DNA-binding defective mutants failed to bind and activate RARE.

<p>(A) Schematic diagram of P/R wild type (WT) and mutants used in the study. B, C, D, E and F, RARA functional regions; DBD, DNA-binding domain. (B) RARE3-tk Luc reporter plasmid and indicated protein expression constructs were transiently cotransfected into 293T cells. <b><i>Renilla</i></b> luciferase plasmid was transfected into cells as internal control. 14 hours after ATRA (10⁻⁶ M) or ethanol vehicle treatment, transactivation was measured. The number refers to fold of transactivity after ATRA treatment. Mean values were obtained from 3 independent experiments with duplicate wells. Error bars indicate standard deviations of triplicate measurements. *p<0.01. (C) Western blotting analysis of wild type and various mutant PML/RARA proteins with anti-RARA in transiently transfected 293T cells. (D) ChIP analysis of wild type or mutant PML/RARA binding to the endogenous <i>RARB</i> promoter in transfected 293T cells, expressed as increase over nonspecific binding. Means ± standard deviation (SD).</p

DNA-binding is required for PML/RARA transformation.

<p>(A) Typical morphology of the second round colonies generated HPCs transduced with the indicated retroviruses. (B) Serial methylcellulose replating assays of HPCs transduced with the indicated retroviruses. Numbers of colonies in the indicated round plating were obtained from three independent experiments with duplicate dishes. Error bars represent standard deviations. (C) Wright-Giemsa staining of primary hematopoietic precursors transduced with MSCV empty vector, the wild-type or indicated mutant PML/RARA showed histological differentiation of transduced HPCs . Cells were harvested from the first round. Arrowheads indicate transformed bone marrow cells. Representative of three independent experiments. Scale bar, 10 µm. (D) Flow cytometry analysis of the corresponding transduced HPCs using three myeloid differentiation markers, c-Kit, Mac-1, and Gr-1. Representative of three independent experiments.</p

Transcriptional regulation of endogenous target genes by PML/RARA mutants.

<p>(A-B) NIH-3T3 cells stably expressing the indicated PML/RARA mutants were treated or not with 10⁻⁶ M ATRA overnight. <i>RARB</i> and <i>Tg II</i> gene expression were analyzed by quantitative RT-PCR. (C-G) The indicated mutant PML/RARA-transduced primary hematopoietic progenitors were treated with or without 10⁻⁶ M ATRA for 14 hours. <i>RARB</i>, <i>Tg II</i>, <i>HCK</i>, <i>PU.1</i> and <i>ID1</i> gene expression was also analyzed by quantitative real-time PCR. All results from quantitative real-time PCR were normalized to <i>β-actin</i>. Results, obtained from three independent experiments, are given as mean ±standard deviations.</p

Concerted Growth and Ordering of Cobalt Nanorod Arrays as Revealed by Tandem in Situ SAXS-XAS Studies

The molecular and ensemble dynamics for the growth of hierarchical supercrystals of cobalt nanorods have been studied by in situ tandem X-ray absorption spectroscopy–small-angle X-ray scattering (XAS-SAXS). The supercrystals were obtained by reducing a Co­(II) precursor under H₂ in the presence of a long-chain amine and a long-chain carboxylic acid. Complementary time-dependent ex situ TEM studies were also performed. The experimental data provide critical insights into the nanorod growth mechanism and unequivocal evidence for a concerted growth–organization process. Nanorod formation involves cobalt nucleation, a fast atom-by-atom anisotropic growth, and a slower oriented attachment process that continues well after cobalt reduction is complete. Smectic-like ordering of the nanorods appears very early in the process, as soon as nanoparticle elongation appears, and nanorod growth takes place inside organized superlattices, which can be regarded as mesocrystals