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.

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.

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.

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.

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.

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.

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.

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.

20S immunoproteasomes remove formaldehyde-damaged cytoplasmic proteins suppressing caspase-independent cell death.

Immunoproteasomes are known for their involvement in antigen presentation. However, their broad tissue presence and other evidence are indicative of nonimmune functions. We examined a role for immunoproteasomes in cellular responses to the endogenous and environmental carcinogen formaldehyde (FA) that binds to cytosolic and nuclear proteins producing proteotoxic stress and genotoxic DNA-histone crosslinks. We found that immunoproteasomes were important for suppression of a caspase-independent cell death and the long-term survival of FA-treated cells. All major genotoxic responses to FA, including replication inhibition and activation of the transcription factor p53 and the apical ATM and ATR kinases, were unaffected by immunoproteasome inactivity. Immunoproteasome inhibition enhanced activation of the cytosolic protein damage sensor HSF1, elevated levels of K48-polyubiquitinated cytoplasmic proteins and increased depletion of unconjugated ubiquitin. We further found that FA induced the disassembly of 26S immunoproteasomes, but not standard 26S proteasomes, releasing the 20S catalytic immunoproteasome. FA-treated cells also had higher amounts of small activators PA28αß and PA28γ bound to 20S particles. Our findings highlight the significance of nonnuclear damage in FA injury and reveal a major role for immunoproteasomes in elimination of FA-damaged cytoplasmic proteins through ubiquitin-independent proteolysis.

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.

Different ATM Signaling in Response to Chromium(VI) Metabolism via Ascorbate and Nonascorbate Reduction: Implications for in Vitro Models and Toxicogenomics.

BACKGROUND: Carcinogenic hexavalent chromium [Cr(VI)] requires cellular reduction to generate DNA damage. Metabolism of Cr(VI) by its principal reducer ascorbate (Asc) lacks a Cr(V) intermediate, which is abundant in reactions with a minor reducing agent, glutathione. Cultured cells are widely used in mechanistic studies of Cr(VI) toxicity; however, they typically contain < 1% of normal Asc levels. Asc deficiency is also expected to diminish protection against reactive oxygen species. OBJECTIVES: We assessed how the presence of Asc in cells affects their stress signaling and survival responses to chromate. METHODS: We investigated the effects of Asc restoration in human lung H460 cells and normal human lung fibroblasts on the activation and functional role of ATM kinase, which controls DNA damage responses involving several hundreds of proteins. RESULTS: Treatment of standard cultures with Cr(VI) strongly activated ATM, as indicated by its automodification at Ser1981 and by phosphorylation of checkpoint kinase 2 (CHK2) and chromatin/transcription regulator KRAB-associated protein 1 (KAP1). Confirming the importance of activated ATM, its inhibition impaired replication recovery and clonogenic survival. In contrast, fully Asc-restored cells lacked ATM activation by Cr(VI), and ATM silencing produced no significant effects on p53 stabilization, apoptosis, replication recovery, or clonogenic survival. Dose dependence studies found a close correlation between ATM activation and the extent of Cr(VI) reduction by glutathione. CONCLUSIONS: Asc restoration in cultured cells dramatically altered their stress responses to Cr(VI) by preventing activation of the oxidant-sensitive ATM network. We suggest that toxicogenomic and other cell response-based approaches likely underestimate Cr(VI) genotoxicity when standard ATM-activating carcinogens are used as references. CITATION: Luczak MW, Green SE, Zhitkovich A. 2016. Different ATM signaling in response to chromium(VI) metabolism via ascorbate and nonascorbate reduction: implications for in vitro models and toxicogenomics. Environ Health Perspect 124:61-66; http://dx.doi.org/10.1289/ehp.1409434.

DNA double-strand breaks by Cr(VI) are targeted to euchromatin and cause ATR-dependent phosphorylation of histone H2AX and its ubiquitination.

