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Nuc1p, CPS-6, EndoG and EXOG are evolutionary conserved mitochondrial nucleases from yeast, Caenorhabditis elegans and humans, respectively. These enzymes play an important role in programmed cell death as well as mitochondrial DNA-repair and recombination. Whereas a significant interest has been given to the cell biology of these proteins, in particular their recruitment during caspase-independent apoptosis, determination of their biochemical properties has lagged behind. In part, biochemical as well as structural analysis of mitochondrial nucleases has been hampered by the fact that upon cloning and overexpression in Escherichia coli these enzymes can exert considerable toxicity and tend to aggregate and form inclusion bodies. We have, therefore, established a uniform E. coli expression system allowing us to obtain these four evolutionary related nucleases in active form from the soluble as well as insoluble fractions of E. coli cell lysates. Using preparations of recombinant Nuc1p, CPS-6, EndoG and EXOG we have compared biochemical properties and the substrate specificities of these related nucleases on selected substrates in parallel. Whereas Nuc1p and EXOG in addition to their endonuclease activity exert 5'-3'- exonuclease activity, CPS-6 and EndoG predominantly are endonucleases. These findings allow speculating that the mechanisms of action of these related nucleases in cell death as well as DNA-repair and recombination differ according to their enzyme activities and substrate specificities. © 2010 Elsevier Inc. All rights reserved.

The objective of the present study was to develop a cellular phenotype assay for nucleotide excision repair (NER), using benzo[a]pyrene diol epoxide (BPDE) as model mutagen. Since in vitro exposure to BPDE may lead to DNA strand breaks resulting from both direct interaction with DNA and incisions introduced by the repair enzymes, we aimed to discriminate between both types of breaks using the comet assay and quantified the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells (PBMCs) with BPDE in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC). The assay was performed with a low (0.5 microM) and a high (2.5 microM) BPDE concentration. The individual NER capacity was defined as the amount of DNA damage induced by BPDE in presence of APC, diminished with the damage induced by BPDE and APC alone. First, the assay was applied to a NER-deficient human fibroblast cell line (XPA-/-) to validate the methodology. Lower repair capacity and a higher amount of BPDE-induced DNA adducts were observed for the XPA-/- fibroblasts as compared to the wild-type fibroblasts. Repeated experiments on PBMCs from four donors showed low intra-individual, intra-experimental and inter-assay variation for both concentrations, indicating the reliability of the method. To assess the inter-individual variation, the assay was applied to PBMCs from 22 donors, comparing the repair capacity after exposure to 0.5 microM (N = 10) and 2.5 microM (N = 12) BPDE. The repair capacity showed a higher inter-individual variation as compared to the intra-individual variation. Moreover, this difference was more pronounced using the low concentration. All these results indicate the adequacy of the method using this low concentration. Further improvement, however, should be recommended by applying the study with low BPDE concentration in a larger population and taking into account the relevant genotypes for NER.

