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. 2025 May 19:16:362-378.
doi: 10.18632/oncotarget.28720.

PRDX1 protects ATM from arsenite-induced proteotoxicity and maintains its stability during DNA damage signaling

Affiliations

PRDX1 protects ATM from arsenite-induced proteotoxicity and maintains its stability during DNA damage signaling

Reem Ali et al. Oncotarget. .

Abstract

Redox regulation and DNA repair coordination are essential for genomic stability. Peroxiredoxin 1 (PRDX1) is a thiol-dependent peroxidase and a chaperone that protects proteins from excessive oxidation. ATM kinase (Ataxia-Telangiectasia Mutated) and the MRN (MRE11-RAD50-NBS1) complex are DNA damage signaling and repair proteins. We previously showed that cells lacking PRDX1 are sensitive to arsenite, a toxic metal that induces DNA single- and double-strand breaks (DSBs). Herein, we showed that PRDX1 interacts with ATM. PRDX1-deleted cells have reduced ATM, MRE11, and RAD50 protein levels, but not NBS1. In control cells treated with arsenite, we observed γH2AX foci formation due to arsenite-induced DSBs, and not from PRDX1-deleted cells. Arsenite caused profound depletion of ATM in PRDX1-deleted cells, suggesting that PRDX1 protects and stabilizes ATM required to form γH2AX foci. Importantly, arsenite pretreatment of PRDX1-deleted cells caused hypersensitivity to chemotherapeutic agents that generate DSBs. Analysis of a clinical cohort of ovarian cancers treated with platinum chemotherapy revealed that tumours with high PRDX1/high ATM or high PRDX1/high MRE11 expression manifested aggressive phenotypes and poor patient survival. The data suggest that PRDX1 can predict responses to chemotherapy, and targeting PRDX1 could be a viable strategy to improve the efficacy of platinum chemotherapy.

Keywords: cell cycle; homologous recombination; protein interaction; protein modification; redox signaling.

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Conflict of interest statement

