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.

ATR activation by Cr-DNA damage is a major survival response establishing late S and G2 checkpoints after Cr(VI) exposure.

Inhalation exposure to hexavalent chromium is known to cause lung cancer and other pulmonary toxicity. Cellular metabolism of chromium(VI) entering cells as chromate anion produces different amounts of reactive Cr(V) intermediates and finally yields Cr(III). Direct reduction of Cr(VI) by ascorbate (Asc), the dominant metabolic reaction in vivo but not in standard cell cultures, skips production of Cr(V) but still permits extensive formation of Cr-DNA damage. To understand the importance of different forms of biological injury in Cr(VI) toxicity, we examined activation of several protein- and DNA damage-sensitive stress responses in human lung cells under Asc-restored conditions. We found that Asc-restored cells suppressed upregulation of oxidant-sensitive stress systems by Cr(VI) but showed a strong activation of the apical DNA damage-responsive kinase ATR. ATR signaling was triggered in late S phase and persisted upon entry of cells into G2 phase. Inhibition of ATR prevented the establishment of late-S and G2 cell cycle checkpoints and did not lead to a compensatory activation of a related kinase ATM. Inactivation of ATR also strongly impaired viability of Cr(VI)-treated lung cells including stem-like cells and revealed a significant formation of toxic Cr-DNA damage at low Cr(VI) doses. Our findings identified a major Cr(VI) resistance mechanism involving sensing of Cr-DNA damage by ATR in late S phase and a subsequent establishment of protective cell cycle checkpoints.

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.

Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron.

Epidemiological studies in chromate production have established hexavalent chromium as a potent lung carcinogen. Inhalation of chromium(VI) most often occurs in mixtures with other metals as among stainless steel welders, which is the largest occupational group with Cr(VI) exposure. Surprisingly, carcinogenicity of Cr(VI)-containing welding fumes is moderate and not consistently higher than that of Cr-free welding. Here, we investigated interactions between chromate and three other metal ions [Fe(III), Mn(II), Ni(II)] that are typically released from stainless steel welding particles. In human lung epithelial cells with physiological levels of ascorbate and glutathione, Cr(VI) was by far the most cytotoxic metal in single exposures. Coexposure with Fe(III) suppressed cytotoxicity and genotoxicity of Cr(VI), which resulted from a severe inhibition of Cr uptake by cells and required extracellular ascorbate/glutathione. Chemically, detoxification of Cr(VI) occurred via its rapid extracellular reduction by Fe(II) that primarily originated from ascorbate-reduced Fe(III). Glutathione was a significant contributor to reduction of Cr(VI) by Fe only in the presence of ascorbate. We further found that variability in Cr(VI) metabolism among common cell culture media was caused by their different Fe content. Ni(II) and Mn(II) had no detectable effects on metabolism, cellular uptake or cytotoxicity of Cr(VI). The main biological findings were confirmed in three human lung cell lines, including stem cell-like and primary cells. We discovered extracellular detoxification of carcinogenic chromate in coexposures with Fe(III) ions and identified the underlying chemical mechanism. Our findings established an important case when exposure to mixtures causes inactivation of a potent human carcinogen.

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.

Chemical mechanisms of DNA damage by carcinogenic chromium(VI).

Hexavalent chromium is a firmly established human carcinogen with documented exposures in many professional groups. Environmental exposure to Cr(VI) is also a significant public health concern. Cr(VI) exists in aqueous solutions as chromate anion that is unreactive with DNA and requires reductive activation inside the cells to produce genotoxic and mutagenic effects. Reduction of Cr(VI) in cells is nonenzymatic and in vivo principally driven by ascorbate with a secondary contribution from nonprotein thiols glutathione and cysteine. In addition to its much faster rate of reduction, ascorbate-driven metabolism avoids the formation of Cr(V) which is the first intermediate in Cr(VI) reduction by thiols. The end-product of Cr(VI) reduction is Cr(III) which forms several types of Cr-DNA adducts that are collectively responsible for all mutagenic and genotoxic effects in Cr(VI) reactions with ascorbate and thiols. Some Cr(V) forms can react with H2O2 to produce DNA-oxidizing peroxo species although this genotoxic pathway is suppressed in cells with physiological levels of ascorbate. Chemical reactions of Cr(VI) with ascorbate or thiols lack directly DNA-oxidizing metabolites. The formation of oxidative DNA breaks in early studies of these reactions was caused by iron contamination. Production of Cr(III)-DNA adducts in cells showed linear dose-dependence irrespective of the predominant reduction pathway and their processing by mismatch repair generated more toxic secondary genetic lesions in euchromatin. Overall, Cr(III)-DNA adduction is the dominant pathway for the formation of genotoxic and mutagenic DNA damage by carcinogenic Cr(VI).

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.

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.