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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of sugar receptors on human myeloid leukemia cells was comparatively assessed by a highly sensitive binding assay, employing a panel of 14 types of neoglycoenzymes (chemically glycosylated Escherichia coli beta-galactosidase). The selected carbohydrate ligands mainly encompass common components of natural glycoconjugates as mono- or disaccharides. The monocytoid cells of the THP-1 line, the very young myeloblasts and the myeloblasts of the lines KG-1a and KG-1, the promyelocytes of the HL-60 line, and the early myeloblasts/erythroblasts of the K-562 line displayed a nonuniform pattern of specific binding with quantitative differences at a fixed, nonsaturating concentration of the probes. Scatchard analysis in four cases corroborated the indication of cell-type-related differences between the various cell lines. To test whether the detectable cellular sugar-binding sites can mediate adhesion to glycoligands, a rather simple model matrix of nitrocellulose-immobilized neoglycoproteins was first used. In comparison to the carbohydrate-free carrier protein significant cell adhesion was observed primarily with neoglycoproteins that exposed galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and fucose moieties among the 11 tested types of carbohydrate residue. Subsequently, human bone marrow stromal cell layers were tested as a model matrix with increased levels of physiological relevance and complexity. Mixtures of carbohydrate and neoglycoprotein were employed as inhibitors of an interaction via lectins between the stromal and the tumor cells. The carbohydrate-dependent alterations of this parameter revealed cell-type-associated properties. Tumor cell binding was significantly decreased for not more than two lines with the effective sugars, namely N-acetylgalactosamine, mannose, fucose, and sialic acid.
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PMID:Carbohydrate-dependent binding of human myeloid leukemia cell lines to neoglycoenzymes, matrix-immobilized neoglycoproteins, and bone marrow stromal cell layers. 816 78

In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli beta-galactosidase (beta-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 x 10(4)) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of beta-gal activity depending on the incubation time and the number of effector cells. Released beta-gal activity was assayed sensitively by using 4-methylumbelliferyl-beta-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.
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PMID:Enzyme release assay of human NK cell activity using beta-galactosidase-expressing K562 target cell line. 836 May 3

Attainment of cell type-specific cytotoxicity with minimal side effects is the ultimate goal of cancer therapy. By employing the prostate-specific antigen promoter (PSAP), we investigated (1) whether PSAP-driven antisense genetic constructs targeting DNA polymerase-alpha and topoisomerase II alpha (Top II alpha), designated PSAP-antipol and PSAP-antitop respectively, could induce death of prostate cancer cells, and (2) whether the cytotoxicity is restricted to cells of prostate origin. A PSAP-driven beta-galactosidase gene, PSAP-LacZ, was also used to estimate the expression of the PSAP-driven transcripts. Lipofection-mediated gene transfers were performed with these 3 constructs and a control plasmid, pCDNA3, in 3 human prostate cancer cell lines (LNCaP, DU-145, PC-3) and 5 other cell lines (Cos-1 [monkey kidney], HL-60 [human myeloid leukemia], Hep G2 [human hepatoma], NCI H460 [human lung cancer] and SW 480 [human colon cancer]). On transfection with PSAP-LacZ, LNCaP, DU-145, and PC-3 showed a 10.8, 1.8, and 1.6 fold increase in beta-galactosidase activity, respectively. The remaining 5 cell lines showed no changes after transfection. Corresponding to the levels of the induced beta-galactosidase activity, LNCaP showed the strongest growth inhibition by the antisense constructs: 36% by PSAP-antipol, 39% by PSAP-antitop and 80% by PSAP-antipol+PSAP-antitop. DU-145 and PC-3 had minimal growth inhibition with PSAP-antipol alone or PSAP-antitop alone. However, when cotransfected with PSAP-antipol and PSAP-antitop, DU-145 and PC-3 displayed 42% and 55% growth inhibition, respectively. In contrast, no cytotoxicity was observed in the remaining 5 cell lines when transfected with PSAP-antipol, PSAP-antitop or both. Therefore, PSAP-driven antisense gene therapy targeting DNA polymerase-alpha and Top II alpha inhibits the growth of human prostate cancer cells and the cytotoxic effect is restricted in cells of prostate origin.
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PMID:Prostate-specific antigen promoter driven gene therapy targeting DNA polymerase-alpha and topoisomerase II alpha in prostate cancer. 871 4

