ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells

Abstract

IntroductionMolecular processes that maintain the stem cell pool and govern the proliferation and differentiation of hematopoietic stem and progenitor cells are widely investigated but incompletely understood. Identification of these genes will facilitate our understanding of hematopoiesis and may be used to improve clinical outcomes.Using differential display-polymerase chain reaction (dd-PCR), we identified a differentially expressed transcript in CD34-enriched populations, with low or absent expression in CD34-depleted populations. Sequencing of this transcript revealed significant homology to oxysterol-binding protein (OSBP). On the basis of ESTs in the GenBank database, a family of up to 8 human genes that code for OSBP-related proteins is postulated to exist. 1 These have been designated OSBP-related proteins (ORPs). A partial sequence for our OSBP-related gene was thus designated ORP-3. 1 Study design CD34 ؉ purificationUmbilical cord blood (UCB) samples were obtained after uncomplicated vaginal or cesarean delivery. Adult bone marrow (ABM) cells were obtained from cell scrapings of discarded rib and femur heads after cardiothoracic or hip-replacement surgery. All samples were donated by volunteers according to approved institutional guidelines. Mononuclear cells were prepared by density-gradient separation through Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden). CD34 ϩ cells were isolated using a MiniMacs bead separation kit (Miltenyi Biotec, Becton Dickinson, Sunnyvale, CA).Flow cytometric analysis of CD34 ؉ populations and fluorescence-activated cell sorting of CD34 ؉ subsets CD34 ϩ cells were labeled with anti-CD38-fluorescein isothiocyanate, anti-CD34-phycoerythrin, and anti-CD45-PECY5 (Coulter Immunotech, Fullerton, CA). CD34 ϩ purity was determined by flow cytometric analysis (FacsCalibur, Becton Dickinson) following modified ISHAGE guidelines 2 using CELLQuest software (Becton Dickinson). When required, CD34 ϩ 38 Ϫ/ dull cells were sorted using a FacsCalibur Cell Concentrator Sorting Module. Culture of umbilical cord blood CD34 ؉ cellsIn selected experiments, isolated CD34 ϩ cells were cultured for 1 week at 37°C in a humidified atmosphere flushed with 5% CO 2 in air, at a concentration of 0.5 ϫ 10 6 cells/mL in ␣ minimum essential medium (Trace, Noble Park, Victoria, Australia) with 20% fetal bovine serum (CSL Biosciences, Parkville, Victoria, Australia), 2 mM L-glutamine, 200 U/mL penicillin-streptomycin (Sigma, St Louis, MO), 20 ng/mL recombinant human stem cell factor (Amgen, Thousand Oaks, CA), 10 ng/mL IL-1B (Endogen, Woburn, MA), 10 ng/mL IL-3, 10 ng/mL IL-6, and 10 ng/mL G-CSF (Amrad, Boronia, Victoria, Australia). dd-PCRRNA was extracted by RNeasy total RNA isolation kit (Qiagen, Clifton Hill, Victoria, Australia) and was DNase treated (Invitrogen, Carlsbad, CA). Reverse transcription and dd-PCR were carried out as described in the RNAimage Differential Display System (GenHunter, Nashville, TN). We used ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Appli...

Mechanisms of resistance to the cytotoxic effects of oxysterols in human leukemic cells

“…This is in contrast to the findings of Metherall et al [25] and Ayala-Torres et al [4] who reported differential expression of these genes in oxysterol-sensitive and -resistant CHO and CEM cells, respectively. Similarly, we observed no significant difference in the expression of the gene for OSBPL3 which was found to be differentially expressed amongst resistant and sensitive primary HPCs from UCB and ABM (respectively) [14,15].…”

Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7

“…By using differential display, Gregorio-King and co-workers reported that ORP3 expression was significantly higher in primitive CD34+ cells than in the more mature CD34-haematopoietic cell population (Gregorio-King et al 2001). In a recent study, we observed that expression of ORP3 mRNA was upregulated about twofold during differentiation of human monocytes into macrophages (Johansson et al 2003).…”

“…Mouse ORP1 was one of the genes that showed significant changes in expression during differentiation of 3T3-L1 adipocytes (Ross et al 2002). Furthermore, expression of one human (ORP3) and two mouse (ORP4 and ORP10) ORPs has been shown to vary during differentiation of haematopoietic precursor cells (Gregorio-King et al 2001;Anniss et al 2002;Covell et al 2003). We have also recently reported that expression of four ORPs (ORP1, ORP3, ORP9 and ORP11) was induced during differentiation of human monocytes into macrophages (Johansson et al 2003).…”

ORP3 Splice Variants and Their Expression in Human Tissues and Hematopoietic Cells

“…3). None of the exon variations (exons 9, 12, or 15) identified here alter the putative dimerization box or leucine zipper region (Gregorio-King et al, 2001). …”

“…In a previous study we identified the ORP3 gene, and found that it is more highly expressed in CD34 positive (CD34 1 ) compared with CD34 negative hematopoietic cells (CD34 2 ) (Gregorio- King et al, 2001). We deduced the complete ORP3 gene from RACE, EST sequences, and homology to genomic clones, to give the "full-length" ORP3, comprising 23 exons and encoding a protein containing 887 amino acids (Accession No: AY008372).…”