Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA-protein crosslinks (DPC) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions.
BackgroundCarcinogenic 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.ObjectivesWe assessed how the presence of Asc in cells affects their stress signaling and survival responses to chromate.MethodsWe 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.ResultsTreatment 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.ConclusionsAsc 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.CitationLuczak 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
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.
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