CD95(FAS/Apo-1) belongs to the family of TNF receptor related molecules and is expressed on many benign and malignant types of cells. CD95 triggering is involved in the apoptotic death of lymphoid cells and may also be important in myelopoiesis. Myeloid leukemia cells are in most cases resistant to CD95 triggering despite expressing the antigen. We reasoned that myeloid leukemic cells can become sensitized to the Fas pathway of cell death if CD95 expression is restored by gene transfer. As a model, we utilized the cell line K562 which does not express CD95 on their cell surface. The gene for CD95 was introduced into K562 cells (by electroporation). As a control, the gene coding for beta-galactosidase was used. The CD95 transfected K562 cells stably expressed CD95, and had a growth pattern comparable to untransfected cells, but still remained resistant to Fas-triggering (tested using recombinant Fas-ligand). Our experiment shows that antigen expression is not a critical determinant of Fas-mediated apoptosis, but further down-stream mechanisms determine the fate of the cells. The transfected cells also had a comparable susceptibility to NK-mediated lysis which shows that the expression of CD95 does not interfere with NK-mediated lysis of target cells.
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PMID:Expression of CD95(FAS) by gene transfer does not sensitize K562 to Fas-killing. 916 3

The c-Fes protein-tyrosine kinase exhibits strong expression in myeloid hematopoietic cells. Previous studies have shown that Fes induces differentiation in the chronic myelogenous leukemia-derived cell line K-562, suggesting that the Fes signal for differentiation is dominant to the Bcr-Abl signal for transformation in these cells. In addition, Fes has been shown to associate with and phosphorylate Bcr on NH2-terminal sequences retained within Bcr-Abl. To determine whether Fes interacts directly with Bcr-Abl, kinase-inactive Bcr-Abl was coexpressed with Fes in 293T cells, and phosphorylation was assessed by anti-phosphotyrosine immunoblotting. Bcr-Abl was strongly phosphorylated by Fes under these conditions, suggestive of direct interaction. Similarly, tyrosine phosphorylation of kinase-inactive Fes was observed after coexpression with active Bcr-Abl. To test for the interaction of Fes with Bcr-Abl under physiological conditions, wild-type and kinase-defective Fes were stably expressed in the cytokine-dependent myeloid leukemia cell line, DAGM. Expression of either form of Fes alone did not affect the proliferation or interleukin 3 dependence of these cells. The DAGM/Fes cells were then infected with Bcr-Abl retroviruses, and their rates of cytokine-independent outgrowth were compared. Fes dramatically suppressed Bcr-Abl-induced DAGM cell outgrowth relative to a cell line expressing beta-galactosidase as a negative control. This effect required Fes tyrosine kinase activity, because the kinase-inactive form of Fes did not affect Bcr-Abl-induced cell outgrowth. The phosphotyrosine content of both wild-type and kinase-inactive Fes was strongly enhanced after coexpression with Bcr-Abl in DAGM cells, similar to the 293T result. Phosphorylation of wild-type Fes correlated with stimulation of Fes tyrosine kinase activity in the presence of Bcr-Abl. These results show that Fes and Bcr-Abl interact in myeloid cells, leading to Fes activation and suppression of Bcr-Abl-induced conversion to cytokine independence.
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PMID:The c-Fes protein-tyrosine kinase suppresses cytokine-independent outgrowth of myeloid leukemia cells induced by Bcr-Abl. 1070 30

Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl.
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PMID:Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck. 1084 48

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