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)

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO by N-methyl-D-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely prevented S-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 microM) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with beta-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-L-cysteine (50 microM). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity via S-nitrosation of the Cys-163 residue of caspase-3.
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PMID:Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. 1006 32

Forced overexpression of wild-type Alzheimer amyloid precursor protein (APP) causes postmitotic neurons to degenerate. Caspase-3 (CPP32) is a principal cell death protease involved in neuronal apoptosis during physiological development and under pathological conditions. Here, we investigated whether APP overexpression activates caspase-3 in human postmitotic neurons using adenovirus-mediated gene transfer. When a recombinant adenovirus vector expressing human wild-type APP695 was infected in vitro into neurally differentiated embryonal carcinoma NT2 cells, only postmitotic neurons underwent severe degeneration. Before neurodegeneration, full-length APP- and Abeta-immunoreactive peptides were accumulated in infected neurons, and caspase-3-like protease activity was markedly elevated. Western blot analysis revealed that activated caspase-3 subunits were generated in APP-accumulating neurons. Such neuronal caspase-3 activation was undetectable in NT2 neurons infected with beta-galactosidase-expressing adenovirus. Addition of the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde to the culture medium significantly reduced the severity of degeneration exhibited by APP-overexpressing neurons. Immunocytochemical analyses revealed that some APP-accumulating neurons contained activated caspase-3 subunits and exhibited the characteristics of apoptosis, such as chromatin condensation and DNA fragmentation. Activation of caspase-3 was also observed in vivo in rat hippocampal neurons infected with the APP-expressing adenovirus. These results suggest that wild-type APP is an intrinsic activator of caspase-3-mediated death machinery in postmitotic neurons.
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PMID:Activation of neuronal caspase-3 by intracellular accumulation of wild-type Alzheimer amyloid precursor protein. 1043 52

Tat proteins (trans-activating proteins) are present in all known lentiviruses and are early RNA binding proteins that regulate transcription. Tat from the human immunodeficiency virus type-1 is a protein comprising 86 amino acids and encoded by 2 exons. The first 72 amino acids are encoded by exon 1 and exhibit full trans-activating activity. The second exon encodes a 14-amino-acid C-terminal sequence that is not required for trans-activation but does contain an RGD motif, which is important in binding to alphavbeta3 and alpha5beta1 integrins. Tat has an unusual property for a transcription factor; it can be released and enter cells freely, yet still retain its activity, enabling it to up-regulate a number of genes. Tat also has an angiogenic effect; it is a potent growth factor for Kaposi sarcoma-derived spindle cells, and, separately, it has been shown to bind to a specific receptor, Flk-1/KDR, on vascular smooth muscle cells, as well as to integrin-like receptors present on rat skeletal muscle cells and the lymphocyte cell line H9. It appears that the basic domain of tat is important, not only for translocation but also for nuclear localisation and trans-activation of cellular genes. As such, targeting of tat protein or, more simply, the basic domain provides great scope for therapeutic intervention in HIV-1 infection. There is also opportunity for tat to be used as a molecular tool; the protein can be manipulated to deliver non-permeable compounds into cells, an approach that already has been employed using ovalbumin, beta-galactosidase, horseradish peroxidase, and caspase-3.
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PMID:HIV-1-trans-activating (Tat) protein: both a target and a tool in therapeutic approaches. 1053 42