APE1/Ref-1 (also called HAP1 or APEX, and here referred to as APE1), the mammalian ortholog of Escherichia coli Xth (exonuclease III), is a vital protein (20) that acts as a master regulator of cellular response to oxidative stress conditions and contributes to the maintenance of genome stability (55, 56). APE1 is involved in both the base excision repair (BER) pathways of DNA lesions, acting as the major apurinic/apyrimidinic (AP) endonuclease, and in transcriptional regulation of gene expression as a redox coactivator of different transcription factors, such as early growth response protein 1 (Egr-1), NF-κB, and p53 (55, 56). These two biological activities are located in two functionally distinct protein domains. In fact, the N-terminal region, containing the nuclear localization signal (NLS) sequence, is principally devoted to redox activity through Cys65, while the C-terminal one exerts enzymatic activity on the abasic sites of DNA (56, 63). The protein C terminus is highly conserved during phylogenesis, while the N terminus is not. Except in mammals, which always show a high sequence conservation (more than 90%), and Danio, Drosophila, Xenopus, and Dictyostelium (presenting a sequence identity of less than 40%), the N-terminal region is mostly absent in other organisms. A third APE1 function, which is regulated by Lys6/Lys7 acetylation (7), is indirect binding to the negative calcium response elements (nCaRE) of some promoters (i.e., PTH and APE1 promoters), thus acting as a transcriptional repressor (12, 30). In different mammalian cell types, the APE1 subcellular distribution is mainly nuclear and is critical for controlling cellular proliferative rate (20, 25, 28). However, cytoplasmic, mitochondrial, and endoplasmic reticulum localization has also been reported (11, 22, 33, 50, 54). Interestingly, cytoplasmic expression of APE1 has been correlated with aggressiveness of different tumors (55, 56), although its role in tumorigenic processes is completely unknown. To date, no subnuclear distribution of APE1 has been reported. APE1 is an abundant protein (about 104 to 105 copies/cell) within eukaryotic cells and has a relatively long half-life (about 8 h). Thus, fine-tuning of the multiple APE1 functions may reside on its posttranslational modifications and on the modulation of its interactome under different conditions. While some posttranslational modifications have a functional role (i.e., Lys6/Lys7 acetylation) (7, 17), little information is available on APE1 protein interacting partners, except for those that are involved in BER function (38). Interestingly, proteolysis at residue Lys31 has recently been related to an enhanced immune cell death mediated by granzymes A and K (16, 23). This proteolytic event reduces APE1 accumulation within nuclei (16, 29) and its interaction with XRCC1 (60) and alters APE1 functions (16, 23). Recently, proteolysis occurring at Asn33 (giving rise to a protein form called NΔ33APE1) has also been described (11), suggesting that removal of the NLS may constitute a general mechanism for redirecting APE1 toward noncanonical subcellular compartments, such as mitochondria (11, 33, 54). Unfortunately, neither has the specific protease responsible for this cleavage been identified in nonimmune cells, nor has the mitochondrial localization signal been mapped yet. Mitochondrial localization of APE1 could be associated with a potential role in mtDNA repair of oxidized bases (11, 33, 54). However, since it is not clear whether NΔ33APE1 maintains its DNA repair activity in vivo (16) or acquires an aspecific endonuclease activity for double-stranded DNA (dsDNA) (66), at present it is not possible to drive any definitive conclusion. Moreover, as the truncated NΔ33APE1 form is associated with an apoptotic phenotype (23), it cannot be excluded that its generation may causatively be involved in the cytotoxic effect, driving proapoptotic triggering directly within mitochondria. The first 42 amino acids of APE1 are highly unordered in the protein crystallographic structure (3, 35), while the remainder of the protein has a globular fold (21). It is therefore presumable that the protein's N terminus is used for interacting with other partners, thus modulating the different APE1 functions. Interestingly, a similar bipartite arrangement for Rrp1, the Drosophila homologue of mammalian APE1, has been described, pointing out a functional role of the unstructured N-terminal domain in modulating protein-protein interactions (42, 52). By using an unbiased proteomic approach, in this work we have identified and characterized a novel APE1 complex. We found that APE1 N terminus is essential for binding to a number of proteins involved in ribosome biogenesis and RNA processing. Among the interacting partners, we focused on the nucleophosmin (NPM1)-APE1 interaction. NPM1 is an abundant protein which specifically resides within the granular region of the nucleolus and has been implicated in a variety of cellular processes, including centrosome duplication, maintenance of the genome's integrity, and ribosome biogenesis (19). NPM1 has a chaperone activity regulated by phosphorylation (51) and an endoribonuclease activity at a specific site of the spacer region between the 5.8S and the 28S rRNAs (43). Here, we demonstrate that the NPM1-APE1 interaction is required for APE1 subnuclear localization and for modulating the cleansing process of rRNA. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus possibly affecting gene expression through posttranscriptional mechanisms.

Poly(ADP-ribosyl)ation is a posttranslational modification of cellular proteins mostly catalysed by poly(ADP-ribose) polymerase-1 (PARP-1) and to a lesser extent by PARP-2. PARP-1 and PARP-2 use NAD+ as substrate in response to cellular exposure to various DNA-damaging agents to form the biopolymer poly(ADP-ribose) (PAR). On the one hand, poly(ADP-ribosyl)ation is a key regulator of genomic stability under conditions of genotoxic stress where PARP-1 plays a major role in DNA repair, transcription regulation and recovery of cells after DNA damage. On the other hand, massive poly(ADP-ribosyl)ation induced by severe acute DNA damage results in rapid depletion of cellular NAD+ and ATP pools, which can lead to cell death.In three sub-projects, this study aimed to explore the role of PARP-1 in DNA repair and cell death using in vitro as well as in vivo models:(i) PARP-1 is involved in a number of pathophysiological conditions such as diabetes, inflammation and stroke, consequently, pharmacological inhibitors of PARP have the potential to elicit beneficial effects in these diseases. In the first part of the present study, a new PARP inhibitor, BYK204165, was examined for inhibition of PAR-synthesis in H2O2-treated 3T3 fibroblasts from Parp-1+/+ and Parp-1-/- mice, where the 100-fold PARP-1 selectivity of the compound was confirmed by its failure to inhibit PARP-2 in both cell lines. The new compound might provide a novel and convenient functional tool toward the assessment of the contribution of PARP-1 and PARP-2 related PAR formation in intact cells, because the enzymatic activity of the two isoforms can be distinguished by use of a selective PARP-1 inhibitor.(ii) Since inhibition of PAR formation generally influences DNA repair mechanisms, the second part of the study explored the consequences of stably overexpressed human PARP-1 (hPARP 1) in Chinese hamster cells (COMF10) on the cytotoxicity induced by alkylating agents (MMS, MNNG) and X-irradiation. Measurements of apoptosis, necrosis, DNA repair and genomic stability were taken as experimental endpoints. Analysis of cell viability after treatment with MMS and MNNG revealed consistently larger fractions of necrotic cells in the COMF10 cells compared to control. Furthermore, DNA repair kinetic measurements after X irradiation of hPARP-1 overexpressing murine lymphoma EL-4 cells, demonstrated acceleration in DNA repair, whereas pharmacological inhibition of PARP by PJ34 delayed and reduced DNA repair capacity.(iii) Finally, it was intended to generate an in vivo system for tissue-specific overexpression of hPARP-1 protein in mice. Therefore, 18 transgenic founder mice were generated by DNA microinjection with a transgene comprising hPARP-1 cDNA under the control of a strong promoter. The transcription is "locked" by a Stop-sequence that can be eliminated in vivo by expression of Cre recombinase as a result of crossing with appropriate transgenic tissue-specific "Cre-deleter" mice (at first T-cell specific). Unexpectedly however, although mRNA transcripts of the hPARP-1 transgene could be found in their offspring, for largely unknown reasons its respective protein expression could not be detected. Its failure on the level of translation possibly could be due to unexpected transcriptional start sites within the transgene. Therefore, an alternative approach should be used for follow-up projects in order to obtain hPARP-1-overexpressing mice.In summary, studies within the work of this thesis contributed to the disposal of a novel and selective PARP-1 inhibitor, which provides a valuable tool to dissect different roles of PARP 1 and PARP-2 in cellular functions (Eltze et al., 2008). Moreover, it was shown that overexpression of hPARP-1 in rodent cells has two important consequences. Its overexpression leads to an increased DNA repair capacity after X-irradiation, but on the other hand also to an increased susceptibility to DNA damage in response to alkylating agents or PARP inhibition, demonstrating the dual role of PARP-1 in mechanisms of DNA repair and cell death (Eltze and Kunzmann et al., submitted). Finally, novel transgenic mice with intended tissue-specific overexpression of hPARP-1 were generated and characterized on a genetic level. Unexpectedly, despite cell culture validation of the expression construct and transgene expression on the mRNA level, no protein expression of the transgene could be detected in these mice for largely unknown reasons. This outcome needs to be considered in future approaches aiming at the generation of hPARP-1 transgenic mice.

