Santiago Mateos

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.

The use of DNA double strand break quantification in radiotherapy

DNA double-strand breaks (DSB) are an important direct consequence of treating cells with ionizingradiation. A variety of evidence points toward DSBs being the key damage type linked to radiation-induced lethality. In particular, the link between DSB and chromosome breakage, which in turn closely correlates with cell death in some cell types, is strongly supportive of this concept. There has been much interest in the possibility of using measures of strand breaks as a pretreatment test of radiation response. This has largely been in the context of assessing inherent cellular sensitivity through damage induction or repair parameters. A number of studies have produced hopeful results, but overall there has been no parameter that can reliably predict radiosensitivity. This may be due to the inadequacies of the assays, but it is more likely to reflect the fact that the radiosensitivity of cells is dictated by a whole series of events; alterations in many of these can alter the overall response. In addition, it is now recognized that cell-signalling pathways form an essential part of the cellular response to damage, and these can be triggered by damage other than DSB. It is therefore possible that while DSBs are clearly important--and they may be the single most important lesion in some types--other damage types may be significant triggers of cell death pathways after ionizing radiation treatment.

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.

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.

Variation in sensitizing effect of caffeine in human tumour cell lines after γ-irradiation

Abstract Background and purpose : We have investigated whether the protective role of the G2 checkpoint has increasing importance when the p53-dependent G1 checkpoint is inactivated. Materials and methods: We have studied the differential effect of caffeine by clonogenic assays and flow cytometry in three human tumour cell lines with different functionality of p53 protein. Results : The radiosensitizing effect of caffeine (2 mM) expressed itself as a significant decrease in surviving fraction at 2 Gy and a significant increase in α-values in RT112 and TE671, both with non-functional p53. However, no radiosensitizing effect was seen in cells with a normal p53 function (MCF-7 BUS). Two millimoles of caffeine also caused important changes in the cell cycle progression after irradiation. MCF-7 BUS showed a G1 arrest after irradiation and an early G2 arrest but those cells that reached the second G2 did not arrest significantly. In contrast, TE671 exhibited radiosensitization by caffeine, no G1 arrest, a G2 arrest in those cells irradiated in G2, no significant accumulation in the second G2 but an overall delay in release from the first cell cycle, which could be abrogated by caffeine. RT112 was similar to TE671 except that the emphasis in a G2 arrest was shifted from the block in cells irradiated in G2 to those irradiated at other cell cycle phases. Conclusion : The data presented confirm that p53 status can be a significant determinant of the efficacy of caffeine as radiosensitizer in these tumour cell lines, and document the importance of the G2 checkpoint in this effect.

DNA double-strand break rejoining in xrs5 cells is more rapid in the G2 than in the G1 phase of the cell cycle

The radiosensitive xrs5 mutant cell line of CHO K1 shows an overall deficiency in DNA double-strand break (dsb) rejoining. However, xrs5 paradoxically shows an apparently normal rate of disappearance of chromatid breaks with time, the kinetics of which is thought to reflect the underlying rejoining of dsb. Nevertheless the yield of chromatid breaks is elevated by four-fold in xrs5. A possible explanation of the paradox might be that xrs5 is proficient in rejoining dsb in the G2 phase of the cell cycle but converts a higher number of dsb into chromatid breaks. In order to test this we have measured the rejoining of dsb in partially synchronised G2 xrs5 cells and compared the kinetics with those of cells synchronised in the G1 phase. Synchronisation of cells was achieved in G2 by release of cells from an aphidicolin block, and in G1 by staurosporine block. Cell synchrony was monitored by cytofluorometry and showed typically a 67% synchronisation of G2 cells and a 91% synchronisation of G1 cells. Rejoining of dsb was measured using neutral filter elution at pH 9.6. G2 cells showed a two-component kinetic with t1/2 values of 9 min and 3.6 h for dsb rejoining. Corresponding t1/2 values for G1 cells were 15 min and approximately 8.8 h. The t1/2 value of 3.6 h found for dsb rejoining in G2 cells is similar to a previously published value for asynchronous parental CHO K1 cells of approximately 4 h. The kinetics of chromatid break rejoining was measured in both xrs5 and CHO K1 following a dose of 0.75 Gy. The kinetics were found to be similar (t1/2 = 2.4 h) in the two cell lines, as previously reported using an equiclastogenic dose.

