Nuria Pastor

Roles of DNA topoisomerases in chromosome segregation and mitosis.

DNA topoisomerases are highly specialized nuclear enzymes that perform topological changes in the DNA molecule in a very precise and unique fashion. Taking into account their fundamental roles in many events during DNA metabolism such as replication, transcription, recombination, condensation or segregation, it is no wonder that the last decade has witnessed an exponential interest on topoisomerases, mainly after the discovery of their potential role as targets in novel antitumor therapy. The difficulty of the lack of topoisomerase II mutants in higher eukaryotes has been partly overcome by the availability of drugs that act as either poisons or true catalytic inhibitors of the enzyme. These chemical tools have provided strong evidence that accurate performance of topoisomerase II is essential for chromosome segregation before anaphase, and this in turn constitutes a prerequisite for the development of normal mitosis. In the absence of cytokinesis, cells become polyploid or endoreduplicated.

EVALUATION OF GENOTOXIC EFFECTS OF HEAVY METALS AND ARSENIC IN WILD NESTLING WHITE STORKS (CICONIA CICONIA) AND BLACK KITES (MILVUS MIGRANS) FROM SOUTHWESTERN SPAIN AFTER A MINING ACCIDENT

Studies of birds from Doñana (southwestern Spain) after the Aznalcóllar mining accident (April 1998) have reported high levels of genetic damage when compared to conspecifics from reference areas. However, potential relationships between DNA damage and metal pollution have not yet been reported. The aim of the present study was to investigate the current levels of Zn, Pb, As, Cu, and Cd and to determine if they were associated with the genetic damage observed in free-living, nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) born in the Doñana area after the mining spill. Blood concentrations of heavy metals and of As were quantified and DNA damage (comet assay) was determined in 258 storks and 132 kites monitored during a four-year period (1999-2002). Correlations between these elements and genetic damage varied between species and throughout years within species. Some elements did not show any relationship with DNA damage (e.g., Pb), whereas others had a significant correlation (e.g., As in storks, and Cu and Cd in kites) or only marginal statistical effects (e.g., Zn and Cd in storks, and As in kites) in some years but not in others. These results suggest that nestling white storks and black kites were affected, in part, by the elements studied, but they alone do not satisfactorily explain the observed DNA damage. Moreover, our results show that species-specific differences should be carefully considered when planning schemes for pollution monitoring, and highlight the need for including the temporal scale into the study of the pollutants effects in the wild.

The Coffee Constituent Chlorogenic Acid Induces Cellular DNA Damage and Formation of Topoisomerase I– and II–DNA Complexes in Cells

Chlorogenic acid (CGA) is a plant polyphenol with known antioxidant properties. Although some studies suggest that CGA has anticancer properties, others indicate that this dietary constituent may cause DNA damage and induce carcinogenic effects. Because CGA is widely consumed in the form of coffee, it is important to further evaluate the putative DNA-damaging activity of CGA. Here we have employed two standard techniques commonly used for DNA damage detection (the comet assay and the γ- H2AX focus assay) and observed that CGA (0.5-5 mM) induces DNA damage in normal and cancer cells. We report for the first time that CGA induces high levels of topoisomerase I- and topoisomerase II-DNA complexes in cells (TARDIS assay). Catalase pretreatment abolished the formation of these topoisomerase-DNA complexes and reduced the cytotoxic activity of CGA, therefore indicating that hydrogen peroxide plays an important role in these activities. Lung cancer cells (A549) were more sensitive than normal lung fibroblasts (MRC5) to the cytotoxic activity of CGA, supporting previous findings that CGA may induce selective killing of cancer cells. Taking into consideration our results and the pharmacokinetic profile of CGA, the possible cancer preventive, carcinogenic and therapeutic potential of this dietary agent are discussed.

both bovine and rabbit lymphocytes conditioned with hydrogen peroxide show an adaptive response to radiation damage

We have carried out experiments to study the possible induction of an adaptive response in cultured bovine and rabbit lymphocytes conditioned with subtoxic doses of hydrogen peroxide after stimulation and subsequently challenged with 1 Gy of X-rays. Peroxide treatment was given at different doses 48 h after the addition of PHA to stimulate the cells. A protective effect of pre-exposure to H2O2 against radiation damage detected as micronuclei in binucleated cells was evident for all the animals tested regardless the dose of H2O2 used, although this effect was in general of greater magnitude in bovine than in rabbit cells. These results lend further support to our previous finding in human lymphocytes that DNA single strand breaks induced by H2O2 (most likely due to the generation of hydroxyl radicals) is the most important lesion to trigger the adaptive response.

