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)

Lymphohematopoiesis occurs in the densely packed environment of the intramedullary spaces. Primitive lymphohematopoietic stem cells exist in close apposition to a variety of supportive cells including both hemopoietic and nonhemopoietic lineages. Using an in vitro long-term Dexter liquid culture system, we have established that a variety of cytokines are produced constitutively by such stromal cells in culture. These cytokines include Steel factor, interleukin-6 (IL-6), and colony-stimulating factor (CSF-1). Granulocyte-CSF and granulocyte-macrophage-CSF mRNA can be detected after refeeding of cultures, although in quiescent cultures message for these factors is difficult to detect. Interleukin-3, IL-4, and IL-5 are not detectable by standard Northern blot analysis or bioassay of condition media. However, IL-3--detectable by reverse-transcriptase PCR and biologic activity--was confirmed by growth of factor-dependent cells on stromal cells with IL-3 antibody blocking of such growth. Stem cells resident on such stromal cells are mirrored by the high proliferative potential colony-forming cell assay and are responsive to a relatively large number of cytokines, with Steel factor being of central importance, appearing to be a critical component of various synergistic combinations. Steel factor allows reduced levels of other factors in such combinations and works early in a temporal sequence. Hematopoietic stem cells can engraft in normal nonmyeloablated hosts. Using a male/female BALB/c transplantation model, we have shown high rates of engraftment into normal animals, out after marrow infusion to 25 months, after marrow infusion and that post-5-fluorouracil bone marrow is quite deficient in such engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro and in vivo studies of stromal niches. 799 65

Although parathyroid hormone-related peptide (PTHRP) is produced by adult T cell leukemia (ATL) cells and causes hypercalcemia in ATL patients, very little is known about the regulation of PTHRP gene expression in the leukemic cells. The present study was undertaken to clarify the role of T cell growth factor, interleukin-2 (IL-2), in the expression of PTHRP gene, using a human T cell leukemia virus type I (HTLV-I)-infected T cell line, MT-2. Recombinant human IL-2 caused a transient increase in the steady state level of PTHRP messenger RNA (mRNA) in MT-2 cells, and a maximal effect was observed at 3-6 h. The effect of IL-2 was dose dependent, with a maximal response being observed at 10(-10) M. A monoclonal antibody against IL-2 receptor (anti-Tac antibody) inhibited the IL-2-induced increase in PTHRP mRNA level. Recombinant human IL-1, IL-3, IL-4, and IL-6 failed to increase PTHRP mRNA level. Nuclear run-off transcription assay showed that the transcription rate of the PTHRP gene was modestly increased by IL-2. In addition, IL-2 caused a substantial increase in the stability of PTHRP mRNA, compared with control cells in which the apparent half-life of PTHRP mRNA was less than 30 min after RNA synthesis was inhibited by the RNA polymerase II inhibitor, dichlorobenzimidazole riboside. The secretion of PTHRP, as determined by both a newly established immunoradiometric assay using recombinant human PTHRP(1-87) as the standard and an RIA using an antibody against PTHRP(109-141), was increased by IL-2 but not by IL-1, IL-3, IL-4, or IL-6. The IL-2-induced increase in PTHRP secretion was completely inhibited by the addition of anti-Tac antibody. These results demonstrate that IL-2 stimulates the production and secretion of PTHRP by HTLV-I-infected T cells through specific binding to IL-2 receptor and that the effect of IL-2 is mediated by a posttranscriptional as well as a transcriptional mechanism. It is suggested that IL-2 may be involved in an auctocrine/paracrine fashion not only in the proliferation of HTLV-I-infected T cells but also in the enhanced production and secretion of PTHRP and thus the development of hypercalcemia in ATL patients.
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PMID:Interleukin-2 increases production and secretion of parathyroid hormone-related peptide by human T cell leukemia virus type I-infected T cells: possible role in hypercalcemia associated with adult T cell leukemia. 809 24

