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)

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.
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PMID:Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines. 749 68

The activity of the c-Mpl ligand hematopoietic progenitors meets criteria expected for thrombopoietin (TPO). Bio-assays have shown that blood TPO levels are inversely related to platelet mass. We sought to identify the molecular basis for this regulation. To determine if TPO mRNA levels respond to platelet demand, RNA from selected organs of mice with high, normal or low platelet counts was subjected to semiquantitative reverse transcriptase-polymerase chain reaction. Although no differences in TPO mRNA levels between control and treated mice could be detected in liver or kidney, TPO-specific bands were more intense after 25 to 30 polymerase chain reaction cycles in marrow-derived mRNA from thrombocytopenic mice. The TPO-specific bands were less intense in thrombocytotic mouse marrow and spleen than control mouse marrow and spleen after 30 cycles. These data support the hypothesis that TPO levels are regulated, at least in part, by modulating mRNA levels in response to platelet demand.
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PMID:Murine thrombopoietin mRNA levels are modulated by platelet count. 757 32

When 15-deoxyspergualin (DSG) was administered into [BALB/c-->C3H/He] bone marrow (BM) chimeras from day 14 to day 25, increased platelet counts were observed from day 25 to day 33. Twofold increase of platelet counts was observed in DSG-treated BM chimeras compared with phosphate buffered saline (PBS)-treated BM chimeras. By using reverse transcriptase-polymerase chain reaction (RT-PCR), several cytokine mRNA expressions were analyzed in order to clarify which cytokines are involved in thrombopoiesis. So far, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), stem cell factor (SCF), and IL-11 have been reported to have potent thrombopoietic activity in vivo. Although some other cytokines such as IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) possess the capacity of thrombopoiesis, megakaryocytopoiesis is more marked in these cytokines. IL-6 mRNA expression was increased in spleen cells from DSG-treated BM chimeras from day 25 to day 32 and in bone marrow cells from day 19 to day 28. LIF mRNA expression was not significantly increased compared with PBS control. Although SCF mRNA expression was increased, the kinetics of increased SCF mRNA expression did not fit the kinetics of increased platelet counts. Increased mRNA expression in other hematopoietic cytokines, such as IL-3, granulocyte-colony stimulating factor (G-CSF) and GM-CSF were also observed, thus suggesting that these cytokines may synergistically support thrombopoiesis in concert with IL-6. These results suggest that IL-6 and other hematopoietic cytokines might induce increased platelet counts, although the involvement of thrombopoietin (TPO) and IL-11 should be analyzed in the future.
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PMID:A novel immunosuppressant 15-deoxyspergualin and thrombopoiesis. 795 88

Thrombopoietin (TPO) is a recently identified hematopoietic growth factor that is essential for the growth and development of megakaryocytes. We have previously shown that TPO induces proliferation of acute myeloblastic leukemia (AML) cells in vitro. In this study, we have examined the expression of TPO and its receptor c-mpl in a series of AML cases and human leukemia cell lines. The mRNA transcripts of TPO were detectable in 18 of 50 AML cases and in some myeloid leukemia cell lines (HEL, M07E and CMK) by means of reverse transcriptase polymerase chain reaction (RT-PCR). In addition, TPO transcripts were coexpressed with c-mpl transcripts in 10 of 50 AML cases and in HEL, M07E and CMK cells. With regard to the French-American-British (FAB) classification, coexpression OF TPO and c-mpl was observed with high frequency in AML cases of M7-type. Despite the TPO expression in a substantial fraction of leukemia cells, biological activity of TPO was not found in the conditioned medium that was obtained from cultivation of TPO mRNA-positive leukemia cells. These results suggest that TPO may not commonly participate in the abnormal growth of AML cells as an extracellular autocrine growth factor.
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PMID:Coexpression of thrombopoietin and c-mpl genes in human acute myeloblastic leukemia cells. 855 44

