Effect of DNA repair inhibitor AsiDNA on the incidence of telomere fusion in crisis.

Telomere fusions lead to a state of genomic instability, and are thought to drive clonal evolution and tumorigenesis. Telomere fusions occur via both Classical and Alternative Non-Homologous End Joining repair pathways. AsiDNA is a DNA repair inhibitor that acts by mimicking a DNA double strand break (DSB) and hijacking the recruitment of proteins involved in various DNA repair pathways. In this study, we investigated whether the inhibition of DSB-repair pathways by AsiDNA could prevent telomere fusions during crisis. The present study showed that AsiDNA decreased the frequency of telomere fusions without affecting the rate of telomere erosion. Further, it indicated that AsiDNA does not impact the choice of the repair pathway used for the fusion of short dysfunctional telomeres. AsiDNA is thought to prevent short telomeres from fusing by inhibiting DNA repair. An alternative, non-mutually exclusive possibility is that cells harbouring fusions preferentially die in the presence of AsiDNA, thus resulting in a reduction in fusion frequency. This important work could open the way for investigating the use of AsiDNA in the treatment of tumours that have short dysfunctional telomeres and/or are experiencing genomic instability.

Combining DNA Barcoding and Chemical fingerprints to authenticate Lavender raw material.

OBJECTIVE: This study was initiated and conducted by several laboratories, 3 of the main cosmetic ingredient suppliers and 4 brands of cosmetics in France. Its objective is to show the interest and robustness of coupling chemical and genetic analyses in the identification of plant species. In this study, the Lavandula genus was used. METHODS: In this study, we used two analytical methods. Chemical analysis from UHPLC (ultra-high-performance liquid chromatography) and genetic analysis from barcoding with genetic markers. RESULTS: Eleven lavender species were selected (botanically authenticated) and analysed. The results show that three chemical compounds (coumaric acid hexoside, ferulic acid hexoside and rosmarinic acid) and three genetic markers (RbcL, trnH-psbA and ITS) are of interest for the differentiation of species of the genus lavandula. CONCLUSION: The results show that the combination of complementary analytical methods is a relevant system to prove the botanical identification of lavender species. This first study, carried out on a plant of interest for cosmetics, demonstrates the need for authentication using a tool combining genetic and chemical analysis as an advance over traditional investigation methods used alone, in terms of identification and authentication reliability.

OBJECTIF: Cette étude a été lancée et menée par plusieurs laboratoires, trois des principaux fournisseurs d'ingrédients cosmétiques et quatre marques de cosmétiques en France. Son objectif est de montrer qu'associer les analyses chimiques et génétiques dans l'identification des espèces végétales présente un intérêt et est une approche solide. Dans cette étude, c'est le genre Lavandula qui a été utilisé. MÉTHODES: Dans cette étude, nous avons fait appel à deux méthodes analytiques. L'analyse chimique, à partir de la chromatographie en phase liquide à haute performance (ultra-high-performance liquid chromatography, UHPLC), et l'analyse génétique en procédant à un codage à barres avec des marqueurs génétiques. RÉSULTATS: Onze espèces de lavande ont été sélectionnées (authentifiées du point de vue botanique) et analysées. Les résultats montrent que trois composés chimiques (acide coumarique hexoside, acide ferulique hexoside et acide rosmarinique) et trois marqueurs génétiques (RbcL, trnH-psbA et ITS) présentent un intérêt pour la différenciation des espèces du genre lavandula. CONCLUSION: Les résultats montrent que la combinaison de méthodes analytiques complémentaires est un système pertinent pour prouver l'identification végétale des espèces de lavande. Cette première étude, réalisée sur une plante qui offre un intérêt pour les cosmétiques, démontre la nécessité de procéder à une authentification à l'aide d'un outil qui conjugue analyse génétique et chimique ; elle représente une avancée par rapport aux méthodes d'investigation traditionnelles utilisées seules, en termes d'identification et de fiabilité de l'authentification.

Potentiation of doxorubicin efficacy in hepatocellular carcinoma by the DNA repair inhibitor DT01 in preclinical models.

