Inactivation of DNA–Dependent Protein Kinase Promotes Heat–Induced Apoptosis Independently of Heat–Shock Protein Induction in Human Cancer Cell Lines

Seisuke Okazawa,Ayako Kariya,Yoshiaki Tabuchi,Yukihiro Furusawa,Takashi Kondo,Mariame Ali Hassan,Ryuji Hayashi,Kazuyuki Tobe,Mie Arai,Michael Sherman

doi:10.1371/journal.pone.0058325

Abstract

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.

Highlights

Malignant tumors are a major problem in the world that in the medical research field the developing of effective therapeutics has always been on the top of research interests for decades
The studies on DNA damage response (DDR) revealed a plethora of molecular targets such as the Ataxia telangiectasia mutated (ATM), Ataxia telangiectasia and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK)
Heat-induced Apoptosis was Enhanced by DNA-PK Inhibitors

Summary

Introduction

Malignant tumors are a major problem in the world that in the medical research field the developing of effective therapeutics has always been on the top of research interests for decades This goal has persisted over years despite the numerous therapeutic tools available (such as surgery, chemotherapy, radiotherapy, hyperthermia [1], high intensity focused ultrasound (HIFU) [2], immunotherapy [3], etc.), and the variety of their strategic combinations [1,4,5]. The studies on DDR revealed a plethora of molecular targets such as the Ataxia telangiectasia mutated (ATM), Ataxia telangiectasia and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) These proteins are members of the phosphoinositol 3-kinase-like kinase (PIKK) family functioning as transducers in DDR to activate multiple proteins involved in cellular response to DNA damage [7–9]

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Methods

Results