Inverse correlation between p53 protein levels and DNA repair efficiency in human fibroblast strains treated with 4-nitroquinoline 1-oxide: evidence that lesions other than DNA strand breaks trigger the p53 response

Abstract

Ionizing radiation-induced stabilization and the resultant transient accumulation of the p53 tumor suppressor protein is impaired in cells from ataxia telangiectasia (AT) patients, indicating a key role for ATM, the gene mutated in AT, upstream in the radiation-responsive p53 signaling pathway. Activation of this pathway is generally assumed to be triggered by DNA strand breaks produced directly following genotoxic stress or indirectly during excision repair of DNA lesions. The aim of this study was to identify the triggering signal for induction of p53 in diploid human dermal fibroblasts treated with 4-nitroquinoline 1-oxide (4NQO), a model environmental carcinogen that produces both DNA strand breaks (like ionizing radiation) and alkali-stable bulky DNA lesions (like UV light). 4NQO treatment of fibroblasts cultured from normal and AT donors and those from patients with the UV-hypersensitivity disorder xeroderma pigmentosum (XP, complementation groups A, E and G) resulted in up-regulation of p53 protein. In normal fibroblasts, there was no temporal relationship between the incidence of DNA strand breaks and levels of p53 protein; >90% of strand breaks and alkali-labile sites were repaired over 2 h following treatment with 1 µM 4NQO, whereas~3 h of post-treatment incubation was required to demonstrate a significant rise in p53 protein. In contrast, exposure of normal fibroblasts to γ-rays resulted in a rapid up-regulation of p53 and the level peaked at 2 h post-irradiation. XP cells with a severe deficiency in the nucleotide excision repair pathway showed abnormally high levels of p53 protein in response to 4NQO treatment, indicating that lesions other than incisionassociated DNA strand breaks trigger p53 up-regulation. We observed a consistent, inverse correlation between the ability of the various fibroblast cultures to induce p53 following 4NQO treatment and their DNA repair efficiencies. Treatment with 0.12 µM 4NQO, for example, caused a >2-fold up-regulation of p53 in excision repair-deficient (AT, XPA and XPG) strains without eliciting any effect on p53 levels in repair-proficient (normal and XPE) strains. We conclude that up-regulation of p53 by 4NQO is mediated solely by an ATM-independent mechanism and that the p53 response is primarily triggered by persistent alkalistable 4NQO-DNA adducts.Abbreviations: AT, ataxia telangiectasia; dThd, thymidine; 4NQO, 4-nitroquinoline 1-oxide; XP, xeroderma pigmentosum.

Metabolic labeling of human cells with tritiated nucleosides results in activation of the ATM-dependent p53 signaling pathway and acceleration of DNA repair

“…In view of the results presented here, we conclude that previous results determining the effects of DNA-damaging agents on end points such as de novo DNA synthesis (a measure of S-phase checkpoint; Mirzayans et al, 1995), S-phase index (a measure of G 1 /S checkpoint; Enns et al, 1999), and recovery of RNA synthesis (a measure of repair of transcription-blocking DNA lesions; Barley et al, 1998;Mirzayans et al, 1999) are not influenced by radiolabeling because these assays involve incubation of the cells with extremely low activities of [ 14 C]dThd (e.g., 0.005 mCi/ml) for 24 h followed by pulse labeling with p10 mCi/ml of [ 3 H]dThd or [ 3 H]Urd for no more than 1 h. Such radiolabeling conditions do not induce p53 (this study), and thus there is no reason to consider the results obtained by these assays compromised. Likewise, the previous results for quantifying repair DNA synthesis by in situ autoradiography are unlikely to be influenced by artefacts such as p53-dependent modulation of repair because this assay involves pulse labeling the cells with high activities of [ 3 H]dThd for 1 h or less which, in the light of our current findings (Figure 2), should have little or no impact on repair.…”

Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks

“…LCL-N cells treated with 20 and 100 mM H 2 O 2 presented 6.970.5 and 21.471 g-H2AX foci, respectively, but these values were markedly reduced 1 h after recovery (Figure 1b), due to a repair activity. By contrast, no foci were seen in response to 4-NQO, a UV-mimetic compound generating base modifications and secondary SSBs, but not DSBs (Brosh et al, 1999;Mirzayans et al, 1999), thus supporting the specificity of g-H2AX for DSBs. The damaging activity of 4-NQO was confirmed by the accumulation of p53 protein (data not shown).…”

p53 Haploinsufficiency Profoundly Accelerates the Onset of Tongue Tumors in Mice Lacking the Xeroderma Pigmentosum Group A Gene

“…The bulky DNA lesions elicited by 4NQO are known to substrate for GGR and 4NQO-DNA damage per se provokes up-regulation of p53. 37,38 The hypersensitivity of human and mouse XPA cells to killing by 4NQO may be associated with increased activity of p53, leading to augmented p53-mediated apoptosis. 37 Although 4NQO-induced NER is p53-dependent, 37,38 it remains ambiguous that the modulating effect of p53 on NER is gene-dosage-dependent.…”