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: UNIPROT:P42574 (caspase-3)
45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated the role of Aurora kinase A (AURKA) in regulating p73-dependent apoptosis using the p53-deficient cancer cell lines H1299, TE7, and HCT116p53(-/-). Overexpression of AURKA led to down-regulation of the TAp73-induced activation of the p53/p73-dependent luciferase reporter plasmid (pG13-luc). The reduction in the TAp73 transcription activity was confirmed by measuring the activity of luciferase reporters for p21/WAF1, and PUMA. The siRNA knockdown of endogenous AURKA reversed these effects and Western blot analysis showed a significant increase in the protein level of TAp73 and its downstream transcription targets, PUMA, NOXA, and p21/WAF1. The coexpression of AURKA together with TAp73 inhibited the activation of the pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids with reduction in the protein levels of TAp73 transcription targets. Treatment with AURKA-selective small molecule inhibitor MLN8054 led to a significant increase in the activities of pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids. This effect was accompanied by a significant increase in the mRNA and protein levels of several TAp73 transcription targets: p21/WAF1, PUMA, and NOXA. Flow cytometry cell cycle analysis, after MLN8054 treatment, showed more than a 2-fold increase in cell death. The apoptotic outcome was corroborated by showing an increase in cleaved caspase-3 protein levels by Western blot. Using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assay, we showed that the expression of dominant-negative mutant TAp73 expression plasmid (p73DD) counteracted the MLN8054-induced cell death. Taken together, our results indicate that AURKA regulates TAp73-dependent apoptosis and highlight the potential of the AURKA inhibitor MLN8054 in treating cancers that are defective in p53 signaling.
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PMID:Aurora kinase A inhibition leads to p73-dependent apoptosis in p53-deficient cancer cells. 1897 45

Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET(1) with Renilla luciferase (RLuc) and coelenterazine h (CLZ, lambda(em) approximately 475 nm) and BRET(2) with the substrate coelenterazine 400a (CLZ400A substrate, lambda(em)=395 nm) as the respective donors. For BRET(1) the acceptor is yellow fluorescent protein (YFP) (lambda(em) approximately 535 nm), a mutant of green fluorescent protein (GFP), and for BRET(2) it is GFP(2) (lambda(em) approximately 515 nm). It is not clear from previous studies which of these systems has superior signal-to-background characteristics. Here we directly compared BRET(1) and BRET(2) by placing two different protease-specific cleavage sequences between the donor and acceptor domains. The intact proteins simulate protein-protein association. Proteolytic cleavage of the peptide linker simulates protein dissociation and can be detected as a change in the BRET ratios. Complete cleavage of its target sequence by thrombin changed the BRET(2) ratio by a factor of 28.9+/-0.2 (relative standard deviation [RSD], n=3) and changed the BRET(1) ratio by a factor of 3.05+/-0.07. Complete cleavage of a caspase-3 target sequence resulted in the BRET ratio changes by factors of 15.45+/-0.08 for BRET(2) and 2.00+/-0.04 for BRET(1). The BRET(2) assay for thrombin was 2.9 times more sensitive compared with the BRET(1) version. Calculated detection limits (blank signal+3sigma(b), where sigma(b)=standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET(1) and 15 pM (0.0005 U) thrombin with BRET(2). The results presented here suggest that BRET(2) is a more suitable system than BRET(1) for studying protein-protein interactions and as a potential sensor for monitoring protease activity.
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PMID:Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage. 1902 7

Recent reports implicate poly(ADP-ribose) polymerase-1 (PARP-1) in the activation of nuclear factor kappaB (NF-kappaB). We investigated the role of PARP-1 in the NF-kappaB signalling cascade induced by ionizing radiation (IR). AG14361, a potent PARP-1 inhibitor, was used in two breast cancer cell lines expressing different levels of constitutively activated NF-kappaB, as well as mouse embryonic fibroblasts (MEFs) proficient or deficient for PARP-1 or NF-kappaB p65. In the breast cancer cell lines, AG14361 had no effect on IR-induced degradation of IkappaBalpha or nuclear translocation of p50 or p65. However, AG14361 inhibited IR-induced NF-kappaB-dependent transcription of a luciferase reporter gene. Similarly, in PARP-1(-/-) MEFs, IR-induced nuclear translocation of p50 and p65 was normal, but kappaB binding and transcriptional activation did not occur. AG14361 sensitized both breast cancer cell lines to IR-induced cell killing, inhibited IR-induced XIAP expression and increased caspase-3 activity. However, AG14361 failed to increase IR-induced caspase activity when p65 was knocked down by siRNA. Consistent with this, AG14361 sensitized p65(+/+) but not p65(-/-) MEFs to IR. We conclude that PARP-1 activity is essential in the upstream regulation of IR-induced NF-kappaB activation. These data indicate that potentiation of IR-induced cytotoxicity by AG14361 is mediated solely by inhibition of NF-kappaB activation.
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PMID:Ionizing radiation-induced NF-kappaB activation requires PARP-1 function to confer radioresistance. 1906 Sep 26