Background Multiple sequence alignments are a fundamental tool for the comparative analysis of proteins and nucleic acids. However, large data sets are no longer manageable for visualization and investigation using the traditional stacked sequence alignment representation. Results We introduce ProfileGrids that represent a multiple sequence alignment as a matrix color-coded according to the residue frequency occurring at each column position. JProfileGrid is a Java application for computing and analyzing ProfileGrids. A dynamic interaction with the alignment information is achieved by changing the ProfileGrid color scheme, by extracting sequence subsets at selected residues of interest, and by relating alignment information to residue physical properties. Conserved family motifs can be identified by the overlay of similarity plot calculations on a ProfileGrid. Figures suitable for publication can be generated from the saved spreadsheet output of the colored matrices as well as by the export of conservation information for use in the PyMOL molecular visualization program. We demonstrate the utility of ProfileGrids on 300 bacterial homologs of the RecA family – a universally conserved protein involved in DNA recombination and repair. Careful attention was paid to curating the collected RecA sequences since ProfileGrids allow the easy identification of rare residues in an alignment. We relate the RecA alignment sequence conservation to the following three topics: the recently identified DNA binding residues, the unexplored MAW motif, and a unique Bacillus subtilis RecA homolog sequence feature. Conclusion ProfileGrids allow large protein families to be visualized more effectively than the traditional stacked sequence alignment form. This new graphical representation facilitates the determination of the sequence conservation at residue positions of interest, enables the examination of structural patterns by using residue physical properties, and permits the display of rare sequence features within the context of an entire alignment. JProfileGrid is free for non-commercial use and is available from http://www.profilegrid.org. Furthermore, we present a curated RecA protein collection that is more diverse than previous data sets; and, therefore, this RecA ProfileGrid is a rich source of information for nanoanatomy analysis.