Hexavalent chromium is a human respiratory carcinogen that undergoes intracellular activation in vivo primarily via reduction with ascorbate. Replication of Cr-adducted DNA triggers mismatch repair that generates toxic DNA double-strand breaks (DSBs) as secondary lesions. Here, we examined the intranuclear distribution of chromate-induced breaks and a central DSB signaling branch targeting histone H2AX. Using ascorbate-restored cells (H460 human lung epithelial cells, normal human lung and normal mouse embryonic fibroblasts (MEFs)), we found that Cr(VI) produced a typical DSB-associated spectrum of H2AX modifications, including its Ser139-phosphorylated (known as γH2AX) and mono- and diubiquitinated forms. However, whereas canonical DSB signaling relies on ATM, the formation of γH2AX and its ubiquitinated products by Cr(VI) was dependent on ATR kinase. Based on the established mode of ATR activation, this suggests that Cr-induced DSB are not blunt-ended and likely contain single-stranded tails. Confocal imaging with markers of active and inactive chromatin revealed a selective formation of Cr-induced DSB in euchromatin of mouse and human cells. In contrast to DSB, Cr-DNA adducts were produced in both types of chromatin. The euchromatin targeting of Cr-induced DSB makes these lesions particularly dangerous by increasing the probability of deleting active tumor suppressors and producing oncogenic translocations. Accumulation of transcription-inhibiting ubiquitinated forms of γH2AX in euchromatin is expected to contribute to the ability of Cr(VI) to suppress upregulation of inducible genes.

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.

Effects of hydroxylated resveratrol analogs on oxidative stress and cancer cells death in human acute T cell leukemia cell line: prooxidative potential of hydroxylated resveratrol analogs.

Resveratrol and its higher hydroxylated analogs have been reported to possess a variety of biological properties including antioxidant as well as prooxidant effects. The antioxidant properties are assumed to enable these compounds to protect cells from oxidative damage, however prooxidant activity are held likely to be responsible for their cytotoxic or pro-apoptotic effects. In present study the effects of resveratrol (Res) and its three derivatives: 3,3',4,4'-tetrahydroxy-trans-stilbene (M6), 3,4,4',5-tetrahydroxy-trans-stilbene (M8) and 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (M12) were investigated on T cell leukemia Jurkat cells. The tested compounds have cytotoxic activity against cancer cells and IC50 values obtained in the Alamar blue assay were: 58.4 µM, 48.1 µM, 33.4 µM for and 13.8 µM for Res, M6, M8, M12, respectively. Furthermore, we also observed an increased activity of caspase 3 and 9, with significantly higher values in cells incubated with M8 and M12 than Res and M6. Cell death was accompanied by loss of mitochondrial potential, oxidative stress, decrease of glutathione level as well as loss of both mRNA expression and activity of superoxide dismutase (MnSOD). Cytotoxic activity may be connected with the formation of short-living prooxidative metabolites as compounds M8 and M12 were very instable in incubation medium. In conclusion, we elucidated the mechanisms responsible for cytotoxicity of hydroxylated resveratrol analogs in leukemia cells which may also apply to other polyphenols.

Uptake, p53 pathway activation, and cytotoxic responses for Co(II) and Ni(II) in human lung cells: implications for carcinogenicity.

Cobalt(II) and nickel(II) ions display similar chemical properties and act as hypoxia mimics in cells. However, only soluble Co(II) but not soluble Ni(II) is carcinogenic by inhalation. To explore potential reasons for these differences, we examined responses of human lung cells to both metals. We found that Co(II) showed almost 8 times higher accumulation than Ni(II) in H460 cells but caused a less efficient activation of the transcriptional factor p53 as measured by its accumulation, Ser15 phosphorylation, and target gene expression. Unlike Ni(II), Co(II) was ineffective in downregulating the p53 inhibitor MDM4 (HDMX). Co(II)-treated cells continued DNA replication at internal doses that caused massive apoptosis by Ni(II). Apoptosis and the overall cell death by Co(II) were delayed and weaker than by Ni(II). Inhibition of caspases but not programmed necrosis pathways suppressed Co(II)-induced cell death. Knockdown of p53 produced 50%-60% decreases in activation of caspases 3/7 and expression of 2 most highly upregulated proapoptotic genes PUMA and NOXA by Co(II). Overall, p53-mediated apoptosis accounted for 55% cell death by Co(II), p53-independent apoptosis for 20%, and p53/caspase-independent mechanisms for 25%. Similar to H460, normal human lung fibroblasts and primary human bronchial epithelial cells had several times higher accumulation of Co(II) than Ni(II) and showed a delayed and weaker caspase activation by Co(II). Thus, carcinogenicity of soluble Co(II) could be related to high survival of metal-loaded cells, which permits accumulation of genetic and epigenetic abnormalities. High cytotoxicity of soluble Ni(II) causes early elimination of damaged cells and is expected to be cancer suppressive.

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.

Role of direct reactivity with metals in chemoprotection by N-acetylcysteine against chromium(VI), cadmium(II), and cobalt(II).

