Papers by Yvette Habraken
Oncogene, 2003
Camptothecin (CPT) and X-ray (XR) generate doublestrand breaks (DSB) that can be processed by hom... more Camptothecin (CPT) and X-ray (XR) generate doublestrand 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-jB. Cells harbouring mutated NBS, hMRE11, BRCA1 or MLH1 were analysed. NBSand hMRE11-deficient cells present a classical kinetic of NF-jB induction after camptothecin treatment. When DSB are generated by XR, NBS-deficient cells exhibit a delayed and strongly reduced level of NF-jB induction, whereas the hMRE11 mutated cells do not induce NF-jB 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-jB 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-jB activation after both CPT and XR. These results indicate that DSB originating from XR or CPT do not induce NF-jB in a unique way. MMR participates in both cascades, whereas the hMRE11/hRAD50/NBS trimer is specifically involved in the response elicited by XR.
Journal of Experimental Medicine, 2008
U ltraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor ... more U ltraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1-4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix-binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peri tumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a benefi cial therapeutic tool for ischemic diseases.
Proceedings of the National Academy of Sciences, 1996
Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large ... more Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that o...
Journal of Biological Chemistry, 1996
DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the hum... more DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/ MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.
Journal of Biological Chemistry, 1995
Saccharomyces cerevisiae Rad2 protein functions in the incision step of the nucleotide excision r... more Saccharomyces cerevisiae Rad2 protein functions in the incision step of the nucleotide excision repair of DNA damaged by ultraviolet light. Rad2 was previously shown to act endonucleolytically on circular singlestranded M13 DNA and also to have a 5 3 3 exonuclease activity (
PLoS ONE, 2011
The innate immune response constitutes the first line of host defence that limits viral spread an... more The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-kB, is a key regulator of IFN-b expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-b and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-b and ISG15.
Journal of Biological Chemistry, 1998
Nucleic Acids Research, 2009
Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the r... more Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly(dA) and oligo(dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo(dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo(dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which ...
Nature Communications
Prolonged cell survival occurs through the expression of specific protein isoforms generated by a... more Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as...
Carcinogenesis, 1991
3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the... more 3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.
Cancer Research, Mar 1, 1991
Treatment of rat hepatoma cells (H4 cells) with various DNA-damaging agents increases the number ... more Treatment of rat hepatoma cells (H4 cells) with various DNA-damaging agents increases the number of O6-methylguanine-DNA-methyltransferase (transferase) molecules per cell. Because the cellular resistance to chloroethylnitrosoureas depends on the number of transferase molecules, we studied the influence of pretreatment with gamma-irradiation, cis-dichlorodiammineplatinum(II), or 2-methyl-9-hydroxyellipticinium on the sensitivity of H4 cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The BCNU resistance depends on the gamma-ray dose and increases with time after irradiation: it is maximum when the drug is added 48 h after irradiation, which corresponds to the maximum enhancement of the transferase activity in the cells. Pretreatment with a single dose of cis-dichlorodiammineplatinum(II) or 2-methyl-9-hydroxyellipticinium also increases the cellular resistance to BCNU. This resistance is not due to a modification of the alkylation of the cellular DNA in the pretreated cells but is related to the increased transferase activity, as it is no longer observed when this activity is depleted by incubating the pretreated cells with the free base O6-methylguanine before BCNU treatment. These results suggest that tumor cells surviving after gamma-irradiation or drug treatment may become resistant to chemotherapy with chloroethylnitrosoureas.
Apoptosis, 2004
Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alo... 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 (H 2 O 2) generated by ascorbatedriven 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 H 2 O 2 , when administered as a bolus, strongly enhances a constitutive basal NF-κB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity.
Carcinogenesis, 1990
HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and ... more HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol. wt corresponding to ethanoguanine by mass spectrometry; NMR spectrometry also supports this structural assignment. The UV and fluorescence spectra are very similar to those of N2,3-ethenoguanine, providing evidence that the ethano bridge is attached between N2 and 3 positions. Proof that the derivative is N2,3-ethanoguanine comes from the fact that it can be converted to N2,3-ethenoguanine by dehydrogenation on a palladium catalyst. The discovery of this new derivative raises to four the number of tricylic derivatives that have been isolated from DNA treated with 2-haloethylnitrosoureas. The new adduct, N2,3-ethanoguanine, is closely related to an etheno adduct formed by chloroacetaldehyde, a metabolite of the human carcinogen vinyl chloride, and may have relevance to either the therapeutic or carcinogenic actions of the 2-haloethylnitrosoureas.
The Journal of Biological Chemistry, May 24, 2007
NF-B is a crucial transcription factor tightly regulated by protein interactions and post-transla... more NF-B is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) ␣-elicited NF-B activation and delays IB␣ cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-B activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-B signaling. This extension is similarly correlated with delayed IB␣ cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNF␣, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of ib␣ mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-B residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNF␣, the presence of TSA provokes several impairments on the ib␣ promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys 14 and-Ser 10 , respectively; (iii) decreased presence of phosphorylated p65-Ser 536 ; and (iv) reduction of IKK␣ binding. The recruitment of these proteins on the icam-1 promoter, another NF-B-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-B pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-B activation by molecular mechanisms specific of the stimulus and the promoter. * This work was supported in part by the Interuniversity Attraction Poles (IAP)5/12 and IAP6/18 programs (Brussels, Belgium) and the Fonds National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Ph.D. student on the IAP5/12 program. 2 Supported by the Télé vie program (FNRS, Brussels, Belgium). 3 Teacher assistant at the University of Liè ge. 4 Postdoctoral researcher from the FNRS.
Oncogene, Nov 11, 2004
Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell ... 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-jB) in HMEC-1 cells. NF-jB is active since it binds to the NF-jB sites of both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) promoters and induces the transcription of several NF-jB 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.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015
Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly... more Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly characterized functions. Here, we established its intracellular localization and shuttling during cell cycle progression and in response to cellular stress. In normal conditions, MAGE-D2 is present in the cytoplasm, nucleoplasm, and nucleoli. Within the latter, MAGE-D2 is mostly found in the granular and the dense fibrillar components, and it interacts with nucleolin. Transfection of MAGE-D2 deletion mutants demonstrated that Δ203-254 leads to confinement of MAGE-D2 to the cytoplasm, while Δ248-254 prevents its accumulation in nucleoli but still allows its presence in the nucleoplasm. Consequently, this short sequence belongs to a nucleolar localization signal. MAGE-D2 deletion does not alter the nucleolar organization or rRNA levels. However, its intracellular localization varies with the cell cycle in a different kinetic than nucleolin. After genotoxic and nucleolar stresses, MAGE-D2 is excluded from nucleoli and concentrates in the nucleoplasm. We demonstrated that its camptothecin-related delocalization results from two distinct events: a rapid nucleolar release and a slower phospho-ERK-dependent cytoplasm to nucleoplasm translocation, which results from an increased flux from the cytoplasm to nucleoplasm. In conclusion, MAGE-D2 is a dynamic protein whose shuttling properties could suggest a role in cell cycle regulation.
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Papers by Yvette Habraken