Atypical genotoxicity of carcinogenic nickel(II): Linkage to dNTP biosynthesis, DNA-incorporated rNMPs, and impaired repair of TOP1-DNA crosslinks.

Cancer is a genetic disease requiring multiple mutations for its development. However, many carcinogens are DNA-unreactive and nonmutagenic and consequently described as nongenotoxic. One of such carcinogens is nickel, a global environmental pollutant abundantly emitted by burning of coal. We investigated activation of DNA damage responses by Ni and identified this metal as a replication stressor. Genotoxic stress markers indicated the accumulation of ssDNA and stalled replication forks, and Ni-treated cells were dependent on ATR for suppression of DNA damage and long-term survival. Replication stress by Ni resulted from destabilization of RRM1 and RRM2 subunits of ribonucleotide reductase and the resulting deficiency in dNTPs. Ni also increased DNA incorporation of rNMPs (detected by a specific fluorescent assay) and strongly enhanced their genotoxicity as a result of repressed repair of TOP1-DNA protein crosslinks (TOP1-DPC). The DPC-trap assay found severely impaired SUMOylation and K48-polyubiquitination of DNA-crosslinked TOP1 due to downregulation of specific enzymes. Our findings identified Ni as the human carcinogen inducing genome instability via DNA-embedded ribonucleotides and accumulation of TOP1-DPC which are carcinogenic abnormalities with poor detectability by the standard mutagenicity tests. The discovered mechanisms for Ni could also play a role in genotoxicity of other protein-reactive carcinogens.

Monoubiquitinated H2B, a Main Chromatin Target of Formaldehyde, Is Important for S-Phase Checkpoint Signaling and Genome Stability.

Formaldehyde (FA) is a human carcinogen with ubiquitous environmental exposures and significant endogenous formation. Genotoxic activity of FA stems from its reactivity with DNA-NH2 groups. Histone lysines are another source of aldehyde-reactive amino groups in chromatin, however, chromatin/histone damage responses to FA and their biological significance are poorly understood. We examined histone posttranslational modifications in FA-treated human lung cells and found that the majority of the most prominent small lysine modifications associated with active or inactive chromatin were unchanged. FA moderately decreased H3K9 and H3K27 acetylation and H2A-K119 monoubiquitination but caused surprisingly severe losses of H2B-K120 monoubiquitination, especially in primary and stem-like cells. H2Aub1 decreases reflected its slower ubiquitination linked to a lower ubiquitin availability due to K48-polyubiquitination of FA-damaged proteins. Depletion of H2Bub1 resulted from its rapid deubiquitination in part by ATXN7L3-associated deubiquitinases and was independent on DNA damage signaling, indicating a direct chromatin damage response. Manipulations of H2Bub1 abundance showed that it was important for robust ATM and ATR signaling, efficient S-phase checkpoint, and suppression of mitotic transmission of unreplicated DNA and formation of micronuclei. Our findings identified H2B deubiquitination as a major FA-induced chromatin damage response that regulates S-phase checkpoint signaling and genome stability.

Sensitive Detection of Histones and γ-H2AX by Immunoblotting: Problems and Solutions.

Histones and their posttranslational modifications (PTMs) are critical regulators of gene expression. Differentiation, environmental stressors, xenobiotics, and major human diseases cause significant changes in histone variants and PTMs. Western blotting is the mainstay methodology for detection of histones and their PTMs in the majority of studies. Surprisingly, despite their high abundance in cells, immunoblotting of histones typically involves loading of large protein amounts that are normally used for detection of sparse cellular proteins. We systematically examined technical factors in the Western-blotting-based detection of human histones with >30 antibodies. We found that under multiple protein transfer conditions, many histone epitopes on polyvinylidene fluoride (PVDF) membranes had a very low antibody accessibility, which was dramatically increased by the addition of a simple denaturation step. Denaturation of membrane-bound proteins also enhanced the specificity of some histone antibodies. In comparison to standard PVDF membranes, the sensitivity of histone detection on standard nitrocellulose membranes was typically much higher, which was further increased by the inclusion of the same denaturation step. Optimized protocols increased by >100-times detection sensitivity for the genotoxic marker γ-H2AX with two monoclonal antibodies. The impact of denaturation and nitrocellulose use varied for different histones, but for each histone, it was generally similar for antibodies targeting N-terminal and C-terminal regions. In summary, denaturation of membrane-bound histones strongly improves their detection by Westerns, resulting in more accurate measurements and permitting analyses with small biological samples.

