Wakako Hiraoka

Ca2+-dependent and Caspase-3–independent Apoptosis Caused by Damage in Golgi Apparatus due to 2,4,5,7-Tetrabromorhodamine 123 Bromide–induced Photodynamic Effects¶

Abstract To clarify the role of the Golgi apparatus in photodynamic therapy–induced apoptosis, its signaling pathway was studied after photodynamic treatment of human cervix carcinoma cell line HeLa, in which a photosensitizer, 2,4,5,7-tetrabromorhodamine 123 bromide (TBR), was incorporated into the Golgi apparatus. Laser scanning microscopic analysis of TBR-loaded HeLa cells confirmed that TBR was exclusively located in the Golgi apparatus. HeLa cells incubated with TBR for 1 h were then exposed to visible light using an Xe lamp. Light of wavelength below 670 nm was eliminated with a filter. Morphological observation of nuclei stained with Hoechst 33342 revealed that apoptosis of cells was induced by exposure to light. Electron spin resonance spectrometry showed that light-exposed TBR produced both singlet oxygen (1O2) and superoxide anion (O2–). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O2– scavenger, but not by NaN3, a quencher of 1O2. Furthermore, TBR-induced apoptosis was inhibited by aurintricarboxylic acid and ZnCl2, which are known as inhibitors of deoxyribonuclease (DNase) γ, and (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, a chelator of Ca2+, but not by acetyl Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3. These results suggested that O2– was responsible for TBR-induced apoptosis, and Ca2+-dependent and caspase-3–independent nuclease such as DNase γ played an important role in apoptotic signaling triggered by Golgi dysfunction.

Confirmation of enhanced expression of heme oxygenase-1 gene induced by ultrasound and its mechanism: analysis by cDNA microarray system, real-time quantitative PCR, and Western blotting

PurposeThe present study was undertaken to reconfirm heme oxygenase-1 (HO-1) induction by ultrasound, and elucidate the mechanism by which this occurs.MethodsAfter exposure of human lymphoma U937 cells to 1 MHz continuous ultrasound (US), gene profiling by using cDNA microarray analysis, cell viability by using the trypan blue dye exclusion test, mRNA expression by using real-time quantitative polymerase chain reaction, and protein expression by using Western blotting were examined. As an indicator of cavitation, hydroxyl radical formation was studied by using electron paramagnetic resonance-spin trapping.ResultsThe cDNA microarray analysis reconfirmed HO-1 induction in human lymphoma U937 cells after exposure to US, and further identified one upregulated and two downregulated genes. When U937 cells were exposed to US for 1 min, HO-1 induction, as examined by real-time quantitative polymerase chain reaction and Western blotting, was observed at intensities higher than the cavitational threshold. When a potent antioxidant, N-acetyl-l-cysteine, was added to the culture medium before or after sonication, the induction was attenuated, indicating that reactive oxygen species are involved in HO-1 induction. A decrease in mitochondrial membrane potential and generation of superoxide anion radicals were also observed in the cells exposed to US.ConclusionWe used a cDNA microarray system to confirm upregulation of the HO-1 gene and to discover new genes that respond to ultrasonic cavitation. Increased intracellular oxidative stress secondary to the sonomechanical effects arising from ultrasonic cavitation is suggested to be the mechanism of enhancement of HO-1 expression.

A novel copper(II) coordination at His186 in full-length murine prion protein

To explore Cu(II) ion coordination by His(186) in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP(C).

Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV, LC/MS/MS and ESR

Abstract To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron–sulphur (Fe−S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF2α and 5-iPF2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe–S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe–S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe–S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe–S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.

Applications of the Spin-Trapping Method in Radiation Biology

Ionizing radiation produces many kinds of free radicals in biologically important molecules through direct or indirect actions. In this chapter we have shown that a method that combined ESR, spin trapping and chromatographic separation made it possible to identify free radicals induced by direct ionization as well as OH-radical reactions in nucleic-acid related and protein-related compounds. In addition, combination with enzymatic digestion expanded the potential of this method to detect free radical production in molecules with high molecular weight such as polynucleotides, RNA and DNA, and polypeptides and proteins. The effects of ionizing radiation on biological molecules underlie the primary processes involved in radiation biology. ESR combined with spin trapping remains a useful method for clarifying these processes in a living organism.