title: Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde.
abstract: 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 (DPCs) 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Ortega-Atienza
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Samantha E
authors.initials: SE
authors.lastname: Green
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA. Electronic address: anatoly_zhitkovich@brown.edu.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Zhitkovich
mesh_terms: D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects*; D005557:Formaldehyde / Q000633:toxicity*; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D005557:Formaldehyde; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D046988:Proteasome Endopeptidase Complex; D006918:Hydroxyurea
keywords: Cell cycle; DNA repair; DNA–protein crosslink; Formaldehyde; Replication
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