Vulnerability of HIF1α and HIF2α to damage by proteotoxic stressors.

Transcription factors HIF1 and HIF2 are central regulators of physiological responses to hypoxia and important for normal functioning of tissue stem cells and maintenance of healthy microvasculature. Even modest decreases in HIF activity exert detrimental effects in tissues although it is unclear what factors can directly impair HIF functions. We hypothesized that the presence of functionally important, large intrinsically disordered regions in HIFα subunits of HIF1/2 could make them structurally vulnerable to protein-damaging conditions. We found that common protein-damaging agents such as endogenous/exogenous aldehydes (formaldehyde, acetaldehyde), moderate heat shock and the environmental toxicant cadmium cause inactivation of HIF1 and HIF2 due to structural damage to HIFα subunits. Aldehydes triggered a rapid and selective depletion of HIF1α and HIF2α, which occurred as a result of enhanced binding of Pro-hydroxylated/VHL-ubiquitinated HIFα by 26S proteasomes. In the absence of proteasomal degradation, aldehyde-damaged HIF1 and HIF2 were transactivation defective and HIFα subunits became insoluble/denatured when their VHL-mediated ubiquitination was blocked. Protein damage by heat shock and cadmium resulted in the insolubility of Pro-nonhydroxylated HIFα. Thus, VHL-dependent ubiquitination of damaged HIFα also acts as means to maintain their solubility, permitting capture by proteasomes. The observed control of HIFα stability at the point of proteasome binding may extend to several posttranslational modifications that occur in the conformationally flexible regions of these proteins. Our findings revealed vulnerability of HIF1 and HIF2 to direct inactivation by protein-damaging agents, which helps understand their tissue injury mechanisms and favorable responses of hypoxic tumors to hyperthermia.

Monoubiquitinated γ-H2AX: Abundant product and specific biomarker for non-apoptotic DNA double-strand breaks.

DNA double-strand breaks (DSBs) are a highly toxic form of DNA damage produced by a number of carcinogens, drugs, and metabolic abnormalities. Involvement of DSBs in many pathologies has led to frequent measurements of these lesions, primarily via biodosimetry of S139-phosphorylated histone H2AX (γ-H2AX). However, γ-H2AX is also induced by some non-DSB conditions and abundantly formed in apoptosis, raising concerns about the overestimation of potential genotoxic agents and accuracy of DSB assessments. DSB-triggered γ-H2AX undergoes RNF168-mediated K13/K15 monoubiquitination, which is rarely analyzed in DSB/genotoxicity studies. Here we identified critical methodological factors that are necessary for the efficient detection of mono- (ub1) and diubiquitinated (ub2) γ-H2AX. Using optimized technical conditions, we found that γ-H2AX-ub1 was a predominant form of γ-H2AX in three primary human cell lines containing mechanistically distinct types of DSBs. Replication stress-associated DSBs also triggered extensive formation of γ-H2AX-ub1. For DSBs induced by oxidative damage or topoisomerase II, both γ-H2AX and γ-H2AX-ub1 showed dose-dependent increases whereas γ-H2AX-ub2 plateaued at low levels of breaks. Despite abundance of γ-H2AX, γ-H2AX-ub1,2 formation was blocked in apoptosis, which was associated with proteolytic cleavage of RNF168. Chromatin damage also caused only the production of γ-H2AX but not its ub1,2 forms. Our results revealed a major contribution of ubiquitinated forms to the overall γ-H2AX response and demonstrated the specificity of monoubiquitinated γ-H2AX as a biodosimeter of non-apoptotic DSBs.

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage.

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA.

Nickel-induced HIF-1α promotes growth arrest and senescence in normal human cells but lacks toxic effects in transformed cells.

