Zinc salts differentially modulate DNA damage in normal and cancer cells.

Zinc plays an essential role in a wide range of cellular processes, including defense against free radicals and maintaining genomic stability. The presence of zinc in some proteins is fundamental for their functioning as transcription factors. Little is known about interaction between zinc and DNA, which can be important in light of reports on the role of zinc in cancer transformation and sometimes contradictory character of these reports. In the present study we studied cyto- and genotoxicity of zinc sulfate (ZnSO(4)) in normal human lymphocytes and human myelogenous leukemia K562 cancer cells in the presence of zinc and hydrogen peroxide (H(2)O(2)). Zinc at concentrations from the range 10-1000 microM decreased the viability of cancer cells and this effect, especially for low concentrations of the element, was much more pronounced than in normal cells. Zinc did not induce DNA damage in normal cells, but did so in cancer cells. We observed a key difference between the action of zinc in normal and cancer cells in the presence of H(2)O(2), since the element exerted a protective effect against cyto- and geno-toxic action of H(2)O(2) in the former, whereas it increased such action in the latter. Zinc inhibited the repair of DNA damage induced by H(2)O(2) in cancer cells. The results suggest that zinc may protect normal cells against DNA-damaging action and increase this action in cancer cells, which indicates the dual action of this element in dependency of target cells and can be useful in cancer therapy.

STI571 reduces NER activity in BCR/ABL-expressing cells.

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.

ATR-Chk1 axis protects BCR/ABL leukemia cells from the lethal effect of DNA double-strand breaks.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C (MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (gamma-H2AX). gamma-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR-Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Nickel(II) affects poly(ADP-ribose) polymerase-mediated DNA repair in normal and cancer cells.

Nickel(II) can be genotoxic, but the mechanism of its genotoxicity is not fully understood and the process of DNA repair may be considered as its potential target. We studied the effect of nickel chloride on the poly(ADP-ribose) polymerase (PARP)-mediated repair of DNA damaged by gamma-radiation and idarubicin with the alkaline comet assay in normal and cancer cells. Our results indicate that nickel chloride at very low, non-cytotoxic concentration of 1 microM can affect PARP-mediated DNA repair of lesions evoked by idarubicin and gamma-radiation. We also suggest that in the quiescent lymphocytes treated with gamma-radiation, nickel(II) could interfere with DNA repair process independent of PARP.

Imatinib (STI571) inhibits DNA repair in human leukemia oncogenic tyrosine kinase-expressing cells.

BCR/ABL oncogene, as a result of chromosome aberration t(9;22), is the pathogenic principle of almost 95% of human chronic myeloid leukemia (CML). Imatinib (STI571) is a highly selective inhibitor of BCR/ABL oncogenic tyrosine kinase used in leukemia treatment. It has been suggested that BCR/ABL may contribute to the resistance of leukemic cells to drug and radiation through stimulation of DNA repair in these cells. To evaluate further the influence of STI571 on DNA repair we studied the efficacy of this process in BCR/ABL-positive and -negative cells using single cell electrophoresis (comet assay). In our experiments, K562 human chronic myeloid leukemia cells expressing BCR/ABL and CCRF-CEM human acute lymphoblastic leukemia cells without BCR/ABL expression were employed. The cells were exposed for 1 h at 37 degrees C to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 5 microM, mitomycin C (MMC) at 50 microM or to gamma-radiation at 15 Gy with or without a 24 h preincubation at 1 microM of STI571. The MTT cells survival after 4 days of culture showed that STI571 enhanced the cytotoxity of the examined compounds in the K562 line. Further it was found, that the inhibitor decreased the efficacy of DNA repair challenged by each agent, but only in the K562 expressing BCR/ABL. Due to the variety of DNA damage induced by the employed agents in this study we can speculate, that BCR/ABL may stimulate multiple pathways of DNA repair. These results extend our previous studies performed on BCR/ABL-transformed mouse cells onto human cells. It is shown that BCR/ABL stimulated DNA repair in human leukemia cells. In conclusion we report that STI571 was found to inhibit DNA repair and abrogate BCR/ABL-positive human leukemia cells therapeutic resistance.

[Eukaryotic DNA polymerases]. / Eukariotyczne polimerazy DNA.

Knowledge about eukaryotic DNA polymerases has increased considerably during recent years. Much have been learnt about both the structures and the functions of "classical" DNA polymerases alpha, beta, delta, epsilon and gamma. New DNA polymerases that possess very unusual functions have been identified. They are able to perform translesional synthesis, take part in somatic hypermutation and prevent some cancers. Much attention has also been devoted to the role of 3'-->5' exonuclease activity in the accuracy of DNA synthesis. On the other hand, it have been shown that there are also negative aspects of the activity of DNA polymerases. Lack of some DNA polymerases or even their altered functions may lead to carcinogenesis and accelerate the process of ageing.

