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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.
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PMID:Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level. 1023 88

We have investigated the expression and functional competence of folate receptor (FR) isoforms on human hematopoietic cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we find that a substantial fraction of low-density mononuclear and CD34(+) cells express both the beta and gamma isoforms of FR. The alpha isoform of FR (the form most commonly found on cancer cells) was surprisingly absent from all hematopoietic cells examined. Compared with KB cells (a human cell line known for its elevated expression of FR-alpha), the abundance of FR-beta on CD34(+) cell surfaces was relatively low (approximately 8% of KB cell levels). Because many antifolates and folic acid-linked chemotherapeutic agents enter malignant cells at least partially via FR endocytosis, it was important to evaluate the ability of FR on CD34(+) cells to bind folic acid (FA). Based on three FR binding assays, freshly isolated CD34(+) cells were found to display no affinity for FA. Thus, regardless of whether steps were taken to remove endogenous folates before receptor binding assays, FR on primitive hematopoietic cells failed to bind 3H-FA, fluorescein isothiocyanate (FITC)-linked FA, or FA-derivatized liposomes. In contrast, analogous studies on KB cells showed high levels of receptor binding for all three FR probes. These studies show that although multipotent hematopoietic progenitor cells express FR, the receptor does not transport significant amounts of FA. Consequently, antifolates and FA-linked chemotherapeutic agents that can be engineered to enter malignant cells exclusively through the FR should not harm progenitor/stem cell function.
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PMID:Expression and functional characterization of the beta-isoform of the folate receptor on CD34(+) cells. 1033 3

We report the cellular characteristics of cells from three patients with de novo acute myelocytic leukaemia (AML) with t(16;21)(p11;q22), two M4 and one M5a according to the FAB classification, and two permanent cell lines with t(16;21)(p11;q22), TSU1621MT and YNH-1. The FUS/ERG fusion mRNA was demonstrated in all cases by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunophenotypes of the AML cells, and YNH-1 and TSU1621MT cell lines with t(16;21) were characterized as CD34+CD33+CD13+CD11b+CD18+CD56+ HLA-DR-/+. Cells from all samples strongly expressed c-kit, granulocyte colony-stimulating factor receptor (G-CSFR), c-fms (macrophage colony-stimulating factor receptor), interleukin-3 receptor alpha chain (IL-3Ralpha), and granulocyte macrophage colony-stimulating factor receptor alpha chain (GM-CSFRalpha), and these data corresponded well to the growth responsiveness to the cytokines. IL-2Ralpha expression was also found in all t(16;21) samples, but IL-2 did not act on the proliferation of the leukaemic cells in in vitro cultures. G-CSF distinctly promoted the proliferation of leukaemic cells of t(16;21) AML, but did not enhance the expression of MPO and neutrophil differentiation of these cells. Our findings indicate that AML cells with t(16;21) preserve stem cell properties such as CD34 and c-kit expression, and suggest that they have the potential to differentiate into a monocytic lineage. The relationship between the unique cellular characteristics (especially CD56 and IL-2Ralpha expression) and FUS/ERG protein remains undetermined.
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PMID:Myeloid differentiation antigen and cytokine receptor expression on acute myelocytic leukaemia cells with t(16;21)(p11;q22): frequent expression of CD56 and interleukin-2 receptor alpha chain. 1035 36