Selective cytotoxic activity of new lipophilic hydroxytyrosol alkyl ether derivatives.

Recent data suggest that hydroxytyrosol, a phenolic compound of virgin olive oils, has anticancer activity. This communication reports the synthesis of decyl and hexadecyl hydroxytyrosyl ethers, as well as the cytotoxic activity of hydroxytyrosol and a series of seven hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 non-malignant lung fibroblasts. Hydroxytyrosyl dodecyl ether (HTDE) showed the highest selective cytotoxicity, and possible mechanisms of action were investigated; results suggest that HTDE can moderately inhibit glycolysis, induce oxidative stress, and cause DNA damage in A549 cells. The combination of HTDE with the anticancer drug 5-fluorouracil induced a synergistic cytotoxicity in A549 cancer cells but not in non-malignant MRC5 cells. HTDE also displayed selective cytotoxicity against MCF7 breast cancer cells versus MCF10 normal breast epithelial cells in the 1-30 μM range. These results suggest that the cytotoxicity of HTDE is more potent and selective than that of parent compound hydroxytyrosol.

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 hydroalcoholic extract from the leaves of Nerium oleander inhibits glycolysis and induces selective killing of lung cancer cells.

Recent evidence suggests that cardiac glycosides might be used for the treatment of cancer. The ornamental shrub Nerium oleander has been used in traditional medicine for treating several disorders including cancer, and extracts from the leaves of this plant have already entered phase I clinical trials. In this communication, we have prepared a hydroalcoholic extract from the leaves of Nerium oleander (containing 4.75 ± 0.32 % of cardenolides) and have assessed its cytotoxic activity in A549 lung cancer cells vs. MRC5 nonmalignant lung fibroblasts. The results showed that the cytotoxicity of the Nerium oleander extract against the cancer cell line was significantly higher than that against the nonmalignant cell line, with a potency and selectivity similar to those of the anticancer drug cisplatin. Pretreatment of A549 cells with the antioxidants N-acetylcysteine and catalase slightly prevented the cytotoxicity of the extract, therefore suggesting that the formation of reactive oxygen species participates in its cytotoxic activity but does not play a major role. Nerium oleander extract-induced cytotoxicity and DNA damage (gamma-H2AX focus formation) were slightly higher in cells lacking BRCA2 (deficient in homologous recombination repair) than in parental cells; this indicates that the induction of DNA damage may also play a role in the cytotoxicity of the extract. Nerium oleander extract induced a marked inhibition of glycolysis (glucose consumption and lactate production) in A549 cells, comparable to that of the glycolysis inhibitor dichloroacetate (currently in clinical development for cancer therapy). Because platinum compounds are widely used in the treatment of lung cancer, we tested the cytotoxicity of several combinations of cisplatin with the extract and found a moderate synergism when Nerium oleander extract was administered after cisplatin but a moderate antagonism when it was added before cisplatin. Our results suggest that extracts from Nerium oleander might induce anticancer effects in patients with lung cancer and support their possible advancement into phase II clinical trials for the treatment of this type of cancer.

The mycotoxin ochratoxin A inhibits DNA topoisomerase II and induces polyploidy in cultured CHO cells

Ochratoxin A (OTA), a known nephrotoxin and carcinogenic mycotoxin, was investigated to examine its effectiveness to induce cytotoxicity and DNA damage (Comet assay), as well as its possible inhibition of topoisomerase II (topo II) catalytic activity in cultured Chinese hamster ovary (CHO) cells. The analysis of OTA-induced DNA strand breaks as well as the flow cytometric assessment of polyploidy has provided evidence that is consistent with the idea of a mixed mode of action of the mycotoxin: in addition to its genotoxic activity, OTA may also interfere with chromosome distribution during cell division.