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

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

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

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

Marked thrombocytosis (over 50 x 10(4)/microl) is frequently seen in patients with hepatoblastoma. Thrombopoietin (TPO), c-mpl ligand, has recently been purified as the major physiological regulator of the thrombopoiesis and is mainly produced in the liver. Since it is possible that TPO participates in thrombocytosis and the tumor growth of this particular hepatic tumor, serum TPO levels in addition to interleukin 1beta (IL-1beta) and IL-6 levels were assessed in seven untreated patients by using a sandwich enzyme-linked immunosorbent assay. High serum TPO levels were observed in all of the examined patients. The level ranged from 3.15 to 11.02 (mean +/- standard deviation; 6.08+/-1.25) fmol/ml. IL-6 levels were also somewhat higher than normal. Platelet counts, however, appeared to correlate more with serum TPO levels (p = 0.1) than with IL-1beta (p = 0.5) and IL-6 (p = 0.2) levels. Furthermore, using the reverse transcriptase polymerase chain reaction method, the expression of c-mpl mRNA was found in five of eight hepatoblastoma tissues as well as TPO mRNA in all eight tissues. These observations suggest that thrombocytosis in hepatoblastoma patients results from the production of cytokine members, including TPO, within tumor tissues. Additionally, it is possible that TPO might act as a type of autocrine and/or paracrine system for cellular growth in this tumor.
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PMID:Thrombopoietin in patients with hepatoblastoma. 976 12

Thrombocytosis is occasionally seen in patients with carcinomas and has been assumed to be attributable to interleukin-6 or granulocyte-macrophage colony-stimulating factor produced by carcinoma cells. In this study, we clarified whether thrombopoietin (TPO) is involved in carcinoma-associated thrombocytosis. Expression of TPO mRNA was observed in the majority of 27 carcinoma cell lines as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). There were 6 PCR products differing in size; sequence analysis showed the full-length TPO mRNA (TPO-1), 12- and 116-bp deleted variants (TPO-2 and TPO-3, respectively), and 3 novel isoforms (197- and 128-bp deleted forms and a 60-bp insert form of TPO-3; named TPO-4, TPO-5, and TPO-6, respectively). Of 27 lines, 24 expressed TPO-1 mRNA with various other isoforms. Culture supernatants of COS-1 cells transfected with TPO-5 or TPO-6 cDNA did not promote the proliferation of TPO-responsive cells, whereas Western blot analysis on the cell lysates demonstrated TPO-5 but not TPO-6 protein, suggesting poor extracellular secretion (TPO-5) or poor protein synthesis (TPO-6). TPO protein was detected in 10-fold concentrated culture supernatants of cells of these carcinoma lines, with a median concentration of 0.38 fmol/mL as evaluated by enzyme-linked immunosorbent assay. High blood TPO levels were observed with a median value of 3.46 fmol/mL (range, 0.34 to 8.67 fmol/mL) in patients with advanced carcinomas associated with thrombocytosis. These results indicate that thrombocytosis in patients with carcinomas might be caused, at least in part, by TPO produced by carcinoma cells.
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PMID:Production of thrombopoietin by human carcinomas and its novel isoforms. 1047 24

CD34(+) cells and megakaryocyte progenitors were lower in marrow from patients after hematological recovery from the first cycle of 5-fluorouracil, leucovorin, adriamycin, cyclophosphamide (FLAC) chemotherapy plus PIXY321 (GM-CSF/interleukin 3; IL-3 hybrid) than in FLAC + GM-CSF or pre-FLAC marrows. Marrow stromal layers, an in vitro model of the marrow microenvironment, express a combination of stimulatory and inhibitory factors that modulate hematopoietic progenitor cell proliferation and differentiation. The TaqMan assay and quantitative reverse transcriptase-polymerase chain reaction were used to measure monocyte chemoattractant protein-1 (MCP-1), melanoma stimulatory growth activity, and monokine inducible by interferon-gamma (Mig) (inhibitory chemokines for primitive or megakaryocyte progenitors) mRNA levels in in vitro PIXY and GM-CSF-treated and patient post-FLAC marrow stromal layers. Chemokine mRNA was increased after in vitro GM-CSF and to a lesser extent after PIXY treatment. MCP-1 mRNA levels were fivefold higher in FLAC + PIXY than in FLAC + GM-CSF layers, and Mig mRNA was elevated in FLAC + GM-CSF layers. Thrombopoietin (TPO), insulin-like growth factor I (IGF-I), and IGF-II (stimulatory factors for primitive and megakaryocyte progenitors) mRNA were also measured. TPO mRNA levels were 30% lower in GM-CSF and PIXY-pretreated than in control layers with no decrease in IGF mRNA. TPO mRNA in stromal layers of patients who developed grade 3 thrombocytopenia (platelets < 20 x 10(9)/l) during the third cycle of FLAC was only 24% of levels in stromal layers of marrow from other post-FLAC patients. Results demonstrate that patient and in vitro treatment had modulatory effects on TPO and chemokine mRNA expression in marrow stromal layers.
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PMID:Thrombopoietin and chemokine mRNA expression in patient post-chemotherapy and in vitro cytokine-treated marrow stromal cell layers. 1100 17

In the present study, the effects of murine bone marrow endothelial cell conditioned medium (ECM) combined with flt3 ligand (FL) or/and thrombopoietin (TPO) on the proliferation of HPP-CFC and CFU-GM were investigated. Both ECM and the concentrated retentate of ECM (MW>10 kD) promoted the growth of CFU-GM and HPP-CFC, and this promoting effect was further enhanced by addition of FL or TPO. Using reverse transcriptase-polymerase chain reaction (RT-PCR) technique, the expression of FL and TPO mRNA was not found in murine bone marrow endothelial cells.
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PMID:[Effects of murine bone marrow endothelial cell conditioned medium in combination with FL and TPO on the growth of HPP-CFC and CFU-GM]. 1193 Feb 13

Usually thyroid cells isolated from tissue obtained by surgery or thyroid cell lines are used to investigate the pathogenesis of autoimmune thyroid diseases. Isolation and cultivation of thyrocytes from fine-needle aspiration biopsy (FNAB) has not yet been published. The aim of this study was to isolate and cultivate thyrocytes from samples of FNAB. FNAB samples were obtained from nine adults and nine children with Hashimoto's thyroiditis (HT). The aspiration material was filtered resulting in small samples of tissue on the surface of the filter membrane. These tissue fragments were digested by collagenase I and dispase II. The yielding cells were cultivated for 3 weeks in Ham's F12 Kaighn's Modification medium in presence of 1 mU/mL bovine thyrotropin (TSH), 10 microg/mL human insulin, 6 microg/mL transferrin, and 10(-8) M hydrocortisone. Finally, isolated thyroid cells were characterized by determination of gene expression of thyrotropin receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) using a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Thyroid cells obtained by FNAB can be maintained over a time period of approximately 3 weeks. Depending on the sample size a final number of 1000-14,000 cells was gained per FNAB. In addition, all cells isolated by the described method expressed TPO mRNA. TSHR mRNA was found in 4 samples, whereas 15 samples were Tg mRNA-positive. There were no differences with respect to the expression TSHR and TPO mRNA between samples from adults and children. The isolation and cultivation of thyroid cells obtained by FNAB has been established. In contrast to surgical specimen, this technique provides an easy access to thyrocytes derived from individual patients allowing repeated sampling to investigate the time progression of the chronic disease or the effect of treatment over time.
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PMID:Isolation of thyroid cells obtained by fine-needle aspiration biopsy. 1618 6


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