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)

To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.
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PMID:Inhibition of melanoma growth by adenoviral-mediated HSV thymidine kinase gene transfer in vivo. 786 Sep 93

The human monoclonal antibody (HuMAb) L92 reacts to an M(r) 43,000 protein associated with human melanoma. To identify the gene encoding its antigenic epitope, a complementary DNA expression library constructed from the human melanoma cell line UCLASO M14 was screened with HuMAb L92. DNA sequence analysis of the isolated clone revealed that the immunoreactive peptide was composed of 10 amino acids (QDLT-MKYQIF). The peptide was expressed in Escherichia coli with beta-galactosidase as a fused protein. There is no homology between the cloned sequence and other reported DNA sequences. Western blot analysis showed that the fused protein had specific binding to HuMAb L92. An antigen-encoding peptide with 10 amino acids was synthesized and tested for its immunoreactivity in vitro. HuMAb L92 reacted specifically to the 10-amino acid peptide in both an antibody-binding inhibition to the M(r) 43,000 protein and a solid-phase enzyme-linked immunosorbent assay. Using several truncated fusion proteins, we found the minimum number of amino acids required for the antibody binding to be 4 (KYQI). These results suggest that the identified peptide sequence encodes the antigenic epitope of the M(r) 43,000 protein.
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PMID:Human monoclonal antibody identified an immunoreactive tetrapeptide sequence (Lys-Tyr-Gln-Ile) in M(r) 43,000 protein of human melanoma. 801 74

We constructed two genes specific to melanogenesis, human tyrosinase (HT) and tyrosinase-related protein-1 (TRP-1) genes, into two separate expression vectors so that the cloned genes were under the control of a human cytomegalovirus promoter and enhancer. Monkey kidney COS-7 cells and human amelanotic and melanotic melanoma cells were then cotransfected by both HT and TRP-1 or transfected individually with each gene. The transfectants were examined for mRNA expression by reverse transcription-mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectants and cotransfectants of the two genes. Both light and electron microscopic observations indicated that degeneration and premature death of melanocytes occurred in HT transfectants, but not in TRP-1 transfectants or in HT and TRP-1 cotransfectants. Cotransfected cells from five cell lines revealed numerous granular reaction products with an anti-TRP-1 antibody and lysosomal granules with electron-dense material. Our melanin assay confirmed the new production of melanin pigments in these cells, indicating that the lysosomal granules would contain melanin pigments. The gene expression studies of lysosomal protein (beta-galactosidase, CD63, Lamp-1, and Lamp-2) revealed a dramatically elevated gene expression of Lamp-1, which is associated with the membrane receptor of lysosomal granules, in HT- and TRP-1-cotransfected cells. Conversely, the treatment of melanoma cells with antisense oligodeoxynucleotides against Lamp-1 resulted in a decreased expression of TRP-1 protein by immunoprecipitation, supporting the observations of the HT and TRP-1 cotransfection study regarding the up-regulation of Lamp-1 expression. We conclude that HT, TRP-1, and Lamp-1 gene products may function together, being expressed as a multiprotein complex within the melanosomal compartment. Specifically, HT and TRP-1 may function together via Lamp-1 by stabilizing the enzyme-protein complex within the melanosome and prevent the premature death of melanocytes due to tyrosinase-mediated cytotoxicity.
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PMID:Cotransfection of genes encoding human tyrosinase and tyrosinase-related protein-1 prevents melanocyte death and enhances melanin pigmentation and gene expression of Lamp-1. 802 May 95

In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca(2+)-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in lambda Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M(r) 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with beta-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca(2+)-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an antibody against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.
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PMID:Identification of a cDNA coding for a Ca(2+)-binding phosphoprotein (p90), calnexin, on melanosomes in normal and malignant human melanocytes. 826 46

Induction of an invasive phenotype by metastatic tumour cells results in part from inappropriate expression of extracellular matrix-degrading enzymes normally involved in embryonic morphogenesis, tissue remodelling, angiogenesis and wound healing. Such enzymes include endoglycosidases that degrade heparan sulfate (HS) in endothelial basement membrane, as well as better characterized proteases. Heparanase, an endo-beta-D-glucuronidase initially detected in B16 melanoma cells, has been described as a M(r) 96,000 glycoprotein with pI of 5.2, and has been immunolocalized to the cell surface and cytoplasm. We have utilized a polyacrylamide-gel-based HS degradation assay to demonstrate that KNRK, a rat kidney fibroblast cell line transformed by v-K-ras, exhibits HS-degrading activity similar to that of B16F10 mouse melanoma cells. To immunoselect heparanase-expressing clones from a KNRK-cell-specific lambda gt11 cDNA library, we have also prepared a rabbit anti-serum directed against a putative amino-terminal peptide of B16F10 cellular heparanase. Lysogens from one clone expressed a beta-galactosidase fusion protein whose staining with peptide anti-serum was inhibited by competition with excess peptide. Dideoxy-mediated sequencing of the insert termini of this recombinant revealed that it represents a rat homologue of M(r) 94,000 glucose-regulated protein (GRP94/endoplasmin), a molecular chaperone that contains the exact amino-terminal sequence previously attributed to heparanase. Our results call into question the specificity of this peptide sequence, as well as previous immunolocalization studies of heparanase carried out using such anti-sera.
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PMID:Immunoselection of GRP94/endoplasmin from a KNRK cell-specific lambda gt11 library using antibodies directed against a putative heparanase amino-terminal peptide. 831 13

