DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation

Alessio Cardinale,Leonardo Lupacchini,Serena Saladini,Francesca Silvagno,Chiara De Dominicis,Irene Ruspantini,Cristiana Mollinari,Daniela Merlo,Marco Papale,Enrico Garaci

doi:10.1007/s11033-021-06934-5

Abstract

BackgroundEmerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair.Methods and resultsWe used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage.ConclusionsTaken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme.Graphical

Highlights

Oxidative stress induces DNA damage, and the unrepaired or improperly repaired DNA lesions increase genomic instability, which can cause cell death, senescence, or dysregulation of cellular functions
In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function of DNA repair
Because post-mitotic cells adopt mainly non-homologous end joining (NHEJ) to repair damaged DNA [37, 38], we evaluated the effect of H2O2 4 h treatment on NGF-differentiated pheochromocytoma line 12 (PC12) cells (Fig. 4), a well-known neuronal model [39,40,41,42], and γH2AX foci were counted after 24 h recovery (Fig. 4B)

Summary

Introduction

Oxidative stress induces DNA damage, and the unrepaired or improperly repaired DNA lesions increase genomic instability, which can cause cell death, senescence, or dysregulation of cellular functions. In addition to its role in DNA DSB repair, DNA-PK has been involved in several pathways including stress response modulation, apoptosis, telomere homeostasis and specific gene transcription [15]. ROS over-production leads to rapid ATM dimerization/activation and downstream ATM signalling modulating cellular metabolism and cell survival, repairing oxidative DNA damage, and inducing antioxidant enzyme expression to maintain redox homeostasis [16, 18]. Our study indicates that DNA-PK may have a crucial role in cellular oxidative stress response and the enhancement of its activity may open new perspectives for the treatment of ROS-related diseases

Materials and methods

Findings

Discussion