The prognosis for patients with esophageal cancer remains poor, prompting the search for new treatment strategies. Overexpression of E2F-1 has been shown to induce apoptosis in several cancer cell types. In the present study, the effect of adenovirus-mediated E2F-1 overexpression on human esophageal cancer cell lines Yes-4 and Yes-6 was evaluated. Cells were treated by mock infection, infection with an adenoviral vector expressing beta-galactosidase (Ad5CMV-LacZ), or E2F-1 (Ad5CMVE2F-1). Western blot analysis confirmed marked overexpression of E2F-1 in Ad5CMVE2F-1-infected cells. Overexpression of E2F-1 resulted in marked growth inhibition and rapid loss of cell viability due to apoptosis, although Yes-6 cells were somewhat more resistant to E2F-1-mediated growth inhibition than Yes-4 cells. Cell cycle analysis revealed that overexpression of E2F-1 led to G2 arrest, followed by apoptotic cell death. p53 expression remained undetectable in both cell lines after E2F-1 overexpression. The apoptosis inhibitor proteins of the Bcl-2 gene family, Bcl-2, Mcl-1, and BcI-XL, decreased at 48 h after infection in Yes-4 cells, but remained unchanged in Yes-6 cells. Levels of retinoblastoma gene product (pRb) declined at 48 h after E2F-1 infection in Yes-4 cells, at which apoptosis predominated, whereas pRb expression remained constant in Yes-6 cells. Expression of p14ARF did not change after E2F-1 infection in either cell line. Involvement of caspase 3 and caspase 6 in E2F-1-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and poly-ADP-ribose polymerase, as well as fragmentation of the caspase 6 substrate, lamin B. These results indicate that the sensitivity of esophageal cancer cells to E2F-1-mediated apoptosis may be related to differential expression of Bcl-2 family member proteins and suggest that the adenovirus-mediated E2F-1 gene therapy may be a promising treatment strategy for the treatment of this disease.
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PMID:Caspase activation and changes in Bcl-2 family member protein expression associated with E2F-1-mediated apoptosis in human esophageal cancer cells. 1077 92

E2F -1 is a transcription factor that regulates cell cycle progression into S-phase. Deregulation of E2F-1 activity has been associated with cellular commitment to apoptosis. Also critical in the regulation of S-phase are the actions of the cyclin dependent kinases, Cdk2 and cdc2. Inhibition of these cyclin dependent kinases has been similarly associated with disrupting orderly S-phase progression and causing subsequent apoptosis in certain cancer cells. In this study, we examine the ability of adenovirus-mediated E2F-1 overexpression to induce apoptosis in human gastric carcinoma cells. Furthermore, we investigate the effect of the cyclin dependent kinase inhibitors, olomoucine and roscovitine, on E2F-1-mediated apoptosis in human gastric carcinoma cells. AGS and SNU-1 gastric adenocarcinoma cells were infected with adenoviral vectors expressing E2F-1 (Ad5CMVE2F-1) or control viruses expressing beta-galactosidase (Ad5CMVLacZ) or lacking a transgene (Ad5). Gastric adenocarcinoma cells were then independently treated with roscovitine or olomoucine. Finally, gastric adenocarcinoma cells were infected with the various adenoviral vectors in combination with roscovitine or olomoucine. E2F-1 overexpression resulted in an 85% reduction in cell viability at 72 h compared to controls. Combining E2F-1 overexpression with roscovitine resulted in >99% reduction in cell viability by 72 h. Overexpression of E2F-1 resulted in premature S-phase entry and G2/M arrest at 24 h, followed by apoptosis by 72 h. Combining E2F-1 overexpression with roscovitine resulted in an earlier G2/M arrest, followed by a more complete, widespread apoptotic response by 24 h. Caspase 3/CPP32 activation and PARP cleavage in response to E2F-1 overexpression, alone and in combination with roscovitine, implicate the caspase cascade in E2F-1-mediated apoptosis of gastric cancer cells. Bax levels also increased in response to E2F-1 gene transfer, alone and in combination with roscovitine. E2F-1 overexpression induces widespread apoptosis in human gastric carcinoma cells. Combining E2F-1 overexpression with cyclin-dependent kinase inhibitors results in an enhanced apoptotic response, causing nearly complete gastric tumor cell death within 72 h. E2F-1 gene therapy in combination with cyclin dependent kinase inhibitors is a potentially active chemogene therapy strategy for the treatment of human gastric cancer.
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PMID:Adenovirus-mediated E2F-1 gene transfer induces an apoptotic response in human gastric carcinoma cells that is enhanced by cyclin dependent kinase inhibitors. 1085 Dec 67

