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)

Melanoma incidence is growing at a faster rate than any other human malignancy. Wild-type (wt) p53 is important in both G(1) and G(2) cell cycle arrest, and cyclin D1 (CD1) is necessary for G(1)-->S progression in melanoma cells. We reported that an adenoviral vector containing wt p53 significantly reduced [(3)H]thymidine uptake in melanoma cells containing mutant but not wt p53. Subsequently we showed that CD1 decreased melanoma proliferation and increased apoptosis. We now extend these findings by evaluating the effect on preformed melanomas of (1) intratumoral therapy with wt p53 alone, (2) wt p53 in combination with antisense (AS) CD1, both short (< or =14 days) and longer term, and (3) doubling the dose or repeat doses of wt p53 or AS CD1. Two melanoma cells lines that metastasize in SCID mice (451 and 1205) were used, one containing a p53 mutation (451) and the other a normal p53 gene sequence (1205). Compared to injection with a control adenoviral vector containing beta-galactosidase (LacZ), intratumoral injection of wt p53 slowed the growth of tumors formed from 451 cells. Using 5 x 10(8) plaque forming units as our standard intratumoral dose, neither doubling the dose of LacZ, p53 or AS CD1, nor repeat doses of the vectors, was as effective as combined therapy with wt p53+AS CD1, which resulted in the shrinkage of all tumors treated and 4/7 (57%) tumors vanished. No tumors treated with wt p53 or AS CD1 alone vanished. Wt p53+AS CD1 treatment resulted in significantly more cells undergoing apoptosis compared to either therapy alone. In summary, combining the separately effective treatment vectors p53 and AS CD1 led to an enhanced growth-suppressive and apoptotic effect, supporting a role for combination gene therapy to treat human malignant melanoma.
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PMID:p53 alone or in combination with antisense cyclin D1 induces apoptosis and reduces tumor size in human melanoma. 1222 20

Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutical strategies. To be considered as a viable option, however, the respective transgene has to be administered with high tumor specificity. Here, we describe novel polyethylenimine (PEI)-based DNA complexes, shielded by covalent attachment of polyethylene glycol (PEG), that make use of epidermal growth factor (EGF) as a ligand for targeting gene delivery to EGF receptor-expressing human hepatocellular carcinoma (HCC) cells. In vitro transfection of luciferase reporter DNA resulted in high levels of gene expression in the human HCC cell lines Huh-7 and HepG2. An excess of free EGF during transfection clearly reduced expression levels, indicating a specific EGF receptor-mediated uptake of the DNA particles. Following intravenous injection into human HCC xenograft-bearing SCID mice, luciferase expression was predominantly found in the tumor, with levels up to 2 logs higher than in the liver, which was the highest expressing major organ. Histologic investigation showed reporter gene expression (beta-galactosidase) localized to tumor cells. Assessing DNA distribution within the tumor by immunofluorescence microscopy, rhodamine-labelled transgene DNA was found to be mainly associated with HCC cells. In the liver, DNA was taken up almost exclusively by Kupffer cells and, as indicated by the low expression, subsequently degraded. In conclusion, we have shown that intravenous injection of PEGylated EGF-containing DNA/PEI complexes allows for highly specific expression of a transgene in human HCC tumors.
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PMID:Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. 1239 20

We have tested the feasibility of muscle-based gene therapy and tissue engineering for urological dysfunction using highly purified muscle-derived cells (MDC) that display stem cell characteristics. We then explored the potential use of these MDC as an alternative therapy for the treatment of impaired detrusor contractility. The MDC were genetically engineered to express the gene encoding beta-galactosidase and injected into the bladder walls of SCID mice. The injected bladders were harvested at various time-points after injection and assayed for beta-galactosidase activity; the presence of myofibers within the injected tissue was determined by detection of fast myosin heavy chain isoform (MyHCs). We have demonstrated that the injected MDC are capable of not only surviving in the lower urinary tract, but also improving the contractility of the bladder following an induced injury. Two potential mechanisms can be used to explain this finding. First, we have observed that some of the beta-galactosidase-expressing cells expressed alpha-smooth muscle actin, suggesting a differentiation into smooth muscle. Second, a stain for acetylcholine receptors (AChRs), which identifies the location of neuromuscular junctions, revealed that the myofibers derived from the doner cells became innervated into the bladder as early as 2 weeks after injection. These results suggest that gene therapy and tissue engineering based on MDC potentially can be used for urological dysfunction.
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PMID:Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction. 1242 14

Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a approximately 50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated beta-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.
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PMID:Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue. 1266 82

Generation of a vascular network is a hallmark of solid tumor growth, and attempts to switch off the tumor angiogenic phenotype are promising. However, this angiogenic potential might also be exploited to obtain incorporation into tumor vessels of genetically modified third-party cells, which could behave as targets of immunologic or pharmacologic attack. With this in mind, we addressed the efficiency and selectivity of third-party cell recruitment into experimental tumors generated in severe combined immunodeficiency mice. The animals were inoculated intraperitoneally with human ovarian carcinoma cell lines and with beta-galactosidase (beta-gal)-transduced human umbilical vein endothelial cell (HUVEC) or human fibroblasts. Transgenic HUVEC were scattered in tumors, but not in normal mouse tissues; immunohistochemical analysis revealed their selective homing to tumor vascular structures, over 50% of which contained beta-gal(+) cells. Injection of beta-gal-transduced human fibroblasts was also associated with transgenic cell incorporation into tumor masses; however, beta-gal(+) fibroblasts did not home to tumor blood vessels and were only localized within the tumor stroma. These findings show that the recruitment of primary third-party cells into the different compartments of experimentally induced tumors is an efficient and selective phenomenon and indicate possible alternative ways of confronting the tumor angiogenic potential in cancer therapy.
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PMID:Recruitment of human umbilical vein endothelial cells and human primary fibroblasts into experimental tumors growing in SCID mice. 1279 79

Adenoviral vectors from which the E1 region has been deleted ([E1(-)] Ad) are known to induce strong immune responses after systemic delivery. In this study we have evaluated liver toxicities in mice after intravenous injection with high doses of [E1(-)] or modified [E1(-), E2b(-)] Ad vectors (both expressing the bacterial beta-galactosidase [lacZ] marker gene) in C57BL/6, BALB/c, and SCID mice. Our data demonstrate a marked reduction in maximal liver toxicities and pathologies (typically noted at 21 days postinjection) with the use of the [E1(-), E2b(-)] modified vector in all strains of mice tested. Our data also demonstrated that despite the use of the [E1(-), E2b(-)] Ad vector, significant liver toxicities were still observed. To address this issue and the fact that the lacZ gene was perceived as a foreign antigen in the immune-competent C57BL/6 and BALB/c mice, we similarly injected mice tolerant of LacZ (lacZ-TG). In contrast to our studies in C57BL/6 and BALB/c mice, LacZ-TG mice exhibited virtually no evidence of hepatotoxicity after intravenous injection with the [E1(-), E2b(-)] vector, in contrast to use of the [E1(-)] Ad vector. Our results demonstrate that the [E1(-), E2b(-)] Ad vector class can reduce liver toxicities typically ascribed to Ad vector-mediated gene transfer after transfer of a highly immunogenic or foreign gene, whereas transfer of a transgene that is perceived as nonforeign by the host can be delivered with virtually no evidence of toxicity. On the basis of a careful review of the literature, these improvements in vector safety rival those noted with other, more significantly modified Ad vectors described to date.
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PMID:Liver toxicities typically induced by first-generation adenoviral vectors can be reduced by use of E1, E2b-deleted adenoviral vectors. 1467 Jan 23

A major challenge for gene therapy is to be able to deliver efficiently the gene of interest to specific cell types. Here we describe a safe and simple effective naked DNA gene delivery method, via inferior vena cava (IVC) injection, to the recipient's kidneys. It was further demonstrated that gene expression was concentrated in the proximal tubular epithelial cells of the cortico-medullary region of the kidney. Confocal microscopy analyses demonstrated the presence of the exogenous DNA in the renal cell membrane 10 min postgene delivery. However, it was only by 30 min that the presence of the exogenous DNA could be detected in the cell cytoplasm and in the nuclei of the renal cells. Stable expression of the beta-galactosidase gene could be detected for up to 35 days and no toxicity or any adverse pathological effect associated with the delivery method could be observed. Importantly, this IVC gene delivery method could promote the targeting of genes to carcinoma established in the kidney of SCID mice. These results provide the first evidence to support that stable gene expression could be achieved in the renal cells of kidney and the established carcinoma in the kidneys following in vivo gene delivery with naked DNA and could therefore provide the potential to design protocols for the gene therapy of the kidney diseases.
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PMID:Systemic administration of naked DNA with targeting specificity to mammalian kidneys. 1564 68