Das für den Menschen essentielle Spurenelement Selen ist Bestandteil von Selenoproteinen. Neben antioxidativen Eigenschaften von Selenoproteinen werden eine Stimulierung von DNA-Reparaturprozessen und eine Induktion von Phase II-Enzymen des Fremdstoffmetabolismus als chemopräventive Wirkmechanismen von Selen diskutiert. Epidemiologische Studien und Untersuchungen zur Krebshäufigkeit in Korrelation zur Selenaufnahme sind jedoch teilweise widersprüchlich und deuten auf komplexe biologische Wirkmechanismen der Selenverbindungen hin, darunter auch prooxidative Wirkungen. Das Ziel dieser Arbeit war es, reduzierbares Natriumselenit und vollständig reduziertes Selenomethionin hinsichtlich möglicher prooxidativer Eigenschaften und daraus resultierender direkter und indirekter genotoxischer Effekte zu vergleichen. Zunächst konnte gezeigt werden, dass sowohl Natriumselenit als auch Selenomethionin in humanen Lungenadenokarzinomzellen (A549) bioverfügbar sind. Außerdem wurde eine ca. 100fach stärkere Zytotoxizität von Natriumselenit gegenüber Selenomethionin nachgewiesen. Natriumselenit führte in zytotoxischen Konzentrationen zu einer signifikanten Bildung von oxidiertem Glutathiondisulfid und zu einer konzentrationsabhängigen und signifikanten Induktion oxidativer DNA-Schäden. Demgegenüber wurden für vollständig reduziertes Selenomethionin keine direkten genotoxischen Effekte nachgewiesen. Die Untersuchungen zur indirekten Genotoxizität erfolgten anhand der Modulierung der Genotoxizität von (+)-anti-Benzo[α]pyrendiolepoxid (BPDE) und UVC-Strahlung. Beide Selenverbindungen führten bereits im nicht-zytotoxischen Konzentrationsbereich zu einer erhöhten Anzahl BPDE-induzierter DNA-Addukte und zu einer Hemmung der Nukleotidexzisionsreparatur (NER). Zudem konnte gezeigt werden, dass sich die Reparaturhemmung durch Natriumselenit in einer Verstärkung der BPDE-induzierten Zytotoxizität auswirkt. Untersuchungen zur enzymatischen Konjugation von BPDE an Glutathion ergaben keine Anzeichen einer verminderten enzymatischen Detoxifizierung von BPDE. Darüber hinaus wurde eine Hemmung der Reparatur UVC-induzierter Photoläsionen durch beide Verbindungen nachgewiesen. Es konnte gezeigt werden, dass die Hemmung der DNA-Reparatur im Fall von Natriumselenit wahrscheinlich aus einer Interaktion mit dem für die NER essentiellen Protein XPA (Xeroderma Pigmentosum Protein A) resultiert. Weitere Untersuchungen sind notwendig, um die Relevanz der Ergebnisse für den intakten Organismus abzuklären., The essential trace element selenium is part of selenoproteins, as glutathione peroxidases. Besides their antioxidative properties, induction of DNA repair processes and induction of phase II enzymes of the metabolism of xenobiotica are discussed. Epidemiological studies and research on cancer incidences in correlation to selenium uptake are still contradicting and suggest complex biological mechanisms. The aim of this study was to analyze inorganic, reducible sodium selenite and organic, fully reduced selenomethionine with respect to pro-oxidative properties and resulting direct and indirect genotoxicity. First of all it could be shown, that sodium selenite and selenomethionine were bioavailable in human lung carcinoma cells (A549). The cytotoxicity induced by sodium selenite was approximately 100-fold more pronounced than the cytotoxicity induced by fully reduced selenomethionine. High concentrations of sodium selenite led to a significant induction of oxidized glutathione disulfide and to an induction of oxidative DNA damage in a concentration-dependant manner. In contrast, selenomethionine showed no direct genotoxicity caused by pro-oxidative effects. Indirect genotoxicity was analysed as modulation of the genotocity induced by UVC and (+)-anti-Beno[α]pyren diolepoxide (BPDE) genotoxicity. Non-cytotoxic concentrations of both selenium compounds led to elevated levels of BPDE-induced DNA adducts and to an inhibition of nucleotide excision repair (NER). Additionally, it was shown that the inhibition of NER caused by sodium selenite resulted in an induction of BPDE-induced genotoxicity as measured by colony forming ability. No influence of either selenium compound on detoxification of BPDE, the conjugation to glutathione, was detectable. To further confirm the inhibition of NER, the repair of UVC-induced DNA photolesions was analysed. Both selenium compounds led to the inhibition of DNA repair after incubation with high, cytotoxic concentrations. Furthermore, in the case of sodium selenite it was shown that the inhibition of NER resulted most likely from an interaction with the xeroderma pigmentosum protein A, which is essential for nucleotide excision repair. More studies are necessary to clarify the physiological relevance of the results for intact organisms.

To investigate the correlation between O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and benefit from temozolomide in patients with recurrent high-grade glioma.A real-time, quantitative, methylation-specific PCR assay was performed on archival tissue blocks from patients treated with temozolomide at the first recurrence.A subgroup of 38 patients who were chemotherapy-naive at recurrence was analysed (22 glioblastoma, 12 anaplastic astrocytoma [AA] and 4 anaplastic oligoastrocytoma [AOA]); none had 1p/19q loss. Among 10 (26%) patients with a hypermethylated MGMT promoter, none experienced disease progression within the first two treatment cycles compared with 12 of 28 (43%) patients with an unmethylated promoter (p=0.016). By Cox multivariate analysis, tumour grade and MGMT promoter methylation correlated with time to progression (p0.05); MGMT promoter methylation correlated with superior overall survival in AA/AOA but not in glioblastoma.MGMT promoter methylation predicted a survival benefit in patients with 1p/19q intact AA/AOA treated with temozolomide at recurrence.

