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See detailSodium nitroprusside-induced osteoblast apoptosis is mediated by long chain ceramide and is decreased by raloxifene.
Olivier, Sabine ULg; Fillet, Marianne ULg; Malaise, Michel ULg et al

in Biochemical Pharmacology (2005), 69(6), 891-901

Release of high levels of nitric oxide (NO) is associated with osteoblastic cell death. The mechanisms of NO-induced cytotoxicity are not well documented and it is presently not known if estrogenic ... [more ▼]

Release of high levels of nitric oxide (NO) is associated with osteoblastic cell death. The mechanisms of NO-induced cytotoxicity are not well documented and it is presently not known if estrogenic compounds prevent this effect. We studied the role of ceramides in cell death induced by the NO donor sodium nitroprusside (SNP) and we tested the possibility that 17beta-estradiol, the anti-estrogen ICI 182.780 and two selective estrogen receptor modulators raloxifene and tamoxifen modify osteoblastic cell apoptosis. SNP dose-dependently decreased MC3T3-E1 osteoblast viability, increased NO production in the culture media and enhanced the release of intracellular ceramides C22 and C24. Cell death induced by SNP was partially inhibited when MC3T3-E1 cells were pretreated with raloxifene and tamoxifen but was not modified when the cells were pretreated with 17beta-estradiol or ICI 182.780. Cell death induced by SNP resulted from apoptosis as demonstrated by Annexin-V and propidium iodide labeling and a reduction of SNP-induced MC3T3-E1 apoptosis was confirmed in the presence of raloxifene and tamoxifen. SNP induction of C22 and C24 production was inhibited by a pretreatment with raloxifene but not with 17beta-estradiol. Moreover, the synthetic ceramide C24 (0.75 and 1microM) decreased MC3T3-E1 cell viability and osteoblast cell death induced by C24 was partially decreased by raloxifene and to a lesser extent by 17beta-estradiol. These data demonstrate that SNP-induced cell death is mediated by the long chain ceramides C22 and C24 and that raloxifene protected osteoblast from apoptosis induced by SNP, an effect that might be relevant to its pharmacological properties on bone remodeling. [less ▲]

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See detailGSK3-Mediated BCL-3 phosphorylation modulates its degradation and its oncogenicity
Viatour, Patrick ULg; Dejardin, Emmanuel ULg; Warnier, Michael et al

in Molecular Cell (2004), 16(1), 35-45

The oncoprotein BCL-3 is a nuclear transcription factor that activates NF-kappaB target genes through formation of heterocomplexes with p50 or p52. BCL-3 is phosphorylated in vivo, but specific BCL-3 ... [more ▼]

The oncoprotein BCL-3 is a nuclear transcription factor that activates NF-kappaB target genes through formation of heterocomplexes with p50 or p52. BCL-3 is phosphorylated in vivo, but specific BCL-3 kinases have not been identified so far. In this report, we show that BCL-3 is a substrate for the protein kinase GSK3 and that GSK3-mediated BCL-3 phosphorylation, which is inhibited by Akt activation, targets its degradation through the proteasome pathway. This phosphorylation modulates its association with HDAC1, -3, and -6 and attenuates its oncogenicity by selectively controlling the expression of a subset of newly identified target genes such as SLPI and CxcI1. Our results therefore suggest that constitutive BCL-3 phosphorylation by GSK3 regulates BCL-3 turnover and transcriptional activity. [less ▲]

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See detailDiscovery of new rheumatoid arthritis biomarkers using SELDI-TOF-MS ProteinChip approach
de Seny, D. M.; Fillet, Marianne ULg; Meuwis, Marie-Alice ULg et al

in Arthritis and Rheumatism (2004, September), 50(9, Suppl. S), 124

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See detailPotentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha (vol 23, pg 6200, 2003)
Adam, Emmanuelle; Quivy, Vincent; Bex, Françoise et al

in Molecular and Cellular Biology (2004), 24(15), 6890

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See detailAscorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death
Verrax, Julien; Cadrobbi, Julie; Marques, Carole et al

in Apoptosis : An International Journal on Programmed Cell Death (2004), 9(2), 223-233

Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most ... [more ▼]

Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity. [less ▲]

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See detailEnhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b(+) myelopoiesis
De Klerck, Bert; Carpentier, Isabelle; Lories, Rick J et al

in Arthritis Research & therapy (2004), 6(3), 220-231

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b(+) myeloblasts. Both disease manifestations are more pronounced in interferon-gamma ... [more ▼]