Nickel is a human carcinogen that acts as a hypoxia mimic by activating the transcription factor HIF-1α and hypoxia-like transcriptomic responses. Hypoxia and elevated HIF-1α are typically associated with drug resistance in cancer cells, which is caused by increased drug efflux and other mechanisms. Here we examined the role of HIF-1α in uptake of soluble Ni(II) and Ni(II)-induced cell fate outcomes using si/shRNA knockdowns and gene deletion models. We found that HIF-1α had no effect on accumulation of Ni(II) in two transformed (H460, A549) and two normal human cell lines (IMR90, WI38). The loss of HIF-1α also produced no significant impact on p53-dependent and p53-independent apoptotic responses or clonogenic survival of Ni(II)-treated transformed cells. In normal human cells, HIF-1α enhanced the ability of Ni(II) to inhibit cell proliferation and cause a permanent growth arrest (senescence). Consistent with its growth-suppressive effects, HIF-1α was important for upregulation of the cell cycle inhibitors p21 (CDKN1A) and p27 (CDKN1B). Irrespective of HIF-1α status, Ni(II) strongly increased levels of MYC protein but did not change protein expression of the cell cycle-promoting phosphatase CDC25A or the CDK inhibitor p16. Our findings indicate that HIF-1α limits propagation of Ni(II)-damaged normal cells, suggesting that it may act in a tumor suppressor-like manner during early stages of Ni(II) carcinogenesis.

Variation in Extracellular Detoxification Is a Link to Different Carcinogenicity among Chromates in Rodent and Human Lungs.

Inhalation of soluble chromium(VI) is firmly linked with higher risks of lung cancer in humans. However, comparative studies in rats have found a high lung tumorigenicity for moderately soluble chromates but no tumors for highly soluble chromates. These major species differences remain unexplained. We investigated the impact of extracellular reducers on responses of human and rat lung epithelial cells to different Cr(VI) forms. Extracellular reduction of Cr(VI) is a detoxification process, and rat and human lung lining fluids contain different concentrations of ascorbate and glutathione. We found that reduction of chromate anions in simulated lung fluids was principally driven by ascorbate with only minimal contribution from glutathione. The addition of 500 µM ascorbate (∼rat lung fluid concentration) to culture media strongly inhibited cellular uptake of chromate anions and completely prevented their cytotoxicity even at otherwise lethal doses. While proportionally less effective, 50 µM extracellular ascorbate (∼human lung fluid concentration) also decreased uptake of chromate anions and their cytotoxicity. In comparison to chromate anions, uptake and cytotoxicity of respirable particles of moderately soluble CaCrO4 and SrCrO4 were much less sensitive to suppression by extracellular ascorbate, especially during early exposure times and in primary bronchial cells. In the absence of extracellular ascorbate, chromate anions and CaCrO4/SrCrO4 particles produced overall similar levels of DNA double-stranded breaks, with less soluble particles exhibiting a slower rate of breakage. Our results indicate that a gradual extracellular dissolution and a rapid internalization of calcium chromate and strontium chromate particles makes them resistant to detoxification outside the cells, which is extremely effective for chromate anions in the rat lung fluid. The detoxification potential of the human lung fluid is significant but much lower and insufficient to provide a threshold-type dose dependence for soluble chromates.

DNA damage and apoptosis in blood neutrophils of inflammatory bowel disease patients and in Caco-2 cells in vitro exposed to betanin.