Type 1 diabetes results from insulin deficiency caused by destruction of pancreatic beta cells. Glucagon-like peptide (GLP)-1 stimulates beta cell growth and differentiation. To determine whether continuous expression of GLP-1 in vivo can regenerate beta cells and remit type 1 diabetes in mice for a prolonged time, we constructed an adenoviral vector containing the cytomegalovirus promoter/enhancer and albumin leader sequence followed by GLP-1 cDNA (rAd-GLP-1). A single administration of rAd-GLP-1 via the tail vein into streptozotocin (STZ)-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in remission of diabetes within 10 days; normoglycemia remained until the experiment was terminated. The number of insulin-positive cells in the pancreas and insulin secretion significantly increased in rAd-GLP-1-treated mice compared with STZ-induced diabetic mice treated with rAd-beta-galactosidase. Glucose tolerance tests in mice that achieved normoglycemia after treatment with rAd-GLP-1 showed that the kinetics of glucose clearance was similar to normal NOD/SCID mice. Treatment of autoimmune diabetic mice with rAd-GLP-1 restored normoglycemia, which was maintained for 1 year when mice were also treated with an immunoregulator to halt the autoimmune response to beta cells. We suggest that regeneration of insulin-producing cells by GLP-1 gene therapy may be a potential method for prolonged control of type 1 diabetes in humans.
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PMID:Prolonged remission of diabetes by regeneration of beta cells in diabetic mice treated with recombinant adenoviral vector expressing glucagon-like peptide-1. 1716 79

CRACM1 (also called Orai1) constitutes the pore subunit of store-operated calcium release-activated calcium channels. A point mutation in the gene encoding CRACM1 is associated with severe combined immunodeficiency disease in humans. Here we generated CRACM1-deficient mice in which beta-galactosidase activity 'reported' CRACM1 expression. CRACM1-deficient mice were smaller in size. Mast cells derived from CRACM1-deficient mice showed grossly defective degranulation and cytokine secretion, and the allergic reactions elicited in vivo were inhibited in CRACM1-deficient mice. We detected robust CRACM1 expression in skeletal muscles and some regions of the brain, heart and kidney but not in the lymphoid regions of thymus and spleen. In contrast, we found CRACM2 expression to be much higher in mouse T cells. In agreement with those findings, the store-operated calcium influx and development and proliferation of CRACM1-deficient T cells was unaffected. Thus, CRACM1 is crucial in mouse mast cell effector function, but mouse T cell calcium release-activated calcium channels are functional in the absence of CRACM1.
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PMID:Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. 1805 70

Directed endodermal differentiation of murine embryonic stem (ES) cells gives rise to a subset of cells with a hepatic phenotype. Such ES cell-derived hepatic progenitor cells (ES-HPC) can acquire features of hepatocytes in vitro, but fail to form substantial hepatocyte clusters in vivo. In this study, we investigated whether this is due to inefficient engraftment or an immature phenotype of ES-HPC. ES cells engrafted into recipient livers of NOD/SCID mice with a similar efficacy as adult hepatocytes after 28 days. Because transplanted unpurified ES-HPC formed teratomas in the spleen and liver, we applied an albumin promoter/enhancer-driven reporter system to purify ES-HPC by cell sorting. RT-PCR analyses for hepatocyte-specific genes showed that the cells exhibited a hepatic phenotype, lacking the expression of the pluripotency marker Oct4, comparable to cells of day 11.5 embryos. Sorted ES-HPC derived from beta-galactosidase transgenic ES cells were injected into fumaryl-acetoacetate-deficient (FAH(-/-)) SCID mice and analyzed after 8 to 12 weeks. Staining with X-gal solution revealed the presence of engrafted cells throughout the liver. However, immunostaining for the FAH protein indicated hepatocyte formation at a very low frequency, without evidence for large hepatocyte cluster formation. In conclusion, the limited repopulation capacity of ES-HPC is not caused by a failure of primary engraftment, but may be due to an immature hepatic phenotype of the transplanted ES-HPC.
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PMID:Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation. 1852 34


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