S-phase sensing of DNA-protein crosslinks triggers TopBP1-independent ATR activation and p53-mediated cell death by formaldehyde

Abstract

We examined genotoxic signaling and cell fate decisions in response to a potent DNA-protein crosslinker formaldehyde (FA). DNA-protein crosslinks (DPC) are poorly understood lesions produced by bifunctional carcinogens and several cancer drugs. FA-treated human cells showed a rapid activation of ATR kinase that preferentially targeted the p53 transcription factor at low doses and CHK1 kinase at more severe damage, producing bell-shaped and sublinear responses, respectively. CHK1 phosphorylation was transient, and its loss was accompanied by increased p53 accumulation and Ser15 phosphorylation. Activation of p53 was insensitive to inhibition of mismatch repair and nucleotide and base excision repair, excluding the role of small DNA adducts in this response. The p53-targeted signaling was transcription-independent, absent in quiescent cells and specific to S-phase in cycling populations. Unlike other S-phase stressors, FA-activated p53 was functional transcriptionally, promoted apoptosis in lung epithelial cells and caused senescence in normal lung fibroblasts. FA did not induce ATR, RAD1 or RPA foci, and p53 phosphorylation was TopBP1-independent, indicating a noncanonical mode of ATR activation. Replication arrest by FA caused a dissociation of ATR from a chromatin-loaded MCM helicase but no PCNA monoubiquitination associated with stalled polymerases. These results suggest that unlike typical DNA adducts that stall DNA polymerases, replication inhibition by bulkier DPC largely results from blocking upstream MCM helicase, which prevents accumulation of ssDNA. Overall, our findings indicate that S-phase-specific, TopBP1-independent activation of the ATR-p53 axis is a critical stress response to FA-DPC, which has implications for understanding of FA carcinogenesis.

Figure 1. Activation of p53 by FA in human cells. (A) Ser15-p53 phosphorylation in IMR90 and H460 cells treated with 200 μM FA for different periods of time. (B) Left panel: p53 responses in IMR90 cells treated with 150 μM FA for 3 h and collected at different post-exposure times. Right panel: dose dependence of p53 responses in IMR90 cells treated with FA for 3 h and collected 6 h later. (C) Induction of p21 by FA in p53−/− and p53+/+ HCT116 cells. (D) Gene expression in H460 cells collected at 3 and 6 h post-FA (means ± SD, n = 3, * - p < 0.05). (E) Stability of p53 and MDM4 in H460 cells treated with 200 μM FA for 3 h and then incubated in the presence of 100 μg/ml cycloheximide (CHX). A representative western blot and means from two independent protein extracts are shown.

Figure 2. Impact of transcription and cell cycle specificity of p53 activation by FA. (A) Transcription inhibitors α-amanitin (20 μg/ml) and DRB (100 μM) do not prevent p53 activation by FA in H460 cells (0 h post-FA collection). (B) Absence of p53 activation by FA in growth-arrested IMR90 cells. (C) Representative images of H460 cells labeled with EdU for 15 min prior to FA addition and immunostained for phosphorylated Ser15-p53 immediately after FA exposure. sh-SCR – nospecific shRNA, sh-p53 - p53-targeting shRNA. (D) Cell cycle specificity of Ser15-p53 phosphorylation in FA-treated H460 and (E) IMR90 cells. S-phase cells were identified by EdU incorporation (15 min labeling before FA), G₁ by CDT1 and G₂ by cyclin B1 immunostaining. Cells were treated with 200 μM FA for 3 h and fixed immediately after exposure. Data are means ± SD from three slides with at least 100 cells scored per each slide.

Figure 3. Role of p53 in FA-induced apoptosis and senescence. (A) Representative FACS profiles of H460 cells stained with Annexin V and 7-AAD at 24 h post-FA treatment. Values are means ± SD for three cell samples. (B) PARP cleavage in H460 cells collected at 24 h post-FA. (C) Expression of pro-apoptotic genes in H460 cells at 6 h after FA exposures (means ± SD from three experiments, *- p < 0.05). (D) Clonogenic survival of H460 cells expressing nonspecific (sh-SCR) and targeting (sh-p53) shRNA (means ± SD from three experiments, * - p < 0.05). (E) Frequency of replication-defective IMR90 cells in populations expressing control and p53-targeting shRNA (means ± SD from three slides with > 100 cells scored per each slide). EdU was added at 24 h after FA exposure and cells were labeled for 48 h. (F) Abrogation of FA-induced senescence in IMR90 cells by p53 knockdown (means ± SD from three slides with > 100 cells scored per each slide). Senescent cells were identified by SA-β-Gal staining at 6 d after FA exposure.

Figure 4. Role of DNA damage-induced kinases in p53 activation. (A) Inhibitors of DNAPK (10 μM NU7026) and ATM (10 μM KU55399) had no effect on p53 responses in FA-treated H460 cells. (B) Caffeine (4 mM) does not inhibit Thr68-CHK2 phosphorylation by ATM in H460 cells treated for 4 h with 1 μM CPT or 20 μg/ml bleomycin (Bleo). (C) Caffeine (3 mM) blocks p53 accumulation and p21 induction in H460 cells treated with 200 μM FA for 3 h and collected 6 h later. (D) Caffeine (3 mM) but not NU7026 or KU55399 abolishes Ser15-p53 phosphorylation and p53 accumulation in IMR90 cells treated with 150 μM FA (6 h post-FA collection). (E) ATR knockdown inhibits Ser15-p53 phosphorylation in H460 cells treated with 200 μM FA (0 and 6 h post-FA collection). (F) FACS profiles of IMR90 and Seckel syndrome fibroblasts immunostained for phosphorylated Ser15-p53. Cells were collected immediately after FA exposures.

Figure 5. Activation of p53 in cells with downregulated repair of small DNA adducts. (A) Normal activation of p53 in IMR90 cells with stable XPA knockdowns (6 h post-FA collection). (B) No effect of DNA polymerase β inhibitors oleanolic and lithocholic acids (50 μM each) on p53 activation (6 h post-FA collection). (C) PCNA monoubiquitination in chromatin after 3-h long treatments with 200 μM FA and 10 mM hydroxyurea or 3 h after 100 J/m² UV-B (302 nm). Soluble proteins were removed by a PBS-0.2% Triton X-100 extraction for 5 min on ice. (D) Normal Ser15-p53 phosphorylation in IMR90 with knockdown of the mismatch repair protein MSH2.

Figure 6. ATR-associated responses in FA-treated cells. (A) Dose-dependent phosphorylation of p53 after FA exposure for 3 h (0 and 6 h post-FA collection times). HBE – human bronchial epithelial cells. (B) ATR, RPA32, RAD1 and phospho-RPA32 foci in the S-phase of IMR90 cells processed immediately after 3 h-long exposures with FA or CPT. S-phase cells were labeled by EdU incorporation for 15 min immediately before FA exposure. (C) FACS profiles of IMR90 cells stained for DNA content and BrdU incorporated during the last 30 min of mock/FA exposures. (D) Impact of TopBP1 depletion on p53 and CHK1 phosphorylation in H460 and (E) IMR90 cells. Cells were collected immediately after 3-h-long FA exposures. (F) Loss of ATR-MCM association in chromatin of FA-treated IMR90 cells (200 μM FA for 3 h, immediate collection).