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b(+) myeloblasts. Both disease manifestations are more pronounced in interferon-gamma receptor knock-out (IFN-gammaR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b(+) splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-gammaR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-kappaB ligand (RANKL) or with tumour necrosis factor-alpha (TNF-alpha). Significantly larger numbers of such cells could be generated from splenocytes of IFN-gammaR KO mice than from those of wildtype mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-alpha-induced osteoclast formation. Splenocyte cultures of IFN-gammaR KO mice also produced more TNF-alpha and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-gammaR KO mice contained comparatively low levels of pro-interleukin-1beta (pro-IL-1beta) and pro-caspase-1, indicating more extensive conversion of pro-IL-1beta into secreted active IL-1beta. These observations provide evidence that all conditions are fulfilled for the expanding CD11b(+) splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-gammaR KO mice to CIA. [less ▲]

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See detailExpression of 25 kDa thiamine triphosphatase in rodent tissues using quantitative PCR and characterization of its mRNA
Lakaye, Bernard ULg; Verlaet, Myriam ULg; Dubail, Johanne ULg et al

in International Journal of Biochemistry & Cell Biology (2004), 36(10), 2032-2041

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a ... [more ▼]

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a specific 25 kDa thiamine triphosphatase (ThTPase). The aim of the present study was to use quantitative PCR, for comparing the 25 kDa ThTPase mRNA expression in various mouse tissues with its enzyme activities. ThTPase mRNA was expressed at only a few copies per cell. The highest amount of mRNA was found in testis, followed by lung and muscle, while the highest enzyme activities were found in liver and kidney. The poor correlation between mRNA levels and enzyme activities might result either from tissue-specific post-transcriptional regulation of mRNA processing and/or translation or from the regulation of enzyme activities by post-translational mechanisms. Purified recombinant human ThTPase was phosphorylated by casein kinase 11, but this phosphorylation did not modify the enzyme activity. However, the characterization of the 3'-untranslated mRNA region revealed a unique, highly conserved, 200-nucleotide sequence that might be involved in translational control. In situ hybridization studies in testis suggest a predominant localization of ThTPase mRNA in poorly differentiated spermatogenic cells. This is the first study demonstrating a cell-specific 25 kDa ThTPase mRNA expression, suggesting that this enzyme might be related to the degree of differentiation or the metabolic state of the cell. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailDownregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy
Volanti, Cédric ULg; Gloire, Geoffrey ULg; Vanderplasschen, Alain ULg et al

in Oncogene (2004), 23(53), 8649-8658

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with ... [more ▼]

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with visible light. In the presence of oxygen, irradiation leads to the production of reactive oxygen species (ROS). A large production of ROS induces the death of cancer cells by apoptosis or necrosis. A small ROS production can activate various cellular pathways. Here, we show that PDT by pyropheophorbide-a methyl ester (PPME) induces the activation of nuclear factor kappa B (NF-kappaB) in HMEC-1 cells. NF-kappaB is active since it binds to the NF-kappaB sites of both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) promoters and induces the transcription of several NF-kappaB target genes such as those of IL-6, ICAM-1, VCAM-1. In contrast, expression of ICAM-1 and VCAM-1 at the protein level was not observed, although we measured an IL-6 secretion. Using specific chemical inhibitors, we showed that the lack of ICAM-1 and VCAM-1 expression is the consequence of their degradation by lysosomal proteases. The proteasome and calpain pathways were not involved. All these observations were consistent with the fact that no adhesion of granulocytes was observed in these conditions. [less ▲]

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See detailCytoplasmic I kappa B alpha increases NF-kappa B-independent transcription through binding to histone deacetylase (HDAC) 1 and HDAC3
Viatour, Patrick ULg; Legrand-Poels, Sylvie; van Lint, Carine et al

in Journal of Biological Chemistry (2003), 278(47), 46541-46548

IkappaBalpha is an inhibitory molecule that sequesters NF-kappaB dimers in the cytoplasm of unstimulated cells. Upon stimulation, NF-kappaB moves to the nucleus and induces the expression of a variety of ... [more ▼]

IkappaBalpha is an inhibitory molecule that sequesters NF-kappaB dimers in the cytoplasm of unstimulated cells. Upon stimulation, NF-kappaB moves to the nucleus and induces the expression of a variety of genes including IkappaBalpha. This newly synthesized IkappaBalpha also translocates to the nucleus, removes activated NF-kappaB from its target genes, and brings it back to the cytoplasm to terminate the phase of NF-kappaB activation. We show here that IkappaBalpha enhances the transactivation potential of several homeodomain-containing proteins such as HOXB7 and Pit-1 through a NF-kappaB-independent association with histone deacetylase (HDAC) 1 and HDAC3 but not with HDAC2, -4, -5, and -6. IkappaBalpha bound both HDAC proteins through its ankyrin repeats, and this interaction was disrupted by p65. Immunofluorescence experiments demonstrated further that IkappaBalpha acts by partially redirecting HDAC3 to the cytoplasm. At the same time, an IkappaBalpha mutant, which lacked a functional nuclear localization sequence, interacted very efficiently with HDAC1 and -3 and intensively enhanced the transactivation potential of Pit-1. Our results support the hypothesis that the NF-kappaB inhibitor IkappaBalpha regulates the transcriptional activity of homeodomain-containing proteins positively through cytoplasmic sequestration of HDAC1 and HDAC3, a mechanism that would assign a new and unexpected role to IkappaBalpha. [less ▲]