Several neurodegenerative disorders are characterized by filamentous inclusions in neurons that selectively degenerate. The role these inclusions play in neuron degeneration is unclear, but this issue can be investigated experimentally in relevant animal models. The NFH/LacZ transgenic (TG) mice overexpress the high-molecular-weight neurofilament (NF) subunit (NFH) fused to beta-galactosidase, and these hybrid proteins aggregate into NF-rich, filamentous neuronal cytoplasmic inclusions (NCIs) that have been implicated in the progressive, age-dependent degeneration in subsets of affected neurons. Thus, these TG mice recapitulate some of the key pathology of neurodegenerative disorders with intraneuronal inclusions. To determine if the NCIs compromise neuron survival following traumatic brain injury (TBI), 3- to 6-month old TG and wild-type (WT) mice were subjected to TBI or sham injury. At 2 weeks post-TBI, the TG group showed increased TUNEL staining and activated caspase-3 immunoreactivity in cells of cerebral cortex, adjacent white matter, and hippocampus underlying the injury site, relative to control mice, but this labeling decreased at 4 weeks and was minimal thereafter. Compared to control mice, by 8 weeks postinjury, the TG mice showed a marked decrease in neuron density and increased gliosis in the hippocampal dentate gyrus and CA3 region as well as in the lateral thalamus, while the few remaining CA3 neurons exhibited cytoskeletal alterations, decreased synaptic protein immunoreactivity, and dissolution of NCIs. The more profound long-term neurodegenerative sequelae of TBI in the NFH/LacZ mice compared to WT mice suggest that the presence of intraneuronal inclusions may impair the recovery and long-term viability of injured neurons.
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PMID:Neurofilament-rich intraneuronal inclusions exacerbate neurodegenerative sequelae of brain trauma in NFH/LacZ transgenic mice. 1096 87

Normal cells in culture divide a certain amount of times and undergo a process termed replicative senescence. Telomere loss is thought to control entry into senescence. Activation of telomerase in tumors bypasses cellular senescence and is thus a requirement for tumor progression. We reported previously the preferential incorporation of 3'-azido-2', 3'-dideoxythymidine (AZT) in telomeric sequences of immortalized cells in culture. In this work, we have investigated the effects of chronic in vitro AZT exposure on F3II mouse mammary carcinoma cells. We demonstrate, for the first time, that AZT-treated tumor cells have a reduced tumorigenicity in syngeneic BALB/c mice. Tumor incidence was reduced and survival was prolonged in animals inoculated with AZT-treated cells when comparing with control counterparts. The number and size of spontaneous metastases were also decreased in animals inoculated with AZT-treated cells. In addition, we present evidence of morphological and biochemical signs of senescence, as shown by the staining for senescence associated beta-galactosidase activity, and induction of programmed cell death, as demonstrated by an increase of caspase-3 activity, in tumor cells exposed to AZT. These data indicate that chronic exposure of mammary carcinoma cells to AZT may be sufficient to induce a senescent phenotype and to reduce tumorigenicity.
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PMID:Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells. 1126 35