OBJECTIVE: This study aimed to explore the antitumour effect of the DNA repair inhibitor, DT01 (the cholesterol conjugated form of Dbait), as an adjunct treatment to enhance the therapeutic efficacy of transarterial chemoembolization (TACE) in pre-clinical models of hepatocellular carcinoma (HCC). METHODS: A rabbit model bearing liver tumours was either left untreated or treated with TACE or with a combination of TACE+DT01. Tumour growth was monitored by ultrasound. These results were further confirmed in mice grafted with an intrahepatic human HCC model treated with doxorubicin (DOX) alone or DOX+DT01. RESULTS: The combination of DT01 with TACE in a rabbit liver model led to a significant decrease in tumour volume (p=0.03). Colour Doppler and immunohistochemical staining revealed a strong decrease in vascularization in the DT01+TACE-treated group preventing the tumour growth restart observed after TACE alone. Similarly, the DT01 combination with DOX led to significant anti-tumour efficacy compared to DOX alone (p=0.02) in the human HCC model. In addition, a significant decrease in vascularization in the group receiving combination DT01 and DOX treatment was observed. CONCLUSIONS: DT01 is well tolerated and may potentiate HCC treatment by enhancing the DNA-damaging and anti-vascularization effect of TACE with doxorubicin. KEY POINTS: • DT01 combined with TACE leads to significant anti-tumour efficacy without additional toxicity. • A potential anti-angiogenic role of DT01 was identified in preclinical models. • DT01 may potentiate HCC treatment by enhancing the efficacy of TACE.

Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

Chemical Compositions and Insecticidal Activities of Alpinia kwangsiensis Essential Oil against Lasioderma serricorne.

The essential oil obtained by hydrodistillation from Alpinia kwangsiensis rhizomes was investigated by GC-MS. A total of 31 components representing 92.45% of the oil were identified and the main compounds in the oil were found to be camphor (17.59%), eucalyptol (15.16%), ß-pinene (11.15%) and α-pinene (10.50%). These four compounds were subsequently isolated and the essential oil and four isolated compounds exhibited potent insecticidal activity against Lasioderma serricorne adults. During the assay, it was shown that the essential oil exhibited both potential contact (LD50 = of 24.59 µg/adult) and fumigant (LC50 = of 9.91 mg/L air) toxicity against Lasioderma serricorne. The study revealed that the insecticidal activity of the essential oil can be attributed to the synergistic effects of its diverse major components, which indicates that oil of Alpinia kwangsiensis and its isolated compounds have potential to be developed into natural insecticides to control insects in stored grains and traditional Chinese medicinal materials.

Colorectal cancer metastasis: the DNA repair inhibitor Dbait increases sensitivity to hyperthermia and improves efficacy of radiofrequency ablation.

PURPOSE: To assess the usefulness of combining hyperthermia with a DNA repair inhibitor (double-strand break bait [Dbait]) and its potential application to radiofrequency ablation (RFA) in a preclinical model of human colorectal cancer. MATERIALS AND METHODS: The local ethics committee of animal experimentation approved all investigations. First, the relevance was assessed by studying the survival of four human colorectal adenocarcinoma cell cultures after 1 hour of hyperthermia at 41°C or 43°C with or without Dbait. Human colon adenocarcinoma cells (HT-29) were grafted subcutaneously into nude mice (n = 111). When tumors reached approximately 500 mm(3), mice were treated with Dbait alone (n = 20), sublethal RFA (n = 21), three different Dbait schemes and sublethal RFA (n = 52), or a sham treatment (n = 18). RFA was performed to ablate the tumor center alone. To elucidate antitumor mechanisms, 39 mice were sacrificed for blinded pathologic analysis, including assessment of DNA damage, cell proliferation, and tumor necrosis. Others were monitored for tumor growth and survival. Analyses of variance and log-rank tests were used to evaluate differences. RESULTS: When associated with mild hyperthermia, Dbait induced cytotoxicity in all tested colon cancer cell lines. Sublethal RFA or Dbait treatment alone moderately improved survival (median, 40 days vs 28 days for control; P = .0005) but combination treatment significantly improved survival (median, 84 days vs 40 days for RFA alone, P = .0004), with approximately half of the animals showing complete tumor responses. Pathologic studies showed that the Dbait and RFA combination strongly enhances DNA damage and coagulation areas in tumors. CONCLUSION: Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.

Bioinspire-Explore: Taxonomy-Driven Exploration of Biodiversity Data for Bioinspired Innovation.

Successful bioinspired design depends on practitioners' access to biological data in a relevant form. Although multiple open-access biodiversity databases exist, their presentation is often adapted to life scientists, rather than bioinspired designers. In this paper, we present a new tool, "Bioinspire-Explore", for navigating biodiversity data in order to uncover biological systems of interest for a range of sectors. Bioinspire-Explore allows users to search for inspiring biological models via taxa (species, genera, etc.) as an entry point. It provides information on a taxon's position in the "tree of life", its distribution and climatic niche, as well as its appearance. Bioinspire-Explore also shows users connections in the bioinspiration literature between their taxon of interest and associated biological processes, habitats, and physical measurements by way of their semantic proximity. We believe Bioinspire-Explore has the potential to become an indispensable resource for both biologists and bioinspired designers in different fields.