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See detailCell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers
Piette, Jacques ULg; Volanti, Cédric ULg; Vantieghem, Annelies et al

in Biochemical Pharmacology (2003), 66(8), 1651-1659

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the ... [more ▼]

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting ("high dose PDT"). When one of these components is limiting ("low dose PDT"), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents. i.e. pyropheophorbide and hypericin. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

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See detailDifferential involvement of the hMRE11/hRAD50/NBS1 complex, BRCA1 and MLH1 in NF-kappa B activation by camptothecin and X-ray
Habraken, Yvette ULg; Jolois, Olivier ULg; Piette, Jacques ULg

in Oncogene (2003), 22(38), 6090-6099

Camptothecin (CPT) and X-ray (XR) generate double-strand breaks (DSB) that can be processed by homologous or nonhomologous recombination. We studied the participation of proteins involved in recombination ... [more ▼]

Camptothecin (CPT) and X-ray (XR) generate double-strand breaks (DSB) that can be processed by homologous or nonhomologous recombination. We studied the participation of proteins involved in recombination pathways and cell cycle control in the signal transduction between DNA damage and NF-kappaB. Cells harbouring mutated NBS, hMRE11, BRCA1 or MLH1 were analysed. NBS- and hMRE11-deficient cells present a classical kinetic of NF-kappaB induction after camptothecin treatment. When DSB are generated by XR, NBS-deficient cells exhibit a delayed and strongly reduced level of NF-kappaB induction, whereas the hMRE11 mutated cells do not induce NF-kappaB at all. This indicates an important role of the hMRE11/hRAD50/NBS complex in the signal transduction initiated by XR. In HCC1937 cells that express a truncated version of BRCA1, XR induces a very rapid and transient NF-kappaB activation, whereas CPT leads to a delayed activation suggesting that BRCA1 modulates the transduction pathways in different manners after these two stresses. Finally, we found that a proficient MMR pathway is essential to the NF-kappaB activation after both CPT and XR. These results indicate that DSB originating from XR or CPT do not induce NF-kappaB in a unique way. MMR participates in both cascades, whereas the hMRE11/hRAD50/NBS trimer is specifically involved in the response elicited by XR. [less ▲]

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See detailPotentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha
Adam, Emmanuelle; Quivy, Vincent; Bex, Françoise et al

in Molecular and Cellular Biology (2003), 23(17), 6200-6209

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappaB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium ... [more ▼]

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappaB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-kappaB-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact kappaB sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-kappaB-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-kappaKB in the nucleus. We showed that the p65 subunit of NF-kappaB was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-kappaB after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the IkappaBalpha inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-kappaB. This delay was due neither to a defect in IkappaBalpha mRNA production nor to a nuclear retention of IkappaBalpha but was rather due to a persistent proteasome-mediated degradation of IkappaBalpha. A prolongation of IkappaB kinase activity could explain, at least partially, the delayed IkappaBalpha cytoplasmic reappearance observed in presence of TNF plus TSA. [less ▲]

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See detailRaloxifene protects Osteoblasts from apoptosis induced by sodium nitroprusside: Potential involvement of ceramide
Olivier, Sabine ULg; Fillet, Marianne ULg; Malaise, Michel ULg et al

in Journal of Bone and Mineral Research (2003, September), 18(Suppl. 2), 136

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See detailChanges in function of iron-loaded alveolar macrophages after in vivo administration of desferrioxamine and/or chloroquine.
Legssyer, Rachida; Josse, Claire ULg; Piette, Jacques ULg et al

in Journal of Inorganic Biochemistry (2003), 94(1-2), 36-42

Both desferrioxamine (DFO) and chloroquine can significantly reduce hepatic iron in experimental animals with iron overload by chelating iron from the low-molecular-weight pool or decreasing iron uptake ... [more ▼]