NF-kappaB activation is exaggerated in neonatal organisms after oxidant and inflammatory insults, but the reason for this and the downstream effects are unclear. We hypothesized that specific phosphorylation patterns of IkappaBalpha could account for differences in NF-kappaB activation in hyperoxia-exposed fetal and adult lung fibroblasts. After exposure to hyperoxia (>95% O(2)), nuclear NF-kappaB binding increased in fetal, but not adult, lung fibroblasts. Unique to fetal cells, phosphorylation of IkappaBalpha on tyrosine 42, rather than serine 32/36 as seen in TNF-alpha-exposed cells, preceded NF-kappaB nuclear translocation. In fetal cells stably transfected with an NF-kappaB-driven luciferase reporter, hyperoxia significantly suppressed reporter activity, in contrast to increased reporter activity after TNF-alpha incubation. Targeted gene profiling analysis showed that hyperoxia resulted in decreased expression of multiple genes, including proapoptotic factors. Transfection with a dominant-negative IkappaBalpha (Y42F), which cannot be phosphorylated on tyrosine 42, resulted in upregulation of multiple proapoptotic genes. In support of this finding, caspase-3 activity and DNA laddering were specifically increased in fetal lung fibroblasts expressing Y42F after exposure to hyperoxia. These data demonstrate a unique pathway of NF-kappaB activation in fetal lung fibroblasts after exposure to hyperoxia, whereby these cells are protected against apoptosis. Activation of this pathway in fetal cells may prevent the normal pattern of fibroblast apoptosis necessary for normal lung development, resulting in aberrant lung morphology in vivo.
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PMID:Hyperoxia-induced NF-kappaB activation occurs via a maturationally sensitive atypical pathway. 1907 56

We have identified TNFSF10 (TRAIL) as a p53-transcriptional target gene. There are two p53 DNA-binding sites in the human TNFSF10 promoter region, at 346 and 625 bp upstream of the transcription start site. A human p53-expressing adenovirus (Ad-p53) induced TRAIL mRNA and protein expression in HCT116 p53-/- human colon cancer cells. A human TRAIL-promoter reporter assay showed increased luciferase activity with the promoter vector that contains two p53 DNA-binding motifs,following Ad-p53 infection, compared to the control adenovirus infection. Using HCT116 cells, gene silencing of TNFSF10 by siRNA suppressed caspase 3 and 7 activity, even after treatment with the DNA-damaging chemotherapeutic agent adriamycin. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells. In vivo, TRAIL expression was induced in mouse natural killer cells at 24 hours after systemic treatment with 5-Fluorouracil. p53-dependent TRAIL induction in natural killer cells after chemotherapy exposure provides a link between the tumor suppressor p53 and the host immune response during cancer therapy as well as a paracrine-mediated cell-extrinsic death response. Our findings provide new mechanistic insights into the signaling of p53-dependent cell death and tumor suppression, including the involvement of the host immune system and natural killer cells in vivo in the anti-tumor efficacy of chemotherapy.
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PMID:TNFSF10 (TRAIL), a p53 target gene that mediates p53-dependent cell death. 1910 33

Spinocerebellar ataxia type 8 (SCA8) involves bidirectional expression of CUG (ATXN8OS) and CAG (ATXN8) expansion transcripts. The pathogenesis of SCA8 is complex and the spectrum of clinical presentations is broad. In the present study, we assessed the SCA8 repeat size ranges in Taiwanese Parkinson's disease, Alzheimer's disease and atypical parkinsonism and investigated the genetic variation modulating ATXN8 expression. Thirteen large SCA8 alleles and a novel ATXN8 -62 G/A promoter SNP were found. There is a significant difference in the proportion of the individuals carrying SCA8 larger alleles in atypical parkinsonism (P = 0.044) as compared to that in the control subjects. In lymphoblastoid cells carrying SCA8 large alleles, treatment of MG-132 or staurosporine significantly increases the cell death or caspase 3 activity. Although expressed at low steady-state, ATXN8 expression level is significantly higher (P = 0.012) in cells with SCA8 large alleles than that of the control cells. The ATXN8 transcriptional activity was significantly higher in the luciferase reporter construct containing the -62G allele than that containing the -62A allele in both neuroblastoma and embryonic kidney cells. Therefore, our preliminary results suggest that ATXN8 gene -62 G/A polymorphism may be functional in modulating ATXN8 expression.
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PMID:SCA8 repeat expansion: large CTA/CTG repeat alleles in neurological disorders and functional implications. 1922 59

Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reporter assays. Bioinformatic analyses indicated two genes Gadd45 beta and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45 beta mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.
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PMID:Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells. 1936 40

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.
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PMID:Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells. 1938 Oct 65

The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.
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PMID:Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549. 1940 23

In evaluating the cytotoxic effects and the mechanisms of the apoptotic and necrotic actions of trimethyltin chloride (TMT) on human hepatoma G2 (HepG2) cells, the present study focused on the involvement of antiproliferation, DNA damage, cell death, apoptosis-related proteins, and p53-dependent transcriptional activity. Twenty-four hour TMT treatments (4-64 microM) induced apoptosis and necrosis in HepG2 cells. Thirty-two micromolar and higher concentration significantly increases cell death. DNA damage was observed at 8 microM. Additionally, TMT increased the activity of cellular caspase-3 and the release of mitochondrial cytochrome c in a concentration-dependent manner. Our data demonstrated that the Bcl-2 family of proteins was involved in the apoptotic process but that p53 expression level was not affected. The results of luciferase reporter assay indicated that TMT-induced apoptosis seemed to adopt a transcription-dependent route, by activating p53 target genes such as PUMA and p21.
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PMID:Apoptotic and necrotic action mechanisms of trimethyltin in human hepatoma G2 (HepG2) cells. 1965 6


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