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 developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional control of the murine sarcoma virus long terminal repeat; and (ii) a eukaryotic expression vector driving transcription of a potential growth inhibitory c-DNA under control of the cytomegalovirus promoter/enhancer. Twenty-four hours after transfection, cellular DNA was labeled for an additional 24 h with 5-bromo-2-deoxyuridine (BrdU) to label cellular DNA. After fixation, transfected cells were identified by histochemical staining with a beta-gal substrate, 6-chloro-3-indolyl-beta-D-galactopyranoside (i.e., Red-Gal). Transfected cells (beta-gal-positive) that traversed S phase (i.e., DNA synthesis) were quantified by indirect immunocytochemical staining for BrdU. Since autoradiography was not required to score for DNA synthesis, the length of experiments was much shorter than previously described growth-arrest assays performed with transiently transfected cells. Experiments with two growth-arrest genes, p53 and the p21 cyclin-dependent kinase inhibitor, demonstrated the utility of this assay.
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PMID:Rapid characterization of growth-arrest genes in transient transfection assays. 754 21

p53 induction and cell cycle arrest occur following DNA damage, possibly to allow repair prior to replication. p21WAF1/CIP1, a cyclin-cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen-interacting protein, is induced by p53 and mediates the cell cycle arrest. To investigate a role for p21 in DNA repair in vivo, we studied the expression of in vitro damaged reporter DNA transfected into p21 +/+ or -/- HCT116 human colon cancer cells. Introduction of UV-damaged or cisplatinum-damaged cytomegalovirus-driven beta-galactosidase reporter DNA into tumor cells revealed a significant decrease (2-5-fold) in reporter expression in p21 -/- versus +/+ cells. In the absence of DNA damage, there was a significant increase (2-3-fold) in the number of 6-TG-resistant colonies derived from p21 -/- versus +/+ cells. Reintroduction of wild-type p21, but not a p21 C-terminal truncation mutant which lacks the proliferating cell nuclear antigen interaction domain, stimulated (2-3-fold) the repair capacity of the p21-deficient cells. We conclude that p21 deficiency is associated with a defect in DNA repair, which could lead to an increased sensitivity of tumor cells to DNA damage.
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PMID:Repair Defect in p21 WAF1/CIP1 -/- human cancer cells. 862 93

Normal cells in a culture enter a nondividing state after a finite number of population doubling, which is termed replicative senescence, whereas cancer cells have unlimited proliferative potential and are thought to exhibit an immmortal phenotype by escaping from senescence. The p21 gene (also known as sdi1), which encodes the cyclin-dependent kinase inhibitor, is expressed at high levels in senescent cells and contributes to the growth arrest. To examine if the p21sdi1 gene transfer could induce senescence in human cancer cells, we utilized an adenoviral vector-based expression system and four human cancer cell lines differing in their p53 status. Transient overexpression of p21sdi1 on cancer cells induced quiescence by arresting the cell cycle at the G1 phase and exhibited morphological changes, such as enlarged nuclei as well as a flattened cellular shape, specific to the senescence phenotype. We also showed that p21sdi1-transduced cancer cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Moreover, the polymerase chain reaction-based assay demonstrated that levels of telomerase activity were significantly lower in p21sdi1-expressing cells compared to parental cancer cells. These observations provide the evidence that p21sdi1 overexpression could induce a senescence-like state and reduce telomerase activity in human cancer cells, suggesting that these novel p21sdi1 functions may have important implications for anticancer therapy.
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PMID:Overexpression of the p21 sdi1 gene induces senescence-like state in human cancer cells: implication for senescence-directed molecular therapy for cancer. 1046 50

Tumor suppressor gene p16 is a cyclin-dependent kinase inhibitor and an important negative cell cycle regulator. The inactivation of p16 appears to be a common event in prostate cancer. Replacement of p16 inhibits prostate tumor cell growth, but the mechanism is not known. Human prostate cancer cell lines PPC-1, which has an inactivated p16, and DU145, which has a nonfunctional retinoblastoma Rb protein (pRb), were used to determine the possible mechanism of p16 mediated growth inhibition. PPC-1 cells treated with 5-aza-2'-deoxycytidine (5-aza-dC), a demethylating agent, induced p16 expression, inhibited cell growth, and induced senescence. Similarly, PPC-1 cells transduced by an adenoviral vector containing the p16 gene (AdRSVp16) produced a p16 protein that suppressed cellular proliferation and induced senescence. Co-staining of AdRSVp16-transduced PPC-1 cells by p16 immunohistochemistry and by beta-galactosidase substrate X-gal showed that the morphologically enlarged cells expressed both p16 and senescence-associated beta-galactosidase. In contrast, AdRSVp16 did not induce senescence in DU145 cells, but did inhibit its growth. However, when wild-type pRb was introduced in DU145 cells, AdRSVp16 was able to induce senescence. Thus, the mechanism by which p16 suppressed prostate cancer was dependent on the pRb functional status of cells whereby p16 caused pRb+ cells to undergo inhibition by senescence, whereas pRb- cells were also inhibited, but not by senescence.
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PMID:p16/MTS1/INK4A suppresses prostate cancer by both pRb dependent and independent pathways. 1071 71

