The Paf oncogene is essential for hematopoietic stem cell function and development

The Paf oncogene is highly expressed in cycling hematopoietic stem cells (HSCs) and is required for the development of long-term HSCs

Purification, Characterization and Receptor Binding of Human Colony-Stimulating Factor-1

Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol

Enforced expression of NUP98-HOXA9 in human CD34(+) cells enhances stem cell proliferation

The t(7;11)(p15:p15) translocation, observed in acute myelogenous leukemia and myelodysplastic syndrome, generates a chimeric gene where the 5' portion of the sequence encoding the human nucleoporin NUP98 protein is fused to the 3' region of HOXA9. Here, we show that retroviral-mediated enforced expression of the NUP98-HOXA9 fusion protein in cord bloodderived CD34(+) cells confers a proliferative advantage in both cytokine-stimulated suspension cultures and stromal coculture. This advantage is reflected in the selective expansion of hematopoietic stem cells as measured in vitro by cobblestone area-forming cell assays and in vivo by competitive repopulation of nonobese diabetic/severe combined immunodeficient mice. N1UP98-HOXA9 expression inhibited erythroid progenitor differentiation and delayed neutrophil maturation in transduced progenitors but strongly enhanced their serial replating efficiency. Analysis of the transcriptosome of transduced cells revealed tip-regulation of several homeobox genes of the A and B cluster as well as of Meis1 and Pim-1 and down-modulation of globin genes and of CAAT/enhancer binding protein a. The latter gene, when coexpressed with AIUP98-HOXA9, reversed the enhanced proliferation of transduced CD34(+) cells. Unlike HOXA9, the NUP98-HOXA9 fusion was protected from ubiquitination mediated by Cullin-4A and subsequent proteasome-dependent degradation. The resulting protein stabilization may contribute to the leukemogenic activitv of the fusion protein

Cyclin C regulates human hematopoietic stem/progenitor cell quiescence

Hematopoietic stem cells (HSCs) can remain quiescent or they can enter the cell cycle, and either self-renew or differentiate. While cyclin C and cdk3 are essential for the transition from the G 0 to G 1 phase of the cell cycle in human fibroblasts, the role of cyclin C in hematopoietic stem/progenitor cells (HSPCs) is not clear. We have identified an important role of cyclin C (CCNC) in regulating human HSPC quiescence, as knocking down CCNC expression in human cord blood (CB) CD34+ cells resulted in a significant increase in quiescent cells that maintain CD34 expression. CCNC knockdown also promotes in vitro HSPC expansion and enhances their engraftment potential in sub-lethally irradiated immunodeficient mice. Our studies establish cyclin C as a critical regulator of the G 0 /G 1 transition of human HSPCs and suggest that modulating cyclin C levels may be useful for HSC expansion and more efficient engraftment

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides

Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation–desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone