In Vitro Anticancer Activity and Mechanism of Action of an Aziridinyl Galactopyranoside.

We recently screened a series of new aziridines ß-D-galactopyranoside derivatives for selective anticancer activity and identified 2-methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-ß-D-galactopyranoside (AzGalp) as the most promising compound. In this article, we explore the possible mechanisms involved in the cytotoxicity of this aziridine and evaluate its selective anticancer activity using cancer cells and normal cells from a variety of tissues. Our data show that AzGalp induces DNA damage (comet assay). Cells deficient in the nucleotide excision repair (NER) pathway were hypersensitive to the cytotoxicity of this compound. These results suggest that AzGalp induces bulky DNA adducts, and that cancer cells lacking a functional NER pathway may be particularly vulnerable to the anticancer effects of this aziridine. Several experiments revealed that neither the generation of oxidative stress nor the inhibition of glycolysis played a significant role in the cytotoxicity of AzGalp. Combinations of AzGalp with oxaliplatin or 5-fluorouracil slightly improved the ability of both anticancer drugs to selectively kill cancer cells. AzGalp also showed selective cytotoxicity against a panel of malignant cells versus normal cells; the highest selectivity was observed for two acute promyelocytic leukemia cell lines. Additional preclinical studies are necessary to evaluate the anticancer potential of AzGalp.

The Cockayne syndrome protein B is involved in the repair of 5-AZA-2'-deoxycytidine-induced DNA lesions.

The Cockayne Syndrome Protein B (CSB) plays an essential role in Transcription-Coupled Nucleotide Excision Repair (TC-NER) by recruiting repair proteins once transcription is blocked with a DNA lesion. In fact, CSB-deficient cells are unable to recover from transcription-blocking DNA lesions. 5-Aza-2'-deoxycytidine (5-azadC) is a nucleoside analogue that covalently traps DNA methyltransferases (DNMTs) onto DNA. This anticancer drug has a double mechanism of action: it reverts aberrant hypermethylation in tumour-suppressor genes, and it induces DNA damage. We have recently reported that Homologous Recombination and XRCC1/PARP play an important role in the repair of 5-azadC-induced DNA damage. However, the mechanisms involved in the repair of the DNMT adducts induced by azadC remain poorly understood. In this paper, we show for the first time the importance of CSB in the repair of azadC-induced DNA lesions. We propose a model in which CSB initiates a signalling pathway to repair transcription blocks induced by incorporated 5-azadC. Indeed, CSB-deficient cells treated with 5-azadC show a delay in the repair of trapped DNMT1, increased levels of DNA damage and reduced survival.

Selective cytotoxic activity and DNA damage by an epoxyalkyl galactopyranoside.

Preclinical Research & Development Several clinically useful anticancer drugs selectively kill cancer cells by inducing DNA damage; the genomic instability and DNA repair defects of cancer cells make them more vulnerable than normal cells to the cytotoxicity of DNA-damaging agents. Because epoxide-containing compounds can induce DNA damage, we have used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the selective cytotoxicity of three epoxyalkyl galactopyranosides against A549 lung cancer cells and MRC-5 lung normal cells. Compound (2S,3S)-2,3-epoxydecyl 4,6-O-(S)-benzylidene-ß-d-galactopyranoside (EDBGP) showed the highest selective anticancer activity and was selected for mechanistic studies. After observing that EDBGP induced cellular DNA damage (comet assay), we found that cells deficient in nucleotide excision repair were hypersensitive to the cytotoxicity of this compound; this suggests that EDBGP may induce bulky DNA adducts. EDBGP did not inhibit glycolysis (glucose consumption and lactate production). Pretreatment of lung cancer cells with several antioxidants did not reduce the cytotoxicity of EDBGP, thereby indicating that reactive oxygen species do not participate in the anticancer activity of this compound. Finally, EDBGP was screened against a panel of cancer cells and normal cells from several tissues, including three genetically modified skin fibroblasts with increasing degree of malignancy. Our results suggest that epoxyalkyl galactopyranosides are promising lead compounds for the development of new anticancer agents.

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination.

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It's well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.

Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid.

Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee.

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.

Alpha, beta-unsaturated lactones 2-furanone and 2-pyrone induce cellular DNA damage, formation of topoisomerase I- and II-DNA complexes and cancer cell death.

The alpha, beta-unsaturated lactones 2-furanone and 2-pyrone are part of the chemical structure of a variety of naturally occurring compounds (e.g., cardenolides, bufadienolides, acetogenins, coumarins, and food-flavoring furanones), some of which have shown anticancer activity and/or DNA damaging effects. Here we report that 2-furanone and 2-pyrone induce cellular DNA damage (assessed by the comet assay and the gamma-H2AX focus assay) and the formation of topoisomerase I- and topoisomerase II-DNA complexes in cells (visualized and quantified in situ by the TARDIS assay). Cells mutated in BRCA2 (deficient in homologous recombination repair) were significantly hypersensitive to the cytotoxic activity of 2-pyrone, therefore suggesting that BRCA2 plays an important role in the repair of DNA damage induced by this lactone. Both lactones were cytotoxic in A549 lung cancer cells at lower concentrations than in MRC5 non-malignant lung fibroblasts. The possible involvement of 2-furanone and 2-pyrone in the anticancer and DNA-damaging activities of compounds containing these lactones is discussed.

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.

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.

The DNA topoisomerase II catalytic inhibitor merbarone is genotoxic and induces endoreduplication.

In the last years a number of reports have shown that the so-called topoisomerase II (topo II) catalytic inhibitors are able to induce DNA and chromosome damage, an unexpected result taking into account that they do not stabilize topo II-DNA cleavable complexes, a feature of topo II poisons such as etoposide and amsacrine. Merbarone inhibits the catalytic activity of topo II by blocking DNA cleavage by the enzyme. While it was first reported that merbarone does not induce genotoxic effects in mammalian cells, this has been challenged by reports showing that the topo II inhibitor induces efficiently chromosome and DNA damage, and the question as to a possible behavior as a topo II poison has been put forward. Given these contradictory results, and the as yet incomplete knowledge of the molecular mechanism of action of merbarone, in the present study we have tried to further characterize the mechanism of action of merbarone on cell proliferation, cell cycle, as well as chromosome and DNA damage in cultured CHO cells. Merbarone was cytotoxic as well as genotoxic, inhibited topo II catalytic activity, and induced endoreduplication. We have also shown that merbarone-induced DNA damage depends upon ongoing DNA synthesis. Supporting this, inhibition of DNA synthesis causes reduction of DNA damage and increased cell survival.

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.

Guanidine-reactive agent phenylglyoxal induces DNA damage and cancer cell death.

BACKGROUND: DNA-damaging compounds (e.g., alkylating agents, cytotoxic antibiotics and DNA topoisomerase poisons) are the most widely used anticancer drugs. The inability of tumor cells to properly repair some types of DNA damage may explain why specific DNA-damaging drugs can selectively kill tumor cells. Phenylglyoxal is a dicarbonyl compound known to react with guanidine groups such as that of the DNA base guanine, therefore suggesting that phenylglyoxal could induce DNA damage and have anticancer activity. METHODS: Cellular DNA damage was measured by the alkaline comet assay and the γH2AX focus assay. Formation of topoisomerase I- and topoisomerase II-DNA complexes was assessed by the TARDIS assay, an immunofluorescence technique that employs specific antibodies to DNA topo I or topo II to detect the protein covalently bound to the DNA in individual cells. Cell growth inhibition and cytotoxicity were determined by XTT, MTT and clonogenic assays. Apoptosis was assessed by the Annexin V flow cytometry assay. RESULTS: Phenylglyoxal induced cellular DNA damage and formation of high levels of topoisomerase I- and topoisomerase II-DNA complexes in cells. These topoisomerase-DNA complexes were abolished by catalase pretreatment and correlated well with the induction of apoptosis. Phenylglyoxal-induced cell death was partially prevented by catalase pretreatment and was higher in lung cancer cells (A549) than in normal lung fibroblasts (MRC5). Mammalian cell lines defective in nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ) were more sensitive to phenylglyoxal than parental cells; this suggests that phenylglyoxal may induce bulky distortions in the shape of the DNA double helix (which are repaired by the NER pathway) and DNA double-strand breaks (which are repaired by HR and NHEJ). CONCLUSION: This report shows that phenylglyoxal is a new DNA-damaging agent with anticancer activity, and suggests that tumor cells with defects in NER, HR and NHEJ may be hypersensitive to the cytotoxic activity of phenylglyoxal.