Gliomas are common malignant tumors of the central nervous system whom oligodendrogliomas and glioblastomas are the major subtypes. Among them, oligodendrogliomas are distinguished by their remarkable sensitivity to chemotherapy, responding dramatically to treatment, contrasting with resistance of glioblastomas. Anaplastic oligodendrogliomas have loss of chromosomal arms 1p and 19q. This genetic alteration is statistically significantly associated with both chemosensitivity and longer recurrence-free survival after chemotherapy. We hypothesized that there are some resistance genes on this area, responsible for chemo-resistance of glioblastomas. Among them we focused on DNA-repair genes because they correct chemotherapy-induced adducts on DNA. We screened these genes with an in vitro chemosensitivity test that we developped, and identified 4 of them as involved in resistance of glioma cells to cisplatin : ercc1, ercc2, mutyh and pnkp. These results were confirmed with the gold standard chemotherapy drug : temozolomide. Expression of these genes were measured in resistant and sensitive human tumors, and the first 3 genes are overexpressed in glioblastomas. Thus, these genes can be considered as innovant targets of a chemo-sensitizing treatment. We entered a pre-clinical trial, financed by LNCC, to test this treatment on mice. We developped sub-cutaneous and intra-cerebral human xenografts of glioma on mice and treated them with siRNA directed to target genes, and with temozolomide. This treatment increased the tumor chemo-sensitivity, reducing tumoral volume. Our preclinical results confirmed both our in vitro data and the potentiality to chemosensitize glioblastoma using siRNA., Les tumeurs primitives du système nerveux central les plus fréquentes sont les gliomes et se classent en deux grandes catégories : les astrocytomes et les oligodendrogliomes. La survie des patients porteurs d'oligodendrogliomes est de 70 % à 5 ans alors qu'elle ne dépasse pas une année en moyenne pour les glioblastomes. La divergence de pronostic observée entre ces deux types de gliomes résulte d'une différence de sensibilité aux traitements conventionnels. La chimiothérapie classique appliquée à ces tumeurs permet en effet d'obtenir une réponse pour 90 % des oligodendrogliomes contrastant avec moins de 10 % pour les glioblastomes. La chimio-sensibilité particulière des oligodendrogliomes peut être la conséquence des altérations géniques survenant lors du processus oncogène. Elle résulterait de l'absence d'expression de gènes de résistance des régions chromosomiques 1p et 19q, gènes ainsi distinctement exprimés entre les deux entités tumorales. Les gènes de réparation de l'ADN peuvent rendre compte de la résistance aux traitements, puisque responsables de la correction des adduits créés par la chimiothérapie. Nous avons recherché grâce à une évaluation fonctionnelle in vitro, quels gènes de réparation de la région 1p/19q sont impliqués dans la correction des adduits du CDDP. Quatre gènes ont ainsi été identifiés : ercc1, ercc2, mutyh et pnkp. Ces résultats ont été vérifiés avec du témozolomide, la drogue de référence dans le traitement des gliomes. L'expression des ces gènes a été mesurée dans des extraits de tumeurs de patients, résistantes (glioblastomes) et sensibles (oligodendrogliomes) à la chimiothérapie, et les 3 premiers sont effectivement surexprimés dans les glioblastomes. Ces gènes définissent ainsi les cibles d'un traitement chimio-sensibilisant. Nous avons entrepris l'étude préclinique de ce traitement basé sur l'utilisation concomitante de siRNA dirigés contre les gènes d'intérêt et de chimiothérapie. Des modèles de gliomes humains sous-cutanés et intra-cérébraux résistants à la chimiothérapie ont été utilisés chez les souris. Le traitement chimio-sensibilisant ciblant ercc1 a significativement augmenté l'effet du témozolomide sur les tumeurs, permettant ainsi de diminuer leur volume. Il pourrait faire prochainement l'objet d'essais cliniques. Nos travaux, menés de l'in silico au stade préclinique, d'une part démontrent la validité de notre hypothèse, c'est-à-dire l'implication des gènes de réparation du 1p/19q dans la chimio-résistance, et conduisent surtout à une opportunité thérapeutique nouvelle de chimio-sensibilisation.

