A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis

Abstract

Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [(3)H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [(3)H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.

Figure 1. Experimental design.

Cells were cultured in the presence of different specific activities of [³H]thymidine. The final concentration of thymidine was maintained constant supplying cold thymidine. After 20 h of incubation, when incorporation reached a plateau, greater than 95% of cells contained labeled nucleotides. Incorporated radioactivity was counted in the trichloroacetic acid (TCA) precipitate. Mutagenesis frequency, DNA sequence, ROS, antioxidant activities, and proteomics were subsequently measured.

Figure 2. [³H]thymidine incorporation induces proteome modifications in CHO cell nuclear proteins.

A typical two-dimensional electrophoresis image of CHO cell nuclear proteins is presented using 100 μg of soluble nuclear proteins. Proteins were visualized by Lava purple staining. Numbered spots were significantly increased or decreased in protein exacts from CHO cells upon [³H]thymidine incorporation. Proteins were identified by mass spectrometry (Table 1, supplementary data S1). Gene ontology of each protein was analyzed (Supplementary data S2), and the expression pattern of three representative proteins upon [³H]thymidine incorporation is presented in Fig. 3.

Figure 3. Characteristic patterns of spots differentially expressed upon [³H]thymidine incorporation.

Each of these three patterns is related to the corresponding spot as presented in table 1 and supplementary data S1 and S2. The values correspond at least to three experiments.

Figure 4. Mutagenesis induced by [³H]thymidine incorporation.

(a) Mutant colonies induced by [³H]thymidine incorporation as indicated by viable ouabain-resistant colonies (locus Na⁺/K⁺ pump ATPase). (b) Mutant colonies induced by [³H]thymidine incorporation as indicated by viable 8-azaadenine-resistant colonies (locus APRT). Scatter plots contain combined data from at least three independent experiments. (c) Mutant colonies induced by ⁶⁰Co external γ radiation with doses delivered as indicated by viable ouabain-resistant colonies (locus Na⁺/K⁺ pump ATPase). (d) Mutant colonies induced by ⁶⁰Co external γ radiation with doses delivered as indicated by viable 6-thioguanine-resistant colonies (locus HPRT).

Figure 5. Mutagenesis analysis.

(a) APRT locus sequence localization of mutations in control (blue) or upon low-dose (red) or high-dose (green) [³H]thymidine incorporation. Number in brackets designates the summation of individual clones for each mutation scored. (b) Types and frequency of mutations in control (black) or upon low-dose (light grey) or high-dose (dark grey) [³H]thymidine incorporation. Up to 110 independent aprt⁻ mutants clones were isolated and amplified for each condition (control, low- and high-doses).

Figure 6. Mutagenesis is induced by oxidative stress from [³H]thymidine incorporation.

(a) Intracellular ROS monitored using carboxy-H2DCFDA fluorescent quantification (flow cytometry) as an oxidative stress probe in control and 4 representative doses on each graph (0, 2 low doses and 2 high doses). (b) The effect of treatment with NAC (20 μM, 1 h prior [³H]thymidine incorporation) on the induction of mutant colonies by [³H]thymidine incorporation (ouabain resistance). Black squares and lines, control mutagenesis induced by [³H]thymidine incorporation (see Fig. 4A). Grey squares and lines, mutagenesis induced by [³H]thymidine incorporation after NAC treatment. (c) The effect of treatment with BSO (5 μM, 12 h prior [³H]thymidine incorporation) on the induction of mutant colonies by [³H]thymidine incorporation (ouabain resistance). Black squares and lines, control mutagenesis induced by [³H]thymidine incorporation (see Fig. 4A). Grey circles and lines, mutagenesis induced by [³H]thymidine incorporation after BSO treatment. Scatter plots present combined data from at least three independent experiments.

Figure 7. BER activities.

(a) hOGG-1 activity. (b) APE-1 activity. Grey arrow denotes low-dose range. Scatter plots present combined data from at least three independent experiments. Grey arrows indicate low-dose range. APE-1 activity increase is significantly linked to [³H]thymidine incorporation (rs = 0.9241 ; p < 0.0001).

Figure 8. Threshold model for cell response to stress from endogenous source.

A threshold of stress (red arrow) from endogenous source is needed to activate detoxification and DNA repair of oxidative damages. Note this threshold of dose is also required to generate γH2AX foci and to induce homologous recombination. Below this threshold detoxification and DNA repair are not activated leading to mutagenesis (black dotted line) induced by the oxidative stress. Dotted line: mutagenesis. *from.