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)

Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding beta-galactosidase (Ad beta Gal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of Ad beta Gal-transduced DC, EC, or myoblasts and were then monitored for anti-beta-galactosidase (anti-beta-Gal) antibody production, induction of gamma interferon-secreting CD8(+) T cells, and protection against melanoma tumor cells expressing beta-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8(+) T-cell response at a level of 50% of the specific response obtained with the Ad beta Gal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a beta-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.
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PMID:Distinct roles of adenovirus vector-transduced dendritic cells, myoblasts, and endothelial cells in mediating an immune response against a transgene product. 1186 57

Melanoma has proven to be resistant to conventional chemotherapy; however,the mechanism of chemoresistance is still unclear. Recent reports show that the transcription factor, E2F-1, may play a role in mediating cytotoxicity of certain chemotherapeutic agents. We have shown in a previous study that adenovirus-mediated overexpression of E2F-1 can efficiently induce apoptosis in melanoma cells. In the present study, the effect of E2F-1 expression on drug sensitivity of melanoma cells was evaluated. Two human melanoma cell lines, SK-MEL-28 and SK-MEL-2, were treated with drugs (etoposide, Adriamycin, roscovitine, cisplatin, 5-fluorouracil, or cycloheximide), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1) at a multiplicity of infection of 1 in vitro. E2F-1 expression was confirmed by Western blot analysis. Sublethal concentrations of each drug alone or infection with Ad-E2F-1 alone produced <5% apoptosis by 3 days posttreatment. Conversely, cotreatment with Ad-E2F-1 and low concentrations of etoposide or Adriamycin markedly sensitized melanoma cells to apoptotic cell death. A slight enhancement of the cytotoxicity of roscovitine was demonstrated in combination with E2F-1 overexpression, but not to cisplatin, 5-fluorouracil, or cycloheximide. Ad-LacZ infection showed no obvious effects on drug sensitivity. Overexpression of p21 can block apoptosis induced by the combination chemogene therapy of Ad-E2F-1 and topoisomerase II poisons and does not require its proliferating cell nuclear antigen-binding ability. The protein synthesis inhibitor cycloheximide also has a cytotoxicity-protective effect against topoisomerase II inhibitor/E2F-1-induced apoptosis and suggests that new protein synthesis is required for this process. Topoisomerase II inhibitors also cooperated with Ad-E2F-1 to enhance antitumor activity in an in vivo model using xenografts in nude mice. When combined with Adriamycin or etoposide, E2F-1 adenovirus therapy resulted in an 87% or 91% decrease in tumor size, respectively, compared with controls (P < 0.002). Our results show that adenovirus-mediated E2F-1 gene transfer can sensitize melanoma cells to some chemotherapeutic agents, particularly topoisomerase II poisons, in vitro and in vivo. These results suggest a new chemosensitization strategy for melanoma gene therapy.
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PMID:Adenovirus-mediated E2F-1 gene transfer sensitizes melanoma cells to apoptosis induced by topoisomerase II inhibitors. 1191 54

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

We investigated whether repeated cationic multilamellar liposome-mediated gene transfers enhanced the transduction efficiency against murine melanoma cell lines and experimental subcutaneous melanoma. In the former, the murine melanoma cell line, B16F10, was transfected by our original cationic multilamellar liposomes containing pVLacZ, which express beta-galactosidase in eukaryotic cells. Cells were exposed to the liposomes in a single, double, or triple procedure during the cell logarithmic proliferative period. We then evaluated the transduction efficiency by X-gal staining and beta-galactosidase assay. The number of positive cells and level of beta-galactosidase activity were significantly increased by repeated exposures compared with a single one. Cells transfected by the fluorescently labeled cationic liposome containing pEGFP-C1 showed both an increased uptake of liposomes and an increased number of EGFP expression cells following repeated exposures. In the latter, murine subcutaneous melanomas, which were made by transplantation of B16F10 in C57BL6 mice, were transfected by same liposomes. Subcutaneous melanomas were exposed to the liposomes in a single, double, or triple procedure. We then evaluated the transduction efficiency by the beta-galactosidase assay. The level of beta-galactosidase activity was significantly increased by repeated exposures compared with a single one. The results indicate that repeated exposures to the liposomes enhanced the transduction efficiency toward murine melanoma cells and experimental subcutaneous melanoma, and may provide a basis for the repeated-exposure protocol for human trials.
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PMID:Repeated cationic multilamellar liposome-mediated gene transfer enhanced transduction efficiency against murine melanoma cell lines. 1223 11