Gesamtdissertation, In einer konsanguinen Familie wurde während einer genetischen Routinediagnostik ein ungewöhnliches Chromosomeninstabilitätsprofil festgestellt. Die vorgestellten Patienten wiesen eine Mikrozephalie, Wachstumsretardierungen, sowie rezidivierende Infektionen auf. Sie verstarben im Kindesalter an einem Lymphom. Hierbei handelt es sich um einen Symptomkomplex, welcher bisweilen bei Patienten mit Mutationen in DNA- Reparaturgenen gefunden wurde. Allerdings zeigten sich bei der Untersuchung der Patientenzellen keine Zunahme mutagen-induzierter Chromosomenbrüche. Im Gegensatz dazu war die spontane Chromosomenbruchrate einheitlich erhöht und manifestierte sich besonders im Auftreten dizentrischer Chromosomen, welche durch die Fusion zweier Telomere unterschiedlicher Chromosomen vor der DNA- Replikation entstehen. Der vorliegende Fall entspricht einem neuen autosomal rezessiven Chromosomeninstabilitätssyndrom. Untersuchung von DNA-Reparatur- und Zellzyklusproteinen bestätigten den Eindruck, dass es sich hierbei um einen DNA-Reparatur kompetenten, jedoch dauerhaft gestressten, zellulären Phänotyp handelt. Das Vorhandensein von Telomerfusionen lässt vermuten, dass das betroffene Gen bei der Aufrechterhaltung und den Schutz der Telomere involviert ist. Unterschiedliche Chromosomeninstabilitätssyndrome, in denen ein DNA-Reparaturgen mutiert ist, zeigen einen mäßigen Anstieg an Telomerfusionen. Dahingegen repräsentiert die hier beschriebene Familie offensichtlich die erste Beschreibung einer Keimbahnmutation in einem Telomerprotein, welches nicht in die DNA-Reparatur involviert ist. Wir haben einen genomweiten SNP- und Mikrosatellitenscan auf Homozygotie durchgeführt. Dabei haben wir eine 17 Mb große Region auf Chromosomen 12q13-14 als wahrscheinlichste Kandidatengenregion identifiziert., During routine genetic diagnosis a consanguineous family with an unusual profile of chromosomal instability was observed. The patients presented with microcephaly, growth retardation and recurrent infections and died of lymphoma during childhood; a symptom-complex often found due to mutations in DNA repair genes. In this case, however, examination of patient cells revealed normal rates of mutagen-induced chromosome breakage. In contrast, spontaneous chromosome breakage was consistently increased and manifested particularly as dicentric chromosomes, which represent the products of telomere fusion between different chromosomes prior to DNA replication. This is a novel pattern of chromosome instability in an autosomal recessive disorder. Examination of DNA- Repair and cell cycle checkpoint proteins confirmed the impression of a DNA- repair-competent but chronically stressed cellular phenotype. The presence of telomere fusions suggests that the underlying gene is likely to be involved in telomere maintenance/protection. Several chromosome instability disorders, in which DNA repair genes are mutated, show a modest increased in telomere fusions, however, the family described here possibly represents the first observation of inherited mutations in a telomeric protein not involved in DNA repair. We have conducted a genome wide SNP and microsatellite scan for homozygosity and have identified a 17Mb region on chromosome 12q13-14 as the most likely location of the causative gene.

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of approximately 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRCI-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.

Aim of this explorative study was to determine the prognostic value of protein expression of the DNA damage repair enzymes ERCC1, hRad51, and BRCA1 for tumour response and survival of non-small-cell lung cancer patients treated with chemotherapy. Patients with either a short or long overall survival were selected from a randomized phase III trial comparing cisplatin-gemcitabine and epirubicin-gemcitabine. Tumour biopsies were assessed for differences in immunohistochemical staining using antibodies against ERCC1, hRad51, and BRCA1. A total of 33 patients were included. A positive nuclear staining for ERCC1, hRad51, and BRCA1 was observed in 44, 12, and 90% of biopsies, respectively. In large cell carcinoma nuclear hRad51 staining was absent. In five biopsies stained for hRad51 an unexpected membrane-like staining was observed; these biopsies showed no nuclear staining. DNA damage repair protein expressions were not significantly different in responders versus non-responders, or in patients with a short or long overall survival. In conclusion, immunohistochemical staining of ERCC1, hRad51, and BRCA1, in tumour biopsies from non-small-cell lung cancer patients was not predictive for tumour response and survival after chemotherapy. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

The HTLV-1 Tax oncoprotein rapidly induces cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) in cells. Tax is thought to have both aneuploidogenic and clastogenic effects. To examine the cellular target for Tax which might mechanistically explain the clastogenic phenomenon, we tested the ability of Tax to induce MN in rodents cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNAPKcs). We found that cells genetically mutated in Ku80 were refractory to Tax's induction of MN while cells knocked-out for DNAPKcs showed increased number of Tax-induced MN. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation) which measures the number of DNA-breaks in cells that contained unprotected 3'-OH ends, we observed that Tax increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells. Taken together, our findings suggest Ku80 as a cellular factor targeted by Tax in engendering clastogenic DNA damage.

In multi-cellular organisms--and hence also in man--aging and dying is the fate of the soma, i.e., the body proper; germ cells are--at least in principle--immortal. As the soma can be considered to be the "service compartment" of the germ cells, it loses its function once a species' phase of reproduction is over. Hence, there is an intimate relationship between reproduction and aging, and therefore also between a species' reproductive strategy and aging: there has been no selection for maintenance of the soma beyond the reproductive phase of life (when there is no reproduction, there is also no natural selection). As a consequence, mechanisms, important for maintenance of the soma during the reproductive phase, increasingly begin to fail once this phase is over, resulting in an accumulation of all kinds of pathology and genetic errors, rendering an individual increasingly more prone to a variety of (internal and external) attacks. In the end, the soma collapses, be it due to organ failure, a neoplasm or to a final external push, e.g., an infection.