Heat shock protein 90α (Hsp90α) is phosphorylated in response to DNA damage and accumulates in repair foci.

DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.

Revisiting Nature's "Unifying Patterns": A Biological Appraisal.

Effective bioinspiration requires dialogue between designers and biologists, and this dialogue must be rooted in a shared scientific understanding of living systems. To support learning from "nature's overarching design lessons" the Biomimicry Institute has produced ten "Unifying Patterns of Nature". These patterns have been developed to engage with those interested in finding biologically inspired solutions to human challenges. Yet, although well-intentioned and appealing, they are likely to dishearten biologists. The aim of this paper is to identify why and propose alternative principles based on evolutionary theory.

Therapeutic approach of human peritoneal carcinomatosis with Dbait in combination with capnoperitoneum: proof of concept.

BACKGROUND: Peritoneal carcinomatosis is an unmet medical need. Laparoscopy offers a unique opportunity to control and to steer the operating environment during surgery by loading carbon dioxide with a therapeutic substance and creating the so-called therapeutic capnoperitoneum. We have treated a human sample of peritoneal carcinomatosis from an endometrial adenocarcinoma ex vivo just after surgery. METHODS: A nontoxic therapeutic agent (Dbait) was aerosolized into a box containing diseased human peritoneum under a pressure of 12 mmHg CO(2). Dbait (noncoding DNA fragments) acts through jamming DNA damage sensing and signaling, ultimately inhibiting DNA repair system of cancer cells. Dbait were coupled to cholesterol molecules to facilitate intracellular uptake, and to Cyanine (Cy5) to allow detection by fluorescence. In a control experiment, the same solution was applied to the other half of the sample using conventional lavage. RESULTS: Physical results revealed fluorescence within the tumor up to 1 mm depth in the therapeutic capnoperitoneum sample and no uptake in the lavage sample. Biological results showed intranuclear phosphorylation of H2AX in the nebulized sample and no activity in the lavage sample. Importantly, tumor nodules showed more activity than the neighbor, normal peritoneum. Detection of histone gamma-H2AX (phosphorylated H2AX) reveals activation of DNA-dependent protein kinase (DNA-PK) by Dbait, which has been shown to be the key step for sensitization to genotoxic therapy. CONCLUSIONS: Dbait are taken up by cancer cells and have a biological activity up to 1 mm depth. Nebulization of the molecule is significantly more effective than conventional lavage. This proof of principle supports the need for clinical studies applying therapeutic capnoperitoneum together with Dbait for treating peritoneal carcinomatosis.

The Clinical Significance of Changes in Serum New Cytokine CYTL1 in Patients with Knee Osteoarthritis.

OBJECTIVE: The present study aims to investigate the clinical significance of changes in the expression of new cytokine-like 1 (CYTL1) in the serum of patients with knee osteoarthritis (KOA). METHODS: A total of 182 patients with KOA, including 84 males and 98 females aged 39-86 with an average age of 66.4 ± 9.7 and an average body mass index (BMI) of 24.9 ± 2.4 kg/m2, were enrolled in the study. The patients were divided into three subgroups: the grade II subgroup (n = 23), grade III subgroup (n = 63), and grade IV subgroup (n = 96) based on severity, as calculated by the Kellgren and Lawrence (K&L) classification system. In addition, 152 volunteers from our health center who came in for physical examination were selected as the control group, including 70 males and 82 females aged 37-82 with an average age of 63.4 ± 9.5 and an average BMI of 24.8 ± 2.2 kg/m2. An enzyme-linked immunosorbent assay was adopted to detect the serum CYTL1 levels, and the correlation between CYTL1 and the severity of KOA was analyzed. RESULTS: The serum level of CYTL1 was significantly lower in the KOA group than in the control group (P < 0.05). In the KOA group, the difference in the serum level of CYTL1 was statistically significant between the subgroups and decreased significantly with an increase in the severity of the disease (F = 54.826, P < 0.001). Therefore, the serum level of CYTL1 was correlated with the severity of the disease, as determined by the K&L classification system (r = -0.613, P < 0.001). CONCLUSION: The serum levels of CYTL1 are strongly correlated with the severity of the disease in patients with KOA and could be a new therapeutic target for KOA.

Small-molecule drugs mimicking DNA damage: a new strategy for sensitizing tumors to radiotherapy.

