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)

We demonstrated recently that constitutive expression of proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in head and neck squamous cell carcinoma is correlated with activation of transcription factor nuclear factor (NF)-kappaB/Rel A (p50/p65), which binds the promoter region within each of the genes encoding this repertoire of cytokines. NF-kappaB can be activated after signal-dependent phosphorylation and degradation of inhibitor-kappaBalpha and has been reported to promote cell survival and growth. In the present study, we expressed a phosphorylation site mutant of inhibitor-kappaBalpha (IkappaBalphaM) in head and neck squamous cell carcinoma lines UM-SCC-9, -11B, and -38 to determine the effect of inhibition of NF-kappaB on cytokine expression, cell survival in vitro, and growth in vivo. After transfection with IKBalphaM, only a few UM-SCC-9 clones were obtained that stably expressed the mutant IkappaB, suggesting that expression of a mutant IkappaBalpha may affect survival of the transfected UM-SCC cell lines. After cotransfection of IkappaBalphaM with a Lac-Z reporter, we found that the number of surviving beta-galactosidase-positive cells in the three cell lines was reduced by 70-90% when compared with controls transfected with vector lacking the insert. In UM-SCC-9 cells that stably expressed IkappaBalphaM, inhibition of constitutive and tumor necrosis factor-a induced NF-kappaB activation, and production of all four cytokines was observed. Although UM-SCC-9 IkappaBalphaM-transfected cells proliferated at the same rate as vector-transfected cells in vitro, a significant reduction in growth of tumor xenografts was observed in SCID mice in vivo. The decreased growth of UM-SCC-9 IkappaBalphaM-transfected tumor cells accompanied decreased immunohistochemical detection of the activated form of NF-kappaB in situ. These results provide evidence that NF-KB and IkappaBalpha play an important role in survival, constitutive and inducible expression of proinflammatory cytokines, and growth of squamous cell carcinoma. NF-kappaB could serve as a potential target for therapeutic intervention against cytokine and other immediate-early gene responses that contribute to the survival, growth, and pathogenesis of these cancers.
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PMID:Expression of a dominant-negative mutant inhibitor-kappaBalpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survival, proinflammatory cytokine expression, and tumor growth in vivo. 1041 12

SCH58500 is an agent for gene therapy of cancer, consisting of a replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5/beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.
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PMID:The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies. 1059 17

Studies of metastasis can be accelerated and provide more mechanistic information using cell lines which reproducibly and aggressively metastasize, and which are accurately and easily detected in tissues at all stages of the metastatic process. Although reporter proteins such as green fluorescent protein (GFP) and beta-galactosidase have improved the tracking of tumor cells in vivo, their measurement has often been limited to visual observation and manual counting. In this study, we exploited the highly sensitive and objective quantitation provided by flow cytometry to characterize, in detail, the sequence of events associated with orthotopic metastasis in a highly aggressive mouse model. Following stable transfection of the MDA-MB-435 breast carcinoma cell line with GFP, we utilized an in vivo selection process to isolate a variant exhibiting increased primary tumor growth and metastasis. As few as one fluorescent tumor cell per 200,000 host cells could be accurately detected in dissociated tissues by flow cytometry, allowing us to demonstrate that metastatic cells migrate to the lungs of SCID mice very early after orthotopic implantation. Tumor burden in lungs increased in a smooth continuous manner, until death approximately eight weeks later. Levels of circulating tumor cells in blood were also detectable at an early timepoint, but remained relatively low throughout the course of secondary tumor development in the lungs. Surgical removal of the primary tumor at various times after inoculation significantly affected lung tumor burden, supporting the concept that circulating tumor cells in blood inefficiently initiate distal metastases. Furthermore, the continuing contribution to metastasis by the primary tumor was independent of tumor mass. The combined characteristics of enhanced orthotopic metastasis and quantitative detection in blood and tissues will make this a useful new model for the characterization of the multi-stage progression of cancer, and the preclinical evaluation of anti-neoplastic therapies.
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PMID:Characterization of spontaneous metastasis in an aggressive breast carcinoma model using flow cytometry. 1076 21

Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses beta-galactosidase as a marker (MHV68(cy-)). MHV68(cy-) grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68(cy-) show deficient growth of the MHV68(cy-) in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68(cy-) virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68(cy-) had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.
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PMID:Murine gammaherpesvirus 68 cyclin D homologue is required for efficient reactivation from latency. 1088 40

