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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

C1498 is an atypical myeloid leukemia that originated in a C57BL/6 mouse and has been used as a model for acute myelogenous leukemia. In studies of the immune response to C 1498, we found that this tumor contained mRNA encoding the canonical NKT cell receptor Vbeta8.2-Valpha14Jalpha281. Although cell-surface phenotypic analysis showed C1498 to be negative for NK1.1, it expressed several other molecules associated with NKT cell populations, such as DX5, CDld, CD69, CD44, CD45RB and B220. RT-PCR demonstrated that C1498 contained CD3epsilon mRNA transcripts, but message was not found for CD4, CD8alpha, or CD8beta. This indicates that C1498 falls within the double negative (CD4-CD8-) NKT cell lineage. RNase protection analysis showed that C1498 expressed mRNA for IL-2, IL-15, and macrophage migration inhibitory factor (MIF). These findings suggest that C1498 should be re-classified as a NKT cell leukemia with atypical myeloid features. It may, therefore, be a novel cell line in which to study NKT cell development and serve as a model for human NKT cell malignancies.
Leuk Lymphoma 2002 Aug
PMID:Characterization of a murine NKT cell tumor previously described as an acute myelogenous leukemia. 1240 Jun 7

TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT-PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs. The encoded TWE-PRIL protein is composed of TWEAK cytoplasmic and transmembrane domains fused to the APRIL C-terminal domain. TWE-PRIL mRNA is expressed and translated in human primary T cells and monocytes, and endogenous TWE-PRIL protein was detected in primary human T lymphocytes and monocytic cell lines. TWE-PRIL is membrane anchored and presents the APRIL receptor-binding domain at the cell surface. It is a biologically active ligand, as it stimulates cycling in T- and B-lymphoma cell lines. Much like membrane-bound and secreted TNF-alpha, the different cellular localizations of TWE-PRIL and APRIL suggest that they exert distinct biological roles.
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PMID:An endogenous hybrid mRNA encodes TWE-PRIL, a functional cell surface TWEAK-APRIL fusion protein. 1241 89

HL is a malignant lymphoma characterized by a small number of malignant HRS cells among a major population of infiltrating reactive cells, e.g., lymphocytes and eosinophils. We previously reported that mast cells are present in HL-affected lymph nodes and therein are the predominant CD30L-expressing cells. The CD30L expressed on mast cells is functionally active and can provide stimulatory signals to HRS cells. Thus, mast cells constitute an important portion of the infiltrating reactive cells that contribute to tumor progression in HL. Control of the recruitment of this previously unrecognized cell and its interactions with tumor cells are essentially unknown. To elucidate if mast cells might be specifically attracted to the tumor area by chemokines produced by HRS cells, we investigated chemokine expression in HL cell lines and in vivo. By RNase protection assay, mRNA expression of several chemokines could be detected in the cell lines. Despite the heterogeneous expression profile exhibited by the cell lines, 4 of 5 expressed CCL5 (RANTES) mRNA. RT-PCR and immunohistochemistry confirmed expression of CCL5 in vivo. Furthermore, secreted CCL5 was detected in conditioned media from 3 of the cell lines. In a migration assay, we found that CCL5 present in conditioned medium could induce mast cell migration. Taken together, our results suggest that CCL5 produced by HRS cells is one mechanism by which mast cells can be attracted into the tumor tissue in HL.
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PMID:Expression of CCL5/RANTES by Hodgkin and Reed-Sternberg cells and its possible role in the recruitment of mast cells into lymphomatous tissue. 1294 94

Bcl-2 family members either negatively or positively regulate the apoptotic threshold of cells. Bcl-xES (extra short), a novel Bcl-x member, possesses a unique combination of BH4 and BH2 domains as well as a COOH-terminal hydrophobic transmembrane anchor domain. Bcl-xES contains sequences of hydrophobic alpha-6 helices but lacks sequences of alpha-5 helices, suggesting that it does not have pore channel-forming activity but functions uniquely as a trapping protein. mRNA expression analysis by reverse transcriptase-polymerase chain reaction and RNase protection assay reveal that Bcl-xES is expressed in a variety of human cancer cell lines and human tumors, including bone marrow from patients with acute lymphoblastic leukemia. Bcl-xES expression is much less pronounced in some specimens of normal human tissues, including the breast, ovary, testis and lung. Stable, transfected human B lymphoma Namalwa variant cells expressing Bcl-xES were derived to investigate its role in apoptosis. Bcl-xES had a preventive effect on cell death induced by tumor necrosis factor-alpha and various concentrations of anticancer drugs, including camptothecin, etoposide and cisplatin. Its protective action on cell death was correlated with the inhibition of mitochondrial cytochrome c release and caspase activation. In a yeast two-hybrid system, Bcl-xES interacted with most Bcl-2 family members, including those containing only a BH3 domain, and with the Ced-4 homolog Apaf-1. Co-immunoprecipitation and gel filtration chromatography experiments suggest that Bcl-xES delays drug-induced apoptosis by disturbing the formation of Bax oligomers and preventing cytochrome c release, but also by interacting with Apaf-1 and inhibiting procaspase-9 activation, thus averting the apoptogenic proteolytic caspase cascade and cell death.
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PMID:Bcl-xES, a BH4- and BH2-containing antiapoptotic protein, delays Bax oligomer formation and binds Apaf-1, blocking procaspase-9 activation. 1504 82

