Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment

Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-α and TNF-α, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional

Discovery of a Family of Genomic Sequences Which Interact Specifically with the <i>c-MYC</i> Promoter to Regulate <i>c-MYC</i> Expression

<div><p>G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III₁ of the <i>c-MYC</i> promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII₁ located upstream of the P1 promoter regulates up-to 80% of <i>c-MYC</i> transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII₁ sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII₁ resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the <i>c-MYC</i> and <i>SOX2</i> promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.</p></div

Effect of Pu27-HS on SOX2 expression in U937 after 3 days exposure.

<p>(A). Bar graph representing RT-qPCR (C_T values) for <i>SOX2</i> (red bars) compared to <i>c-MYC</i> (gray bars) expression in 4 leukemia cell lines and PBMC from 2 donors. (B) Western blot for SOX2 protein expression in 4 leukemia cell lines and PBMC. Bar graph for the quantification of SOX2 protein normalized to α-tubulin. (C). Evaluation of <i>SOX2</i> expression compared to c-MYC after 72h exposure to 10μM Pu27-HS. Data represent the average of fold change for treated compared to untreated +/- SD of 3 independent experiments (*<i>p</i><0.05). (D). Evaluation of SOX2 protein expression in U937 by Western Blot, representative blot is shown. Quantification of SOX2 normalized to α-Tubulin, bar graph shows the average of intensity of the bands for 2 separate experiments.</p

Localization of the different Pu27 homologous sequences.

<p>Localization of the different Pu27 homologous sequences.</p

Localization and biophysical analysis of the Pu27 homologous oligonucleotides sequences.

<p>(A). Chromosome localization of the Pu27-HS. (B). Circular dichroism spectra for 14 members of the Pu27 family of oligonucleotides showing peaks at 260nm representative of the presence of parallel G-quadruplex. (C). Electromobility shift assays (EMSA), ³²P-labeled oligonucleotides were run on a polyacrylamide gel that shows unique band for each of the oligonucleotides that migrated.; one representative Phospho imager picture is shown. (D). EMSA for competition assay.³²P-labeled Pu27 was run in the presence of a 134bp double strand target sequence (TS) containing the NHEIII₁ of the <i>c-MYC</i> promoter +/- different concentrations of cold Pu27; Kodak image is shown. (E). EMSA for competition assay applied to 14 Pu27-HS ³²P-labeled incubated with 134bp target sequence +/- cold Pu27 at 1nM.</p

Growth inhibition assay for four different leukemia cell lines exposed to the Pu27-HS.

<p><b>(</b>A) U937, (B) HL-60, (C) Raji, and (D) Mol-4: 5000cells/well were exposed to 5 or 10μM of Pu27 homologous oligonucleotides sequences for 5 days then evaluate for growth inhibition in MTT assay. Data report the average+ SD of at least 3 different experiments (*<i>p</i><0.05).</p

Expression of c-MYC in four leukemia cell lines and 2 healthy donors PBMC.

<p><b>(</b>A). Bar graph representing RT-qPCR for relative expression (C_T values) of <i>c-MYC</i> in 4 leukemia cell lines and PBMC. (B) Western blot for c-MYC protein expression. (C). Bar graph for the quantification of protein normalized to α-tubulin.</p

Quadruplex-forming oligonucleotide targeted to the VEGF promoter inhibits growth of non-small cell lung cancer cells.

BACKGROUND:Vascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression. RESULTS:Our experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death. CONCLUSION:Our data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression

Effect of Pu27-HS on c-MYC expression in U937 after 3 days exposure.

<p>(A). Evaluation of c-MYC gene expression by RT-qPCR after 72h exposure to 10μM Pu27-HS oligonucleotides. Data represent the average in fold change compared to untreated +/- SD of 3 independent experiments (*<i>p</i><0.05). (B). Evaluation of c-MYC protein expression by Western Blot, representative blot is shown. (C). Quantification of c-MYC normalized to β-Tubulin, bar graph shows the average of intensity of the band for 3 separate experiments (*<i>p</i><0.05).</p

Sequences of Pu27 and Pu27- homologous G-quadruplex forming oligonucleotides.

<p>Sequences of Pu27 and Pu27- homologous G-quadruplex forming oligonucleotides.</p