In der vorliegenden Arbeit wurden allgemeine und funktionelle Untersuchungen zur großen Untereinheit des humanen Replikationsfaktors C (RFC1) durchgeführt. RFC1 ist als Untereinheit des heteropentameren RFC-Komplexes essentiell an der ATP-abhängigen Beladung des Prozessivitätsfaktors PCNA (Proliferating cell nuclear antigen) an 3Ž-Enden von Primer/Template-Strukturen im Rahmen der DNA-Replikation, wie auch an PCNA-vermittelten DNA-Reparaturprozessen beteiligt. Neben diesen gut charakterisierten Funktionen als essentieller DNA-Replikations- und Reparaturfaktor weisen vereinzelte Untersuchungen auf eine Funktion von RFC1 als transkriptionellen Cofaktor hin, wobei jedoch die funktionelle Bedeutung dieser Beobachtungen in den meisten Fällen bisher nicht näher charakterisiert wurde. Zu Beginn der Arbeit vorliegende Daten ließen auf eine primär transkriptionell regulierte Expression von RFC1 schließen. Die Ergebnisse verschiedener Untersuchungen zur mRNA-Expression von RFC1 bzw. des murinen mRFC1-Gens in unterschi

The hepatocyte growth factor (HGF) receptor, encoded by the MET oncogene, is expressed in ∼70% of human ovarian carcinomas and overexpressed in 30% of cases. Because HGF is known to protect cells from apoptosis, we investigated whether receptor expression modifies ovarian cancer cell response to chemotherapy. The apoptotic effect of the front-line chemotherapeutic drugs paclitaxel and cisplatin on cells treated with HGF was studied. In ovarian cancer cell lines, pretreatment with HGF surprisingly enhances the apoptotic response to low doses of paclitaxel and cisplatin. HGF empowers specifically the intrinsic apoptotic pathway, whereas it protects cells from extrinsic Fas-induced apoptosis. Chemotherapy sensitization is specific for HGF because another growth factor (e.g., epidermal growth factor) increases ovarian cancer cell survival. In nonovarian cancer cell models, as expected, HGF provides protection from drug-induced apoptosis. These data show that HGF sensitizes ovarian carcinoma cells to low-dose chemotherapeutic agents. This suggests that HGF may be used to improve response to chemotherapy in a set of human ovarian carcinomas molecularly classified based on the MET oncogene expression.

A number of studies implicate reactive oxygen intermediates in the induction of DNA damage and apoptosis. Recent studies suggest that the human T-cell leukemia virus type 1 (HTLV-1) Tax protein induces oxidative stress and apoptotic T-cell death. Activation of the T-cell receptor/CD3 pathway enhances the Tax-mediated oxidative and apoptotic effects. Tax-mediated apoptosis and oxidative stress as well as activation of nuclear factor-kappaB can be potently suppressed by antioxidants. This review focuses on Tax-dependent changes in the intracellular redox status and their role in Tax-mediated DNA damage and apoptosis. The relevance of these observations to HTLV-1 virus-mediated T-cell transformation and leukemogenesis are discussed.

The mechanism of radiosensitization by gemcitabine (2',2'-dinuoro-2'-deoxycytidine, dFdC) is not exactly known. We investigated the possible role of inhibition of the repair of DNA double-strand breaks by dFdC by measuring the extent of radiosensitization in different cell lines deficient and proficient in components of nonhomologous end-joining and in the parental cell lines. Different cell lines were incubated with 0.5 and 5 muM dFdC for 4 h. Cells deficient in DNA-dependent protein-kinase catalytic subunit (V3) showed sensitization similar to that of wild-type cells (AA8) and complemented cells (V3+YAC). Ku80-deficient cells (xrs5 and xrs6) showed even more radiosensitization by dFdC as compared with wild-type CHO-K1. However, Ku80-complemented cell lines (xrs5+huKu80 and xrs6+haKu80) did not show radiosensitization. The differences in dFdC-mediated radiosensitization were not attributable to different changes in deoxynucleotide triphosphate levels and cell cycle distribution. We conclude that a functional nonhomologous end-joining pathway is not required for dFdC-mediated radiosensitization.

The second Caucasian xeroderma pigmentosum patient (XP42RO) belonging to complementation group F (XP-F) is described, Mild ocular photophobia was present from childhood, and acute skin reactions occurred upon exposure to sunlight. Basal and squamous cell carcinomas developed after his twenty-seventh year, In his late forties progressive neurologic symptoms emerged, which included intellectual decline, mild chorea and ataxia, and marked cerebral and cerebelar atrophy, Such neurologic abnormalities are very unusual in XP-F, Similar symptoms have been described in only one of 17 other XP-F individuals. His approximate to 5-fold reduced activity of nucleotide excision repair in cultured cells, combined with moderately affected cell survival and DNA replication after UV exposure, are typical of XP-F., The recent cloning of the XPF gene allowed a molecular genetic analysis of this unusual patient, XP42RO, representing the second case studied in this respect, turned out to be homozygous for a point mutation in the XPF gene, causing an R788-->W substitution in the encoded protein, Surprisingly, this mutation had also been found in one allele of the other unrelated Caucasian XP-F case. The amount of mutated XPF protein is strongly reduced in cells from XP42RO, presumably due to a conformational change. Biochemical, genetic, and clinical data all indicate the presence of considerable residual repair activity, strongly suggesting that the R788W mutation is leaky.