Despite the marked expansion of leukemic cells observed in the hematopoietic system of chronic myeloid leukemia (CML) patients, there is clinical and experimental evidence that normal nonclonal cells persist in the bone marrow (BM) and peripheral blood (PB) of patients in the early chronic phase. In this study, we attempt to select the benign progenitor-enriched population from the PB of CML patients. The CD34+ cells isolated from the PB of 12 CML patients in the chronic phase were treated with low doses (5 or 10 micrograms/mL) of 5-fluorouracil (5-FU). We expanded these cells for 7 days in liquid cytokine-mediated cultures. This expansion in the presence of interleukin-1 (IL-1) plus stem cell factor (SCF) plus IL-3 or leukemia inhibitory factor (LIF) plus SCF plus IL-3 seemed at least to preserve the initial clonogenic potential of CD34+ and 5-FU-resistant CD34+ cells. For the presence of BCR-ABL, mRNA from each of the 12 patients was studied by reverse-transcriptase-polymerase chain reaction (RT-PCR) on 10-15 pooled CFU-GM colonies plucked from methylcellulose cultures of starting and expanded populations. Although all PCR results were positive for colonies harvested before liquid culture, we were able to identify BCR-ABL-negative colonies from an expanded CD34+ population cultured in the presence of recombinant cytokines in 11 of 12 patients studied. 5-FU pretreatment of CML CD34+ cells markedly reduced their clonogenic potential and growth factor-mediated cell proliferation but favored higher frequency of BCR-ABL-free colonies. In conclusion, these data show that 5-FU-resistant CD34+ cells from the PB of CML patients contain normal progenitor cells, which can be selected and expanded in short-term cytokine-mediated cultures.
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PMID:5-Fluorouracil-resistant CD34+ cell population from peripheral blood of CML patients contains BCR-ABL-negative progenitor cells. 854 39

Interleukin-11 is a stromal cells derived cytokine which stimulates the proliferation of primitive haemopoietic progenitor cells. For this paper we have studied the constitutive expression of IL-11 mRNA in a panel of wellknown leukaemic cell lines and samples from AML patients at diagnosis. Moreover, the same cellular populations were evaluated for their proliferative response to recombinant-human-(r-hu). IL-11 alone and combined with r-hu-IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCF, c-kit ligand). The colony-forming ability of HL60, K562, KG1 cells and eight fresh AML cell populations was assessed by a clonogenic assay in methylcellulose. In eight additional AML cases the number of S-phase leukaemic cells induced by IL-11 was determined by the bromodeoxyuridine (BRDU) incorporation assay after 3d of liquid culture. IL-11, as single cytokine, did not stimulate the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions. In contrast, the proliferation of the leukaemic cells in response to IL-3, GM-CSF and SCF was enhanced by co-incubation with IL-11, and this effect was reversed in blocking experiments by the anti-IL-11 Moab. When tested on primary AML samples, IL-11 alone showed little, if any, proliferative activity. However, it increased the IL-3-dependent blast colony formation in eight out of eight cases and GM-CSF in seven cases. IL-11 also augmented synergistically the number of CFU-L stimulated by SCF in seven cases. A combination of three factors (IL-11, SCF and IL-3) yielded optimal colony formation. The BRDU studies showed the significant increase of AML cells in S-phase when IL-11 was combined with SCF, whereas the two CSF had no activity on their own. Positive interaction was also observed when IL-11 was added to IL-3 supplemented cultures in five out of eight cases tested. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) demonstrated the constitutive expression of IL-11 mRNA in all the cell lines and 11/12 AML samples studied at diagnosis. These results indicate that IL-11 is expressed in leukaemic myeloid cells and that their proliferation is regulated by the cytokine which acts as a synergistic factor.
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PMID:Interleukin-11 (IL-11) acts as a synergistic factor for the proliferation of human myeloid leukaemic cells. 854 68

