Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.
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PMID:Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines. 131 45

The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.
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PMID:Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions. 870 Aug 62

TR6 (decoy receptor 3 (DcR3)) is a new member of the tumor necrosis factor receptor (TNFR) family. TR6 mRNA is expressed in lung tissues and colon adenocarcinoma, SW480. In addition, the expression of TR6 mRNA was shown in the endothelial cell line and induced by phorbol 12-myristate 13-acetate/ionomycin in Jurkat T leukemia cells. The open reading frame of TR6 encodes 300 amino acids with a 29-residue signal sequence but no transmembrane region. Using histidine-tagged recombinant TR6, we screened soluble forms of TNF-ligand proteins with immunoprecipitation. Here, we demonstrate that TR6 specifically binds two cellular ligands, LIGHT (herpes virus entry mediator (HVEM)-L) and Fas ligand (FasL/CD95L). These bindings were confirmed with HEK 293 EBNA cells transfected with LIGHT cDNA by flow cytometry. TR6 inhibited LIGHT-induced cytotoxicity in HT29 cells. It has been shown that LIGHT triggers apoptosis of various tumor cells including HT29 cells that express both lymphotoxin beta receptor (LTbetaR) and HVEM/TR2 receptors. Our data suggest that TR6 inhibits the interactions of LIGHT with HVEM/TR2 and LTbetaR, thereby suppressing LIGHT- mediated HT29 cell death. Thus, TR6 may play a regulatory role for suppressing in FasL- and LIGHT-mediated cell death.
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PMID:A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. 1031 73

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal and spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promoter elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the EphA3 gene promoter region. A region of 86 bp located at -348 bp to -262 bp upstream from the transcription start site was identified as the basal promoter. This region was shown to be active in both EphA3-expressing and -nonexpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive restriction enzymes and bisulfite sequencing of genomic DNA, sites of DNA methylation were identified in hematopoietic cell lines which correlated with their levels of EphA3 gene expression. We showed that EphA3 was not methylated in normal tissues but that a subset of clinical samples from leukemia patients showed extensive methylation, similar to that observed in cell lines. These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors.
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PMID:Cloning and characterization of EphA3 (Hek) gene promoter: DNA methylation regulates expression in hematopoietic tumor cells. 1049 21

The calcium-responsive transactivator (CREST) is required for the normal development of neuronal dendritic trees. Here we report that CREST is localized to sub-nuclear structures in the rat neuroendocrine pheochromocytoma PC12 cells. A yellow fluorescence protein-CREST fusion protein was expressed in HEK 293 and PC12 cells and the recombinant protein was exclusively targeted to nuclear bodies. A similar result was obtained with a Flag-tagged CREST. Deleting the N-terminal 148 or the C-terminal 79 amino acid sequences had no effect on targeting, whereas removing 164 amino acid residues from the C-terminus abolished nuclear body localization. We found that CREST did not co-localize with promyelocytic leukaemia oncoprotein (PML) body and was not targeted to PML bodies. Overexpression of CREST markedly increased the number of nuclear bodies positive for the histone acetyltransferase CREB binding protein (CBP). Double immunofluorescence staining of Flag-CREST and CBP suggested that CREST and CBP had a high degree of co-localization within the nuclear bodies. Deletion of the CBP binding domain of CREST inhibited the recruitment of CBP to CREST nuclear bodies. These results suggest that CBP recruitment to nuclear bodies by CREST may play an important role in CREST-mediated calcium-responsive transactivation, and CREST nuclear body may function as an assembly site for activators/co-activators in gene transcription control.
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PMID:The calcium-responsive transactivator recruits CREB binding protein to nuclear bodies. 1548 21