Inflammatory bowel diseases (IBD) are chronic, relapsing, inflammatory disorders of the gastrointestinal tract, and continuing colonic inflammation is considered an important risk factor in the development of colorectal cancer. Our previous studies showed that beetroot (Beta vulgaris var. rubra) products and their major component betanin modulate the reactive oxygen species (ROS) production and DNA damage in 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated human polymorphonuclear neutrophils of healthy volunteers. The aim of the present study was to evaluate the effects of betanin on the oxidative DNA damage and apoptosis in neutrophils isolated from blood of patients with inflammatory bowel disease--ulcerative colitis (UC) and Crohn's disease (CD). The results were compared with those obtained in colon carcinoma-derived Caco-2 cells. Betanin treatment at the concentration of 100 µM for 24 h increased DNA damage assessed by comet assay in IBD patients' neutrophils. A similar effect although less pronounced was observed in Caco-2 cells. Treatment of Caco-2 cells with H2O2 caused a 4-fold increase of DNA strand breaks in comparison to untreated cells, but pre-treatment with betanin reduced DNA damage in these cells. Betanin also induced procaspase-3 cleavage and caspase-3 activity accompanied by the loss of mitochondrial transmembrane potential, indicating its pro-apoptotic activity. These results suggest that betanin may support mechanisms that lead to the release of ROS and apoptotic cell death. In this way betanin may exert anti-inflammatory and potentially cancer preventive activity.

Monitoring Cr intermediates and reactive oxygen species with fluorescent probes during chromate reduction.

Cr(VI) genotoxicity is caused by products of its reductive metabolism inside the cells. Reactive oxygen species (ROS) and Cr(V,IV) intermediates are potential sources of oxidative damage by Cr(VI). Here, we investigated seven fluorescent probes for the detection of ROS and non-ROS oxidants in Cr(VI) reactions with its main reducers. We found that Cr(V)-skipping metabolism of Cr(VI) by ascorbate in vitro gave no responses with all tested dyes, indicating nonreactivity of Cr(IV) and absence of ROS. Cr(VI) reduction with glutathione (GSH) or Cys strongly enhanced the fluorescence of dichlorofluorescein (DCF) and dihydrorhodamine 123 (DHR123) but produced minimal fluorescence with dihydroethidium and no increases with aminophenylfluorescein and CellRox Green, Orange, and Red. Several tests showed that Cr(VI)-thiol reactions lacked ROS and that Cr(V) caused oxidation of DCF and DHR123. DCF reacted only with free Cr(V), whereas DHR123 detected both the free Cr(V) and Cr(V)-GSH complex. We estimated that Cr(VI)-GSH reactions generated approximately 75% Cr(V)-GSH and 25% free Cr(V), whereas Cys reactions appeared to produce only free Cr(V). DHR123 measurements in H460 cells showed that reduction of Cr(VI) was complete within 20 min postexposure, but it lasted at least 1 h without GSH. Cells with restored ascorbate levels exhibited no DCF or DHR123 oxidation by Cr(VI). Overall, our results demonstrated that Cr(VI) metabolism with its biological reducers lacked ROS and that DHR123 and DCF responses were indicators of total and free Cr(V), respectively. CellRox dyes, dihydroethidium and aminophenylfluorescein, are insensitive to Cr(V,IV) and can be used for monitoring ROS during coexposure to Cr(VI) and oxidants.

Chromium(VI) causes interstrand DNA cross-linking in vitro but shows no hypersensitivity in cross-link repair-deficient human cells.

Hexavalent chromium is a human carcinogen activated primarily by direct reduction with cellular ascorbate and to a lesser extent, by glutathione. Cr(III), the final product of Cr(VI) reduction, forms six bonds allowing intermolecular cross-linking. In this work, we investigated the ability of Cr(VI) to cause interstrand DNA cross-links (ICLs) whose formation mechanisms and presence in human cells are currently uncertain. We found that in vitro reduction of Cr(VI) with glutathione showed a sublinear production of ICLs, the yield of which was less than 1% of total Cr-DNA adducts at the optimal conditions. Formation of ICLs in fast ascorbate-Cr(VI) reactions occurred during a short reduction interval and displayed a linear dose dependence with the average yield of 1.3% of total adducts. In vitro production of ICLs was strongly suppressed by increasing buffer molarity, indicating inhibitory effects of ligand-Cr(III) binding on the formation of cross-linking species. The presence of ICLs in human cells was assessed from the impact of ICL repair deficiencies on Cr(VI) responses. We found that ascorbate-restored FANCD2-null and isogenic FANCD2-complemented cells showed similar cell cycle inhibition and toxicity by Cr(VI). XPA-null cells are defective in the repair of Cr-DNA monoadducts, but stable knockdowns of ERCC1 or XPF in these cells with extended time for the completion of cross-linking reactions did not produce any sensitization to Cr(VI). Our results together with chemical and steric considerations of Cr(III) reactivity suggest that ICL generation by chromate is probably an in vitro phenomenon occurring at conditions permitting the formation of Cr(III) oligomers.

