Misao Hachiya

Catalase Regulates Cell Growth in HL60 Human Promyelocytic Cells: Evidence for Growth Regulation by H2O2

Abstract Hachiya, M. and Akashi, M. Catalase Regulates Cell Growth in HL60 Human Promyelocytic Cells: Evidence for Growth Regulation by H2O2. Radiat. Res. 163, 271–282 (2005). Reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are generated constitutively in mammalian cells. Because of its relatively long life and high permeability across membranes, H2O2 is thought to be an important second messenger. Generation of H2O2 is increased in response to external insults, including radiation. Catalase is located at the peroxisome and scavenges H2O2. In this study, we investigated the role of catalase in cell growth using the H2O2-resistant variant HP100-1 of human promyelocytic HL60 cells. HP100-1 cells had an almost 10-fold higher activity of catalase than HL60 cells without differences in levels of glutathione peroxidase, manganese superoxide dismutase (MnSOD), and copper-zinc SOD (CuZnSOD). HP100-1 cells had higher proliferative activity than HL60 cells. Treatment with catalase or the introduction of catalase cDNA into HL60 cells stimulated cell growth. Exposure of HP100-1 cells to a catalase inhibitor resulted in suppression of cell growth with concomitant increased levels of intracellular H2O2. Moreover, exogenously added H2O2 or depletion of glutathione suppressed cell growth in HL60 cells. Extracellular signal regulated kinase 1/2 (ERK1/2) was constitutively phosphorylated in HP100-1 cells but not in HL60 cells. Inhibition of the ERK1/2 pathway suppressed the growth of HP100-1 cells, but inhibition of p38 mitogen-activated protein kinase (p38MAPK) did not affect growth. Moreover, inhibition of catalase blocked the phosphorylation of ERK1/2 but not of p38MAPK in HP100-1 cells. Thus our results suggest that catalase activates the growth of HL60 cells through dismutation of H2O2, leading to activation of the ERK1/2 pathway; H2O2 is an important regulator of growth in HL60 cells.

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis is mediated by tumor necrosis factor alpha in human monocytic U937 cells.

12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-α (TNFα) in U937 cells, and exogenously added TNFα induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFα antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFα and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFα in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFα is an autocrine-stimulating factor for the induction of apoptosis in these cells.

The accident at the Fukushima Daiichi Nuclear Power Plant in 2011.

A huge earthquake struck the northeast coast of the main island of Japan on 11 March 2011, triggering a tsunami with more than 10-m-high waves hitting the area. The earthquake was followed by numerous sustained aftershocks. The earthquake and aftershocks left almost 16,000 people dead and more than 2,800 missing (as of 11 March 2014). The earthquake affected the Fukushima Daiichi Nuclear Power Plant (NPP) of Tokyo Electric Power Company (TEPCO), causing serious damage to the NPP and resulting in large amounts of radioactive materials being released into not only controlled areas but also the environment. Damage was caused to the cooling systems of the NPP, although they automatically shut down after the earthquake. The trouble with the cooling systems led to hydrogen explosions and core meltdown. The major nuclides released on land were ¹³¹I, ¹³⁴Cs, and ¹³⁷Cs. The release of these radioactive materials resulted in contamination of first responders and workers and also a high ambient dose of radiation around the NPP. The local hospital system, including that for radiation emergency medicine, was dysfunctional. Hospitals that had been designated as radiation emergency facilities were not able to function because the earthquake and tsunami had caused damage to their facilities; some of these were located within a 20-km radius of the NPP and in the evacuation areas. Local fire department personnel were also ordered to evacuate. Fukushima prefecture changed the screening level required for decontamination from 13,000 to 100,000 cpm, with decontamination by wiping being performed for over 13,000 cpm. However, as hospitals and fire departments had to abide by lower levels than that of the prefecture for receiving or transporting contaminated patients, these personnel could not accept or transport contaminated people from the NPPs. In addition, hospitals not designated as radiation emergency facilities would not receive patients from the NPPs because of concerns about the health effects of radiation. From this disaster, it was learned that basic knowledge of radiation and its effects is extremely important for health care providers.

Acceleration of Regeneration of Mucosa in Small Intestine Damaged by Ionizing Radiation Using Anabolic Steroids

Abstract Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.

Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.

Role of TNFα in regulation of myeloperoxidase expression in irradiated HL60 promyelocytic cells

Irradiation increases the generation of reactive oxygen intermediates, including hydrogen peroxide (H(2)O(2)). Myeloperoxidase (MPO), a heme-containing glycoprotein located in the primary granules of polymorphonuclear leukocytes and monocytes, reacts with H(2)O(2) and halide ion and produces a more potent microbicidal oxidant, hypochlorous acid (HOCl). Human HL60 promyelocytes constitutively had high levels of MPO protein and mRNA. Irradiation decreased the levels of MPO transcripts; the decrease in MPO transcripts by irradiation occurred in an almost dose-dependent manner. HL60 cells produce tumor necrosis factor alpha (TNFalpha), and irradiation markedly increased the TNFalpha production in these cells; in turn, TNFalpha decreased the levels of MPO transcripts in these cells. Furthermore, treatment of cells with anti-TNFalpha antibody blocked the reduction of MPO by irradiation. We also found that irradiation decreased the levels of the MPO mRNA with concomitant increased levels of TNFalpha mRNA in differentiation-induced HL60 cells and human THP-1 monocytic cells. Irradiation reduced the rate of MPO transcription but had only a slight effect on the half-life of MPO mRNA in HL60 cells. Our results suggest that irradiation reduces the steady-state levels of MPO mRNA mainly at transcriptional level and the endogenous production of TNFalpha is required for the reduction by irradiation in HL60 cells.

Anti-cancer agent OK432 induces manganese superoxide dismutase in human granulocytes

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in scavenging O2‐.OK432, a Streptococcal preparation, has anti‐tumor activity and is also known to be a biological‐response modifier. In this study, we have examined the regulation of MnSOD gene by OK432 in human granulocytes. Granulocytes had a low activity of MnSOD; OK432 increased MnSOD mRNA levels in a dose‐dependent manner and its activity in granulocytes. OK432 also induced the MnSOD mRNA in HL60 promyelocytes, while not affecting the levels of MnSOD mRNA in human fibroblasts. Pre‐treatment with either actinomycin D or cycloheximide inhibited the induction of MnSOD mRNA by OK432. OK432 increased the levels of interleukin‐I (IL‐I) and tumor‐necrosis‐factor‐α (TNFα) mRNA in granulocytes. Furthermore, the induction of MnSOD mRNA by OK432 was blocked by anti‐IL‐I antibody but not by anti‐TNF antibody. These results suggest that the induction of MnSOD mRNA by OK432 is regulated at the transcriptional level, and OK432 induces MnSOD mRNA, at least in part, through production of IL‐I in granulocytes.

EARLY INTAKE OF RADIOCESIUM BY RESIDENTS LIVING NEAR THE TEPCO FUKUSHIMA DAI -ICHI NUCLEAR POWER PLANT AFTER THE ACCIDENT.PART 1: INTERNAL DOSES BASED ON WHOLE-BODY MEASUREMENTS BY NIRS

AbstractThe Tokyo Electric Power Company’s Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident in 2011 resulted in a release of radionuclides into the environment (131I: 142.9 PBq, 137Cs:12.4 PBq). This study presents the results of internal doses to 174 residents living near the FDNPP at the time of the accident based on whole-body (WB) measurements performed by the National Institute of Radiological Sciences (NIRS) during the period between 27 June and 28 July 2011. The 174 subjects consisted of 125 adults (≥18-y) and 49 children (<18-y) and included 90 persons of Namie town, one of the municipalities heavily contaminated with the radionuclides. The number of subjects with significant detection of both 134Cs and 137Cs was relatively small: 28.8% for the adults and 4.1% for the children. A significant gender difference in the Cs detection rate (males > females) was observed in the adults but not the children. In this study, the committed effective dose (CED) from 134Cs and 137Cs was calculated based on individual WB contents (134Cs) corrected against body size, the observed body content ratio of 137Cs to 134Cs, and the assumed intake scenario (namely, acute inhalation of Type F compounds on 12 March 2011 when the first explosive event occurred at the site of the FDNPP). The 90th-percentile CED value for the adults was around 0.1 mSv and the maximum CED (0.63 mSv) was found in an elderly male. Comparable CED results were obtained in other WB measurements subsequently performed by the Japan Atomic Energy Agency (JAEA) in a similar manner to that of the NIRS, suggesting that the contribution of ingestion to the WB content observed would be trivial for most of the JAEA subjects. The intake ratio of 131I to 134Cs was evaluated to be 3~5 based on the 131I thyroid measurement data of Tokonami et al. Using the average intake ratio of 3.8, the resulting median and maximum thyroid-equivalent doses to the adult subjects of this study were estimated at 3.5 mSv and 84 mSv, respectively.

Medical Management of the Consequences of the Fukushima Nuclear Power Plant Incident

A huge earthquake struck the northeast coast of the main island of Japan on March 11, 2011, triggering a tsunami with 14–15 meter‐high waves hitting the area. The earthquake was followed by numerous sustained aftershocks. The earthquake affected the nuclear power plant (NPP) in Fukushima prefecture, resulting in large amounts of radioactive materials being released into the environment. The major nuclides released on land were 131I, 134Cs, and 137Cs. Therefore, almost 170 000 people had to be evacuated or stay indoors. Besides the NPP and the telecommunications system, the earthquake also affected infrastructures such as the supplies of water and electricity as well as the radiation monitoring system. The local hospital system was dysfunctional; hospitals designated as radiation‐emergency facilities were not able to function because of damage from the earthquake and tsunami, and some of them were located within a 20 km radius of the NPP, the designated evacuation zone. Local fire department personnel were also asked to evacuate. Furthermore, the affected hospitals had not established their evacuation plans at that time. We have learned from this “combined disaster” that the potential for damage to lifelines as well as the monitoring systems for radiation in case of an earthquake requires our intense focus and vigilance, and that hospitals need comprehensive plans for evacuation, including patients requiring life support equipment during and after a nuclear disaster. There is an urgent need for a “combined disaster” strategy, and this should be emphasized in current disaster planning and response.

Exogenously-added copper/zinc superoxide dismutase rescues damage of endothelial cells from lethal irradiation

The vascular endothelium is important for the early and late effects observed in lethally irradiated tissue and organs. We examined the effects of exogenously added superoxide dismutase on cell survival and angiogenesis in lethally irradiated human primary umbilical vein endothelial cells. Cell survival was significantly improved in superoxide dismutase-treated cells; the addition of superoxide dismutase to cells after irradiation was also effective for increased survival, as it was before irradiation. Moreover, treatment of cells with superoxide dismutase enhanced the phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal regulated kinases 1 and 2 in human primary umbilical vein endothelial cells. The addition of superoxide dismutase to cells after irradiation attenuated the reduction of angiogenesis by irradiation, and inhibition of the mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal regulated kinases signaling pathway abrogated the rescue effect of superoxide dismutase. Our results suggest that superoxide dismutase rescues human primary umbilical vein endothelial cells from endothelial dysfunction caused by irradiation via a pathway requiring activation of mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal regulated kinases 1 and 2.