Although overexpression of E2F-1 can induce apoptosis in a variety of tumor cell lines, the mechanisms by which E2F-1 induces apoptosis remain ambiguous. In this study, we examine the ability of E2F-1 to induce apoptosis in colon cancer and the molecular mechanisms underlying E2F-1-mediated apoptosis. HT-29 and SW-620 colon adenocarcinoma cells (both mutant p53) were treated by mock infection or adenoviral vectors Ad5CMV (empty vector), Ad5CMVLacZ (beta-galactosidase), and Ad5CMVE2F-1 (E2F-1) at multiplicity of infection of 100. Western blot analysis confirmed marked overexpression of E2F-1 in both cell lines. By 5 days after infection, E2F-1 overexpression resulted in >25-fold reduction in cell growth and >90% loss of cell viability in both cell lines. Cell cycle analysis of Ad-E2F-1-infected cells revealed an increase in G(2)/M and sub-G(1) populations. By in situ terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling analysis, evidence of apoptosis was observed including internucleosomal DNA fragmentation and the formation of apoptotic bodies. In addition, caspase-3 and poly(ADP-ribose) polymerase apoptotic fragments were detected by 48 h after treatment with Ad-E2F-1. Of mechanistic importance, overexpression of E2F-1 caused a G(2)/M arrest followed by increased levels of c-Myc and p14(ARF) proteins. Additionally, expression of the antiapoptotic Bcl-2 family member Mcl-1 was down-regulated in E2F-1-overexpressing cells. In conclusion, E2F-1 overexpression initiates apoptosis and suppresses growth in HT-29 and SW620 colon adenocarcinoma cells. Overexpression of E2F-1 triggers apoptosis and is associated with up-regulation of c-Myc and p14(ARF) proteins and down-regulation of Mcl-1. Therefore, E2F-1 is a potentially active gene therapy agent for the treatment of colon cancer.
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PMID:E2F-1 up-regulates c-Myc and p14(ARF) and induces apoptosis in colon cancer cells. 1170 81

Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than normal tissues. These impart resistance to radiotherapy and anticancer chemotherapy, as well as predisposing to increased tumor metastases. To develop a potentially therapeutic protein drug highly specific for solid tumors, we constructed fusion proteins selectively stabilized in hypoxic tumor cells. A model fusion protein, oxygen-dependent degradation (ODD)-beta-galactosidase (beta-Gal), composed of a part of the ODD domain of hypoxia-inducible factor-1alpha fused to beta-Gal, showed increased stability in cultured cells under a hypoxia-mimic condition. When ODD-beta-Gal was further fused to the HIV-TAT protein transduction domain (TAT(47-57)) and i.p. injected to a tumor-bearing mouse, the biologically active fusion protein was specifically stabilized in solid tumors but was hardly detected in the normal tissue. Furthermore, when wild-type (WT) caspase-3 (Casp3(WT)) or its catalytically inactive mutant was fused to TAT-ODD and i.p. injected to a tumor-bearing mouse, the size of tumors was reduced by the administration of TAT-ODD-Casp3(WT) but not by TAT-ODD-mutant Casp3. TAT-ODD-Casp3(WT) did not cause any obvious side effects on tumor-bearing mice, suggesting specific stabilization and activation of the fusion protein in the hypoxic tumor cells. These results suggest that the combination of protein therapy using a cytotoxic TAT-ODD fusion protein with radiotherapy and chemotherapy may provide a new strategy for annihilating solid tumors.
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PMID:Antitumor effect of TAT-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells. 1192 18

The delivery of proteins across the blood-brain barrier is severely limited by the proteins' size and biochemical properties. Eleven-amino acid human immunodeficiency virus TAT protein is able to cross cell membranes even when coupled with larger peptides. We evaluated whether TAT-Bcl-X(L) fusion protein is protective in focal ischemia. Mice underwent 30 or 90 minutes of intraluminal middle cerebral artery thread occlusion. TAT-Bcl-X(L), TAT-beta-galactosidase, or TAT-GFP (0.6 nmol each) were applied intravenously over 10 minutes either 1 hour before or immediately after ischemia. Additional animals received no TAT protein infusions. We show that the brain tissue is progressively transduced with TAT proteins within 3 to 4 hours after intravenous delivery. We provide evidence that TAT-Bcl-X(L) treatment reduces infarct volume and neurological deficits after long ischemic insults lasting 90 minutes, when applied both before and after ischemia. After short insults, lasting only 30 minutes, TAT-Bcl-X(L) further diminishes the number of caspase-3-reactive and DNA fragmented cells and increases the number of viable neurons in the striatum. Our results indicate that TAT fusion proteins are elegant and powerful tools that might be of clinical interest for stroke treatment, because factors may be intravenously applied. Thus, fusion proteins may open fascinating perspectives for future research.
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PMID:Intravenous TAT-Bcl-Xl is protective after middle cerebral artery occlusion in mice. 1240 59


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