HMGB1 Carried by Small Extracellular Vesicles Potentially Plays a Role in Promoting Acquired Middle Ear Cholesteatoma.

Cholesteatoma is a specific medical condition involving the abnormal, non-cancerous growth of skin-like tissue in the middle ear, potentially leading to a collection of debris and even infections. The receptor for advanced glycation (RAGE) and its ligand, high-mobility box 1 (HMGB1), are both known to be overexpressed in cholesteatoma and play a potential role in the pathogenesis of the disease. In this study, we investigated the role of small extracellular vesicles (sEVs) in carrying HMGB1 and inducing disease-promoting effects in cholesteatoma. No significant differences in the concentration of isolated sEVs in the plasma of cholesteatoma patients (n = 17) and controls (n = 22) were found (p > 0.05); however, cholesteatoma-derived sEVs carried significantly higher levels of HMGB1 (p < 0.05). In comparison to sEVs isolated from the plasma of controls, cholesteatoma-derived sEVs significantly enhanced keratinocyte proliferation and IL-6 production (p < 0.05), potentially by engaging multiple activation pathways including MAPKp44/p42, STAT3, and the NF-κB pathway. Thus, HMGB1(+) sEVs emerge as a novel factor potentially promoting cholesteatoma progression.

The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils.

The aim of this study was to evaluate the effect of betanin, one of the beetroot major components, on ROS production, DNA damage and apoptosis in human resting and stimulated with phorbol 12-myristate13-acetate polymorphonuclear neutrophils, one of the key elements of the inflammatory response. Incubation of neutrophils with betanin in the concentration range 2-500 µM resulted in significant inhibition of ROS production (by 15-46%, depending on the ROS detection assay). The antioxidant capacity of betanin was most prominently expressed in the chemiluminescence measurements. This compound decreased also the percentage of DNA in comet tails in stimulated neutrophils, but only at the 24 h time point. In resting neutrophils an increased level of DNA in comet tails was observed. Betanin did not affect the activity of caspase-3, in resting neutrophils, but significantly enhanced the enzyme activity in stimulated neutrophils. The western blot analysis showed, however, an increased level of caspase-3 cleavage products as a result of betanin treatment both in resting and stimulated neutrophils. The results indicate that betanin may be responsible for the effect of beetroot products on neutrophil oxidative metabolism and its consequences, DNA damage and apoptosis. The dose and time dependent effects on these processes require further studies.

NAD+ metabolism controls growth inhibition by HIF1 in normoxia and determines differential sensitivity of normal and cancer cells.

The hypoxia-induced transcription factor HIF1 inhibits cell growth in normoxia through poorly understood mechanisms. A constitutive upregulation of hypoxia response is associated with increased malignancy, indicating a loss of antiproliferative effects of HIF1 in cancer cells. To understand these differences, we examined the control of cell cycle in primary human cells with activated hypoxia response in normoxia. Activated HIF1 caused a global slowdown of cell cycle progression through G1, S and G2 phases leading to the loss of mitotic cells. Cell cycle inhibition required a prolonged HIF1 activation and was not associated with upregulation of p53 or the CDK inhibitors p16, p21 or p27. Growth inhibition by HIF1 was independent of its Asn803 hydroxylation or the presence of HIF2. Antiproliferative effects of hypoxia response were alleviated by inhibition of lactate dehydrogenase and, more effectively, by boosting cellular production of NAD+, which was decreased by HIF1 activation. In comparison to normal cells, various cancer lines showed several fold-higher expressions of NAMPT, which is a rate-limiting enzyme in the main biosynthetic pathway for NAD+. Inhibition of NAMPT activity in overexpressor cancer cells sensitized them to antigrowth effects of HIF1. Thus, metabolic changes in cancer cells, such as enhanced NAD+ production, create resistance to growth-inhibitory activity of HIF1 permitting manifestation of its tumor-promoting properties.Abbreviations: DMOG: dimethyloxalylglycine, DM-NOFD: dimethyl N-oxalyl-D-phenylalanine, NMN: ß-nicotinamide mononucleotide.

