Biomedical subjects

Nabeel R Yaseen

Publications and source records attributed to Nabeel R Yaseen.

7 recordsLinked to original sources

PubMed2006

NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.

NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. We examined its effect on differentiation, proliferation, and gene expression in primary human CD34+ hematopoietic cells. Colony-forming cell (CFC) assays in semisolid medium combined with morphologic examination and flow cytometric immunophenotyping revealed that NUP98-HOXA9 increased the numbers of erythroid precursors and impaired both myeloid and erythroid differentiation. In continuous liquid culture, cells transduced with NUP98-HOXA9 exhibited a biphasic growth curve with initial growth inhibition followed by enhanced long-term proliferation, suggesting an increase in the numbers of primitive self-renewing cells. This was confirmed by a dramatic increase in the numbers of long-term culture-initiating cells, the most primitive hematopoietic cells detectable in vitro. To understand the molecular mechanisms underlying the effects of NUP98-HOXA9 on hematopoietic cell proliferation and differentiation, oligonucleotide microarray analysis was done at several time points over 16 days, starting at 6 hours posttransduction. The early growth suppression was preceded by up-regulation of IFNbeta1 and accompanied by marked up-regulation of IFN-induced genes, peaking at 3 days posttransduction. In contrast, oncogenes such as homeobox transcription factors, FLT3, KIT, and WT1 peaked at 8 days or beyond, coinciding with increased proliferation. In addition, several putative tumor suppressors and genes associated with hematopoietic differentiation were repressed at later time points. These findings provide a comprehensive picture of the changes in proliferation, differentiation, and global gene expression that underlie the leukemic transformation of human hematopoietic cells by NUP98-HOXA9.

Antigens, CD34↗

PubMed2006

Ultrastructural nuclear import assay.

Electron microscopy (EM) has been used for several decades to study the mechanisms of nuclear transport. In early studies of nuclear import, gold-conjugated nuclear proteins were microinjected into cells and followed by EM. As the components of the nuclear pore complex (NPC) and soluble mediators of nuclear import were cloned and characterized, gold-conjugated antibodies were utilized to sublocalize the components of the nuclear transport machinery by immuno-EM. Further, gold-conjugated recombinant proteins were used to probe permeabilized cells or isolated nuclear envelopes and characterize binding sites for these proteins at the NPC. More recently, recombinant gold-conjugated nuclear proteins were used in in vitro nuclear import assays to help dissect the mechanisms of nuclear import. We have used this ultrastructural nuclear import assay to study the nuclear import of the transcription factor PU.1. The results showed that this import requires energy but is carrier-independent. In the presence of energy, gold-conjugated PU.1 shifted to the nuclear side of the NPC and the inside of the nucleus. In conjunction with biochemical assays, these results indicated that this shift involved Ran-dependent binding of PU.1 to NUP153, a nucleoporin situated at the nuclear side of the NPC. Here we describe in detail the methods used in the ultrastructural nuclear import assay including preparation of recombinant protein, gold conjugation, in vitro nuclear import assay, electron microscopy, and data analysis.

Active Transport, Cell Nucleus↗

PubMed2005

Carrier-independent nuclear import of the transcription factor PU.1 via RanGTP-stimulated binding to Nup153.

PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, we show that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increased dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.

Active Transport, Cell Nucleus↗

PubMed2004

Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography.

Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.

Chromatography, High Pressure Liquid↗

PubMed2004

CD45-associated protein inhibits CD45 dimerization and up-regulates its protein tyrosine phosphatase activity.

CD45, a receptor-like protein tyrosine phosphatase (PTP), plays an essential role in lymphocyte development and immune responses. Recent evidence suggests that dimerization of CD45 down-regulates its function. However, the mechanisms by which CD45 dimerization is regulated remain unclear, and there is no direct evidence that the PTP activity of CD45 dimers is less than that of monomers. CD45 in lymphocytes associates with CD45-AP (CD45-associated protein). Here we show that T cells from CD45-AP-null mice have a much higher level of CD45 dimers than those of wild-type mice, suggesting that CD45-AP inhibits CD45 dimer formation. This was confirmed with the use of a novel CD45-AP-null T-cell line, ALST-1, that we established from a spontaneous thymic tumor found in a CD45-AP-null mouse. Transfected CD45-AP inhibited CD45 dimer formation in ALST-1 cells in proportion to the amount of CD45-AP expressed. Finally, with the use of microsomal fractions from both mouse thymocytes and ALST-1 transfectants, the PTP activity of CD45 was found to be significantly lower in CD45-AP-negative cells than in CD45-AP-positive cells. Therefore, our results support a model in which binding of CD45-AP to inactive CD45 dimers converts them to active monomers.

Animals↗

PubMed2003

The oncogene Nup98-HOXA9 induces gene transcription in myeloid cells.

The nucleoporin Nup98 gene is frequently rearranged in acute myelogenous leukemia (AML). In most cases this results in fusion of the N terminus of Nup98 to the DNA binding domain of a homeodomain transcription factor. The prototype of these fusions, Nup98-HOXA9, is associated with human AML and induces AML in mouse models. To understand the mechanisms by which Nup98-HOXA9 causes AML, we expressed it in myeloid cells and identified its target genes using high density oligonucleotide microarrays. The analysis was performed in triplicate and was confirmed by quantitative real time PCR. Of the 102 Nup98-HOXA9 target genes identified, 92 were up-regulated, and only 10 were down-regulated, suggesting a transcriptional activation function. A similar analysis of wild-type HOXA9 revealed 13 target genes, 12 of which were up-regulated, and 1 was down-regulated. In contrast, wild-type Nup98 had no effect on gene expression, demonstrating that the HOXA9 DNA binding domain is required for gene regulation. Co-transfection experiments using a luciferase reporter linked to the promoter of one of the Nup98-HOXA9 target genes confirmed up-regulation at the transcriptional level by Nup98-HOXA9 but not by either HOXA9 or Nup98. These data indicate that Nup98-HOXA9 is an aberrant transcription factor whose activity depends on the HOXA9 DNA binding domain but has a stronger and wider transcriptional effect than HOXA9. Several of the genes regulated by Nup98-HOXA9 are associated with increased cell proliferation and survival as well as drug metabolism, providing insights into the pathogenesis and epidemiology of Nup98-HOXA9-induced AML.

Animals↗

PubMed2003

Biphenotypic acute leukemia with coexpression of CD79a and markers of myeloid lineage.

Acute leukemias demonstrating immunophenotypic features of more than 1 cell lineage are referred to as acute leukemias of ambiguous lineage in the new World Health Organization classification system. A subtype of leukemia of ambiguous lineage is biphenotypic acute leukemia in which the malignant cell population expresses markers of 2 different lineages, most commonly myeloid and either B- or T-lymphoid lineages. This entity has been defined by a scoring system proposed by the European Group for the Immunological Characterization of Acute Leukemias (EGIL), with various markers assigned a score of 2, 1, or 0.5 depending on their specificity for myeloid or lymphoid lineage. Those cases having a score greater than 2 for the myeloid and either the B- or T-lymphoid lineages are biphenotypic acute leukemia in this system. One marker, CD79a, has been so clearly associated with acute lymphoblastic leukemia (ALL) by some researchers that its expression in the presence of blast markers is considered indicative of B-ALL. We describe an unusual case of acute leukemia meeting the criteria for biphenotypic acute leukemia in which CD79a expression was observed in the blast population.

Aged↗