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)

After a limited number of population doublings (PDs), cultures of normal mammalian diploid cells undergo an irreversible growth arrest known as replicative senescence [1]. As well as contributing to cellular ageing, senescence is viewed as an important mechanism of tumour suppression by preventing the emergence of immortal cell clones [2-4]. Senescent cells have a number of characteristics that distinguish them from cycling or quiescent cells including elevated levels of two cyclin-dependent kinase (Cdk) inhibitors, p16INK4a and p21CIP1 [5-11]. Here, we demonstrate that both of these Cdk inhibitors, as well as other members of their protein families (the INK4 and CIP/KIP families, respectively [12]), induce several facets of the senescent phenotype when ectopically expressed in young human diploid fibroblasts. These include a reduced proliferative capacity, an altered size and shape, the presence of underphosphorylated retinoblastoma protein (pRb), increased expression of plasminogen activator inhibitor (PAI-1) and the appearance of senescence-associated beta-galactosidase (SA-beta-gal) activity [2,3,13-15]. A 20 amino acid peptide from p16INK4a that inhibits Cdks active in the G1 phase of the cell cycle [16] produces similar effects in a dose-dependent manner suggesting that, in primary fibroblasts, inhibition of G1-specific Cdk activity is sufficient to induce phenotypic changes that normally occur at the end of their finite lifespan.
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PMID:Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. 951 19

Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was observed to result in a four-fold increase in both secreted and cell-associated PAI-1 antigen over a four day period. Subcellular fractionation of PMA-treated MEG-01 cells on 45% self-forming Percoll gradients was employed to separate low density membrane and Golgi-rich fractions from a high density granule-containing region. A subsequent 30-60% pre-formed Percoll gradient was employed to remove contaminating lysosomes from the PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these MEG-01 granules share a similar size distribution (350-600 nm) and morphology to platelet alpha-granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was approximately 10% of the levels present in isolated platelet alpha-granules. To elevate PAI-1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI-1 and beta-galactosidase resulted in low transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular PAI-1 by 31-fold (1,200 ng/10(6) cells at 10 MOI) in comparison to mock-infected cells. Pulse-chase experiments demonstrated that SFV/PAI-1 mediated gene expression could enhance PAI-1 storage 6-9-fold, reaching levels present within platelets. To document the ability of PAI-1 to be stored in a rapidly releasable form in MEG-01 cells, we isolated platelet-like particles from the media conditioned by the cells and examined secretagogue-induced release of PAI-1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secretion of PAI-1 following treatment with ADP in comparison to particles incubated in the absence of secretagogue. These results suggest that SFV mediated gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha-granule proteins.
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PMID:Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01. 1152 53

Mad family proteins have an antagonistic action on Myc-dependent cell proliferation and transformation. We isolated a human cDNA clone, human Mad4 (hMad4), encoding a polypeptide of 209 amino acid residues, exhibiting 90% identity with mouse Mad4. Northern blot analysis shows that hMad4 probe hybridizes to a 3.8 kb message; its expression is highest in quiescent human WI38 fibroblasts. Among tissues, hMad4 mRNA is most abundant in brain, lung, and muscle. Consistent with other members of the Mad family, hMad4 can repress the transactivation activity of Myc/Max heterodimers on an E-box chloramphenicol acteyl transferase (CAT) reporter plasmid; inhibition of both proliferation and clonogenic formation of hMad4-infected cells correlates with the in vitro reporter repression. Moreover, infection of young human fibroblasts induces a replicative senescence-like state. This phenotype was accompanied by s-beta-galactosidase and PAI-1 expression. These results suggest that hMad4 might be an important regulator of replicative senescence in human cells.
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PMID:hMad4, c-Myc endogenous inhibitor, induces a replicative senescence-like state when overexpressed in human fibroblasts. 1276 91