CONFLICTS OF INTEREST

Authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
(A)Western blot showing ATM, RAD50, NBS1, MRE11 and H2AX protein levels in HEK293 control and HEK293_PRDX1 KO cells. (B) Western blot showing ATM, RAD50, NBS1, MRE11 and H2AX protein levels in HeLa control and HeLa_PRDX1 KO cells. (C) Clonogenic survival assay showing ATM inhibitor AZ31 sensitivity in HEK293 control and HEK293 PRDX1_KO cells. (D) Clonogenic survival assay showing ATM inhibitor KU55933 sensitivity in HEK293 control and HEK293 PRDX1_KO cells. Survival fraction statistical analysis was performed using a two-way ANOVA test. * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
(A) Western blot showing ATM, RAD50, NBS1, MRE11, and H2AX protein levels in HEK293 control and HEK293_PRDX1 KO cells treated with arsenite at the indicated doses. (B) Western blot showing ATM, RAD50, MRE11, and H2AX protein levels in HeLa control and HeLa_PRDX1 KO cells treated with arsenite at the indicated doses. Briefly, cells were plated overnight, then treated with freshly prepared arsenite doses in PBS. After treatment, cells were incubated with 100 mM N- ethylmaleimide (NEM) for 10 mins on ice, then cells were scrapped and collected by centrifugation. Cell pellets were resuspended in lysis buffer (20 mM Tris- HCL pH 7.4, 100 mM NaCl, 0.5% NP-40, EDTA, 5 mg/ml NEM, and 1X protease and phosphatase inhibitor cocktail) and sonicated. Samples were spun in a microcentrifuge at 13,000 rpm for 10 min at 4°C. Proteins were quantified by micro-BCA and lysates were analyzed by western blot.
Figure 3
Figure 3
(A) Western blot showing ATM, RAD50, MRE11, and H2AX protein levels in HEK293 control and HEK293_PRDX1 KO cells following incubation with MG132 and treatment with arsenite. Cells were plated overnight and then treated with the proteasome inhibitor MG132 (25 μM) for 3 h. Cells were treated with 100 μM or 2.5 mM arsenite for 10 min or left untreated. Cells were washed with 100 mM NEM in PBS and scrapped. Lysates were analyzed by immunoblotting. (B) Relative ATM mRNA expression levels by RT-qPCR in HEK293 control and HEK293_PRDX1 KO cells treated with 100 μM or 2.5 mM arsenite for 10 min. (C) Relative MRE11 mRNA expression levels by RT-qPCR in HEK293 control and HEK293_PRDX1 KO cells treated with 100 μM or 2.5 mM arsenite for 10 min. GADPH was used for normalization. (D) Clonogenic survival assay showing the sensitivity of ATM inhibitor AZ31 plus arsenite combination in HEK293 control and HEK293_PRDX1 KO cells. 500 Cells were plated in 6-well plates overnight and treated with AZ31 (2 μM) for 30 min. Then cells were treated with different arsenite doses in PBS for another 30 min. After incubation cells were topped with fresh media and left to form clones for 10 days. Plates were stained with crystal violet Methanol mixture. Survival fraction statistical analysis was performed using a two-way ANOVA test, ** p < 0.01.
Figure 4
Figure 4
(A) Representative photomicrographic images showing HEK293 control and HEK293 PRDX1 KO cells treated with the indicated doses of arsenite. (B, C) Quantification of ATM and γH2AX nuclear fluorescence, respectively, in control cells by ImageJ Software. (D, E) Quantification of ATM and γH2AX nuclear fluorescence, respectively, in HEK293_PRDX1 KO cells by ImageJ Software. Statistical analysis was performed using one-way ANOVA. The error bars represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
(A) Quantification of γH2AX positive cells by flow cytometry in HEK293 control and HEK293 PRDX1 KO cells treated with 100 μM or 2.5 mM arsenite for 30 min. (B) Quantification of γH2AX positive cells by flow cytometry in HeLa control and HeLa PRDX1 KO cells treated with 100 μM or 2.5 mM arsenite for 30 min. Cells were plated in 6-well plates overnight and treated with arsenite in PBS (100 μM or 2.5 mM arsenite for 30 min) then cells were washed and left to recover in fresh media for 16 h. Cells were fixed in 70% ethanol for 30 min and stained with propidium iodide and FITC -γH2AX. Cells were analyzed by flow cytometry, and data analysis was performed in FlowJo software. (C) Co-immunoprecipitation shows the interaction between PRDX1 and ATM, MRN complex in HeLa and HEK293 control cells. HEK293 PRDX1 KO cells were used as a negative control. Statistical analysis was performed using one-way ANOVA. The error bars represent the mean ± SD., ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
(A) Cell cycle analysis by flow cytometry software in HEK293 control and HEK293 PRDX1 KO cells treated with arsenite. (B) Cell cycle analysis by flow cytometry software in HeLa control and HeLa PRDX1 KO cells treated with arsenite. Cells were plated in 6-well plates overnight, the following day cells were treated with arsenite in PBS (100 μM or 2.5 mM arsenite for 30 min). Cells were washed and left to recover in fresh media for 16 h. Cells were fixed in 70% ethanol for 30 min and stained with propidium iodide and FITC -γH2AX. Cells were analyzed by flow cytometry and data analysis was performed in FlowJo software. (C) Daunorubicin sensitivity in HEK293 control and HEK293 PRDX1 KO cells pre-treated with arsenite followed by clonogenic survival assay. (D) Cisplatin sensitivity in HEK293 control and HEK293 PRDX1 KO cells pre-treated with arsenite followed by clonogenic survival assay. Cell cycle statistical analysis was performed using two-way ANOVA. The error bars represent the mean ± SD., * p < 0.05, ** p < 0.01, *** p < 0.001. Survival statistical analysis was performed using two-way ANOVA, ** p < 0.01.
Figure 7
Figure 7
Kaplan-Meier survival analysis shows ovarian cancer progression-free survival and (A) PRDX1 nuclear expression. (B) PRDX1 cytoplasmic expression and (C) PRDX1 nuclear/cytoplasmic expression. (D) Kaplan-Meier survival analysis shows ovarian cancer progression-free survival and PRDX1/MRE11 co-expression. (E) Kaplan-Meier survival analysis shows ovarian cancer progression-free survival and PRDX1/ATM co-expression.

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