Delia C. Tang

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development.

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.

Hydroxyurea exerts bi-modal dose-dependent effects on erythropoiesis in human cultured erythroid cells via distinct pathways

Summary. Hydroxyurea (HU) has been shown to increase the proportion of fetal haemoglobin (HbF) in most sickle cell patients. A low‐dosage regimen increased total haemoglobin (Hb) levels in some thalassaemia intermedia patients by preferentially increasing β‐globin biosynthesis. To further characterize these apparent dose‐dependent effects of HU, we examined erythroid cells exposed to HU (5–100 µmol/l) in two‐phase liquid culture. Low doses (from 5 to 25 µmol/l) increased Hb levels by up to 2·7‐fold, and a high dose (100 µmol/l) increased Hb levels when added at d 3–6 of phase II, with no significant changes in response to HU during the late stage of phase II culture (≥ 9 d). HU exposure during d 0–3 of phase II culture increased the number of erythroid colonies to a maximum of fivefold at 5 µmol/l HU. GATA‐1 mRNA was downregulated at a high dose and GATA‐2 was dose dependently upregulated over a lower dosage range. Treatment with 100 µmol/l HU dramatically upregulated the death receptor DR‐5, caspase 3, as determined by cDNA microarray analysis. In contrast, 10 µmol/l HU modestly upregulated mRNA levels of the early growth response gene. Our results suggest that HU exerts concentration‐dependent effects on HbF production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.

Thrombopoietin has a differentiative effect on late-stage human erythropoiesis.

To further explore the mechanism of the effect of thrombopoietin (TPO) on erythropoiesis, we used a two‐phase culture system to investigate the effect of TPO on late‐stage human erythroid lineage differentiation. In serum‐free suspension and semisolid cultures of human peripheral blood derived erythroid progenitors, TPO alone did not produce benzidine‐positive cells. However, in serum‐containing culture, TPO alone stimulated erythroid cell proliferation and differentiation, demonstrated by erythroid colony formation, production of benzidine‐positive cells and haemoglobin (Hb) synthesis. Monoclonal anti‐human erythropoietin antibody and anti‐human erythropoietin receptor antibody completely abrogated the erythroid differentiative ability of TPO in the serum‐containing systems. This implied that binding of EPO and EPO‐R was essential for erythropoiesis and the resultant signal transduction may be augmented by the signals emanating from TPO–c‐Mpl interaction. Experiment of withdrawal of TPO further demonstrated the involvement of TPO in late‐stage erythropoiesis. RT‐PCR results showed that there was EPO‐R but not c‐Mpl expression on developing erythroblasts induced by TPO in serum‐containing system. Our results establish that TPO affects not only the proliferation of erythroid progenitors but also the differentiation of erythroid progenitors to mature erythroid cells.

Influence of lineage‐specific cytokines on commitment and asymmetric cell division of haematopoietic progenitor cells

Summary. We examined the influence of cytokines on erythroid‐ and myeloid‐lineage development of AC133+ cells during primary and secondary cultures. Cells cultured for 14 d in liquid medium containing erythropoietin (EPO) were amplified 831‐fold with 98·2% erythroid cells. A similar culture exposed to granulocyte colony‐stimulating factor (G‐CSF) grew 1350‐fold with 97·4% myeloid cells. To assess whether the cells with EPO inducement could respond at this point to G‐CSF signal, or vice versa, the EPO‐stimulated population was re‐grown with G‐CSF, constituting 95·2% myeloid, of 5075‐fold, cells after 14 d of re‐culture. Conversely, reculture of the G‐CSF‐stimulated population with EPO resulted in a 4083‐fold growth with 81·4% erythroid cells. Semisolid culture containing EPO orG‐CSF showed that some individual colonies had self‐ renewal potential after 14 d culture and could be induced todevelop into a different lineage. Analysis of primitive markers, CD34 and Notch1, or lineage markers, EPO‐R and CD13, by single‐cell reverse transcription polymerase chain reaction showed that individual colonies of 2–16 cells contained at least one CD34‐positive cell with expression ofNotch1 and co‐expression of EPO‐R and CD13 appeared on either CD34‐positive or CD34‐negative cells. In situ hybridization with the same cell surface markers in cell populations confirmed the asymmetric cell division and co‐expression from single cell data. The study provides a useful model for the analysis of multipotential progenitor development, and indicates that progenitor cells co‐express genes from different lineage pathways before commitment and that cytokines influence lineage commitment.

Activation of the human delta-globin gene promoter in primary adult erythroid cells

Restoration of the CCAAT box or insertion of an erythroid Krüppel‐like factor (EKLF) binding site in the delta promoter activates its expression in several erythroid cell lines. We extended these studies using a novel primary human adult erythroid cell (hAEC) system to investigate these effects at the late erythroblast stage. Restoration of the CCAAT box at −70 bp, or insertion of an EKLF binding site at −85 bp or −95 bp in the promoter significantly increased delta globin gene expression in hAEC. Our results demonstrate that the altered CCAAT box (CCAAC) and the lack of an EKLF binding site in δ‐globin contribute to its low level of expression in the hAEC model as well.