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions.

Decitabine (5-aza-2′-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1–DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML.

5-Aza-2′-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair

5-Aza-2′-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.

Tea flavanols inhibit cell growth and DNA topoisomerase II activity and induce endoreduplication in cultured Chinese hamster cells

Tea polyphenols are promising chemopreventive anticancer agents, the properties of which have been studied both in vitro and in vivo, providing evidence that - within this group of compounds - the tea flavanols are able to inhibit carcinogenesis, an effect that in some cases could be correlated with increased cell apoptosis and decreased cell proliferation. Of four main tea flavanols, namely (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (+)-catechin (CA) and (-)-epicatechin (EC), it was found that EGCG was the most potent to inhibit dose dependently the topoisomerase II (TOPO II) catalytic activity isolated from hamster ovary AA8 cells. In the range of concentrations that caused TOPO II inhibition, a high level of endoreduplication, a rare phenomenon that consists in two successive rounds of DNA replication without intervening mitosis, was observed, while neither micronuclei nor DNA strand breaks (Comet assay) were detected at the same doses. We propose that the anticarcinogenic effect of tea flavanols can be partly explained by their potency and effectiveness to induce endoreduplication. Concerning such an induction, maximum effect seems to require a pyrogallol structure at the B-ring. Additional substitution with a galloylic residue at the C3 hydroxyl group leads to further augmentation of the effect. Thus, we suggest that the chemopreventive properties of tea flavanols can be at least partly due to their ability to interfere with the cell cycle and block cell proliferation at early stages of mitosis.

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

An uncommonly high yield of spontaneous endoreduplication is a feature of the CHO mutant EM9, besides its defective repair of single, as well as double-DNA strand-breaks and its extraordinarily elevated yield of sister chromatid exchanges (SCEs) after bromodeoxyuridine (BrdU) incorporation into DNA. Since the nuclear enzyme topoisomerase II (topo II) has been reported to be responsible for the segregation of daughter chromosomes during mitosis, in the present investigation we have made use of the bisdioxopiperazine ICRF-193, a topo II catalytic inhibitor that interferes with the normal turnover of the enzyme. In order to see whether both EM9 cells and its parental cell line AA8, which show differences in the spontaneous frequency of endoreduplicated cells are or not equally sensitive to the topo II catalytic inhibitor, both cell lines have been treated with a range of doses of the bisdioxopiperazine. Our results show that both cell lines respond to the treatment entering in an endoreduplication cycle, but the EM9 cells are extremely sensitive to the inhibition of topo II.

A comparative study of genotoxic effects of anti-topoisomerase II drugs ICRF-193 and bufalin in Chinese hamster ovary cells

With the ultimate purpose of testing the existence of possible differences in the effectiveness of the topoisomerase II catalytic inhibitor ICRF-193 (a bisdioxopiperazine) and the enzyme suppressor bufalin (a bufadienolide from toad venom) we have carried out a series of experiments aimed at inducing cytotoxicity as well as DNA and chromosome damage in transformed CHO cells. In order to assess any possible influence of DNA repair capacity of the treated cells on the final outcome, we have made use of the repair-defective CHO mutant EM9, which shows a defect in DNA single- and double-strand breaks repair for comparison with its repair-proficient parental line AA8. Our results seem to indicate that, while both ICRF-193 and bufalin suppress cell growth and result in a clear inhibition of topoisomerase II catalytic activity, only ICRF-193 has been shown as able to induce both chromosome and DNA damage, with a more pronounced effect in the CHO mutant EM9 than in the repair-proficient line AA8.

Testing the SCE mechanism with non-poisoning topoisomerase II inhibitors

There are controversial theoretical models about a possible involvement of DNA topoisomerase II (topo II) in the molecular mechanism of sister chromatid exchanges (SCEs). In order to clarify the role of this enzyme, if any, in such recombinational event, CHO parental AA8 and mutant EM9 cells, which shows and extremely high baseline frequency of SCE, have been treated with different doses of the non-poisoning topoisomerase inhibitors, ICRF-193 and bufalin. The frequencies of SCEs after the treatments have been determined and the inhibitory effect of these compounds has been assessed using a topo II activity assay. The results indicate that ICRF-193 and bufalin effectively inhibit topo II activity in AA8 and EM9 cell lines. ICRF-193 induced a moderate increase in the frequency of SCEs in both types of cells, while bufalin did not modify the level of SCEs in any of them. The results are discussed taking into account the apparently unlike mechanisms of inhibition of topo II by ICRF-193 and bufalin.