title: Inactivation of DNA-dependent protein kinase promotes heat-induced apoptosis independently of heat-shock protein induction in human cancer cell lines.
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.
authors.affiliation: First Department of Internal Medicine, Graduate School of Medicine Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
authors.forename: Seisuke
authors.initials: S
authors.lastname: Okazawa
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Furusawa
authors.forename: Ayako
authors.initials: A
authors.lastname: Kariya
authors.forename: Mariame Ali
authors.initials: MA
authors.lastname: Hassan
authors.forename: Mie
authors.initials: M
authors.lastname: Arai
authors.forename: Ryuji
authors.initials: R
authors.lastname: Hayashi
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Tabuchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kondo
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Tobe
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D020868:Gene Silencing; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D015203:Reproducibility of Results
chemical_list: D006360:Heat-Shock Proteins; D047428:Protein Kinase Inhibitors; D051747:DNA-Activated Protein Kinase