We have previously reported that in adult mouse bone marrow, CD34low/- c-kit+ Sca-1+ lineage markers negative (Lin-) (CD34-KSL) cells represent hematopoietic stem cells with long-term marrow repopulating ability whereas CD34+ c-kit+ Sca-1+ Lin- (CD34+KSL) cells are progenitors with short-term reconstitution capacity. To further characterize cells in those two populations, relative expression of various genes were examined by reverse transcriptase polymerase chain reaction (RT-PCR). In CD34-KSL cells, none of the genes studied was found to be expressed with the exception of GATA-2, IL-1R alpha, IL-2R gamma, AIC-2B, c-kit, EPO-R, and c-mpl. In contrast, expression of GATA-1 and all cytokine receptor genes examined except IL-2R beta, IL-7R alpha and IL-9R alpha were found in CD34+KSL. The difference between these two populations was also shown in single cell culture analysis of these cells. When cells were clone-sorted and cultured in the presence of SCF, IL-3 and EPO, CD34-KSL cells required much more time to undergo the first cell division than CD34+KSL cells. Dormancy and random fashion of cell division by CD34-KSL cells were also evident by the analysis of the second cell division, which was found to be delayed and unsynchronous compared with CD34+KSL cells. Clonal culture analysis showed that CD34-KSL cells were more potent in proliferation and multilineage differentiation capacities than CD34+KSL cells. In a paired-daughter cell experiment, 75% of CD34-KSL and 50% of CD34+KSL paired-daughter-derived colonies were nonidentical with wide variety of lineage combinations. Taken together, these data support our previous notion that CD34-KSL cells are at higher rank in hematopoietic hierarchy than CD34+KSL cells. In addition, our results using highly enriched stem cell population directly obtained from mouse bone marrow support the proposed stochastic nature of lineage commitment.
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PMID:Further characterization of CD34-low/negative mouse hematopoietic stem cells. 1037 11

Human CD34(+) hematopoietic progenitor cells obtained from bone marrow (BM), umbilical cord blood (UCB), and mobilized peripheral blood (MPB) were purified and investigated for the expression of the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1). CXCR4 was found present on the cell surface of all CD34(+) cells, although it was expressed at lower density on MPB with respect to BM CD34(+) cells. Freshly isolated and in vitro-cultured CD34(+) cells also coexpressed SDF-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Of interest, CD34(+)/CD38(+) committed progenitor cells, unlike primitive CD34(+)/CD38(-) cells, expressed SDF-1 mRNA. Supernatants from in vitro-cultured CD34(+) cells contained substantial (3 to 8 ng/mL) amounts of SDF-1 by enzyme-linked immunosorbent assay and induced migration of CD34(+) cells. Because CD34(+) cells express low levels of CD4, the primary receptor of the human immunodeficiency virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV strains, we investigated the susceptibility of CD34(+) cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a conventional RT activity in culture supernatants and by real-time PCR for HIV DNA in CD34(+) cells exposed to both laboratory adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the anti-CD4 Leu3a monoclonal antibody (MoAb) to either human CD4(+) T cells or CD34(+) cells. In contrast, sgp120 interfered with an anti-CXCR4 MoAb binding to human T lymphocytes, but not to CD34(+) cells. However, CXCR4 on CD34(+) cells was downregulated by SDF-1. These results suggest that CXCR4 and its ligand SDF-1 expressed in CD34(+) progenitors may play an important role in regulating the local and systemic trafficking of these cells. Moreover, these findings suggest multiple and potentially synergistic mechanisms at the basis of the resistance of CD34(+) cells to X4 HIV infection, including their ability to produce SDF-1, and the lack of CXCR4 internalization following gp120 binding to CD4.
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PMID:Human CD34(+) cells express CXCR4 and its ligand stromal cell-derived factor-1. Implications for infection by T-cell tropic human immunodeficiency virus. 1038 99

Herein, we show that CD34, c-kit double-positive (CD34(+)c-kit(+)) cells from the aorta-gonad-mesonephros (AGM) region of the developing mouse are multipotent in vitro and can undergo both B-lymphoid and multimyeloid differentiation. Molecular analysis of individual CD34(+)c-kit(+) cells by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) shows coactivation of erythroid (beta-globin) and myeloid (myeloperoxidase [MPO]) but not lymphoid-affiliated (CD3, Thy-1, and lambda5) genes. Additionally, most cells coexpress the stem cell-associated transcriptional regulators AML-1, PU.1, GATA-2 and Lmo2, as well as the granulocyte colony-stimulating factor receptor (G-CSF-R). These results show that the CD34(+)c-kit(+) population from the AGM represents a highly enriched source of multipotent hematopoietic cells, and suggest that limited coactivation of distinct lineage-affiliated genes is an early event in the generation of hematopoietic stem and progenitor cells during ontogeny.
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PMID:Functional and molecular analysis of hematopoietic progenitors derived from the aorta-gonad-mesonephros region of the mouse embryo. 1047 73

Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph(+)/BCR-ABL(+)) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G(0) cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA. CD34(+) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE(+)) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.
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PMID:Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. 1047 35

Receptor tyrosine kinases (RTK) play a significant role in the signal transduction of normal, and malignant hematopoietic cells. We have previously shown that Axl, a novel RTK, is mainly expressed in leukemias of myeloid origin, and that its expression may be associated with cells of monocytic origin. Since expression of certain RTKs in cancer may be associated with different biology and survival, we investigated whether the expression of Axl is associated with clinical characteristics and survival in acute myeloid leukemia (AML). RNA from 54 patients with AML treated in a cooperative group trial was analyzed in a retrospective and blinded manner using a semi-quantitative reverse transcriptase polymerase chain reaction-based assay with primers specific for the Axl gene. Axl expression was found in 19 out of the 54 cases (35%). Axl expression was not detected more frequently in patients of older age, specific FAB categories, or cases with extramedullary disease. However, there existed a correlation between Axl and bcl-2 expression levels. AML cells with high bcl-2 expression showed higher Axl expression (r = 0.32; P = 0.02), and furthermore, Axl transcript numbers were also higher in AML with high CD34 expression (n = 38, r = 0.42; P = 0.008). No significant difference between leukemias expressing and not expressing Axl was found with regard to complete remission rate. However, quantitative Axl expression was associated with worse progression-free and overall survival. Higher Axl levels had worse prognosis for progression-free (beta: 0.68, s.e.: 0.28, P = 0.015) and overall survival (beta: 0.61, s.e.: 0.31, P = 0.05) using multivariate Cox models adjusted for age, Auer rods and leukocyte counts. In conclusion, in this retrospective analysis, no difference with regard to clinical characteristics at diagnosis was found between AML patients whose leukemia cells show Axl expression vs patients whose cells are Axl negative. The association between Axl and bcl-2 and Axl and CD34 expression in de novo AML needs further investigation. Similarly, the negative impact of Axl levels on outcome should be confirmed in a larger cohort.
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PMID:Axl expression is associated with adverse prognosis and with expression of Bcl-2 and CD34 in de novo acute myeloid leukemia (AML): results from a multicenter trial of the Swiss Group for Clinical Cancer Research (SAKK). 1048 85

We report on a 16-year-old boy with B cell acute lymphocytic leukemia presenting marked leukocytosis (388,000/microliter) and resistance to multidrug chemotherapy. Karyotypical analysis revealed a novel t(3;15)(q27;q2?2) chromosomal abnormality. Because 3q27 is known to be a locus of the bcl-6 gene, which is frequently involved in B cell malignancies, molecular biological analyses were performed. Although no rearrangement was detected in 5 genes including bcl-6 on 3q27 and 2 genes on 15q2, reverse transcriptase-polymerase chain reaction procedures detected relatively strong mRNA expression of the bcl-6, smrp, dvl3, and tpml genes. These results indicate that immature leukemic cells with CD10 and CD34 positivity and rearrangement of the T cell receptor beta gene may coexist with relatively mature subpopulations that are positive for CD19 and CD20 surface markers, bcl-6 expression, and rearrangement of the gene for immunoglobulin kappa.
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PMID:[B cell acute lymphocytic leukemia with marked leukocytosis and t(3;15)(q27;q2?2)]. 1048 45

The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34(+) cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34 and different levels of c-kit (++,+,Lo/-), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34KitLo+/- cells transduced with c-kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-beta1 and tumor necrosis factor-alpha. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.
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PMID:Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-alpha and transforming growth factor-beta1. 1049 4


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