PURPOSE: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. EXPERIMENTAL DESIGN: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. RESULTS: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of "false" DNA damage signaling and repair inhibition. CONCLUSIONS: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger "false" signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

Preclinical Studies Comparing Efficacy and Toxicity of DNA Repair Inhibitors, Olaparib, and AsiDNA, in the Treatment of Carboplatin-Resistant Tumors.

Purpose: Carboplatin is used to treat many cancers, but occurrence of drug resistance and its high toxicity remain a clinical hurdle limiting its efficacy. We compared the efficacy and toxicity of DNA repair inhibitors olaparib or AsiDNA administered alone or in combination with carboplatin. Olaparib acts by inhibiting PARP-dependent repair pathways whereas AsiDNA inhibits double-strand break repair by preventing recruitment of enzymes involved in homologous recombination and non-homologous end joining. Experimental Design: Mice with MDA-MB-231 tumors were treated with carboplatin or/and olaparib or AsiDNA for three treatment cycles. Survival and tumor growth were monitored. Toxicities of treatments were assayed in C57BL/6 immunocompetent mice. Circulating blood hematocrits, bone marrow cells, and organs were analyzed 10 and 21 days after end of treatment using flow cytometry and microscopy analysis. Resistance occurrence was monitored after cycles of treatments with combination of AsiDNA and carboplatin in independent BC227 cell cultures. Results: Olaparib or AsiDNA monotherapies decreased tumor growth and increased mean survival of grafted animals. The combination with carboplatin further increased survival. Carboplatin toxicity resulted in a decrease of most blood cells, platelets, thymus, and spleen lymphocytes. Olaparib or AsiDNA monotherapies had no toxicity, and their combination with carboplatin did not increase toxicity in the bone marrow or thrombocytopenia. All animals receiving carboplatin combined with olaparib developed high liver toxicity with acute hepatitis at 21 days. In vitro, carboplatin resistance occurs after three cycles of treatment in all six tested cultures, whereas only one became resistant (1/5) after five cycles when carboplatin was associated to low doses of AsiDNA. All selected carboplatin-resistant clones retain sensitivity to AsiDNA. Conclusion: DNA repair inhibitor treatments are efficient in the platinum resistant model, MDA-MB-231. The combination with carboplatin improves survival. The association of carboplatin with olaparib is associated with high liver toxicity, which is not observed with AsiDNA. AsiDNA could delay resistance to carboplatin without increasing its toxicity.

Repellent activities of essential oils rich in sesquiterpenoids from Saussurea amara (L.) DC. and Sigesbeckia pubescens Makino against two stored-product insects.

The essential oils (EOs) from aerial parts of Saussurea amara (L.) DC. (SAEO) and Sigesbeckia pubescens Makino (SPEO) were analyzed for their chemical composition by GC-MS, and their repellent activities against adults of the red flour beetle, Tribolium castaneum Herbst, as well as the booklouse, Liposcelis bostrychophila Badonnel, were evaluated for the first time. Results of GC-MS analysis indicated that both SAEO and SPEO were characterized by high content of sesquiterpenoids (relative content > 70%) including oxygenated sesquiterpenoids. The two oil samples and their major component caryophyllene oxide exerted beneficial repellent effects on T. castaneum and L. bostrychophila at 2 and 4 h post-exposure. At 4 h post-exposure, the PR value of caryophyllene oxide could still reach 92% (class V) against T. castaneum at minimum testing concentration of 3.15 nL/cm2, and this compound was observed to result in the greatest repellency (PR = 100%) against L. bostrychophila at 12.63 nL/cm2. This work confirmed the potent repellent efficacy of SAEO and SPEO for controlling pest damage and suggested their potential to be developed into botanical repellents.

Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP Inhibitors.

Purpose: Cancer treatments using tumor defects in DNA repair pathways have shown promising results but are restricted to small subpopulations of patients. The most advanced drugs in this field are PARP inhibitors (PARPi), which trigger synthetic lethality in tumors with homologous recombination (HR) deficiency. Using AsiDNA, an inhibitor of HR and nonhomologous end joining, together with PARPi should allow bypassing the genetic restriction for PARPi efficacy.Experimental Design: We characterized the DNA repair inhibition activity of PARPi (olaparib) and AsiDNA by monitoring repair foci formation and DNA damage. We analyzed the cell survival to standalone and combined treatments of 21 tumor cells and three nontumor cells. In 12 breast cancer (BC) cell lines, correlation with sensitivity to each drug and transcriptome were statistically analyzed to identify resistance pathways.Results: Molecular analyses demonstrate that olaparib and AsiDNA respectively prevent recruitment of XRCC1 and RAD51/53BP1 repair enzymes to damage sites. Combination of both drugs increases the accumulation of unrepaired damage resulting in an increase of cell death in all tumor cells. In contrast, nontumor cells do not show an increase of DNA damage nor lethality. Analysis of multilevel omics data from BC cells highlighted different DNA repair and cell-cycle molecular profiles associated with resistance to AsiDNA or olaparib, rationalizing combined treatment. Treatment synergy was also confirmed with six other PARPi in development.Conclusions: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Clin Cancer Res; 23(4); 1001-11. ©2016 AACR.