In a 16-patient study, cultured fibroblast populations from normal skin were able to replicate an average of 14.8 +/- 2.2 times before becoming senescent, while fibroblast populations from the ulcer bed reached the end of their replicative life span after 7.2 +/- 1.9 population doublings (p= 0.001). Fibroblast populations from 10 of 16 pressure ulcers became senescent after fewer than five population doublings, whereas when populations of fibroblasts from adjacent normal skin were studied, only 2 of 16 became senescent within this same time period. In addition, only an occasional fibroblast from normal skin stained positively for senescence-associated beta-galactosidase compared to approximately 50% of equally aged ulcer bed fibroblasts (p = 0.0060). Senescent ulcer bed fibroblasts secreted significantly more plasmin than early passage ulcer bed fibroblasts (p= 0.0237), nearly six times as much plasmin as early passage normal skin fibroblasts (p < 0.0001), three and a half times the level of normal skin fibroblasts of the same age (11.52 +/- 4.58 microg/mg protein; p= 0.0003), and more than one and a half times the level of senescent normal skin fibroblasts (p= 0.0525). Senescent pressure ulcer fibroblasts generated significantly more plasminogen activator inhibitor-1 (1179.27 +/- 25.37 ng/mg protein) than normal skin fibroblasts of the same age (132.16 +/- 16.20 ng/mg protein; p = 0.0357). Also, senescent ulcer bed fibroblasts produced higher levels of transforming growth factor-beta1, but these were not significantly different from senescent normal skin fibroblasts. Although senescent ulcer fibroblasts produce elevated levels of plasminogen activator inhibitor-1 and transforming growth factor-beta1, the ratio of these factors to plasmin levels suggests that this may have little influence on extracellular matrix synthesis or maintenance in the chronic wound. These data show that cultured fibroblasts from most patient pressure ulcers profile a wound environment that is associated with an increasing population of senescent fibroblasts; however, factors within the chronic wound environment that promote cellular senescence remain unclear. We have proposed that a prolonged inflammatory response may be a contributing factor to the chronic wound condition.
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PMID:Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-beta1. 1565 39

Discodermolide is a microtubule stabilizing agent that suppresses dynamic instability and blocks cells in mitosis. Selection of A549 nonsmall cell lung carcinoma cells with increasing concentrations of discodermolide yielded a clone that proliferated in 8 nM. When these cells were exposed to any concentration greater than 8 nM, replication ceased and the cells developed a flattened, enlarged, granular morphology. Accelerated senescence was demonstrated by a functional beta-galactosidase activity at pH 6. When parental A549 cells were treated with IC50-concentrations of doxorubicin, Taxol or discodermolide, the latter two drugs quickly produced aberrant mitosis. However, discodermolide, but not Taxol, also produced a large increase in senescence-associated beta-galactosidase activity and altered levels of known senescence markers. Although some of these differences between Taxol and discodermolide were dose dependent, only discodermolide produced a doxorubicin-like induction of a senescence phenotype, including a senescence-associated beta-galactosidase activity, up-regulation of PAI-1 and p66Shc, and a strong, sustained, Erk1/2 activation. This research provides insights into the mechanism of action of discodermolide and provides the first demonstration of a microtubule stabilizing agent that inhibits tumor cell growth with a powerful induction of accelerated senescence.
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PMID:The microtubule stabilizing agent discodermolide is a potent inducer of accelerated cell senescence. 1571 Nov 27

