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)

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.
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PMID:Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent. 1091 34

Studies in fibroblasts have shown that H2O2, as a model for oxidative damage, leads to a G1 growth arrest phenotypically similar to senescence. These observations as well as the observation that bladder cancer is associated with deletions of CDKN2, a gene important in normal senescence, led us to examine normal urothelial cell response to H2O2. We hypothesized that low dose H2O2 exposure would lead to p16 and/or p14arf mediated senescence. We show that H2O2 leads to endogenous beta-galactosidase expression similar to senescence, but instead of G1 arrest, it leads to G2/M growth arrest without induction of either p16 or p14arf. Lack of p21 induction and a similar G2/M growth arrest in E6 immortalized uroepithelial cells suggests that this response is independent of p53 as well. An increased level of cdc2 tyrosine-15 phosphorylation following H2O2 treatment suggests that the observed growth arrest is mediated by a G2 checkpoint mechanism.
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PMID:A G2/M growth arrest response to low-dose intermittent H2O2 in normal uroepithelial cells. 1093 79

Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation of epithelial cells. Serial subculture of NHOKs to the postmitotic stage also induces terminal differentiation. However, the detailed mechanisms of both differentiation processes remain substantially unknown. To investigate the molecular differences in these processes, NHOKs were induced to differentiate by exposure to 1.2 mM of calcium and by serial subculture to the postmitotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and the expression of senescence-associated beta-galactosidase (SA-beta-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were also determined. Both calcium treatment and serial subculture to the postmitotic stage notably elevated the cellular involucrin. The percentage of SA-beta-gal-positive cells was significantly elevated by the continued subculture, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16(INK4A) protein was progressively increased by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both differentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subculture. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation, but that the differentiation processes are not identical, because calcium-induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16(INK4A).
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PMID:Concurrence of replicative senescence and elevated expression of p16(INK4A) with subculture-induced but not calcium-induced differentiation in normal human oral keratinocytes. 1097 54

Our objective was to determine the efficacy of adenoviral-mediated gene therapy with wild-type p53 or p21 in human breast cancer cells and investigate interactions with radiation and chemotherapy. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435, both with p53 mutations, were transduced with adenoviral vectors containing wild-type p53 (Ad5CMV-p53) or p21/WAF1/Cip1 (Ad5CMV-p21), and the effects on growth were determined. Infection was combined with low-dose (1.4 - 3.7 Gy) irradiation to see if this would improve transduction efficiency and enhance numbers of cells killed. Transduction with either vector resulted in expression of p21WAF1/cip1 and growth inhibition, although Ad5CMV-p53 transduction produced greater growth inhibition than did Ad5CMV-p21. The cell lines differed in sensitivity to the vectors. The Ad5CMV-p53 vector in a multiplicity of infection (MOI) of 125 resulted in 50% to 80% inhibition of MDA-MB-231, while MOI 250 of the same vector resulted in 27% inhibition of MDA-MB-435. Infection with Ad5CMV-p21 produced modest growth inhibition in both cell lines (< or = 40% at MOI 200), although protein expression was detected at lower viral doses. Low dose gamma-irradiation (1.4 to 3.7 Gy) was used to try and improve the rate of gene transfer. Modest increases in transduction efficiency and duration of expression of a vector containing beta-galactosidase occurred in irradiated breast cancer cells. Radiation 24 hr before transduction with Ad5CMV-p53 increased the proportions of apoptotic MDA-MB-231 cells. The cells transduced with Ad5CMV-p21 were arrested in G1, yet when they were irradiated before adenoviral transduction, the overexpression of p21 protected the cells from the cytotoxic effects of the radiation. Clonogenic assays showed that Ad5CMV-p21 reduced the sensitivity of MDA-MB-231 to VP-16 and paclitaxel. Combining these drugs with Ad5CMV-p53 did not consistently or significantly decrease clonogenic survival.
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PMID:Adenoviral-mediated gene therapy with Ad5CMVp53 and Ad5CMVp21 in combination with standard therapies in human breast cancer cell lines. 1104 64

