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 (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.

Formaldehyde Is a Potent Proteotoxic Stressor Causing Rapid Heat Shock Transcription Factor 1 Activation and Lys48-Linked Polyubiquitination of Proteins

“…14e18 The results of these investigations were contradictory even for the same cell model, and one study has found that the sensitivity of specific mutants depended on the exposure protocol. 14 Genotoxic signaling triggered by FA in human cells occurred only in the S phase, 19,20 which is consistent with the stalling of replication helicases by DNA-attached proteins. 21 However, FA-induced activation of the transcription factor p53 20 and the DNA damage-responsive kinase ataxia-telangiectasia mutated (ATM) 22 showed bellshaped dose dependence, suggesting that FA-treated cells may experience another major stress that becomes dominant at certain exposure conditions even in S-phase cells.…”

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

“…H460 cells express the wild-type p53 transcription factor and displayed normal stress responses to various carcinogens, such as ionizing radiation (Zhang et al, 2006), chromium(VI) (Luczak et al, 2016) and formaldehyde (Ortega-Atienza et al, 2015; Wong et al, 2012). We have previously characterized p53 activation and mechanisms of cell death by Ni(II) ions in this cell line (Green et al, 2013; Wong et al, 2013a), making it well-suited for dissecting potential toxicological effects of HIF-1α.…”

Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

“…Studies in mammalian cells suggested that the proteasome participates in DPC removal (Baker et al, 2007;Desai et al, 1997;Lin et al, 2008;Mao et al, 2001;Quiñones et al, 2015;Zecevic et al, 2010). Proteasome inhibition prevents the removal of different types of DPCs including trapped topoisomerases I and II and DNA polymerase β (Desai et al, 1997;Lin et al, 2008;Mao et al, 2001;Quiñones et al, 2015), and sensitizes cells to formaldehyde treatment (Ortega-Atienza et al, 2015). Additionally, DPC polyubiquitylation was observed in the case of covalent topoisomerase I cleavage complexes (Desai et al, 1997).…”