Extensor digitorum longus muscles (EDL) of SCID mice were induced to undergo degeneration-regeneration subsequent to orthotopic, whole-muscle transplantation. Two days after transplantation some of these muscles received injections of primary myoblasts derived from EDL muscles of transgenic mice, which express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter and enhancer. Nine weeks after transplantation, regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received no further treatment and to unoperated EDL muscles in order to determine the effect of myoblast transfer on muscle regeneration. Many myofibers containing donor derived myonuclei could be identified in the regenerated muscles that had received exogenous myoblasts. The mass of the muscles subjected to transplantation only was significantly less (31% less) than that of unoperated muscles. The addition of exogenous myoblasts to the regenerating EDL resulted in a muscle mass similar to that of unoperated muscles. The absolute twitch and tetanic tensions and specific twitch and tetanic tensions of transplant-only muscles were 28%, 36%, 32%, and 41%, respectively, of those of unoperated muscles. Myoblast transfer increased the absolute twitch and tetanic tensions of the regenerated muscles by 65% and 74%, respectively, and their specific twitch and tetanic tensions were increased by 41% and 48%, respectively. These data suggest a possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss.
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PMID:Enhancement of adult muscle regeneration by primary myoblast transplantation. 1097 36

Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP-26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in alphaMEM containing ascorbate-2-phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT-PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neo(r)) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by beta-galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral-marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions.
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PMID:Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo. 1116 57

The formation of ectopic bone within skeletal muscle is a widely observed phenomenon. However, the source of the osteoprogenitor cells responsible for ectopic bone formation remains unknown. This study was designed to test for osteogenic differentiation among cells isolated from skeletal muscle tissue. Different subpopulations of cells derived from an adult mouse skeletal muscle were tested for induction of alkaline phosphatase activity after exposure to bone morphogenetic protein-2 in vitro. A responsive subpopulation was identified, transduced with a retrovirus encoding for beta-galactosidase (Rv-lacZ) and an adenoviral construct encoding for one bone morphogenetic protein-2, and injected into the hindlimb of immune compromised (severe combined immunodeficient, or SCID) mice. The injected cells appeared to actively participate in the ectopic bone formation. The existence of lacZ-positive muscle-derived cells colocalized with osteocalcin-producing cells within lacunae of newly formed bone matrix suggests osteoblast and osteocyte differentiation. Although a specific cell was not isolated, these data support the contentions that osteoprogenitor cells reside within skeletal muscle and that muscle may represent a source other than bone marrow for the harvest of these cells.
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PMID:Osteoprogenitor cells within skeletal muscle. 1119 54

Recombinant adenoviral (rAd) vectors are capable of mediating high-efficiency gene transfer in vivo. Under conditions requiring systemic administration, however, the use of rAd vectors can be problematic due to the presence of circulating anti-adenovirus antibodies developed either through natural infection or during the course of treatment. We developed a passive immunization model in SCID/Beige mice to assess the effect of human and mouse anti-adenovirus antibodies on systemic administration of a rAd vector expressing beta-galactosidase (rAd-betagal). In this model, the in vitro neutralizing activity of human or mouse antibodies used for passive immunization correlated well with inhibition of transduction of the liver following i.v. administration of rAd-betagal. Depletion of antibodies to individual adenovirus structural proteins (hexon, penton, fiber) by affinity chromatography demonstrated that antibodies to each of the three virion components contributed to neutralization of infectivity in vitro and to inhibition of transduction in vivo. Depletion of antibodies against all three structural proteins from human or mouse immune serum prior to passive immunization restored in vivo transduction activity to levels comparable to those obtained with nonimmune serum. Our data suggest that depletion of both murine and human anti-adenoviral antibodies can restore transduction in vivo during systemic rAd gene therapy in hosts previously exposed to adenovirus.
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PMID:Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy. 1135 81

Although gene therapy has been suggested to be a novel strategy to treat hepatocellular carcinoma (HCC), no study showing the clinical feasibility of vectors to treat HCC has been reported. In this preclinical study, we show evidence indicating that hemagglutinating virus of Japan (HVJ) liposomes are a feasible vector to treat HCC in a clinical setting using ganciclovir (GCV) and herpes simplex virus thymidine kinase (HSV-tk), which is driven by the cytomegalovirus immediate early enhancer/promoter (plasmid pcDNA3/HSV-tk). In in vitro experiments, almost complete tumor cell regression was achieved with the optimal GCV concentration (100 microg/mL) and more than 1/3 regression was seen even with a 20% transduction ratio using HuH7 HCC cells stably transformed by HSV-tk. HVJ liposomes showed a 19.7% (mean) transduction rate of the lacZ gene in a relatively large mass of more than 300 mm3 in vivo, which is a clinically detectable size, implanted into SCID mice. Moreover, a single HSV-tk injection of HVJ liposomes followed by GCV treatment inhibited tumor growth at least within a week, and repeat administration was more effective. Furthermore, subcutaneous injection of an HVJ liposomes vehicle induced no apparent inflammatory response in C3H/HeN mice, whereas lacZ gene transfection resulted in inflammatory pathology, suggesting a lower immunogenicity of the HVJ envelope protein than those of bacteria-derived plasmid DNA or the beta-galactosidase gene product. From these findings, we conclude that HVJ liposomes are a clinically safe and effective gene transfer vector to treat HCC.
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PMID:Preclinical and therapeutic utility of HVJ liposomes as a gene transfer vector for hepatocellular carcinoma using herpes simplex virus thymidine kinase. 1139 77

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.
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PMID:Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice. 1173 59


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