Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Philadelphia (Ph) or BCR/ABL-negative cells with immature phenotype (CD34-positive, DR-negative) can be recovered from patients with chronic myeloid leukemia (CML) in chronic phase. We used the technique described by Berardi et al (Science 1995; 267: 104-108) to select stem cells from marrow or blood of CML patients at diagnosis or during treatment with alpha-interferon. Mononuclear cells (MNC), and in some experiments CD34+ cells, were maintained for 7 days in the presence of 5-fluorouracil (5-FU), stem cell factor and interleukin-3. The number of viable cells recovered after culture was between 7.4 and 70.2 for 10(6) cells plated. These cells exhibited the following phenotype: CD34+, CD117+, CD38-, lineage-, and were able to generate cobblestone areas and secondary colonies in long-term culture (LTC), with a frequency similar to that of cells selected from normal marrow. Study by fluorescence in situ hybridization of LTC cells or secondary colonies showed no evidence of BCR/ABL rearrangement. Reverse transcriptase polymerase chain reaction studies on pooled LTC cells or secondary colonies were also negative. By contrast, LTC cells or secondary colonies obtained from CML CD34+ cells without culture in the presence of 5-FU were always positive for BCR/ABL rearrangement. Finally, 5-FU selected cells were able to engraft NOD/SCID mouse, as human cells were detected in blood and marrow 10 weeks post transplantation, which were BCR/ABL negative by RT-PCR. This method of culture makes it possible to select constantly BCR/ABL-negative cells with capacities of development in LTC assay and of NOD/SCID mouse engraftment.
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PMID:Selection of BCR/ABL-negative stem cells from marrow or blood of patients with chronic myeloid leukemia. 1040 Apr 13

Human immunodeficiency virus (HIV) entry is mediated not only by the CD4 receptor, but also by interaction with closely related molecules that act as membrane coreceptors. We have analyzed mRNA expression and/or cell membrane exposition of the coreceptors most widely used by diverse HIV-1 strains (CXCR4, CCR5, and CCR3) on purified hematopoietic progenitor cells (HPCs) induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), and monocytic (Mo) lineages. Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytofluorimetric analysis showed the presence of both CXCR4 and CCR5 in quiescent HPCs, but failed to detect CCR3-specific transcripts. Chemokine expression in HPC progenies showed that CXCR4 receptor is detected on the majority of MKs from early to late stages of maturation, whereas it is moderately decreased in the Mo lineage. In the G pathway, two distinct cell populations, CXCR4(+) and CXCR4(-), were observed: morphological analysis of the sorted populations showed that the CXCR4(+) cells were largely eosinophils and the CXCR4(-) were granulocytes of the neutrophilic series. Furthermore, in the E pathway, CXCR4 was almost completely absent. CCR5 expression is restricted to Mo cultures, ie, approximately 30% to 80% cells throughout all monocytopoietic differentiation/maturation stages. Finally, CCR3 mRNA is always absent in all the unilineage cultures. Evaluation of CD4 expression by flow cytometry on both quiescent HPCs and differentiating unilineage precursors showed that the CD4 receptor is present on approximately 15% of the starting CD34(+) HPC population, highly expressed in the Mo lineage up to 80% at terminal maturation, present on 20% to 30% of maturing Mks, and not detectable in either the E or G lineage. Expression of CD4 receptor together with CXCR4 and/or CCR5 coreceptor in the four lineages correlates with hematopoietic precursor susceptibility to T-lymphotropic and macrophage (M)-tropic HIV strains infection: (1) CD4(-) G and E cells were resistant to both M-tropic and T-lymphotropic strains; (2) HPC-derived Mks were susceptible to T-tropic, but resistant to M-tropic, infection; (3) Mo differentiating cells efficiently replicate both HIV strains. Furthermore, we showed that the CXCR4 and CCR5 ligands (stromal-derived factor 1 and macrophage-inflammatory protein-1alpha [MIP-1alpha], MIP-1beta and RANTES, respectively) inhibit HIV replication in both maturing Mo and Mk cells. Taken together, our data show a lineage-specific modulation of chemokine receptor/coreceptor during hematopoietic cell differentiation and extend previous observations on the relationship between the expression of HIV receptor/coreceptors, susceptibility, and chemokine-mediated resistance to HIV infection.
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PMID:Lineage-specific expression of human immunodeficiency virus (HIV) receptor/coreceptors in differentiating hematopoietic precursors: correlation with susceptibility to T- and M-tropic HIV and chemokine-mediated HIV resistance. 1093 97

