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)

Our previous studies conducted in pancreatic cancer models established in nude mice and hamsters revealed that cloned somatostatin receptor subtype 2 (sst2) gene expression induced both antioncogenic and local antitumor bystander effects in vivo. In the present study, in vivo gene transfer of sst2 was investigated in two transplantable models of primary and metastatic pancreatic carcinoma developed in hamsters. LacZ reporter or mouse sst2 genes were expressed by means of two different delivery agents: an adenoviral vector and a synthetic polycationic carrier [linear polyethylenimine (PEI)]. sst2 was injected into either exponentially growing pancreatic primary tumors or hepatic metastases, and then transgene expression and tumor progression were investigated 5-6 days after gene transfer. Molecular mechanisms involved in the inhibition of tumor growth were also analyzed. Both adenovirus- and PEI-mediated in vivo gene transfer in primary pancreatic tumors induced an increase of beta-galactosidase activity and expression of sst2 transgene nRNA (100% and 86% of tumors for adenovirus and PEI vector, respectively). Adenoviral vector-based sst2 gene transfer resulted in significant reduction of pancreatic tumor growth (P < 0.05). Using PEI vector, both pancreatic primary tumor growth and metastatic tumor growth were also significantly slackened as compared with both LacZ-treated and untreated control groups (P < 0.02). Moreover, the proliferative index decreased significantly (P < 0.005), whereas apoptosis increased (P < 0.005) in tumors transferred with sst2 gene. The increase of apoptosis correlated with an activation of the caspase-3 and poly(ADP-ribose) polymerase pathways. We concluded that in both primary and metastatic pancreatic cancer models, the synthetic gene delivery system can achieve in vivo sst2 gene transfer and results in a significant antitumor effect characterized by an increase of apoptosis and an inhibition of cell proliferation. This new strategy of gene therapy allows the restoration of expression of an antioncogenic molecule and could be promising for the treatment of advanced pancreatic cancer.
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PMID:Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models. 1241 37

The inhibition of apoptosis is generally believed to be a major determinant of resistance to chemotherapy. However, recent findings have shown that caspase inhibitors do not protect cancer cells from death by cytotoxic agents, but may switch drug-induced apoptosis to an alternative 'default death'. The primary goals of this study were to determine the major characteristics of the 'default death' and the mechanism by which this switch is activated. For this purpose, we first investigated putative cell death modes induced by doxorubicin. Molecular markers associated with these death modes were utilized to identify the default death resulting from the inhibition of apoptosis. Our findings demonstrated that doxorubicin induced at least three distinct types of cell death, senescence, apoptosis and a type of necrosis, which were concentration dependent. Specific molecular markers such as p21/WAF1, activated caspase-3 and activated Akt were associated with these death modes. The pan-caspase inhibitor (Q-VD-OPH) greatly reduced doxorubicin-induced caspase-3 activation but did not protect cells against drug toxicity. The combination of doxorubicin and Q-VD-OPH caused an increased expression of p21/WAF1 and senescence -associated -beta-galactosidase activity, but did not alter Akt activation. Collectively, these findings suggest that the inhibition of apoptosis may lead to an increased expression of cell cycle inhibitors and cellular senescence.
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PMID:Caspase inhibition switches doxorubicin-induced apoptosis to senescence. 1274 3

We conducted a study in mice to reevaluate and clarify many aspects of the early survival of muscle cells following transplantation. Male mouse muscle cells (primary-cultures and T-antigen-immortalized clones) labeled with [14C]thymidine and beta-galactosidase were injected into female muscles. Each label was detected in the muscles after different time periods. TUNEL, alizarin red, and immunodetection of active caspase-3 were done in muscle sections. The donor cell labels disappeared from the muscles following donor cell death, but this was not instantaneous and even if the donor cells were killed before transplantation, the first 6 hours were not enough to clear [14C]thymidine and Y chromosome. Using the cell pellet before injection as the 100% baseline for cells injected to evaluate cell death can lead to misinterpretations: the Y-chromosome band was 5-fold stronger than that of a muscle injected with cells, irrespective of whether the cells were previously killed or not. There was no evidence of an immediate massive donor cell death. Necrosis (detected by alizarin red) and apoptosis (detected by active caspase-3) were present among the donor myoblasts following transplantation. Necrosis seemed to be the most important mechanism during the first hours. T-antigen immortalized cells died earlier and more massively than primary-cultured cells, but the surviving cells proliferated more. Indeed, they seemed to exhibit more apoptosis and they triggered a more rapid CD8+ cell infiltration. As a result of our findings, many concepts concerning the early donor cell death following myoblast transplantation must be reconsidered.
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PMID:Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms. 1453 84