Both desferrioxamine (DFO) and chloroquine can significantly reduce hepatic iron in experimental animals with iron overload by chelating iron from the low-molecular-weight pool or decreasing iron uptake by the transferrin-transferrin receptor cycle, respectively. However, no previous studies have investigated whether combination therapy of these two drugs would further decrease the tissue iron overload as well as iron-induced toxicity. Chloroquine administration, 15 mg/kg, 5x/week, to rats during the iron loading regime, 10 mg/kg, 3x/week for 4 weeks, significantly decreased both hepatic (54%) and macrophage iron content (24%). However when administered in combination with desferrioxamine, 10 mg/kg, 3x/week for 2 weeks at the cessation of iron loading, no further reduction of hepatic iron content was noted while the iron content of the macrophages significantly increased, possibly indicating the flux of ferrioxamine through these cells. Further studies are warranted to investigate the speciation of iron within these macrophages. Macrophages isolated from chloroquine-treated iron loaded rats showed a reduction in latent NFkappaB activation and a significant increase in lipopolysaccharide-stimulated nitrite release by comparison to these parameters in iron loaded macrophages. Co-administration of chloroquine and desferrioxamine normalised the latent activity of NFkappaB to that of control macrophages as well as increasing LPS-stimulated NO release towards control values. However, DFO alone did not have any significant effect upon either of these parameters. Such results may have important relevance for the reduced immune function of iron loaded macrophages isolated from thalassaemia patients receiving chelation therapy and their propensity to increased infection. [less ▲]

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See detailMechanisms involved in exogenous C2- and C6-ceramide-induced cancer cell toxicity.
Fillet, Marianne ULg; Bentires-Alj, Mohamed; Deregowski, Valérie et al

in Biochemical Pharmacology (2003), 65(10), 1633-42

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the ... [more ▼]

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the apoptosis of HCT116 and OVCAR-3 cancer cells, exogenous C2-, C6-, and C16-ceramides were used to mimic the endogenous lipid increase that follows a large variety of stresses. C2- and C6-ceramides (cell-permeable ceramide analogs), but not C16-ceramide, induced nuclear factor-kappaB (NF-kappaB) DNA-binding, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and mitochondrial cytochrome c release, indicating that apoptosis occurs through the caspase cascade and the mitochondrial pathway. No difference in survival was observed between control cells and cells expressing mutated IkappaBalpha and treated with the permeable ceramides. This suggests that, at least in these cell lines, stable NF-kappaB inhibition did not modify the ceramide-induced cytotoxicity pathway. C6-ceramide also induced a double block in G1 and G2, thus emptying the S phase. [less ▲]

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See detailSynergistic activation of human immunodeficiency virus type 1 promoter activity by NF-kappa B and inhibitors of deacetylases: Potential perspectives for the development of therapeutic strategies
Quivy, Vincent; Adam, Emmanuelle; Collette, Yves et al

in Journal of Virology (2002), 76(21), 11091-11103

The transcription factor NF-kappaB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment ... [more ▼]

The transcription factor NF-kappaB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-kappaB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number Of kappaB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IkappaBalpha. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-kappaB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression. [less ▲]

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See detailPhosphorylation of varicella-zoster virus IE63 protein by casein kinases influences its cellular localization and gene regulation activity
Bontems, Sébastien ULg; Di Valentin, Emmanuel ULg; Baudoux, Laurence et al

in Journal of Biological Chemistry (2002), 277(23), 21050-21060

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined ... [more ▼]

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level. [less ▲]

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See detailActivation of the NF-kappaB transcription factor by Camptothecin
Habraken, Yvette ULg; Piette, Jacques ULg

Poster (2002, January 30)

We used Camptothecin (CPT) a DNA topoisomerase I inhibitor to introduce double-strand breaks (DSB) in DNA. CPT and derivatives presently used in chemotherapy are known to have maximal cytotoxicity for ... [more ▼]

We used Camptothecin (CPT) a DNA topoisomerase I inhibitor to introduce double-strand breaks (DSB) in DNA. CPT and derivatives presently used in chemotherapy are known to have maximal cytotoxicity for cells in S phase. We have shown that the activation of NF-κB and its transcriptional activity are enhanced in S phase HeLa cells. The CPT-induced activation of NF- κB is slow but stable. However, the composition of the complex evolves with time from mostly p50/p65 after 2 h to almost exclusively p52 after 24 h. The signal transduction progresses through the activation of the IKK complex, the phosphorylation of IκBα on S32 and S36 and the degradation of the inhibitor by the 26S proteasome. The transient expression of a kinase inactive mutant of NIK abolishes the activation of NF-κB by CPT indicating that this kinase could play a role upstream of the IKK complex whereas a dominant negative form of MEKK1 has no effect. To better understand the early steps of the signaling cascade initiated by the DSB, we compare the induction of NF-κB in both NBS-/- and NBS+/+ fibroblasts. NBS turn out to be implicated in NF-κB activation by X-rays, it does not seems to be important for activation by CPT supporting the idea that the components of the signaling cascade are not identical. [less ▲]

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