The in vivo effect of G-CSF on the maturation of mouse bone marrow megakaryocytes was studied by monitoring the DNA contents. Megakaryocytes were first identified by a specific 1C2 monoclonal antibody against mouse platelets and megakaryocytes and DNA contents of these cells were measured by propidium iodine. Megakaryocytes of mice transgenic for human G-CSF had a modal DNA class of 8N, showing a striking contrast to the previous reports that normal mouse megakaryocytes from most strains have 16N DNA content as a modal class. Daily 10 micrograms administration of G-CSF to mice for three to five days affected the DNA distribution pattern of bone marrow megakaryocytes, with a higher proportion of cells having 8N DNA contents. This G-CSF treatment, however, did not influence the peripheral blood platelet count or bone marrow megakaryocyte number. Administration of G-CSF along with thrombopoietin (TPO) reduced the proportion of megakaryocytes, with 32N DNA, the DNA class that was increased by TPO. Finally, the presence of mRNA for the mouse G-CSF receptor was demonstrated in two megakaryoblastic cell lines by reverse transcriptase polymerase chain reaction. These results indicated that G-CSF may have a suppressive effect on the maturation of mouse bone marrow megakaryocytes when monitored by the DNA polyploidy. Although further study is clearly necessary, the presence of mRNA for the G-CSF receptor in megakaryocytic lineage strongly suggests the direct action of G-CSF on this cell lineage.
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PMID:Overexpression of granulocyte colony-stimulating factor in vivo decreases the level of polyploidization of mouse bone marrow megakaryocytes. 882 Sep 58

Thrombopoietin (TPO) is the major regulator of platelet production in vivo and is the ligand for the MPL receptor. In an effort to determine the distribution of TPO and MPL in the different hematopoietic cell types and in various types of tissue, we examined the mRNA expression of this ligand-receptor pair in two series of human leukemia-lymphoma cell lines and of solid tumor cancer cell lines using northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. At the northern blot mRNA level, 8/15 (53%) megakaryocytic and 3/11 (27%) erythroid leukemia cell lines expressed MPL mRNA; except for one positive monocytic cell line, the remaining 78 pre B-cell, B-cell, plasma cell, T-cell, NK cell, myeloid, monocytic and Hodgkin/anaplastic large cell lymphoma (ALCL)-derived cell lines were negative. No MPL message was detected in any of the 23 solid tumor cell lines established from 21 different tumors. In order to examine whether a low level of MPL expression could be detected, 51 leukemia cell lines were investigated with the RT-PCR technique. By this technique, MPL message was seen in many more cell types: 13/26 (50%) of non-erythromegakaryocytic cell lines and in nearly all megakaryocytic (14/15, 93%) and erythroid (10/11, 91%) cell lines. Thus, the highest expression of MPL clearly occurs in cells with megakaryocytic differentiation; furthermore, expression of MPL appears to be restricted to hematopoietic cell types. TPO mRNA expression was examined by RT-PCR and found in 9/11 (82%) of the solid tumor cell lines (derived from colon, endometrium, kidney, liver, ovary, retinoblastoma and urinary bladder cancers). Among the leukemia-lymphoma cell lines, TPO mRNA was detected by RT-PCR in most plasma cell, myeloid, megakaryocytic and erythroid cell lines, but not in pre B-cell, B-cell or T-/NK-cell lines. The results reported here extend the observations of MPL and TPO expression in normal cells to the whole spectrum of hematological cell types and to an array of different tissue types, both exemplified by their malignant counterparts.
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PMID:Expression of thrombopoietin and thrombopoietin receptor MPL in human leukemia-lymphoma and solid tumor cell lines. 896 Jan 8