The role of the DNA hypermethylating agent Budesonide in the decatenating activity of DNA topoisomerase II.

Catenations between sister chromatids result from DNA replication and must be resolved to ensure proper chromatid segregation in mitosis. Functionally active Topoisomerase II (Topo II), through its mechanism of concerted breaking and rejoining of double stranded DNA, is required to carry out this fundamental process. In previous studies we have shown that modifications in DNA sequence by halogenated pyrimidines and by the demethylating agent 5-azacytidine leads to malfunction of Topo II that results in an increased yield of endorreduplicated cells as a result of segregation failure. In the present work we have evaluated the possible influence of the methylating agent Budesonide to modify the frequency of endoreduplicated cells in AA8 Chinese hamster cell population. Our results seem to indicate that when Budesonide was administered for two consecutive cell cycles did induce an increase in the yield of endoreduplicated cells, as previously observed for the hypomethylating agent 5-azaC. We have also examined the possible relationship between extensive hypermethylation induced by Budesonide in DNA and stabilization of cleavable complexes by m-AMSA. Taken as a whole, our results show that the degree of methylation in DNA correlates with the effectiveness of m-AMSA to stabilize the Topo II-DNA complexes and to induce DNA cleavage. These findings evidence for the first time the functional importance of DNA hyper- and hypomethylation changes as epigenetic factors able to modulate Topo II activity for proper chromosome segregation.

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.

Cytotoxicity and mitotic alterations induced by non-genotoxic lithium salts in CHO cells in vitro.

Aneugenic compounds are able to cause chromosome missegregation during mitosis which results in aneuploidy in cells that are able to survive. Aneuploidy is considered a key early condition in the progression from a normal cell into a cancerous cell. The possible toxicity of therapeutic lithium has raised concern because lithium salts are currently widely prescribed as an efficient treatment of manic-depressive disorders and numerous undesirable side effects of long-term treatment have been reported to date. We have observed a dose-dependent cytotoxic effect of both Li2CO3 and LiCl in AA8 CHO cells, while no genotoxic damage was detected. Mitotic abnormalities such as multipolar anaphases and lagging chromosomes leading to the presence of micronuclei in the next interphase were frequently observed after treatment with lithium salts. Thus, the effectiveness of both lithium salts to induce alterations in the normal segregation of chromosomes could be ascribed to interference with proteins involved in the organization and/or function of the mitotic apparatus.

DNA demethylation protects from cleavable complex stabilization and DNA strand breakage induced by the topoisomerase type I inhibitor camptothecin.

Methylation of cytosine in CpG sequences of the DNA in mammalian cells is an epigenetic feature regulated very exactly that bears importance for events like gene expression, DNA replication, transcription and genetic imprinting. Changes in the DNA methylation pattern, both hypermethylation and hypomethylation, have been observed in the carcinogenic process. These changes, in general, influence the DNA conformation in such a way that certain proteins are disturbed in their interactions with the molecule. In this paper, we investigated in cultured Chinese hamster ovary cells the influence of hypomethylation induced by the substitution of 5-aza-2'-deoxycytidine for cytidine in DNA on topoisomerase type I (topo I) function, measured as the capacity of the enzyme inhibitor camptothecin (CPT) to stabilize the topoisomerase-DNA complexes and to induce DNA strand breakage. Our results demonstrate that the degree of methylation in DNA correlates with the effectiveness of CPT to stabilize the topo I-DNA complexes and to induce DNA cleavage. A protective effect of hypomethylation, as a whole, has been observed.

The high rate of endoreduplication in the repair deficient CHO mutant EM9 parallels a reduced level of methylated deoxycytidine in DNA.