Lithium affects development of various organisms and cell fate through the inhibition of glycogen synthase kinase-3 beta and induction of the Wnt/beta-catenin signaling pathway. In this study, we investigated the effects of lithium on primary bovine aortic endothelial cells (BAEC). Lithium treatment of BAEC induced beta-catenin stabilization but failed to activate the transcriptional activity of the beta-catenin/T-cell factor complex. Lithium caused a sustained G(2)/M cell cycle arrest without affecting cell viability. Reversibility of this cell cycle arrest occurred up to 3 days after treatment but was reduced thereafter. Lithium-treated BAEC exhibited a senescent-like morphology with an increase in cells positive for the senescence-associated-beta-galactosidase activity. Lithium also increased the expression of p21(Cip), a cyclin-dependent kinase inhibitor, both at the protein and RNA levels. No change in p21(Cip) mRNA stability was observed, whereas the transcriptional activity of a p21(Cip) promoter-luciferase construct containing p53 binding sites was increased after lithium treatment. Furthermore, lithium caused increased transcription of a reporter gene under the control of a promoter containing the p53 consensus binding sites both in transiently transfected BAEC and in a stably transfected fibroblast cell line. Lithium caused accumulation of p53 protein in BAEC without affecting p53 mRNA levels. Finally, up-regulation of p21(Cip) in response to lithium did not occur in mouse embryonic fibroblasts that were null for p53 alleles, confirming the dependence on a p53 pathway for this lithium effect. These findings demonstrate for the first time that lithium induces also stabilization of the tumor suppressor p53 and reveal a new mechanism that may contribute to the neuroprotective effects of lithium.
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PMID:Lithium inhibits cell cycle progression and induces stabilization of p53 in bovine aortic endothelial cells. 1133 98

Cellular senescence, initially observed during subculturing of normal diploid fibroblasts, can also be induced by chronic exposure to cellular stress, such as UV light, oxidative stress, or DNA damaging agents. Here we demonstrate that stable expression of an activated form of MKK6 (MKK6EE), a direct activator of the stress-induced p38(HOG) mitogen-activated protein kinase pathway, is sufficient for inducing features of senescence including a flattened, vacuolated, and irregular morphology, staining for acidic beta-galactosidase, and accumulation of age-associated pigments. Consistent with the senescent phenotype, p38(HOG) activation induces a G(1) cell cycle arrest, which is permanent and irreversible after 4 days. MKK6EE also induces biochemical features of senescence in a p38-dependent manner, including enhanced expression of p21(CIP), a cyclin-dependent kinase inhibitor. Microarray analysis of MKK6EE cells showed a pattern of gene expression noted previously in Werner Syndrome and senescent fibroblasts. These results define p38(HOG) as an intracellular pathway that activates a senescence checkpoint in tumor cells and may play a role in Ras- or stress-induced senescence.
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PMID:Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence. 1220 64

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.
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PMID:Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line. 1229 82

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.
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PMID:Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence. 1241 54

Based on the fact that aberrant overexpression of some growth factor receptors was observed in a variety of human cancer cells, a novel nonviral gene delivery system GE7, which contains a 16-amino-acid ligand for identifying EGF receptor was constructed for tumor-targeted gene therapy. Intravenous administration of GE7 system revealed that it has the ability to target beta-galactosidase (beta-gal) reporter gene into murine hepatoma (Hepa) cells. Owing to the limited antitumor effects elicited by a single-gene transfer, recent efforts to treat malignancy using combined gene therapy have been accomplished with varying degrees of success. In this study, the human cyclin-dependent kinase inhibitor gene p21(WAF-1) and the murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injection of the EGF R targeted GE7/DNA complex into murine hepatoma-bearing mice. The results demonstrated that combined administration of p21(WAF-1) and GM-CSF could remarkably inhibit the growth of subcutaneously transplanted hepatoma Hepa cells, and significantly increase the survival rate of tumor-bearing mice. The activities of natural killer (NK) cells and specific cytotoxic T lymphocytes (CTL) were clearly enhanced after combined gene therapy. In vitro experiments showed that p21(WAF-1) gene transfer exhibited a suppressive function on the growth of Hepa cells and the expression of H-2K(b) and B7-1 molecules on Hepa cells increased significantly after combined genes delivery. All these results suggested that the GE7 system was able to target therapeutic genes efficiently to cancer cells, which showed high EGF R expression. The cotransfer of p21(WAF-1) and GM-CSF genes apparently inhibited the growth of tumors through (a) the arrest of tumor cell growth and (b) the enhancement of systemic antitumor immunity.
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PMID:Systemic genetic transfer of p21WAF-1 and GM-CSF utilizing of a novel oligopeptide-based EGF receptor targeting polyplex. 1283 33

To understand the role of the cell cycle regulatory protein in the control of smooth muscle cell (SMC) proliferation, we tested the overexpression of p21Waf1, a cyclin-dependent kinase inhibitor, in human normal (MS9) and immortalized SMCs (ISS10) transfected with ori-minus simian virus 40 DNA, using an adenovirus-mediated system. In MS9, overexpression of p21Waf1 resulted in the inhibition of cell cycle progression at the G1/S boundary without apoptosis. On the other hand, in ISS10, overexpression of p21Waf1 induced marked apoptosis. In these cells, immunohistochemistry revealed that overexpressed p21Waf1 was localized in the nucleus. No differential expression pattern of either p53 or SV40T was observed in p21Waf1- and control gene (beta-galactosidase)-infected cells. Old-passaged ISS10 cells eventually showed growth arrest and a senescent-like phenotype. Immunohistochemistry revealed that p21Waf1 was localized in the cytoplasm of the early-passaged cells, but was found in the nucleus of the old-passaged cells. Our data suggested that nuclear accumulation of p21Waf1 plays a role in the cell death of immortalized SMC, which carries dysfunction of the cell cycle regulatory proteins such as p53. This culture model may be useful for studying the process of SMC proliferation, cell death, senescence, and cell cycle regulation.
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PMID:Overexpression of p21Waf1 induces apoptosis in immortalized human vascular smooth muscle cells. 1456 87


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