Contact and Repellant Activities of Zerumbone and Its Analogues from the Essential Oil of Zingiber zerumbet (L.) Smith against Lasioderma serricorne.

The contact toxicity and repellent activities of the essential oil extracted from the rhizomes of Zingiber zerumbet (L.) Smith (Zingiberaceae) was evaluated against cigarette beetles (Lasioderma serricorne). The essential oil obtained by hydrodistillation was investigated by GC-FID and GC-MS. The main constituents of the essential oil were zerumbone (40.2%), α-caryophyllene (8.6%), humulene epoxide II (7.3%), camphene (5.9%) and fenchene (4.7%). Zerumbone and its analogues totally are accounting for 60.3% of the essential oil. It was found that the essential oil possessed contact toxicity against L. serricorne adults with a LD50 value of 48.3 µg/adult. α-Caryophyllene (LD50 = 13.1 µg/adult) exhibited stronger contact toxicity against L. serricorne than humulene oxide (LD50 = 31.2 µg/adult), ß-caryophyllene (LD50 = 35.5 µg/adult) and zerumbone (LD50 = 42.4 µg/adult). Moreover, α-caryophyllene possessed strong repellent activity (Class IV and V, respectively) against the beetles at 78.63 nL/cm2, after 2 and 4 h treatment. The results indicate that zerumbone and its analogues might be developed into natural insecticides or repellents for control of cigarette beetles, but their bioactivities are affected by their structures.

Benzoquinoquinoxaline derivatives stabilize and cleave H-DNA and repress transcription downstream of a triplex-forming sequence.

Oligopyrimidine*oligopurine sequences with potential to form intramolecular triple helix structures (H-DNA) have been found mainly in high eukaryote genomes. However, the natural occurrence and function of H-DNA remains elusive largely because we lack appropriate reagents to demonstrate the formation of these structures in cells. We examined whether a triple-helix specific stabilizing compound, benzoquinoquinoxaline (BQQ), and its 1,10-phenanthroline derivative can be efficiently utilized to study the formation and stabilization of an intramolecular triple-helical DNA structure in growing Escherichia coli cells and in vitro. Cell uptake of BQQ was confirmed by fluorescence microscopy. A plasmid carrying an H-DNA forming sequence upstream of a reporter gene was used to assess the effects of H-DNA formation and stabilization in growing cells. The presence of the H-DNA forming sequence dramatically repressed beta-lactamase expression, and sub-growth-inhibitory doses of BQQ caused a further 40% reduction. Most importantly, repression was dependent on the triple-helix forming sequence and correlated with the addition of BQQ. As the abundance of the H-DNA forming plasmid was not affected by the addition of BQQ, the dose-dependent reduction at the protein level observed here is likely caused by repression of transcription. Finally, the triple-helix specific interaction of BQQ with the target DNA sequence was demonstrated using a triple-helix directed cleavage assay by BQQ-1,10-phenanthroline conjugate in vitro.

Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited.

BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

Stimulation of D-loop formation by polypurine/polypyrimidine sequences.

Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We compared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.

Position- and orientation-specific enhancement of topoisomerase I cleavage complexes by triplex DNA structures.

Topoisomerase I (Top1) activities are sensitive to various endogenous base modifications, and anticancer drugs including the natural alkaloid camptothecin. Here, we show that triple helix-forming oligonucleotides (TFOs) can enhance Top1-mediated DNA cleavage by affecting either or both the nicking and the closing activities of Top1 depending on the position and the orientation of the triplex DNA structure relative to the Top1 site. TFO binding 1 bp downstream from the Top1 site enhances cleavage by inhibiting religation and to a lesser extent DNA nicking. In contrast, TFO binding 4 bp downstream from the Top1 site enhances DNA nicking especially when the 3' end of the TFO is proximal to the Top1 site. However, when the orientation of the triplex is inverted, with its 5' terminus 4 bp downstream from the Top1 site, religation is also inhibited. These position- and orientation-dependent effects of triplex structures on the Top1-mediated DNA cleavage and religation are discussed in the context of molecular modeling and effects of TFO on DNA twist and mobility at the duplex/triplex junction.