Protein kinase C (PKC), a key component of the signaling pathways leading to proliferation and differentiation, consists of a family closely related serine/threonine protein kinases. The mRNA expression of these PKC isoforms has been characterized during hematopoietic differentiation. Using the reverse-transcriptase polymerase chain reaction technique, we have analyzed the levels of isoform transcripts in bone marrow CD34(+) hematopoietic progenitors and their progeny differentiated along erythroid, megakaryocyte, or granulocyte/monocyte lineages, upon exposure to growth factors. In contrast with isoforms alpha, beta(I), beta(II), delta, and epsilon, ubiquitously expressed, isoforms theta, eta/L, zeta, and iota/lambda exhibited a lineage-restricted expression. These qualitative changes, which allow to distinguish the erythroid and megakaryocyte phenotypes from the granulocyte/monocyte phenotype, include zeta exclusively upregulated in granulocytes/monocytes and theta, eta/L, and iota/lambda exclusively expressed in megakaryocytes and erythroblasts. In contrast, erythroblasts and megakaryocytes, which supposedly share a common bipotential progenitor, displayed only quantitative changes. These results evidence the selective expression of PKC isoforms at transcriptional and/or posttranscriptional levels in hematopoietic progenitors induced to differentiate, which may suggest a differential contribution of individual isoforms to cellular signaling.
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PMID:Differential expression of protein kinase C isoform transcripts in human hematopoietic progenitors undergoing differentiation. 1051 25

"Simple" variants of the t(8;21) translocation involving chromosome 8 and a chromosome other than number 21 are rare. To our knowledge, only t(3;8)(q29;q22), t(8;11)(q22;q13), t(8;16)(q22;q24), t(8;20)(q22;p13), and t(8;22) have been reported in the literature. This paper describes for the first time two patients with acute myelogenous leukemia with a consistent t(8;19)(q22;q13) translocation. Their myelograms were compatible with the FAB-M2 subtype. The blasts from case 2 expressed CD34, CD33, CD13, and CD19. Karyotype analyses were performed on bone marrow cells using R- and G-banding at presentation. A t(8;19)(q22;q13) translocation was found in 28/30 metaphases for case 1 and in 23/25 metaphases for case 2. The latter case also had a deletion of chromosome 9, del(9)(q12q22) as an additional abnormality. Reverse transcriptase-polymerase chain reaction study revealed no AML1/ETO fusion transcript in case 2. Dual-color fluorescence in situ hybridization (FISH) assay using two probes (BAC92 and YAC412A4) convincingly demonstrated that the chromosomal material from 8q was translocated onto 19q rather than 19p in case 2. Thus, we consider t(8;19)(q22;q13) a true "simple" variant of t(8;21), and assume that a fusion gene resulting from the t(8;19) may contain the ETO gene located at 8q22 and an unknown partner gene from 19q13, which probably is a new transcription factor, whose molecular entity warrants further study.
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PMID:Two cases of AML (M2) with a t(8;19)(q22;q13): a new cytogenetic variant. 1074 98

Caspase-3/CPP32, a member of the interleukin-1 converting enzyme (ICE) family, is considered an executioner protease in mammalian cells during apoptosis. Although expression and activation of caspase-3/CPP32 protein have been studied in many tissues and leukemia cell lines, this has not been explored in primitive hematopoietic CD34(+) cells. In this study, we evaluated expression and activation of caspase-3/CPP32 protein in CD34(+) cells from cord blood (CB) during apoptosis induced by growth factor deprivation. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and flow cytometry analysis were used in this study to determine the expression of caspase-3/CPP32 in CD34(+) CB cells during apoptosis. Our results demonstrated that caspase-3/CPP32 mRNA was constitutively expressed at a very low level in freshly isolated CD34(+) cells. Expression of caspase-3/CPP32 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3/CPP32 proenzyme was detected in the freshly isolated CD34(+) cells and after 3 days expansion with cytokines. Within 12 hours after growth factor withdrawal from expanded cells caspase-3/CPP32 was activated and a cleavage 20 kDa protein was detected; a poly(ADP-ribose) polymerase (PARP) was cleaved by activated caspase-3/CPP32. Activation of caspase-3/CPP32 and apoptosis upon growth factor withdrawal were inhibited/reduced by the caspase inhibitors, z-VAD-fmk and DEVD-CHO. These results demonstrate that caspase-3/CPP32 is involved in apoptosis of primitive CB CD34(+) cells but may not be the only mechanism involved.
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PMID:Expression and activation of caspase-3/CPP32 in CD34(+) cord blood cells is linked to apoptosis after growth factor withdrawal. 1098 91

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)
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PMID:Donor stromal cells from human blood engraft in NOD/SCID mice. 1109 86

Laminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed alpha, beta, and gamma chains. Combinations of 11 characterized laminin subunits (alpha 1-alpha 5, beta 1-beta 3, and gamma 1-gamma 3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase-polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34(+) cell lines. Antibodies against the beta 1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells. (Blood. 2000;96:4194-4203)
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PMID:Characterization and functional analysis of laminin isoforms in human bone marrow. 1111 Jun 91