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER. published

In normal human fibroblasts, repair of(6-4)PP in the active adenosine deaminase (ADA) gene occurs with similar rate in the transcribed and non-transcribed strand of the ADA gene, and removal of (6-4)PP from the active ADA gene is faster than from the inactive X-chromosomal 754 locus. Heat shock decreased the rate of repair of the active ADA gene down to the level of inactive genes, whereas the rate of repair of the inactive 754 locus was not affected.

Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes, To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts with different repair capacities and different transcriptional status of the adenosine deaminase gene, i.e. normal human cells, human cells carrying an inactive copy of the adenosine deaminase gene and xeroderma pigmentosum complementation group C fibroblasts, The results indicate that repair of active genes is impaired by inhibition of two repair pathways: (i) a global repair system involved in the repair of CPDs in potentially active genes; and (ii) the transcription-coupled repair pathway responsible for the accelerated repair of the transcribed strand. Since X-ray-induced DNA damage is also preferentially removed from the transcribed strand of active genes, selective inhibition of repair of radiation-induced DNA damage in active genes may play a key role in radiosensitization due to hyperthermia.

Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in controlling cell survival or death. As a consequence, therapeutic interventions with PARP-1 inhibitors are sought in different pathological conditions such as cancer, cardiovascular and inflammatory diseases, as well as brain ischemia. In the first part of this work, as a continuation of our efforts in the field, we report the design, synthesis and biological appraisal of novel potent PARP-1 inhibitors. A crystallization experiment is carried out to ascertain the mode of binding to PARP-1 of the most potent compound, namely 2-((dimethylamino)methyl)-9-hydroxythieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), whilst molecular modeling studies are performed to infer the role of water molecules in ligand binding. In the second part of the work, we discuss the results of HYDAMTIQ in models of brain ischemia as well as its preliminary physicochemical and pharmacokinetic characterization. Collectively, the data obtained qualify HYDAMTIQ as a novel lead candidate for advancement to clinical settings of brain ischemia.

Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vivo is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells lacking this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor α, γ-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP−/− thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching andV(D)Jrecombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.

Budding yeast telomeres are reversibly bound at the nuclear envelope through two partially redundant pathways that involve the Sir2/3/4 silencing complex and the Yku70/80 heterodimer(1,2). To better understand how this is regulated, we studied the role of SUMOylation in telomere anchoring. We find that the PIAS-like SUMO E3 ligase Siz2 sumoylates both Yku70/80 and Sir4 in vivo and promotes telomere anchoring to the nuclear envelope. Remarkably, loss of Siz2 also provokes telomere extension in a telomerase-dependent manner that is epistatic with loss of the helicase Pif1. Consistent with our previously documented role for telomerase in anchorage(3), normal telomere anchoring in siz2 Delta is restored by PIF1 deletion. By live-cell imaging of a critically short telomere, we show that telomeres shift a way from the nuclear envelope when elongating. We propose that SUMO-dependent association with the nuclear periphery restrains bound telomerase, whereas active elongation correlates with telomere release.

Background: Inherited pathogenic variants in BRCA1 and BRCA2 are the most common causes of hereditary breast and ovarian cancer (HBOC). The risk of developing breast cancer by age 80 in women carrying a BRCA1 pathogenic variant is 72%. The lifetime risk varies between families and even within affected individuals of the same family. The cause of this variability is largely unknown, but it is hypothesized that additional genetic factors contribute to differences in age at onset (AAO). Here we investigated whether truncating and rare missense variants in genes of different DNA-repair pathways contribute to this phenomenon. Methods: We used extreme phenotype sampling to recruit 133 BRCA1-positive patients with either early breast cancer onset, below 35 (early AAO cohort) or cancer-free by age 60 (controls). Next Generation Sequencing (NGS) was used to screen for variants in 311 genes involved in different DNA-repair pathways. Results: Patients with an early AAO (73 women) had developed breast cancer at a median age of 27 years (interquartile range (IQR); 25.00–27.00 years). A total of 3703 variants were detected in all patients and 43 of those (1.2%) were truncating variants. The truncating variants were found in 26 women of the early AAO group (35.6%; 95%-CI 24.7 - 47.7%) compared to 16 women of controls (26.7%; 95%-CI 16.1 to 39.7%). When adjusted for environmental factors and family history, the odds ratio indicated an increased breast cancer risk for those carrying an additional truncating DNA-repair variant to BRCA1 mutation (OR: 3.1; 95%-CI 0.92 to 11.5; p-value = 0.07), although it did not reach the conventionally acceptable significance level of 0.05. Conclusions: To our knowledge this is the first time that the combined effect of truncating variants in DNA-repair genes on AAO in patients with hereditary breast cancer is investigated. Our results indicate that co-occurring truncating variants might be associated with an earlier onset of breast cancer in BRCA1-positive patients. Larger cohorts are needed to confirm these results.

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more abundant and fitter mitochondria could help to preserve mitochondrial functions upon irradiation.

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of similar to 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH+4 are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRC1-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.