Early hematopoietic progenitors expressing the CD34+ phenotype can be harvested from the peripheral blood of normal individuals. We have optimized the liquid culture of human CD34+ peripheral blood progenitors (PBPs) to achieve differentiation into a population of cells consisting almost entirely of eosinophil progenitors and maturing eosinophils. Growth of CD34+ PBPs for 28 days in the presence of the combination of IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 resulted in an almost 250-fold increase in cell number, yielding a population that contained 83% maturing eosinophils. The residual population consisted of basophils and mast cells (3% by acidic toluidine blue staining, 15.2% by flow cytometric assay for binding to high-affinity IgE receptor) and immature cells. This provides an opportunity to examine the kinetics of the acquisition of specialized mature eosinophil characteristics during eosinophil differentiation. Several host-defense and bioactive proteins are found almost exclusively in eosinophil granules. In addition, stimulated eosinophils, like neutrophils, produce copious amounts of toxic oxygen radicals. We used our culture system and the sensitive technique of reverse-transcriptase polymerase chain reaction to analyze the kinetics of production of messenger RNA transcripts encoding several eosinophil proteins, including five eosinophil granule proteins and four subunit peptides of the superoxide-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in small numbers of differentiating eosinophils from peripheral blood CD34+ cells. Freshly isolated CD34+ PBPs contained transcripts for the ubiquitously present housekeeping protein phosphoglucokinase but contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. On day 3 of culture, no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased several fold during the entire culture period. Similar kinetics were seen for all but one of the NADPH oxidase protein transcripts. However, transcripts for the p67phox NADPH oxidase protein were not detected until day 7, and functional oxidase activity did not appear until day 12. From that point, oxidase activity increased dramatically over the culture period. These studies demonstrate that commitment of CD34+ PBPs to the eosinophil lineage occurs very early, by day 3, but that further events in differentiation must take place before the appearance of histologically staining eosinophil granules and acquisition of functional oxidase capacity.
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PMID:Early commitment to the eosinophil lineage by cultured human peripheral blood CD34+ cells: messenger RNA analysis. 875 12

We report that I-Ab-restricted T cell clones, elicited by influenza infection of C57BL/10 mice and specific for the hemagglutinin peptide HA1 186-205, express class II. They respond to peptide stimulation by IL release (IL-3 or IFN-gamma) without a requirement for APC but do not proliferate. Moreover, surface expression of class II requires de novo synthesis in the presence of the stimulatory peptide and is inhibited by coculture with TCR-specific Ab, or brefeldin A or cycloheximide. Clonotypic specificity of peptide induction was confirmed by failure of other allele specific peptides to enhance class II expression. Addition of the viral peptide to T cells induced homotypic adhesion, which provides a physical basis for stabilization of class II-peptide complexes at the cell surface. Extinction of class II expression was evident in the corresponding T cell hybridomas, which might account for the failure to report class II expression by murine T cells. Control studies indicated that class II was not passively acquired from APC by demonstrating 1) failure of processed Ag to induce class II expression, 2) allo-class II (Ak) was not acquired by coculture with peptide and semisyngeneic (H-2 b/k) APC, 3) absence of class II expression by a NP peptide-specific Th2 clone under identical culture conditions, and most significantly, 4) reverse-transcriptase PCR amplification and surface expression of class II using highly purified preparations of FACS-selected CD4+ class II- cells cocultured with the stimulatory peptide.
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PMID:Viral peptide specific induction of MHC class II expression by murine T cell clones. 880 37

Here we review our recent experience addressing the role of SCF in multiple myeloma (MM). We first investigated the proliferation of MM cell lines and bone marrow samples from myeloma patients in response to rh-SCF alone and combined with Interleukin-6 (IL-6), IL-3, and IL-3/GM-CSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (>90%) by immunomagnetic depletion of T, myeloid, monocytoid and NK cells. The number of S-phase cells was evaluated after 3 days of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. All the experiments were performed in serum-free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL-6, IL-3 and PIXY-321. Conversely, SCF addition resulted in 2.4-fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S-phase. The c-kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti-SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the MO7e proliferation assay. These results suggest that an autocrine proliferative loop may be operative in MT3 cell line. When tested on fresh myeloma samples, SCF increased the number of S-phase plasma cells (4.7 +/- 1.6% vs 3.4 +/- 1.3% in control cultures; p = 0.02). Significant proliferation was also induced by IL6, IL-3 and PIXY-321. The addition of SCF significantly enhanced the proliferation of myeloma cells responsive to IL-6. Preliminary experiments performed on circulating plasma cells and myeloma precursors further supported the role of SCF on the proliferation of the neoplastic clone in MM.
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PMID:C-kit ligand (SCF) in human multiple myeloma cells. 883 3

Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth muscle cell repertoire, and whose in vivo counterpart is that of myoid cells found in adult and fetal human bone marrow cords. We studied the cytokine expression by reverse-transcriptase polymerase chain reaction (RT-PCR) from pooled fast-growing clones from 10 different bone marrow samples. RT-PCR indicated that 30 cytokines (out of 42 studied) were expressed by CDCLs (20 after medium renewal and hydrocortisone renewal, three after addition of interleukin I beta (IL-1 beta) and seven in only part of the CDCL layers examined). The cytokines expressed comprised mediators known to be involved in the maintenance of early and late hematopoiesis (IL-1 alpha and IL-beta, IL-6, IL-7, IL-8, IL-11 and IL-13; colony-stimulating factors, thrombopoietin, erythropoietin, stem cell factor, fit 3-ligand, hepatocyte cell growth factor, tumor necrosis factor alpha, leukemia inhibitory factor, transforming growth factors beta 1 and beta 3; and macrophage inflammatory protein 1 alpha), angiogenic factors (fibroblast growth factors 1 and 2, vascular endothelial growth factor) and mediators whose usual target (and source) is the connective tissue-forming cells (platelet-derived growth factor A, epidermal growth factor, transforming growth factors alpha and beta 2, oncostatin M and insulin-like growth factor 1), or neuronal cells (nerve growth factor). The cytokines not expressed were lymphokines (IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, and IL-12 and interferon gamma) or mediators synthesized by macrophages (inhibin, activin, platelet-derived growth factor B, and IL-1 receptor antagonist). This study complements the description of the phenotype of the myoid cells, confirming that these cells are the marrow connective tissue-forming cells; moreover, this work suggests that stromal control of hematopoiesis is multifactorial and that myoid cells are involved in the control of marrow angiogenesis and innervation.
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PMID:The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. 909 Jul 90

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-6 on clonogenic growth of blast-cell progenitors from 19 immunologically defined CD10-positive B-lineage acute lymphoblastic leukemias (ALL) coexpressing (My+ALLs) or not (My-ALLs) myeloid antigens have been studied. Our results demonstrate that GM-CSF was able to support the clonogenic growth of blast cells from My+ALLs, being totally ineffective on My-All samples. Accordingly, both alpha and beta chains of GM-CSF receptor (R) were expressed by My+ALL blasts, as investigated by reverse-transcriptase polymerase chain reaction (RT-PCR). Colony cells from GM-CSF-stimulated My+ALL cultures displayed the same immunophenotype as primary leukemic cells at diagnosis (CD10+, CD19+, CD22+), and retained the expression of myeloid-associated antigens and of GM-CSF-R transcripts. Moreover, My+ALL blasts showed a preferential sensitivity to the growth-promoting activity of IL-3 and IL-6, as compared with My-ALL cells. In addition to rearrangements of the JH region of immunoglobulin genes, My+ALL cells showed aberrant rearrangements of gamma (three cases) and beta (two cases) T-cell receptor genes, as well as of bcr sequences (three cases). Our data, showing an unexpected cross-lineage response of My+ALLs to GM-CSF, and their preferential stimulation by IL-3 and IL-6, as compared with My-ALLs, further support the concept that My+ALLs represent a separate entity with unique biological features.
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PMID:Human granulocyte-macrophage colony-stimulating factor supports the clonogenic growth of B-lineage acute lymphoblastic leukemias expressing myeloid antigens. 942 72

In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-alpha. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1alpha, IL-1beta, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-alpha, TGF-beta, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-gamma transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+ CD11c+ CD3- DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.
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PMID:The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation. 946 23


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