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)

Primary, malignant brain tumors show an extensive infiltrative invasion into surrounding normal brain. At present, little information is available regarding the local invasive behavior of human brain tumors and until now no animal model suitable to mimic human gliomas has been reported. To identify the infiltrative behavior of an established glioblastoma cell line (SNB19), we achieved a stable transfection of the SNB19 cell line with beta-galactosidase (lac-Z) plasmid. The stable beta-galactosidase-expressing cells were then injected intracerebrally into nude mice in an attempt to follow its pattern of spread. The mice were sacrificed at 3, 4, and 6 weeks postinjection. We could detect tumor formation in all of the animals, and the tumor size increased gradually over the 6 week time period. Three weeks after injection, tumor cells showed characteristic infiltrative invasion along the corpus callosum. We also observed tumor-cell invasion into the anterior commissure in some animals, and each tumor cell could be identified by lac-Z expression as visualized by its blue color. Further invasion was identified at 4 and 6 weeks postinjection. Our results suggest that this model could be used to study the molecular mechanisms involved in the invasion of gliomas so that appropriate therapeutic intervention strategies could be designed.
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PMID:Invasive pattern of lac-Z-transfected human glioblastoma cells in nude mice brain. 901 6

Up-regulation of vascular endothelial growth factor (VEGF) expression is a major event leading to neovascularization in malignant gliomas. Hypoxia is believed to be the crucial environmental stimulus for this up-regulation. To critically assess this hypothesis, we asked whether the mechanisms defined previously for hypoxia-induced VEGF expression in vitro are similarly involved and sufficient for up-regulation of VEGF gene expression in vivo, using a lacZ reporter gene under the control of VEGF regulatory sequences in an experimental glioma model. Inclusion of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5' regulatory sequences used in the hybrid gene produced weak beta-galactosidase staining in a special tumor cell subtype, the so-called perinecrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 binding site abolished reporter gene expression in the PNP cells, indicating that transcriptional activation of VEGF expression in gliomas is mediated by HIF 1. Inclusion of 3' untranslated sequences from the VEGF gene in the reporter constructs resulted in an increased beta-galactosidase staining in the PNP cells, suggesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma cells growing as solid tumors. Combination of the 5' flanking region including the HIF 1 site along with 3' untranslated sequences produced increased levels of beta-galactosidase expression in PNP cells. EF 5 immunostaining for regions of low oxygen partial pressure covered the same PNP cells that were stained for beta-galactosidase. Collectively, the data provide experimental evidence that VEGF gene expression is activated in a distinct tumor cell subpopulation, the perinecrotic palisading cells of gliomas, by two distinct hypoxia-driven regulatory mechanisms.
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PMID:Up-regulation of vascular endothelial growth factor expression in a rat glioma is conferred by two distinct hypoxia-driven mechanisms. 928

Cells of a human glioblastoma line were stably transfected with a glial fibrillary acidic protein (GFAP) promoter sequence/lacZ reporter gene. Following this modification, they produced Escherichia coli beta-galactosidase constitutively in amounts that could be measured through their conversion of an added fluorophore into a product readily estimated by fluorimetry. Human interferons (IFN) selectively and in a dose-dependent manner reduce the formation of beta-galactosidase in this system. We have used it as the basis for a novel assay that is sensitive (4-40 pg/ml), precise, completed in 30 h, and applicable to both type I and type II human IFNs. Statistical analysis showed interassay relative standard deviations ranging from 5% to 11%, and most individual assays revealed potencies with limits of error within 85%-115%. Neither partially trypsin-digested IFN nor the other cytokines and mitogens we tested reacted in this system, except for tumor necrosis factor-alpha (TNF-alpha). The high selectivity was further shown by the loss of response to IFN in the presence of the appropriate specific anti-IFN or anti-IFN-gamma receptor antibodies.
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PMID:The beta-gal interferon assay: a new, precise and sensitive method. 971 60