Assessment of training and selected factors on speed and quality of performing different tasks on the endoscopic simulator.

INTRODUCTION: One of the most significant challenges nowadays is to educate and predict the predispositions of young surgeons taking into consideration that every ability has its own learning curve. AIM: To determine the influence of selected factors and examine the shape and the length of the learning curve in performing simple tasks on an endoscopic simulator. MATERIAL AND METHODS: Twenty students took part in 4 training sessions with a one-week break between sessions. They were training 12 min and performed three tasks at every session on the endoscopic simulator. To identify whether selected factors influence the time of completing tasks, the participants were asked to fill in questionnaires. All participants also completed the Minnesota Manual Dexterity Test (MMDT) to assess hand-eye coordination. RESULTS: Our research reveals that regardless of activities performed in free time, the shape of the learning curve was logarithmic. Improvement after the fourth session ranged from 50% to 75%. Performing specific activities in the free time did not influence the results achieved on the simulator. No statistically significant correlation between MMDT results and the time to accomplish each task was found. CONCLUSIONS: This study has shown that the length of the learning curve of performing simple tasks is quite short and the shape is logarithmic. It suggests that more complex exercises should be included in the training programme.

p53 Activation by Cr(VI): A Transcriptionally Limited Response Induced by ATR Kinase in S-Phase.

Cellular reduction of carcinogenic chromium(VI) causes several forms of Cr-DNA damage with different genotoxic properties. Chromate-treated cultured cells have shown a strong proapoptotic activity of the DNA damage-sensitive transcription factor p53. However, induction of p53 transcriptional targets by Cr(VI) in rodent lungs was weak or undetectable. We examined Cr(VI) effects on the p53 pathway in human cells with restored levels of ascorbate that acts as a principal reducer of Cr(VI) in vivo but is nearly absent in standard cell cultures. Ascorbate-restored H460 and primary human cells treated with Cr(VI) contained higher levels of p53 and its Ser15 phosphorylation, which were induced by ATR kinase. Cr(VI)-stimulated p53 phosphorylation occurred in S-phase by a diffusible pool of ATR that was separate from the chromatin-bound pool targeting DNA repair substrates at the sites of toxic mismatch repair (MMR) of Cr-DNA adducts. Even when more abundantly present than after exposure to the radiomimetic bleomycin, Cr(VI)-stabilized p53 showed a much more limited activation of its target genes in two types of primary human cells. No increases in mRNA were found for nucleotide excision repair factors and a majority of proapoptotic genes. A weak transcription activity of Cr(VI)-upregulated p53 was associated with its low lysine acetylation in the regulatory C-terminal domain, resulting from the inability of Cr(VI) to activate ATM in ascorbate-restored cells. Thus, p53 activation by ascorbate-metabolized Cr(VI) represents a limited genome-protective response that is defective in upregulation of DNA repair genes and proapoptotic transcripts for elimination of damaged cells.

Vitamin C increases DNA breaks and suppresses DNA damage-independent activation of ATM by bleomycin.

Bleomycin is a redox-active drug with anticancer and other clinical applications. It is also frequently used as a tool in fundamental research on cellular responses to DNA double-strand breaks (DSBs). A conversion of bleomycin into its DNA-breaking form requires Fe, one-electron donors and O2. Here, we examined how a major biological antioxidant ascorbate (reduced vitamin C), which is practically absent in standard cell culture, impacts cellular responses to bleomycin. We found that restoration of physiological levels of vitamin C in human cancer cells increased their killing by bleomycin in 2D cultures and 3D tumor spheroids. Higher cytotoxicity of bleomycin occurred in cells with normal and shRNA-depleted p53. Cellular vitamin C enhanced the ability of bleomycin by produce DSBs, which was established by direct measurements of these lesions in three cell lines. Vitamin C-restored cancer cells also showed a higher sensitivity to killing by low-dose bleomycin in combination with inhibitors of DSB repair-activating ATM or DNA-PK kinases. The presence of ascorbate in bleomycin-treated cells suppressed a DSB-independent activation of the ATM-CHK2 axis by blocking superoxide radical. In vitro studies detected a greatly superior ability of ascorbate over other cellular reducers to catalyze DSB formation by bleomycin. Ascorbate was faster than other antioxidants in promoting two steps in activation of bleomycin. Our results demonstrate strong activation effects of vitamin C on bleomycin, shifting its toxicity further toward DNA damage and making it more sensitive to manipulations of DNA repair.