During maturation, thymocytes interact directly and indirectly with different cell types of the thymic microenvironment. Such a cellular communication has been basically ascribed to soluble factors and surface receptors. However, little attention has been given to cellular communication mediated by gap junctions. The existence of these intercellular channels in the immune system remained a controversial issue since the 1970s until recently, when a growing body of evidence has indicated their presence and physiological roles in the immune system. In this work, we investigated whether thymocytes express gap junction-forming proteins (connexins, Cx) and are capable of forming functional intercellular channels. Using RT-PCR, we demonstrated that thymocytes express the mRNA for two Cx isoforms: Cx30.3 and Cx43, but not for Cx26, Cx30, Cx31, Cx31.1, Cx32, Cx33, Cx36, Cx37, Cx40, Cx45, Cx46, and Cx50. In addition, the presence of Cx30.3 and Cx43 was confirmed using different techniques (RNase protection assay, western blot and immunofluorescence). However, despite the expression of these two Cxs, we did not detect functional homocellular coupling between thymocytes or between EL-4 cells (a Cx43 expressing thymic lymphoma-derived cell line) or heterocellular coupling between thymocytes and thymic epithelial cells (TEC) or between EL-4 and TEC in unstimulated conditions. Concluding, in this study, we described for the first time the expression of connexins in thymocytes, which may constitute a new molecule having a functional role in thymocytes maturation.
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PMID:Characterization of connexin 30.3 and 43 in thymocytes. 1523 37

Epigenetic silencing of downstream components of the transforming growth factor beta pathway including the cyclin-dependent kinase inhibitors (CDKIs) p15INK4B, p27KIP1 and p21CIP1 is implicated in the pathogenesis of some hematological malignancies. Loss of p15INK4B expression due to promoter methylation occurs frequently in human T-cell acute lymphoblastic leukemia (T-ALL) but the expression and methylation status of p27KIP1 remains to be characterized in T-ALL or T-cell lymphoblastic lymphoma (T-LBL). As well, while some have reported a high frequency of p21CIP1 methylation in ALL patient samples others have found the gene to be unmethylated in this disease and the relationship between p21CIP1 expression and promoter methylation has not been examined in T-LBL. Using RNase protection assays (RPA) and methylation specific PCR (MSP), we found p27KIP1 to be expressed and its promoter unmethylated in 20 of 20 (100%) and 28 of 28 (100%) T-LBL/ALL samples, respectively. In contrast, p21CIP1 mRNA was absent in 7 of 14 (50%) T-LBL biopsies and 5 of 6 (83%) T-ALL cell lines. However, like p27KIP1 there was no evidence of p21CIP1 promoter methylation by MSP or temporal temperature gradient electrophoresis (TTGE) analysis of 35 CpG sites in any of the 28 T-LBL/ALLs analyzed. Similar to T-ALL, we found p15INK4B mRNA was absent in 13 of 14 (93%) T-LBL biopsies and its promoter methylated in 6 of 10 (64%) cases. Our results indicate that p21CIP1 mRNA is absent in human T-LBL biopsies and T-ALL cell lines at a high frequency. However, unlike p15INK4B, reduced p21CIP1 expression in T-LBL/ALL is independent of dense promoter-associated CpG methylation. In contrast to some hematological malignancies p27KIP1 methylation does not appear to contribute significantly to T-LBL/ALL pathogenesis.
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PMID:Methylation status of cyclin-dependent kinase inhibitor genes within the transforming growth factor beta pathway in human T-cell lymphoblastic lymphoma/leukemia. 1547 71