The tie gene encodes a receptor tyrosine kinase that together with its thus far unidentified ligand appears to play a distinct role in the regulatory pathway of early hematopoiesis and angiogenesis. Here, we attempted to define the possible involvement of tie in the pathobiology of hematopoietic malignancies by examining tie mRNA expression in human leukemia and lymphoma cells. We used a large panel of 93 well-characterized human continuous leukemia-lymphoma cell lines as model systems for the various hematopoietic cell lineages. At the Northern blot level, none of the 27 lymphoid leukemia or lymphoma-derived cell lines (originating from four B-precursor leukemia, four B-cell leukemia, four B-cell non-Hodgkin's lymphoma, two myeloma, two Burkitt lymphoma, four T-cell leukemia, five Hodgkin lymphoma, two anaplastic large cell lymphoma) tested expressed tie transcripts, whereas 23/42 (55%) of the myeloid cell lines analyzed expressed tie mRNA: in detail, 15 of 20 (75%) megakaryocytic, five of 11 (45%) erythroid, three of seven (43%) myelocytic and none of four monocytic cell lines were tie mRNA positive. In the reverse transcriptase-polymerase chain reaction analysis, which can detect very low levels of mRNA expression, all 12 myeloid cell lines and 19 of 39 (48%) lymphoid cell lines were positive. In experiments aimed at inducing cellular differentiation over an incubation period of 4 days, the phorbol ester PMA strongly enhanced tie mRNA expression in one erythroid and in one myelocytic cell line, but (like thrombopoietin) down-regulated tie mRNA expression in two megakaryocytic cell lines. Taken together these results indicate that tie is predominantly expressed in leukemia cells derived from the myeloid cell lineages (and here in particular in megakaryoblastic cells) and not in lymphoid leukemia cells. These observations provide some evidence for the hypothesis that tie is a receptor for a regulatory factor involved in normal and plausibly also leukemic hematopoiesis.
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PMID:Expression of tie receptor tyrosine kinase in human leukemia cell lines. 930 79

We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FACS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained within the CD34(+) cell fraction. In the present study, the frequency of stromal progenitors in both the CD34+ and CD34- subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34+, CD38-, HLA-DR-, CDw90+ phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34-, CD38-, HLA-DR-, CDw90+ phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34+ fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34+ or CD34- bone marrow cells using reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34+ or CD34- cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34+ bone marrow cells, although the differences between CD34+ and CD34- stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/mL SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment.
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PMID:Thrombopoietin is synthesized by bone marrow stromal cells. 934 28

We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of >/= 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3-challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, approximately 5 x 10(3) tissue culture infectious dose50/mL/10(6) cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that approximately 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.
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PMID:Productive human immunodeficiency virus-1 infection of purified megakaryocytic progenitors/precursors and maturing megakaryocytes. 945 52

CXCR4 is the receptor for the alpha-chemokine stromal cell-derived factor 1 (SDF-1) and has been shown to be expressed on a diversity of leukocytes. In this report, the expression of the CXCR4 receptor in cells of megakaryocytic lineage and the role of SDF-1 in megakaryocytopoiesis were investigated. Using flow cytometry in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we observed that bone marrow CD34(+), CD61(+) cells, blood platelets, and megakaryocytic leukemia cell lines all expressed the CXCR4 receptor. To examine the expression of the CXCR4 receptor on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-Meg]), CXCR4-positive and -negative CD34(+) populations were separated from bone marrow and cultured in a plasma clot culture system. A subpopulation of the CFU-Meg was found in the CXCR4-positive fraction. The functional significance of CXCR4 expression on cells of the megakaryocytic lineage was examined by studying the effects of SDF-1alpha on migration and proliferation of megakaryocyte progenitor cells in vitro. We found that SDF-1alpha potently induced megakaryocyte progenitor migration and significantly enhanced adhesion of mature marrow megakaryocytes to endothelium. No marked effects of SDF-1alpha alone or in combination with thrombopoietin and stem cell factor/kit ligand on megakaryocyte production in vitro were noted. These results demonstrate for the first time that the CXCR4 alpha-chemokine receptor is expressed on cells of the megakaryocytic lineage from progenitors to platelets and that its ligand SDF-1alpha may modulate several aspects of megakaryocytopoiesis.
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PMID:The alpha-chemokine receptor CXCR4 is expressed on the megakaryocytic lineage from progenitor to platelets and modulates migration and adhesion. 968 Mar 41


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