Recent advances in molecular biology have permitted significant progress toward the treatment of malignant brain tumors using gene transduction methods. Adenovirus vectors have recently been shown to transduce genes successfully into brain tumor cells both in vitro and in vivo. We have investigated the feasibility of gene transduction for brain tumors using adenovirus vectors. To evaluate in vitro transduction rate by adenovirus vectors, rat 9L gliosarcoma cells or human glioblastoma cells were infected with recombinant replication-deficient adenovirus vectors containing the E. coli beta-galactosidase gene (Adex-CALacZ) and stained with X-Gal. We observed a multiplicity of infection (MOI)-dependent rate. Approximately 100% transgene expression was achieved at a MOI of 5 after seven days of incubation. To evaluate transgene expression in a rat brain tumor model, AdexCALacZ was stereotactically injected into established rat 9L brain tumors. Intratumoral injection of AdexCALacZ resulted in high transgene expression in tumor cells. Although injection of AdexCALacZ in the normal basal ganglia resulted in broad and diffuse transduction into endogenous neural cells, direct intratumoral injection resulted in transduction that was relatively restricted to the tumor cells as well as some neighboring normal cells. Transduction rates were relatively elevated at the margin of the tumor. Our results suggest that adenovirus vectors might be a feasible method to transfer therapeutic genes into malignant brain tumors.
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PMID:[Gene transduction for experimental brain tumors using recombinant adenovirus vector]. 975 66

The oligodendrocyte-type-2 astrocyte lineage (O-2A) comprises a progenitor cell that is able to differentiate into an oligodendrocyte or astrocyte in vitro. The lineage was originally identified in the neonatal rat central nervous system but evidence suggests that the equivalent O-2A lineage also exists in humans. Apart from its putative and widely studied role in glial repair, this cell type could potentially be involved in malignant glioma formation. In this study we demonstrate that a rat O-2A progenitor cell line carrying the bacterial beta-galactosidase reporter gene and transformed with the c-myc and H-ras oncogenes which has lost its differentiation capacity in vitro generates glioma-like growth after stereotactic injection into the adult rat brain. Tumour pathology was similar to human glioblastoma, suggesting that one of the pathways in the generation of human glioblastomas may be the transformation of adult O-2A progenitor cells. Parallel studies demonstrated the presence of a DNA-binding protein complex, termed APprog, in a panel of human glioma cell lines. This protein was initially identified in O-2A progenitor cells and not their differentiated progeny. These data lead us to propose that APprog could be used as an indicator of the lineage origin of gliomas.
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PMID:Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells transformed with c-myc and H-ras form high-grade glioma after stereotactic injection into the rat brain. 977 21

For glioma- and glioblastoma-specific gene expression, we utilized a nestin regulatory element whose activity was evaluated by the reporter gene lacZ. Nestin is a 38-kDa intermediate filament protein, and is expressed specifically in the neuroepithelial stem cells. Nestin is detected in gliomas and glioblastomas, but not in normal brain tissue. We constructed a nestin gene regulator by placing nestin's second intron before the 5' upstream region (2iNP). To obtain enhanced expression of this tissue-specific regulator, we utilized the adenovirus double-infection method with a Cre-loxP on/off switching system. We constructed a 'regulator' vector, Ax2iNPNCre, which expresses Cre recombinase under the control of the nestin regulatory element, 2iNP. A 'reporter' vector, AxCALNLNZK, expresses lacZ under the control of a strong CAG promoter when the stuffer sequence has been removed by Cre recombinase at a pair of loxP sites. We used seven human glioma/glioblastoma cell lines: U251, KG-1C, NGM5, U87 MG, LN-Z308, NP-2 and T98G. Of these, nestin was expressed highly in U251 and KG-1C, less in NGM5, and undetectably in the other four lines. With the use of the two adenovirus vectors, we found X-gal staining and high nestin regulator-promoted beta-galactosidase activities in four of the seven glioma/glioblastoma cell lines. Staining was strong in U251, KG-1C and NGM5, and less in U87 MG. LacZ expression was nearly undetectable in the non-glioma cell line, HeLa, but a little in COS-7. The adenovirus double-infection method, which uses a nestin regulator, is applicable for glioma/glioblastoma-specific expression.
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PMID:Glioma/glioblastoma-specific adenoviral gene expression using the nestin gene regulator. 1080 92

BACKGROUND: The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated beta-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. RESULTS: The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated beta-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. CONCLUSION: Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.
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PMID:Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats. 1198 28