It has been recently proposed that hypomethylation of DNA induced by 5-azacytidine (5-azaC) leads to reduced chromatid decatenation that ends up in endoreduplication, most likely due to a failure in topo II function [S. Mateos, I. Domínguez, N. Pastor, G. Cantero, F. Cortés, The DNA demethylating 5-azaC induces endoreduplication in cultured Chinese hamster cells, Mutat. Res. 578 (2005) 33-42]. The Chinese hamster mutant cell line EM9 has a high spontaneous frequency of endoreduplication as compared to its parental line AA8. In order to see if this is related to the degree of DNA methylation, we have investigated the basal levels of both endpoints in AA8 and EM9, as well as the effect of extensive 5-azaC-induced demethylation on the production of endoreduplication. Based on the correlation between the levels of DNA methylation and indices of endoreduplication we propose that genomic DNA hypomethylation in EM9 cell line is probably an important factor that bears significance in relation to the high basal level of endoreduplication observed in this cell line.

Genotoxicity detected in wild mice living in a highly polluted wetland area in south western Spain.

A field study was carried out in the south of the Iberian Peninsula in an industrial area in the neighbourhood of Huelva city, SW Spain, and in a natural area (Doñana National Park) for comparison, to estimate the genetic risk induced by environmental pollution in wild mice. Genotoxic effects in a sentinel organism, the Algerian mice (Mus spretus) free living in the industrial area were compared with animals of the same species living in the natural protected area. The single cell gel electrophoresis, or Comet assay, was performed as a genotoxicity test in peripheral blood of mice. Our results clearly show that mice free living in the contaminated area bear a high burden of genetic damage as compared with control individuals. The results suggest that the assessing of genotoxicity levels by the Comet assay in wild mice can be used as a valuable test in pollution monitoring and environmental conservation.

Protection of halogenated DNA from strand breakage and sister-chromatid exchange induced by the topoisomerase I inhibitor camptothecin.

The fundamental nuclear enzyme DNA topoisomerase I (topo I), cleaves the double-stranded DNA molecule at preferred sequences within its recognition/binding sites. We have recently reported that when cells incorporate halogenated nucleosides analogues of thymidine into DNA, it interferes with normal chromosome segregation, as shown by an extraordinarily high yield of endoreduplication, and results in a protection against DNA breakage induced by the topo II poison m-AMSA [F. Cortés, N. Pastor, S. Mateos, I. Domínguez, The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes, DNA Repair 2 (2003) 719-726; G. Cantero, S. Mateos, N. Pastor; F. Cortés, Halogen substitution of DNA protects from poisoning of topoisomerase II that results in DNA double-strand breaks (DSBs), DNA Repair 5 (2006) 667-674]. In the present investigation, we have assessed whether the presence of halogenated nucleosides in DNA diminishes the frequency of interaction of topo I with DNA and thus the frequency with which the stabilisation of cleavage complexes by the topo I poison camptothecin (CPT) takes place, in such a way that it protects from chromosome breakage and sister-chromatid exchange. This protective effect is shown to parallel a loss in halogen-substituted cells of the otherwise CPT-increased catalytic activity bound to DNA.

Modulation of radiation response by inhibiting topoisomerase II catalytic activity.

Due to the essential role played by DNA topoisomerases (topos) in cell survival, the use of topoisomerase inhibitors as chemotherapeutic drugs in combination with radiation has become a common strategy for the treatment of cancer. Catalytic inhibitors of these enzymes would be promising to improve the effectiveness of radiation and therefore, it appears reasonable to incorporate them in combined modality trials. In this work, we have investigated the capacity of both ICRF-193 and Aclarubicin (ACLA), two catalytic inhibitors of topoisomerase II (Topo II), to modulate radiation response in Chinese hamster V79 cell line and its radiosensitive mutant irs2. We also have explored potential mechanisms underlying these interactions. Experiments were performed in the presence and absence of either ICRF-193 or ACLA, and topo II activity was measured using an assay based upon decatenation of kinetoplast DNA (kDNA). For the combined experiments cells were incubated for 3 h in the presence of various inhibitor concentrations and irradiated 30 min prior to the end of treatments and cell survival was determined by clonogenic assay. DNA-damaging activity was measured by single-cell gel electrophoresis. Our results demonstrate that combinations of catalytic inhibitors of topo II and radiation produce an increase in cell killing induced by ionising radiation. The mechanism of radiation enhancement may involve a direct or indirect participation of topo II in the repair of radiation-induced DNA damage.