Although several observations show local T cell recognition of retinal Ag, there has been no direct demonstration that the APC were retinal derived, rather than recruited. In this study, CD45(+) cells isolated from immunologically quiescent murine retina were tested in vitro for functional evidence of Ag presentation to naive and Ag-experienced CD4 T cells specific for beta-galactosidase. Because CD45(+) cells from brain have been reported to be efficient APC, they were included for comparison. Measures of activation included changes in CD4, CD25, CD44, CD45RB, CD62L, CD69, caspase-3 activation, CFSE dilution, size, number of cells recovered, and cytokine production. Retinal CD45(+) cells gave no evidence of Ag-dependent TCR ligation in naive T cells, unlike splenic APC and CD45(+) cells from brain, which supported potent responses. Instead, addition of retinal CD45(+) cells to cocultures of naive 3E9 T cells plus splenic APC reduced the yield of activated T cells and cytokine production by limiting T cell activation at early time points. Ag-experienced T cells responded weakly to Ag presented by retinal CD45(+) cells. Activating the retinal cells with IFN-gamma, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-beta did not reveal suppressed retinal APC activity. Because retina lacks tissue equivalents of meninges and choroid plexus, rich sources of dendritic cells in brain, cells from retina may better represent the APC activity of fresh, adult CNS parenchymal and perivascular cells. The activity of the retinal CD45(+) cells appears to be directed to limiting T cell responses.
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PMID:The antigen-presenting activity of fresh, adult parenchymal microglia and perivascular cells from retina. 1515 73

The E1 protein of hepatitis C virus (HCV) shows the ability to induce cell lysis by the alteration of membrane permeability when expressed in Escherichia coli cells. This function seems to be an intrinsic property of a C-terminal hydrophobic region of E1 as permeability changes and cell lysis can be blocked by mutagenesis of specific amino acids in this domain. To establish whether the expression of E1 protein and its C-terminal domain was able to induce cell death also in eukaryotic cell, we cloned HCV sequences expressing the full-length E1 (E383), the C-terminal domain (SVP) and a mutant lacking the C-terminal region (E340) in the pRC/CMV expression vector. HepG2 cell line was co-transfected with empty vector or HCV expression plasmids and a reporter vector that expressed beta-galactosidase (beta-gal) to visualize co-transfected blue cells. At 60 h after transfection, the loss of blue cells, considered as a measure of cell death, was 31.5 and 64.3% for the E1 and SVP clones. On the contrary, the number of blue cells after transfection with E340 plasmid was similar to that observed with the control vector. The analysis by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay revealed an increased number of apoptotic cells at 48 h after transfection with E1 and SVP clones. Furthermore, cells transfected with SVP revealed a typical internucleosomal DNA fragmentation and the activation of caspase-3-like proteases as the specific inhibitor Ac-DEVD-CHO peptide partially blocked SVP apoptosis. These data indicate that the intracellular expression of HCV E1 protein and its C-terminal domain induces an apoptotic response in human hepatoma cell line.
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PMID:The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death. 1517 86

Most brain tumors consist of transformed glia cells and are highly vascularized by capillary endothelial cells. The aim of the present study therefore was to deliver pro-apoptotic caspase-3 into malignant C6 glioma and immortalized rBCEC4 brain endothelial cells to induce cell death. Both cell lines were transfected with a reporter protein (beta-galactosidase) using lipid-mediated gene transfer (FuGENE6) or using the novel protein delivery reagent BioPORTER. beta-Galactosidase protein was successfully delivered into both cells, the protein expression peaked around day 2 and was transient. Delivery of caspase-3 induced TUNEL-positive cell death of both cell types. As a control, caspase-3 was also delivered to non-neoplastic primary astrocytes and endothelial cells and induced cell death. In conclusion BioPORTER-protein delivery of pro-apoptotic molecules may provide a potent tool to cause death of the cells in brain tumors, however, this method is limited due to its toxicity to non-malignant cells.
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PMID:Protein delivery of caspase-3 induces cell death in malignant C6 glioma, primary astrocytes and immortalized and primary brain capillary endothelial cells. 1569 Jan 27

Gene therapy may be a promising approach for treatment of brain ischemia. In this study, we examined the effect of postischemic gene transfer of midkine, a heparin-binding neurotrophic factor, using a focal brain ischemia model with the photothrombotic occlusion method. At 90 min after induction of brain ischemia in spontaneously hypertensive rats, a replication-deficient recombinant adenovirus encoding mouse midkine (AdMK, n=7) or a control vector encoding beta-galactosidase (Adbetagal, n=7) was injected into the lateral ventricle ipsilateral to ischemia. At 2 days after ischemia, we determined infarct volume by 2,3,5-triphenyltetrazolium chloride staining. There were no significant differences in cerebral blood flow 1 h after ischemia between AdMK and Adbetagal groups. Infarct volume of AdMK group was 51+/-27 mm3, which was significantly smaller than that of Adbetagal group (86+/-27 mm3, P<0.05). TUNEL-positive and cleaved caspase-3-positive cells in the periischemic area of AdMK-treated rats were significantly fewer than those in Adbetagal-treated rats, suggesting that the reduction of infarct volume by midkine was partly mediated by its antiapoptotic action. Thus, gene transfer of midkine to the ischemic brain may be effective in the treatment of brain ischemia.
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PMID:Postischemic gene transfer of midkine, a neurotrophic factor, protects against focal brain ischemia. 1570 67