hSNF5, the smallest member of the SWI/SNF chromatin remodeling complex, is lost in most malignant rhabdoid tumors (MRT). In MRT cell lines, reexpression of hSNF5 induces G1 cell cycle arrest, elevated p16INK4a, and activated replicative senescence markers, such as beta-galactosidase (beta-Gal) and plasminogen activator inhibitor-1. To compare the replicative senescence caused by hSNF5 in A204 cells to normal cellular senescence, we examined the activation of both p16INK4a and p21CIP/WAF1. Analogous to normal cellular senescence, both p16INK4a and p21CIP/WAF1 were up-regulated following hSNF5 restoration. Furthermore, we found that hSNF5 bound the p16INK4a and p21CIP/WAF1 promoters, suggesting that it directly regulates transcription of these genes. Using p16INK4a RNA interference, we showed its requirement for the replicative senescence caused by hSNF5 but not the growth arrest. Instead, p21CIP/WAF1 remained activated by hSNF5 in the absence of high p16INK4a expression, apparently causing the growth arrest in A204. Interestingly, we also found that, in the absence of p16INK4a, reexpression of hSNF5 also increased protein levels of a second cyclin-dependent kinase (CDK) inhibitor, p18INK4c. However, our data show that lack of hSNF5 does not abrogate cellular responsiveness to DNA damage or growth-inhibitory factors. In summary, our studies suggest that hSNF5 loss may influence the regulation of multiple CDK inhibitors involved in replicative senescence.
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PMID:Loss of the hSNF5 gene concomitantly inactivates p21CIP/WAF1 and p16INK4a activity associated with replicative senescence in A204 rhabdoid tumor cells. 1628 6

Vascular ageing is accelerated in patients with diabetes. However, the underlying mechanism remains unclear. Here, we show that high glucose induces activation of apoptosis signal-regulating kinase 1 (ASK1), an apoptosis-inducing signal that mediates endothelial cell senescence induced by hyperglycemia. High glucose induced a time-dependent increase in the levels of ASK1 expression and its activity in human umbilical vein endothelial cells (HUVECs). Incubation of endothelial cells with high glucose increased the proportion of cells expressing senescence-associated beta-galactosidase (SA-beta-gal) activity. However, transfection with an adenoviral construct including a dominant negative form of ASK1 gene significantly inhibited SA-beta-gal activity induced by high glucose. In addition, infection with an adenoviral construct expressing the constitutively active ASK1 gene directly induced an increase in the levels of SA-beta-gal activity. Activation of the ASK1 signal also enhanced plasminogen activator inhibitor-1 (PAI-1) expression in HUVECs. Induction of senescent endothelial cells in aortas and elevation of plasma PAI-1 levels were observed in streptozotocin (STZ) diabetic mice, whereas these changes induced by STZ were attenuated in ASK1-knockout mice. Our results suggest that hyperglycemia accelerates endothelial cell senescence and upregulation of PAI-1 expression through activation of the ASK1 signal. Thus, ASK1 may be a new therapeutic target to prevent vascular ageing and thrombosis in diabetic patients.
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PMID:Apoptosis signal-regulating kinase 1 mediates cellular senescence induced by high glucose in endothelial cells. 1673 28

Yeast Sir2 plays critical roles in gene silencing, stress resistance and longevity. Mammalian Sirt1 NAD(+)-dependent protein deacetylase, the closest homolog of Sir2, regulates cell cycle, cellular senescence, apoptosis and metabolism, by functional interactions with a number of biological molecules such as p53. To investigate a role of Sirt1 in endothelial dysfunction and premature senescence, we examined the effects of Sirt1 inhibition in human umbilical vein endothelial cells (HUVEC). Sirt1 inhibition by sirtinol, which is a 2-hydroxy-1-napthaldehyde derivative, or siRNA for Sirt1-induced premature senescence-like phenotype, as judged by increased senescence-associated beta-galactosidase (SA-beta-gal) activity, sustained growth arrest and enlarged and flattened cell morphology at 10 days after the treatment. Sixty-four percent of sirtinol (60 mumol/L)-treated HUVEC was SA-beta-gal-positive, whereas only 17% of vehicle-treated cells were positive. Sirt1 inhibition by sirtinol or Sirt1 siRNA increased PAI-1 expression and decreased both protein expression and activity of eNOS. Treatment with sirtinol or Sirt1 siRNA increased acetylation of p53, while p53 expression was unaltered. Impaired epidermal growth factor-induced activation of mitogen-activated protein kinases was associated with Sirt1 inhibition-induced senescence-like growth arrest. Conversely, overexpression of Sirt1 prevented hydrogen peroxide-induced SA-beta-gal activity, morphological changes and deranged expression of PAI-1 and eNOS. These results showed that Sirt1 inhibition increased p53 acetylation and induced premature senescence-like phenotype in parallel with increased PAI-1 and decreased eNOS expression. Our data suggest that Sirt1 may exert protective effects against endothelial dysfunction by preventing stress-induced premature senescence and deranged expression of PAI-1 and eNOS.
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PMID:Sirt1 modulates premature senescence-like phenotype in human endothelial cells. 1791 62