The antioxidant N-acetylcysteine (NAC) is widely used for the assessment of the role of reactive oxygen species (ROS) in various biological processes and adverse drug reactions. NAC has been found to effectively inhibit the toxicity of carcinogenic metals, which was attributed to its potent ROS-suppressive properties. However, the absence of redox activity among some metals and findings from genetic models suggested a more diverse, smaller role of oxidative stress in metal toxicity. Here, we examined mechanisms of chemoprotection by NAC against Cd(II), Co(II), and Cr(VI) in human cells. We found that NAC displayed a broad-spectrum chemoprotective activity against all three metals, including suppression of cytotoxicity, apoptosis, p53 activation, and HSP72 and HIF-1α upregulation. Cytoprotection by NAC was independent of cellular glutathione. NAC strongly inhibited the uptake of all three metals in histologically different types of human cells, explaining its high chemoprotective potential. A loss of Cr(VI) accumulation by cells was caused by NAC-mediated extracellular reduction of chromate to membrane-impermeative Cr(III). Suppression of Co(II) uptake resulted from a rapid formation of Co(II)-NAC conjugates that were unable to enter cells. Our results demonstrate that NAC acts through more than one mechanism in preventing metal toxicity and its chemoprotective activity can be completely ROS-independent. Good clinical safety and effectiveness in Co(II) sequestration suggest that NAC could be useful in the prevention of tissue accumulation and toxic effects of Co ions released by cobalt-chromium hip prostheses.

Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma.

The development of cervical cancer requires genetic and epigenetic factors which result in the persistence of a malignant phenotype. Cervical cancer exhibits also some unique differences from other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue with over-expression of HIF-1 (hypoxia-inducible factor-1) transcription factor, which targets the transcription of over 70 genes involved in many aspects of cancer biology. One of the genes, which could be induced by HIF-1 is chemokine (C-X-C motif) receptor 4 (CXCR4). CXCR4 could also be epigenetically regulated by methylation of CpG dinucleotides located in the promoter region. Here, we examined the CXCR4, DNMT3A, DNMT3B and DNMT1 transcript levels in cancer tissue (n=30) and non-cancer, normal uterine cervical tissue (n=30) from a Polish cohort. We also compared the methylation status of CXCR4 promoter region in cancer and normal tissue samples. Our result showed significantly higher levels of CXCR4, DNMT3A, DNMT3B and DNMT1 transcript (p=0.0058, 0.0163, 0.0003 and <0.0001, respectively) levels in cancer tissue as compared to normal samples. We did not observe DNA methylation in the CXCR4 promoter region in either control or cancer tissue samples. CXCR4 has a functional hypoxia response element (HRE) in the promoter region, located -1.3 kb from the transcription start site. Our work shows for the first time that HIF-1A could promote the induction of CXCR4 gene expression (Spearman's correlation coefficient = 0.515, p=0.003) in patients with primary advanced uterine cervical carcinoma.

Increased expression of HIF-1A and its implication in the hypoxia pathway in primary advanced uterine cervical carcinoma.

The development of cervical cancer exhibits some unique differences compared to other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue. Lack of oxygen (hypoxia) induces the HIF-1 (hypoxia-inducible factor-1) transcription factor, which is a heterodimer composed of a constitutively expressed ß subunit and a hypoxia-inducible α-subunit. HIF-1A targets the transcription of over 70 genes involved in many aspects of cancer biology. In well-oxygenated environments, the HIF-1A subunit is hydroxylated, recognized and marked for proteosomal destruction by an E3 ubiquitin ligase, the von Hippel-Lindau protein (pVHL) complex. Under hypoxic stress, proline hydroxylase (PHD) activity is diminished, and stabilized HIF-1A is involved in the activation of the tissue response to hypoxia. Here, we examined the HIF-1A and VHL transcript levels and HIF-1A protein levels in cancerous tissue (n=30) and non-cancerous, normal uterine cervical tissue (n=30). We also compared the methylation status of HIF-1A and of the VHL promoter regions in cancerous and normal tissue samples. Significantly higher levels of HIF-1A and VHL transcripts (p<0.0001 and p=0.0042, respectively) and of HIF-1A protein (p=0.0037) were detected in cancerous tissue compared to normal samples. We did not observe DNA methylation in the HIF-1A and VHL promoter region in either control or cancerous tissue samples. VHL has a functional hypoxia response element (HRE) in the promoter region, and the induction of this HRE acts within a negative feedback loop to limit the hypoxic HIF-1A response. Our findings may suggest that HIF-1A could promote its own degradation by the induction of VHL gene expression (Spearman correlation coefficient, 0.515; p=0.003). Our study shows for the first time that this increase in VHL expression could be HIF-1A-dependent and serves within a negative feedback pathway during hypoxia to regulate the cell-specific oxygen threshold for HIF-1A activation.