Although the majority of circulating leukemic cells in chronic lymphocytic leukemia (CLL) are in G0/early G1, recent studies have shown that these cells have undergone multiple cell divisions. In this study, we have determined whether there are abnormalities in cell cycle control in CLL by examining the three cyclin D isoforms in 43 patients and correlating the findings with clinical features. Cyclin D mRNA was measured by a sensitive RNase protection assay and the order of expression in CLL cells was D3 > D2 > D1. The mean cyclin D1 and D3 mRNA levels were 4 to 6-fold higher in CLL cells than in normal peripheral blood B cells. In contrast, the levels of cyclin D2 mRNA were similar in CLL and normal B cells. Expression of the cyclin D isoforms was two- to four-fold greater in normal T cells than B cells, and the order of expression for both cell types was D2 > D3 > D1. The relative overexpressions of cyclins D1 and D3 in CLL were unrelated to gene amplification, as assessed by Southern blotting, but structural changes in the genes were seen in four patients. Both cyclin D1 and D3 mRNA levels correlated positively with lymphocyte doubling time (LDT) and inversely with Rai stage and duration of disease. In addition, a significant correlation was observed between cyclin D mRNA levels and survival, with patients having high levels of cyclin D1, and to a lesser extent cyclin D3, mRNA having the best survival. Thus, cyclin D1 and D3 are relatively overexpressed in CLL cells and patients with higher levels have low stage disease, long LDT and prolonged survival. Further studies should evaluate the predictive value of cyclin D measurements in comparison to other prognostic markers in CLL.
Leuk Lymphoma 2005 Sep
PMID:Cyclin D expression in chronic lymphocytic leukemia. 1610 4

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.
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PMID:In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. 1743 94

To clarify whether p53 mutation could be involved in the pathogenesis of various subtypes of lymphoma, we investigated 62 Japanese cases of non-Hodgkin's lymphomas (NHLs) for p53 gene mutations and their relationship with the expression of p53 protein. Mutations in exons 5-9 of the p53 gene were screened for using the non-isotopic RNase cleavage assay (NIRCA) and confirmed by direct sequencing, followed by immunohistochemical analysis for p53 protein. Missense and/or nonsense mutations of p53 were detected in 3 (10.7%) of 28 diffuse large B-cell lymphomas (DLBLs) and 2 (15.4%) of 13 T-cell NHLs (15.4%). A single missense mutation at codon 157 (Val to Phe) in exon 5 and at codon 273 (Arg to Pro) in exon 8 was found respectively in 2 DLBLs and in one peripheral T-cell lymphoma (unspecified). In these 3 cases harbouring a missense mutation, overexpression of p53 protein was observed in more than 80% of tumour cells. Double transversion mutations comprising of a missense mutation at codon 167 (Gln to His) in exon 5 and a nonsense mutation at codon 183 (Ser to stop codon) in exon 5 were detected in one DLBL that had apparently transformed from follicular lymphoma and in one advanced adult T-cell lymphoma (ATL). In these two cases harbouring p53 nonsense mutation, no cells positive for p53 protein immunostaining were detected, as well as lymphomas without p53 mutation.
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PMID:Mutation of the p53 tumour suppressor gene and overexpression of its protein in 62 Japanese non-Hodgkin's lymphomas. 1760 75

Targeted RNases (TRs) are immunoenzymes with ribonucleases as cytotoxic effector domains, which are less immunogenic as plant or bacterial toxin components of classical immunotoxins. In this study, we show the generation and production of the first entirely human TR (huTR) directed against CD30+ lymphomas. The scFv-Fc-RNase construct was produced in human embryonic kidney (HEK) 293T cells, yielding up to 4 mg/L soluble protein after purification by protein A affinity chromatography. Size exclusion chromatography revealed a homodimer of the predicted molecular mass. Surface plasmon resonance analysis revealed an affinity to CD30 of KD of less than 1 nM for both the scFv-Fc and the scFv-Fc-RNase proteins. Internalization of the scFv-Fc-RNase protein by CD30+ Karpas-299 cells was demonstrated by confocal microscopy. Proliferation of the CD30+ lymphoma cell line Karpas-299 was strongly inhibited by CD30-specific huTR protein (IC50=3.3 nM). The huTR is a promising candidate for the immunotherapy of CD30+ lymphomas because of its expected low immunogenicity, good production yields, and potent effector function upon target cell binding and internalization. Its modular design is set to target other internalizing tumor antigens using different antibody domains.
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PMID:Human antibody RNase fusion protein targeting CD30+ lymphomas. 1823 Jul 57


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