Gene therapy protocols for cancer usually involve removal of tumor cells, culture in vitro to allow gene transfer, and subsequent reintroduction in vivo. Targeting therapeutic genes to tumor cells in situ requires an accuracy of gene delivery that currently is not possible with the use of existing techniques. To overcome these limitations we have used two promoters, which are preferentially active in melanocytic cells, to direct gene expression specifically to melanoma cells both in vitro and in vivo. Here we describe experiments showing that as little as 769 base pairs of the 5'-flanking regions of the tyrosinase, and 1.4 kilobase pair of the tyrosinase-related protein 1, genes are sufficient to direct expression of the beta-galactosidase gene to both human and murine melanoma cells and melanocytes, while not permitting expression in a range of other cell types in vitro. These promoters showed high levels of activity in 12 of 14 murine and human melanoma cell lines tested but showed only basal levels of activity, similar to that of a promoterless construct, in a range of 12 other cell types. Cell type specificity is maintained when the construct is delivered to cells either by physical means or by inclusion of the cell type-specific expression cassette into a retroviral vector. Direct injection of DNA, encoding the beta-galactosidase gene expressed from either promoter, into established B16 melanomas or Colo 26 tumors in syngeneic mice resulted in extensive transduction of tumor cells in the B16 melanomas (approximately 10% of tumor cells expressing 10 days after DNA injection), whereas no blue-staining cells were seen in the Colo 26 tumors. The reporter gene was expressed in melanoma cells and in some normal melanocytes but not in other surrounding normal tissue. We propose that the combination of a tissue-specific promoter driving a therapeutic gene, with delivery of such a construct directly to sites of tumor growth in vivo, either by direct DNA injection or by retroviral infection, may provide significantly enhanced safety for gene therapy for solid tumors.
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PMID:In vitro and in vivo targeting of gene expression to melanoma cells. 843 71

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryotic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression of the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix, in the HPV-negative cervical cancer line HT-3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a beta-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from the transfected cells with 14 C-labeled chloramphenicol. The CMV-promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-, SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only the SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells. In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression.
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PMID:Differential promoter activity in benign and malignant human cells of skin origin. 858 24

An established melanoma cell line (MM96L) was transfected with selectable plasmid constructs encoding either whole SV40 large T antigen, or beta-galactosidase fusions with the retinoblastoma protein (Rb)-binding region of SV40 large T antigen and a nonbinding mutant derivative of it. Both of the beta-galactosidase fusions also encoded the large T nuclear targeting signal. Transcription of inserted genes was regulated through a Zn+2-inducible metallothionein IA promoter, which provides tight but not absolute control of expression. Only the wild-type large T segment fusion was functionally active in the binding of Rb protein. Stable lines derived from primary transfectants with the expression plasmid encoding the mutant large T segment fusion showed a normal FACS scan profile, a normal growth rate, and (upon induction) high levels of nuclear staining for beta-galactosidase. However, cells transfected with the wild-type (Rb-binding) large T segment fusion grew slowly, with surviving clones assuming a predominantly tetraploid karyotype and relatively much lower levels of beta-galactosidase activity upon Zn+2 induction. The latter cells, but not those transfected with the corresponding non-Rb-binding fusion construct, also exhibited elevated cell death and apoptosis in response to the inducer Zn+2. These results implied that expression of an Rb-binding protein has deleterious effects on the melanoma cell line growth and may reflect a role for Rb of a related pocket protein in maintaining the differentiation state of these transformed cells.
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PMID:A melanoma cell line sensitive to expression of a fusion protein binding the retinoblastoma protein family pocket domain. 863 90

To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.
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PMID:Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma. 875 51

The efficacy of a recombinant vaccinia virus (rvv-mGM-CSF) expressing murine granulocyte-macrophage colony stimulating factor (GM-CSF) for use in cancer gene therapy was evaluated. C57BL/6 mice with established B16-F10 melanoma were treated by s.c. injection of irradiated B16 cells infected with two different recombinant vaccinia virus (rvv) constructs. Mice treated with rvv-mGM-CSF vaccine survived longer (p < 0.05), were free of palpable tumors (> 4 mm) longer (p < 0.02), and had smaller mean tumor volumes (p < 0.005) compared to those treated with irradiated B16 cells infected with a control rvv (rvv-lacZ) expressing Escherichia coli beta-galactosidase or irradiated uninfected B16 cells. The vaccine appeared to be B16 tumor cell specific, because there was no therapeutic effect when heterologous but syngeneic (H-2b) colon adenocarcinoma cells, MC-38 infected with rvv-mGM-CSF were used as vaccine. In this model, rvv expressing interleukin-2 (IL-2) was ineffective. In addition, experimental lung metastasis of B16 tumor cells was significantly inhibited by rvv-mGM-CSF vaccine compared to several control vaccines when the vaccine was applied either by i.p. route (p < 0.006) or by s.c. injection (p < 0.0008). B16 cells expressing mGM-CSF after infection with rvv-mGM-CSF or transduction with a retroviral vector, were equally effective (p > 0.14) as vaccines against lung metastasis. Inhibition of metastasis was also B16 tumor cell specific. These data suggest that this approach of cancer gene therapy has a potential for use in cancer patients.
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PMID:Cancer gene therapy using tumor cells infected with recombinant vaccinia virus expressing GM-CSF. 889 77


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