Global Gene Expression Signatures in CD34+ Hematopoietic Cells Following Exposure to 16,16-Dimethylprostaglandin E2.

Abstract 2523 Poster Board II-500 Prostaglandin E2 (PGE2), a member of the eicosanoid family, is a local signaling molecule released from mammalian blood vessel walls and other hematopoietic niches. PGE2 produces tissue specific effects by interacting with G protein receptors in cell membranes. It has been found to be an important regulator of hematopoietic stem cell (HSC) number during embryogenesis and is required for the development of HSCs (North et al., Nature 447:1007–1011, 2007). Additionally, PGE2 enhances HSC engraftment in competitive transplantation assays (North et al., Nature 447:1007–1011, 2007) through alterations in the survival, proliferation and homing of transplanted cells (Hoggatt et al., Blood 113:5444–5455, 2009). Immunoselected CD34+ cells were isolated from the leukapheresis products of both human and non-human primates following cytokine mobilization with G-CSF. CD34+ cells were incubated in X-Vivo 10™ with either 50μM or no PGE2 for one hour at 37°C on RetroNectin™ coated plates. After one hour incubation, media was replaced with X-Vivo 10™ supplemented with 10ng/mL SCF and IL-6 and cells were kept at 37°C and collected either prior to or at 2, 6, 12 and 24 hours following PGE2 exposure. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA was co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method and gene regulation was analyzed at each time point using an Array scatter plot comparison. Statistical analysis was done using Array Class Comparison analysis and pathway analysis was carried out using Ingenuity Pathway Analysis. Many of the significantly up regulated genes were associated with pathways activated by PGE2, including genes involved in the inflammatory response, signal transduction, and G proteins. Interestingly the human CD34+ cells showed at 2 hours a 21.8-fold increase of 3′,5′-cyclic AMP phosphodiesterase mRNA consistent with prior observations in zebrafish and murine cells (Goessling et al. Cell 136: 1136–1148). Pathways that increased at 2 hours include molecular mechanisms of cancer, and those that were down regulated include CCR5, CTLA4, and leukocyte extravasation signaling. At 6 hours the molecular mechanisms of cancer pathway predominated even more (diminishing by 12 hours) while oxidative phosphorylation was down regulated. Genes involved in cAMP and WNT signaling are observed to be up regulated at earlier time points but diminish with time. These results correlate with protein expression observations that have been made in the zebrafish and murine models following PGE2 treatment (Goessling et al. Cell 136: 1136–1148) and are consistent with the observation that PGE2 may influence the self renewal of HSCs. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties.

Identification and characterization of a virus-inducible non-coding RNA in mouse brain

Infection of mice with Japanese encephalitis virus or Rabies virus results in the activation of a gene encoding a novel, non-coding RNA (ncRNA) in the mouse central nervous system. This transcript, named virus-inducible ncRNA (VINC), is identical to a 3.18 kb transcript expressed in mouse neonate skin (GenBank accession no. AK028745) that, together with a number of unannotated cDNAs and expressed sequence tags, is grouped in the mouse unigene cluster Mm281895. VINC is expressed constitutively in early mouse embryo and several adult non-neuronal mouse tissues, as well as a murine renal adenocarcinoma (RAG) cell line. Northern blotting of nuclear and cytoplasmic RNAs revealed that VINC is localized primarily in the nucleus of RAG cells and is thus a novel member of the nuclear ncRNA family.

IN SILICO SEARCH FOR NATURAL ANTISENSE TRANSCRIPTS REVEALS THEIR DIFFERENTIAL EXPRESSION IN HUMAN TUMORS

We created an algorithm that allows high-throughput mapping of sense-antisense (SA) pairs of transcripts. By this method we mapped approximately 32 000 SA pairs of human mRNAs. Collected SA pairs were divided into three groups: SA pairs based on two or more UniGene clusters (17% of all sense-antisense pairs), SA pairs based on ESTs that belong to the same UniGene cluster (42%), and SA pairs formed by UniGene cluster and non-unique unclustered transcripts (41%). To study expression patterns of natural SA pairs we created a software application "Antisense Cluster Filter". We retrieved tissue expression data for all the transcripts forming identified SA pairs, including clustered and unclustered ones. After that, we selected 108 SA pairs represented by transcripts differentially regulated in human tumors. For each of these SA pairs one of the transcripts was expressed only in tumors, another one was expressed both in non-malignant and malignant tissues. Indicated SA pairs may represent a new class of tumor markers. An example of the tumor-specific natural antisense to C3orf4 mRNA is detailed.