Endothelial cell (EC) senescence and dysfunction occurring after chronic injury and inflammation are highly associated with the development and progression of cardiovascular diseases. However, the factors involved in the establishment of EC senescence remain poorly understood. We have previously shown that lithium, an inhibitor of glycogen synthase kinase (GSK)-3beta and activator of the Wnt/beta-catenin signaling pathway, induces an EC senescent-like phenotype. Herein, we show that lithium induces a rapid and pronounced up-regulation of the matrix metalloproteinase (MMP)-1, an inflammation and senescent cell marker, at the mRNA and protein levels, whereas the induction of two other senescent cell markers is either weak (interleukin-8) or delayed (plasminogen activator inhibitor-1). Lithium effect on MMP-1 expression is also specific among other MMPs and not mediated by GSK3beta inhibition. Lithium affects MMP-1 expression mainly at the transcriptional level but neither the AP1/Ets regulatory sites nor the redox sensitive (-1607/2G) site in MMP-1 promoter are involved in lithium-dependent MMP-1 regulation. However, down-regulation of p53, a target of lithium in EC, dampens both basal and lithium-induced MMP-1 expression, which further links MMP-1 up-regulation with the establishment of cell senescence. Although increased MMP-1 levels are usually associated with angiogenesis in enabled proliferative EC, the exogenous addition of activated MMP-1 on lithium- arrested EC increases the number of EC positive for the senescent-associated-beta-galactosidase marker. Conversely, down-regulation of MMP-1 expression by small interfering RNAs blunts the lithium-dependent increase in senescent-associated-beta-galactosidase positive cells. Altogether our data indicate that lithium-induced MMP-1 may participate in the reinforcement of EC senescence and reveal a novel mechanism for lithium-induced tissue remodeling.
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PMID:Enhanced endothelial cell senescence by lithium-induced matrix metalloproteinase-1 expression. 1940 40

Diabetic nephropathy is the commonest cause of end-stage renal disease. Inordinate kidney growth and glomerular hyperfiltration at the very early stages of diabetes are putative antecedents to this disease. The kidney is the only organ that grows larger with the onset of diabetes mellitus, yet there remains confusion about the mechanism and significance of this growth. Here we show that kidney proximal tubule cells in culture transition to senescence in response to oxidative stress. We further determine the temporal expression of G(1) phase cell cycle components in rat kidney cortex at days 4 and 10 of streptozotocin diabetes to evaluate changes in this growth response. In diabetic rats we observe increases in kidney weight-to-body weight ratios correlating with increases in expression of the growth-related proteins in the kidney at day 4 after induction of diabetes. However, at day 10 we find a decrease in this profile in diabetic animals coincident with increased cyclin-dependent kinase inhibitor expressions. We observe no change in caspase-3 expression in the diabetic kidneys at these early time points; however, diabetic animals demonstrate reduced kidney connexin 43 and increased plasminogen activator inhibitor-1 expressions and increased senescence-associated beta-galactosidase activity in cortical tubules. In summary, diabetic kidneys exhibit an early temporal induction of growth phase components followed by their suppression concurrent with the induction of cyclin-dependent kinase inhibitors and markers of senescence. These data delineate a phenotypic change in cortical tubules early in the pathogenesis of diabetes that may contribute to further downstream complications of the disease.
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PMID:Transition of kidney tubule cells to a senescent phenotype in early experimental diabetes. 2050 38


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