The leukemia and lymphoma disease locus Evi12 was mapped to the noncoding region of a novel gene, Gnn (named for Grp94 neighboring nucleotidase), that is located immediately upstream of the Grp94/Tra1 gene on mouse chromosome 10. The Gnn gene is conserved in mice and humans. Expression of fusion constructs between GFP and Gnn cDNA isoforms in HEK-293 cells showed that Gnn proteins are located mainly in the cytoplasm. Immunoblotting experiments demonstrated the presence of multiple Gnn protein isoforms in most organs, with the lowest levels of expression of the protein detected in bone marrow and spleen. The Evi12-containing leukemia cell line NFS107 showed high levels of expression of a approximately 150-kDa Gnn isoform (Gnn107) that was not observed in control cell lines. Overexpression may be due to the viral insertion in Evi12. The Gnn107 protein is probably encoded by a Gnn cDNA isoform that is expressed exclusively in NFS107 cells and that includes sequences of TU12B1-TY, a putative protein with homology to 5'-nucleotidase enzymes. Interestingly, using Affymetrix gene expression data of a cohort of 285 patients with acute myeloid leukemia (AML), we found that GNN/TU12B1-TY expression was specifically increased in two AML clusters. One cluster consisted of all AML patients with a t(8;21) translocation, and the second cluster consisted of AML patients with a normal karyotype carrying a FLT3 internal tandem duplication. These findings suggest that we identified a novel proto-oncogene that may be causally linked to certain types of human leukemia.
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PMID:The common viral insertion site Evi12 is located in the 5'-noncoding region of Gnn, a novel gene with enhanced expression in two subclasses of human acute myeloid leukemia. 1582 39

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.
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PMID:[Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells]. 1669 44

All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1-CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG-CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG-CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b-GFP or GFP-Vps4B(E235Q), a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP-CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP-CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.
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PMID:CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway. 1685 78

The human multidrug resistance gene MDR1 encodes the protein product P-glycoprotein (P-gp). P-gp is an integral membrane protein which mediates ATP-dependent substrate efflux. We recently discovered a novel G --> T variant at 1199 nucleotide position of MDR1 which exhibits a 2.3% allelic frequency in leukemia patients. The functional effects of this MDR1-G1199T variant were evaluated with recombinant HEK cells that stably express the wild-type, G1199A, or G1199T variant of the MDR1 protein, P-gp, at comparable levels. A panel of cytotoxic P-gp substrates comprising doxorubicin, vinblastine, vincristine, paclitaxel, or topotecan (a poor P-gp substrate) was used to evaluate the functional impact of G1199 variations. Compared to MDR1(wt), MDR1(G1199A) exhibited an increased resistance to doxorubicin, paclitaxel, vinblastine, and vincristine. In contrast, MDR1(G1199T) reduced resistance to (1/4) that of MDR1(wt) for all drugs except topotecan. Expression of MDR1 exhibits some degree of resistance to topotecan, but 1199 variation has no impact. These data were consistent with the variation in intracellular doxorubicin concentrations measured in MDR1 recombinant cells. Our results suggest that patients with the novel MDR1-G1199T variant may exhibit a lower degree of MDR1 dependent chemoresistance, and those with the G1199A polymorphism may exhibit a higher degree of resistance, compared with MDR1 wild-type patients.
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PMID:A novel MDR1 G1199T variant alters drug resistance and efflux transport activity of P-glycoprotein in recombinant Hek cells. 1691 72

Porcine endogenous retroviruses (PERV) are a major concern when porcine tissues and organs are used for xenotransplantation. PERV has been shown to infect human cells in vitro, highlighting a potential zoonotic risk. No pathology is associated with PERV in its natural host, but the pathogenic potential might differ in the case of cross-species transmission and can only be inferred from knowledge of related gammaretroviruses. We therefore investigated the integration features of the PERV DNA in the human genome in vitro in order to further characterize the risk associated with PERV transmission. In this study, we characterized 189 PERV integration site sequences from human HEK-293 cells. Data showed that PERV integration was strongly enhanced at transcriptional start sites and CpG islands and that the frequencies of integration events increased with the expression levels of the genes, except for the genes with the highest levels of expression, which were disfavored for integration. Finally, we extracted genomic sequences directly flanking the integration sites and found an original 8-base statistical palindromic consensus sequence [TG(int)GTACCAGC]. All these results show similarities between PERV and murine leukemia virus integration site selection, suggesting that gammaretroviruses have a common pattern of integration and that the mechanisms of target site selection within a retrovirus genus might be similar.
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PMID:Porcine endogenous retrovirus integration sites in the human genome: features in common with those of murine leukemia virus. 1692 52


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