Treatment failure after radiation therapy of prostate cancer (PC) could be a significant problem. Our objective is to design genetic radiosensitizing strategies for the treatment of PC. Cells from individuals with the genetic disorder ataxia telangiectasia (AT) are hypersensitive to ionizing radiation. Therefore, we examined whether attenuation of the AT gene product, AT mutated (ATM), in PC cells could result in an increased intrinsic radiosensitivity. A p53-mutant PC cell line, PC-3 was infected with adenoviral vectors, expressing antisense ATM RNA to various domains of the ATM gene. Immunoblot analyses of cellular extracts from antisense ATM-transfected PC-3 cells showed attenuated expression of the ATM protein within 2 days of viral infection. Compared with cells infected with an adeno-beta-galactosidase vector, antisense ATM-transfected PC-3 cells showed aberrant control of S-phase cell-cycle checkpoints after exposure to ionizing radiation. Under these conditions, the intrinsic radiosensitivity of the PC-3 cells was enhanced. Consequently antisense ATM gene therapy could serve as a paradigm for strategies that target the cellular survival mechanisms of an irradiated tumor cell and may provide therapeutic benefit to patients undergoing radiation therapy for PC.
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PMID:Adenovirus-mediated antisense ATM gene transfer sensitizes prostate cancer cells to radiation. 1105 87

The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli beta-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 x 10(9) plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.
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PMID:Adenovirus-mediated tumor suppressor p53 gene therapy for anaplastic thyroid carcinoma in vitro and in vivo. 1109 36

Suzuki, K., Mori, I., Nakayama, Y., Miyakoda, M., Kodama, S. and Watanabe, M. Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening. Normal human diploid cells irradiated with X rays showed permanent cell cycle arrest and exhibited senescence-like phenotypes including the expression of senescence-associated beta-galactosidase (SA-beta-gal). X irradiation caused persistent phosphorylation of TP53 at Ser 15 and accumulation of the TP53 protein, followed by the induction of CDKN1A (also known as p21(Waf1/Cip1)) and CDKN2A (also known as p16), preceded the expression of SA-beta-gal. NCI-H1299 human lung carcinoma cells, in which no TP53 protein was expressed, were irradiated with X rays with or without the exogenous expression of TP53 gene. Although induction of TP53 protein alone could induce SA-beta-gal expression, the frequency of SA-beta-gal-positive cells was significantly increased when TP53-induced H1299 cells were exposed to X rays. The mean terminal restriction fragment length in normal human cells was approximately 12 kb and did not change in SA-beta-gal-positive cells. These results indicate that ionizing radiation induces senescence-like growth arrest that is dependent on TP53 function but independent of telomere shortening. Our findings suggest that cells harboring irreparable DNA damage are programmed to undergo premature senescence to maintain the integrity of the genome.
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PMID:Radiation-induced senescence-like growth arrest requires TP53 function but not telomere shortening. 1112 Dec 42

Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.
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PMID:Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. 1112 81

Virus-like particles (VLPs) composed of recombinant capsid protein L1 and L2 of human papillomavirus type 16 were conjugated with polylysine (PL) and gene transfer was performed using VLP-PL conjugates to allow the expression of targeted gene. When HeLa cells were incubated with VLP-PL conjugate coupled with plasmid cytomegalovirus beta-galactosidase (pCMVbeta-gal), about 10% of cells were transfected and demonstrated beta-galactosidase activity. Hence chloramphenicol acetyltransferase activity was also expressed significantly in VLP-PL-plasmid simian virus 2 chloramphenicol acetyl transferase (pSV2CAT)-transfected cells, VLP-PL conjugate was tested whether it could transfer a tumor suppressor gene, pCMVp53, to HeLa cells and the exogenously provided p53 gene complexed to VLP-PL conjugate was detected from HeLa cells by polymerase chain reaction (PCR) analysis. Interestingly, additional increase of transfection efficiency was demonstrated in the presence of poloxamer 407 when C-33A cells were transfected with VLP-PL-pCMVbeta-gal complex. The result support the notion that VLP-PL conjugate may be a promising vector to transfer genetic materials into cancer cells and poloxamer 407 can be used for enhancing the transfection efficiency of VLP-PL conjugate.
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PMID:Improvement of gene transfer to cervical cancer cell lines using non-viral agents. 1112 65

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.
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PMID:Apoptosis-linked in vivo regulation of the tissue transglutaminase gene promoter. 1117 60


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