Previous studies have demonstrated that the c-kit encoded tyrosine kinase receptor and its ligand, steel factor (SLF), are critical for normal blood cell development. We have reported that transduction of the c-kit gene into single hematopoietic progenitor cells (HPC), CD34(+++) cells, from cord blood (CB) enhances erythroid colony formation via a SLF-dependent mechanism. We therefore decided to evaluate the impact on cell proliferation of co-transducing c-kit and SLF cDNAs into these cells. CD34(+++) cells were sorted as a population or as 1 cell/well for cells expressing the highest levels of CD34 and different levels of c-kit. Cells were then prestimulated with granulocyte macrophage (GM)-colony stimulating factor (CSF), interleukin (IL)-3, IL-6, erythropoietin (Epo) in the presence and absence of various concentrations of SLF. Cells were then transduced with SLF and/or c-kit cDNAs, and then assayed for colony formation with the same cytokine combination. At a single cell level, co-transduction with c-kit and SLF genes significantly enhanced colony formation compared with individual gene transduction, especially by erythroid and multipotential progenitors that responded to stimulation by added cytokines. Little or no growth was seen with the c-kit- and/or SLF-transduced cells without addition of cytokines. The degree of enhancement effected by co-transduction inversely correlated with the degree of expression of c-kit protein before transduction. Optimal enhancing effects were noted in CD34(+++) kit(Lo/-) cells co-transduced with both c-kit and SLF cDNAs. Reverse transcriptase-polymerase chain (RT-PCR) analysis of SLF mRNA expression in CD34(+++) cells and enzyme-linked immunoadsorbent assay (ELISA) measurement of secreted SLF protein demonstrated that the transduced SLF cDNA was expressed and soluble SLF was released in medium cultured with SLF gene transduced MACS-separated CD34(+) cells in the presence, but not in the absence, of IL-3, GM-CSF, IL-6, and Epo. These results demonstrate the enhancement of the proliferation of growth factor responsive HPC that express transduced c-kit and SLF genes.
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PMID:Co-transduction of cDNAs for c-kit and steel factor into single CD34+ cord blood cells further enhances the growth of erythroid and multipotential progenitors. 1117 93

The earliest stages of lymphoid commitment from human pluripotent hematopoietic stem cells have not been defined. A clonogenic subpopulation of CD34(+)CD38(-) cord blood cells were identified that expressed high levels of the CD7 antigen and possessed only lymphoid potential. CD34(+)CD38(-)CD7(+) (CD7(+)) cells uniformly coexpressed CD45RA and HLA-DR; c-kit and Thy-1 expression was absent to low. Clonal analysis demonstrated that single CD7(+) cells could generate B cells, natural killer cells, and dendritic cells but were devoid of myeloid or erythroid potential. In contrast, control CD34(+)CD38(-)CD7(-) (CD7(-)) cells generated both lymphoid and myelo-erythroid cells. The lymphoid potential (generation of lymphoid progeny in bulk and single cell cultures) of CD7(+) cells was equivalent to that of the pluripotent CD7(-) cells. RNA expression studies showed that CD7(+) cells expressed PU.1 and GATA-3, but did not express Pax-5, terminal deoxynucleotide transferase, or CD3epsilon. In contrast to the previously described murine common lymphoid progenitor, the alpha chain of the receptor for interleukin-7 was not detected by fluorescence-activated cell sorting analysis or RNA polymerase chain reaction in CD7(+) cells. These studies identify a clonogenic lymphoid progenitor with both B-cell and natural killer cell lineage potential with a molecular profile that suggests a developmental stage more primitive than previously identified lymphoid progenitors. The CD7(+) phenotype distinguishes primitive human lymphoid progenitors from pluripotent stem cells, thus allowing the study of regulation of early human lymphopoiesis and providing an alternative to pluripotent stem cells for genetic manipulation and transplantation. (Blood. 2001;97:3683-3690)
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PMID:Identification of a novel, human multilymphoid progenitor in cord blood. 1138 3

Nonhuman primates are useful large animal model systems for the in vivo study of hematopoietic stem cell biology. To better understand the degree of similarity of the hematopoietic systems between humans and baboons, and to explore the relevance of such studies in nonhuman primates to humans, this study was designed to compare the global gene expression profile of bone marrow CD34(+) cells isolated from these 2 species. Human complementary DNA (cDNA) filter arrays containing 25 920 human cDNAs were surveyed for this purpose. The expression pattern and relative gene abundance of the 2 RNA sources were similar, with a correlation coefficient of 0.87. A total of 15 970 of these cDNAs were expressed in human CD34(+) cells, of which the majority (96%) varied less than 3-fold in their relative level of expression between human and baboon. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed that expression was comparable between the 2 species. No species-restricted transcripts have been identified, further reinforcing the high degree of similarity between the 2 populations. A subset of 1554 cDNAs, which are expressed at levels 100-fold and greater than background, is described, which includes 959 expressed sequence tags and uncharacterized cDNAs, and 595 named genes, including many that are clearly involved in hematopoiesis. The cDNAs reported here represent a selection of some of the most highly abundant genes in hematopoietic cells and provide a starting point to develop a profile of the transcriptosome of CD34(+) cells.
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PMID:Highly abundant genes in the transcriptosome of human and baboon CD34 antigen-positive bone marrow cells. 1141 67


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