The histone acetyltransferases p300 and cAMP-responsive element-binding protein-binding protein (CBP) are required for the execution of critical biological functions such as proliferation, differentiation, and apoptosis. Both proteins are believed to regulate the activity of a large number of general and cell-specific transcription factors. Here we demonstrate a dramatic decrease in the total cellular levels of p300 and CBP with increasing population doublings of human normal melanocytes. We show that one consequence of p300 depletion is transcriptional down-regulation of the cyclin E gene, caused by deacetylation of histones at its promoter. The cyclin E promoter was activated by p300 and the histone deacetylase inhibitor trichostatin A. Conversely, the cyclin E promoter was repressed by wild-type Retinoblastoma tumor suppressor p105 protein (pRB) and by a dominant negative p300 mutant (DN p300) that lacks histone acetyltransferase activity. We also provide evidence of the alternative recruitment of p300 and histone deacetylase 1 to the cyclin E promoter in proliferating and senescent melanocytes, respectively. The biological significance of these results was established by showing that block of p300 activity by overexpression of DN p300 or by Lys-CoA, a specific chemical inhibitor of p300, resulted in growth inhibition, down-regulation of cyclin E, and activation of the senescence-associated beta-galactosidase marker in human melanocytes and melanoma cells. Together, these results provide evidence for the essential role of p300 in the regulation of proliferation and senescence in cells from melanocytic origin.
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PMID:Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes. 1241 52

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.
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PMID:Therapeutic immune response induced by electrofusion of dendritic and tumor cells. 1271 34

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.
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PMID:Inhibition of c-Myc oncoprotein limits the growth of human melanoma cells by inducing cellular crisis. 1282 59

Although the renin angiotensin system (RAS) is a major regulator of vascular homeostasis, the role of the RAS in tumor angiogenesis is little understood. Here we show that host angiotensin II (ATII) type 1 (AT1) receptor plays an important role in angiogenesis and growth of tumor cells engrafted in mice. Subcutaneous B16-F1 melanoma-induced angiogenesis as assessed by tissue capillary density and microangiography was prominent in WT mice but was reduced in AT1a receptor-deficient (AT1a-/-) mice. Consequently, tumor growth rate was significantly slower, and the mouse survival rate was greater, in AT1a-/- mice than in WT mice. Tumor growth was also reduced in WT mice treated with TCV-116, a selective blocker of AT1 receptor. Because the beta-galactosidase gene was inserted into the AT1a gene locus in AT1a-/- mice, the site of beta-galactosidase expression represents the AT1a receptor expression in these mutant mice. In tumor-implanted AT1a-/- mice, the major site of the beta-galactosidase expression was macrophages in tissues surrounding tumors. Moreover, the number of infiltrated macrophages was significantly lower in AT1a-/- mice than in WT mice, and double-immunofluorescence staining revealed that these macrophages expressed VEGF protein intensively. Therefore, the host ATII-AT1 receptor pathway supports tumor-associated macrophage infiltration, which results in enhanced tissue VEGF protein levels. The host ATII-AT1 receptor pathway thereby plays important roles in tumor-related angiogenesis and growth in vivo.
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PMID:Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth. 1284 60

A number of reports have described the effects of oxidative stress on tumor growth. Therefore, these experiments were designed to test the hypothesis that overexpression of extracellular superoxide dismutase (ecSOD) would inhibit the growth of tumors arising from s.c. implantation of syngenic B16-F1 melanoma cells. C57BL/6 mice were infected i.m. with adenovirus containing either beta-galactosidase (Ad.lacZ) as control or the secreted extracellular isoform of SOD (Ad.ecSOD) 3 days before s.c. implantation of B16-F1 tumor cells. Serum SOD activity was elevated nearly approximately 5-fold over control animals. Two weeks after implantation, B16-F1 tumor size was 65% smaller in mice infected with Ad.ecSOD in comparison with mice infected with Ad.lacZ. However, the presence of SOD did not affect growth rates of B16-F1 cells in vitro. Consistent with smaller tumor volume, tumors from Ad.ecSOD-infected mice also expressed less vascular endothelial growth factor (VEGF). Moreover, in vitro studies using B16-F1 cells confirm that SOD blunts oxidant-dependent VEGF expression. Importantly, CD31 expression and vessel density were markedly reduced in tumors from Ad.ecSOD-infected mice compared with controls. These data suggest that tumor oxidative stress may facilitate tumor vascularization and thus promote tumor growth.
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PMID:Secretion of extracellular superoxide dismutase from muscle transduced with recombinant adenovirus inhibits the growth of B16 melanomas in mice. 1457 88

Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the beta-galactosidase (betagal) gene into M3-GM cells. Their administration activated betagal-specific, L(d)-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-betagal injection sites revealed the presence of cells presenting the respective L(d)-binding betagal peptide epitope. Based on their capacity to activate betagal-specific CTL, they were identified as being CD11c(+) dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.
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PMID:Granulocyte-macrophage colony-stimulating factor-based melanoma cell vaccines immunize syngeneic and allogeneic recipients via host dendritic cells. 1460 18


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