This study investigated the anticancer activity and related mechanisms of neolignans, especially threo, erythro-manassantin A (compound 2), which are isolated from Saururus chinensis, in PC-3 cells. Compound 2 strongly inhibited the proliferation of PC-3 cells in a dose-dependent manner. Different cell morphologies were observed depending on the concentration of compound 2, which suggested different growth inhibitory mechanisms. DNA flow cytometry indicated that both low and high concentrations of compound 2 induced the arrest of PC-3 cells in G1 phase. Western blot analyses showed that hyperphosphorylated Rb and E2F-1 were decreased, whereas hypophosphorylated Rb was increased. The cells treated with compound 2 at 200 ng/ml showed shrinkage morphologically, and the staining of annexin V-FITC revealed apoptotic cell death of these cells. The induction of apoptosis was accompanied by the cleavage of caspase-3, -8, and -9, as well as the downregulation of the Bcl-2 and the upregulation of Bax. By contrast, at low compound 2 concentration (1 ng/ml), the cells arrested in G1 showed characteristic changes in morphology, such as an enlarged, flattened cell shape; the majority strongly expressed SA-beta-galactosidase activity. The number of cells undergoing apoptosis was negligible, and no poly(ADP-ribose) polymerase (PARP) cleavage was observed. The increase of p21 was noticed. However, it appeared to be transient rather than sustained. The protein p27 may be important for maintaining the senescence machinery induced by compound 2 because p27 expression was increased at low concentration compared with that at high concentration. In conclusion, compound 2 showed a significant growth inhibitory effect in PC-3 cells via two different mechanisms, i.e., apoptosis at high concentration and senescence at low concentration.
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PMID:Neolignans from Saururus chinensis inhibit PC-3 prostate cancer cell growth via apoptosis and senescence-like mechanisms. 1614 81

Activation of nuclear factor-kappaB (NF-kappaB) can promote or inhibit apoptosis. Oxidative stress is an important mechanism by which certain anticancer drugs kill cancer cells, and is also one of the mechanisms that activate NF-kappaB. We therefore examined hepatic expression of the NF-kappaB monomer p65 in human hepatocellular carcinoma (HCC) tissue samples from eight patients and compared it with their respective samples of surrounding liver tissues. We also studied the effect of NF-kappaB inhibition in human HCC cells exposed to oxidative stress, by infecting HuH7 cells with a recombinant adenovirus carrying mutant IkappaBalpha (mIkappaBalpha). Cultured HuH7 cells were infected with mIkappaBalpha or beta-galactosidase (beta-Gal) for 24 hr followed by treatment with increasing concentrations of H2O2. Cytotoxicity, NF-kappaB translocation, NF-kappaB DNA binding, cell proliferation, and apoptosis were determined. The monomer p65 was overexpressed in six of eight human HCC tissues. In HuH7 cells, introduction of mIkappaBalpha potently inhibited the translocation, activation, and DNA binding of NF- kappaB. In control (beta-Gal-infected) HuH7 cells, exposure to H2O2 produced a dose-dependent increase in apoptosis, regardless of NF-kappaB status. mIkappaBalpha-mediated inhibition of NF-kappaB activation sensitized HuH7 cells to H2O2-induced inhibition of cell growth, and further promoted cell death. Addition of H2O2 (200-500 microM) to control or mIkappaBalpha-infected HuH7 cells enhanced caspase-3 activity and cleavage. Adenovirus-mediated transfer of mIkappaBalpha potently inhibits NF-kappaB activity in HuH7 cells, and this enhances oxidative stress-induced cell killing.
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PMID:Constitutive activation of NF-kappaB in human hepatocellular carcinoma: evidence of a cytoprotective role. 1654 77

Apoptosis plays an important role in maintaining the normal function of various tissues and organs in different species. Caspase-3 is a terminal caspases which plays an important role in the execution of apoptosis in all vertebrates. It was cloned from zebra fish embryos and its properties were identified through Western blotting and biological activity. In the cells over-expressing caspase-3, Western blotting with an anti-His-tag antibody confirmed the presence of caspase-3 in the three bands that were proposed to correspond to the precursor form (33 kDa), the mature forms processed at the prodomain alone (29 kDa, large subunit) and small sub unit (13 kD). Fish kidney cells were transiently co-transfected with the beta-galactosidase reporter gene and either vector alone (mock), pZCASP3His (caspase-3) or pZCASP3His mutant (caspase-3 mutant). After 72 h following transfection of fish kidney cells, 35% of cells transfected with the zebra fish caspase-3 construct, pZCASP3His, showed apoptotic morphology when compared with cells transfected with the mock vector or an expression construct (pZCASP3His mutant) encoding the caspase-3 mutant lacking Cys. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. A thrombin cleavage recognition site was positioned at the fusion junction to release the caspase-3 from the fusion protein. Phylogenetic analysis showed that the cloned zebra fish caspase was a member of the caspase-3 subfamily with approximately 60% identity with caspase-3 from Xenopus, chicken and mammals. We have obtained structural information by X-ray crystallography. Orthorhombic crystals of the caspase-3 that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0 -7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 36.07 A, b = 38.80 A, c = 135.20 A.
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PMID:Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis. 2289 63


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