The coxsackie adenovirus receptor (CAR) has become of interest for gene therapy due to its crucial function in adenoviral cell entry. In clinical trials with adenoviral vectors, dexamethasone is applied to reduce side effects such as inflammatory reactions or emesis. By using a beta-galactosidase-expressing adenovirus (AdGal), we observed that dexamethasone treatment resulted in decreased adenoviral gene transfer into human cancer cells. Expression of CAR and integrin alpha5beta1 was transcriptionally downregulated by dexamethasone as shown for HeLa cervical cancer cells and U87MG glioblastoma cells. TNFalpha increased CAR expression in HeLa and ovarian cancer cells but decreased CAR expression in U87MG cells. In all tested cancer cell lines, TNFalpha induced a significant increase in the expression of adenovirus-binding integrins alpha5beta1, alphavbeta3 and alphavbeta5. Pretreatment with TNFalpha increased AdGal gene transfer into cancer cells and enhanced the cytotoxic effect of a p53-expressing adenovirus. In contrast, TGFbeta reduced CAR expression level and adenoviral gene transfer into OV-UL-2 ovarian cancer cells. Confocal immunofluorescence analysis revealed localization of CAR at cell-cell adhesions in several human cancer cell lines and disruption of cell-cell contacts increased adenoviral gene transfer into human cancer cells. In clinical cancer gene therapy, efficiency of adenoviral gene delivery could be altered by cell adhesion, TNFalpha, TGFbeta, and dexamethasone.
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PMID:CAR is a cell-cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFalpha, and TGFbeta. 1257 26

Historically, in vivo imaging methods have largely relied on imaging gross anatomy. More recently it has become possible to depict biological processes at the cellular and molecular level. These new research methods use magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared optical imaging, scintigraphy, and autoradiography in vivo and in vitro. Of primary interest is the development of methods using MRI and PET with which the progress of gene therapy in glioblastoma (herpes simplex virus-thymidine kinase) and Parkinson's disease can be monitored and graphically displayed. The distribution of serotonin receptors in the human brain and the duration of serotonin-receptor antagonist binding can be assessed by PET. With PET, it is possible to localize neurofibrillary tangles (NFTs) and beta-amyloid senile plaques (APs) in the brains of living Alzheimer disease (AD) patients. MR tracking of transplanted oligodendrocyte progenitors is feasible for determining the extent of remyelinization in myelin-deficient rats. Stroke therapy in adult rats with subventricular zone cells can be monitored by MRI. Transgene expression (beta-galactosidase, tyrosinase, engineered transferrin receptor) can also be visualized using MRI. Macrophages can be marked with certain iron-containing contrast agents which, through accumulation at the margins of glioblastomas, ameliorate the visual demarcation in MRI. The use of near-infrared optical imaging techniques to visualize matrix-metalloproteinases and cathepsin B can improve the assessment of tumor aggressiveness and angiogenesis-inhibitory therapy. Apoptosis could be detected using near-infrared optical imaging representation of caspase 3 activity and annexin B. This review demonstrates the need for neurohistological research if further progress is to be made in the emerging but burgeoning field of molecular imaging.
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PMID:Molecular imaging: Bridging the gap between neuroradiology and neurohistology. 1502 22

We have identified an 85 kb BAC clone, 346J21, that carries a cell senescence gene (SEN16), previously mapped to 16q24.3. Transfer and retention of 346J21 in breast cancer cell lines leads to growth arrest after 8-10 cell doublings, accompanied by the appearance of characteristic senescent cell morphology and senescence-associated acid beta-galactosidase activity. Loss of transferred BAC results in reversion to the immortal growth phenotype of the parental cancer cell lines. BAC 346J21 restores senescence in the human breast cancer cell lines, MCF.7 and MDA-MB468, and the rat mammary tumor cell line LA7, but not in the human glioblastoma cell line T98G. We postulate that inactivation of both copies of SEN16 is required for the immortalization of breast epithelial cells at an early stage of tumorigenesis. Positional mapping of 346J21 shows that SEN16 is distinct from other candidate tumor suppressor genes reported at 16q24.
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PMID:Functional identification of a BAC clone from 16q24 carrying a senescence gene SEN16 for breast cancer cells. 1555 27


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