Non-homologous DNA end joining repair in normal and leukemic cells depends on the substrate ends.

Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5' complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5' overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes.

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 microM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H(2)O(2)-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.

Genotoxicity of acrylamide in human lymphocytes.

Acrylamide is used in the industry and can be a by-product in a high-temperature food processing. It is reported to interact with DNA, but the mechanism of this interaction is not fully understood. In the present study, we investigated the DNA-damaging potential of acrylamide (ACM) in normal human lymphocytes using the alkaline-, neutral- and 12.1 versions of the comet assay and pulsed-field gel electrophoresis. We also investigated effect of acrylamide on caspase-3 activity as well as its influence on the repair process of hydrogen peroxide-induced DNA damage. Acrylamide at 0.5-50 microM induced mainly alkali-labile sites. This damage was repaired during a 60-min repair incubation. Post-treatment of the damaged DNA with repair enzymes: thymine glycol DNA N-glycosylase (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II (Alk A), recognizing alkylated bases, caused an increase in the extent of DNA damage, indicating the induction of oxidative and alkylative DNA base modifications by acrylamide. Pre-treatment of the lymphocytes with N-tert-butyl-alpha-phenylnitrone (PBN), a spin trap, as well as vitamins C and E decreased the DNA-damaging effect of acrylamide, which suggest that free radicals/reactive oxygen species may be involved in this effect. Acrylamide impaired the repair of DNA damaged by hydrogen peroxide and increased the activity of caspase-3, which may indicate its potential to induce apoptosis. Our results suggest that acrylamide may exert a wide spectrum of diverse effects on DNA of normal cells, including mostly DNA base modifications and apoptosis. Acrylamide may also impair DNA repair. Free radicals may underline these effects and some dietary antioxidants can be considered as protective agents against genotoxic action of acrylamide. As normal lymphocytes contain cyp2e1 and P450, engaged in the bioactivation of ACM to glicidamide it is uncertain whether acrylamide causes all of measured effect per se or this is the result of the action of its metabolites.

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571.

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.

Free radical scavengers can modulate the DNA-damaging action of alloxan.

Alloxan can generate diabetes in experimental animals and its action can be associated with the production of free radicals. It is therefore important to check how different substances often referred to as free radical scavengers may interact with alloxan, especially that some of these substance may show both pro- and antioxidant activities. Using the alkaline comet assay we showed that alloxan at concentrations 0.01-50 microM induced DNA damage in normal human lymphocytes in a dose-dependent manner. Treated cells were able to recover within a 120-min incubation. Vitamins C and E at 10 and 50 microM diminished the extent of DNA damage induced by 50 microM alloxan. Pre-treatment of the lymphocytes with a nitrone spin trap, alpha-(4-pyridil-1-oxide)- N-t-butylnitrone (POBN) or ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which mimics glutathione peroxides, reduced the alloxan-evoked DNA damage. The cells exposed to alloxan and treated with formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results confirmed that free radicals are involved in the formation of DNA lesions induced by alloxan. The results also suggest that alloxan can generate oxidized DNA bases with a preference for purines and contribute to their alkylation.

Analysis of the G/C polymorphism in the 5'-untranslated region of the RAD51 gene in breast cancer.

The breast cancer suppressor proteins BRCA1 and BRCA2 interact with RAD51, a protein essential for maintaining genomic stability by playing a central role in homology-dependent recombinational repair of the DNA double-strand breaks. Therefore, genetic variability in the RAD51 gene may contribute to the appearance and/or progression of breast cancer. A single nucleotide polymorphism in the 5'- untranslated region of RAD51 (a G to C substitution at position 135, the G/C polymorphism) is reported to modulate breast cancer risk. We investigated the distribution of genotypes and frequency of alleles of the G/C polymorphism in breast cancer. Tumor tissues were obtained from postmenopausal women with node-negative and node-positive breast carcinoma with uniform tumor size. Blood samples from age matched healthy women served as control. The G/C polymorphism was determined by PCR-based MvaI restriction fragment length polymorphism. The distribution of the genotypes of the G/C polymorphism did not differ significantly (P > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no differences in the genotype distribution and allele frequencies between node-positive and node-negative patients. There were no significant differences between distributions of the genotypes in subgroups assigned to histological grades according to Scarf-Bloom-Richardson criteria and the distribution predicted by Hardy-Weinberg equilibrium (P > 0.05). Our study implies that the G/C polymorphism of the RAD51 gene may not be directly involved in the development and/or progression of breast cancer and so it may not be useful as an independent marker in this disease.