Immunohistochemical detection of N-homocysteinylated proteins in humans and mice.

N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.

Cytotoxic activity of 3,3',4,4',5,5'-hexahydroxystilbene against breast cancer cells is mediated by induction of p53 and downregulation of mitochondrial superoxide dismutase.

The phytochemical resveratrol, which is found in grapes and red wine, has been reported to have a variety of biological properties. It was shown in our previous research that introduction of additional hydroxyl groups into the stilbene structure increases the biological activity of resveratrol. In this study, the activity of 3,3',4,4',5,5'-hexahydroxystilbene (M8) was investigated in ZR-75-1, MDA-MB-231 and T47D human breast cancer cells. For evaluation of cytotoxic activity of M8, clonogenic and cell proliferation assays were used. The IC50 values obtained in the clonogenic assay were 0.846 microM for T47D, 8.53 microM for ZR-75-1 cells and 25.5 microM for MDA-MB-231, while IC50 values obtained in the cell proliferation assay were significantly higher: 90.1 microM, 98.4 microM, 127.8 microM for T47D, ZR-75-1 and MDA-MB-231 cells, respectively. Compound M8 caused the activation of caspase-8 in MDA-MB-231 cells (marker of extrinsic apoptotic pathway), while activities of caspase-9 (marker of intrinsic apoptotic pathway) and caspase-3 were increased in all 3 tested cell lines. Activation of caspase-9 and caspase-3 was connected with loss of mitochondrial potential and increase of p53, which could have an impact on downregulation of mitochondrial superoxide dismutase (MnSOD) seen in our experiments. MnSOD is a key enzyme providing antioxidative defense in mitochondria - the cellular center of reactive oxygen species' generation. Downregulation of MnSOD can therefore cause a significant decrease of antioxidant defense in cancer cells. An increase of oxidative stress conditions was suggested by loss of reduced glutathione in tested cells. Since cancer cells are usually under permanent oxidative stress, additional increased ROS generation as a result of the interaction of M8 with the mitochondrial respiratory chain and a decrease in oxidative defense can therefore be a promising method for selective elimination of cancer cells.

The effect of resveratrol, its naturally occurring derivatives and tannic acid on the induction of cell cycle arrest and apoptosis in rat C6 and human T98G glioma cell lines.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a potent chemopreventive and potentially cancer therapeutic agent. Since rapid metabolism limits resveratrol bioavailability, derivatives less prone to metabolic transformation are being sought and tested. We evaluated the effect of resveratrol, and its analogs (pterostilbene and 3,5,4'-trimethoxystilbene) along with tannic acid, on cell cycle and apoptosis in rat C6 and human T98G glioma cells. At concentration ranges both lower and higher than IC50 calculated based on MTT assay, all these polyphenols affected the cell cycle distribution. However, resveratrol and pterostilbene increased the percentage of the cells in S phase, while trimethoxystilbene (TMS) caused a massive accumulation of cells at the G2/M phase of the cell cycle. Tannic acid had no effect on cell cycle distribution in C6 cells, but increased the number of dead cells in both glioma cell lines. The ability to induce apoptosis by tannic acid and stilbenes was confirmed by phosphatidylserine externalization, the loss of mitochondrial membrane potential and the level of cleaved caspase-3. The apoptosis rate was most significantly increased by TMS and this was related to p53 induction. These results indicate that methoxylated stilbenes are efficient inhibitors of glioma cell proliferation and apoptosis inducers and might be considered adjuvants in glioma therapy.