p53 変異型ヒト口腔がん細胞における高LET 放射線によるp53 非依存Akt 生存シグナルの抑制

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.博士（医学）・甲第598号・平成25年3月15日Copyright © 2012 Elsevier Inc. All rights reserve

ケッカン ナイヒ サイボウ ニ オケル テイ グルコース ジョウタイ ニ ヨル ミトコンドリア ユライ カッセイ サンソ シュ ノ サンセイ ゾウカ ト シボウサン サンカ ノ カンヨ : トウニョウビョウ ガッペイショウ ノ アラタ ナ メカニズム ノ カノウセイ

博士(医学）熊本大

Differential activation of astrocytes and microglia after spinal cord injury in the fetal rat

Background: Because the immature spinal cord was nerve growth permissive, we examined glial reactions that influence regeneration of the spinal cord in a fetal rat spinal cord injury model. Methods: Three, 7, 21, and 35 days after intrauterine surgery, offsprings were killed and the thoracic and lumbar spinal cords were carefully removed from the spinal column and then cut into 10 m longitudinal sections. These sections were stained with hematoxylin-eosin, anti-glial fibrillary acidic protein antibody (GFAP) as a marker of astrocytes, and anti-complement CR3 antibody (OX-42) as a marker of microglia. A cordotomy model in young adult rat was utilized as a control. Results: In the present study, collagen fibers and scar formation were seen in the severed spinal cords of mature rats, but scar formation was not seen in the fetal rat cordotomy group, regardless of spinal continuity. In the Control group, biological activity of GFAP-positive cells increased over time. In the fetal rat cordotomy model, activity elevated slightly immediately after cordotomy, and disappeared shortly thereafter. In the Control group, OX-42 positive macrophage-like cells proliferated over time. However, in the fetal rat cordotomy model, OX-42 positive macrophage-like cells were recognized on postoperative days 3 and 7, and then disappeared. At 5 mm from the cordotomy site, reactive microglia were recognized in the white matter of Control group spinal cords, but these microglia were not recognized in the fetal rat cordotomy model. Conclusions: In the present study, collagen fibers and scar formation were seen in the severed spinal cords of adult rats, but scar formation was not seen in the fetal rat cordotomy group. Lack of inflammation and scar formation thus appear advantageous for regeneration of the fetal spinal cord. Between fetal and mature rats, chronological changes in the immunohistochemical reactions of astrocytes and microglia following cordotomy were compared, and the results confirmed many differences. The results of the pres

Hyperglycemia Induces Cellular Hypoxia through Production of Mitochondrial ROS Followed by Suppression of Aquaporin-1.

We previously proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) generation is a key event in the development of diabetic complications. Interestingly, some common aspects exist between hyperglycemia and hypoxia-induced phenomena. Thus, hyperglycemia may induce cellular hypoxia, and this phenomenon may also be involved in the pathogenesis of diabetic complications. In endothelial cells (ECs), cellular hypoxia increased after incubation with high glucose (HG). A similar phenomenon was observed in glomeruli of diabetic mice. HG-induced cellular hypoxia was suppressed by mitochondria blockades or manganese superoxide dismutase (MnSOD) overexpression, which is a specific SOD for mtROS. Overexpression of MnSOD also increased the expression of aquaporin-1 (AQP1), a water and oxygen channel. AQP1 overexpression in ECs suppressed hyperglycemia-induced cellular hypoxia, endothelin-1 and fibronectin overproduction, and apoptosis. Therefore, hyperglycemia-induced cellular hypoxia and mtROS generation may promote hyperglycemic damage in a coordinated manner

AQP1 overexpression decreased high glucose-induced mitochondrial ROS generation and high glucose-induced phenomena.

<p>(A) Mitochondrial reactive oxygen species (mtROS) generation in aquaporin-1 (AQP1) overexpression cells. Cells were incubated under indicated conditions, and treated with 300 nM CM-H₂XRos for 15 min. Relative intensity of fluorescence of CM-H₂XRos was measured. Black bars = control adenovirus; white bars = AQP1 adenovirus. *P < 0.05 compared with 5.5 mM glucose and control adenovirus; #P < 0.05 compared with 5.5 mM glucose and AQP1 adenovirus; †P < 0.05 compared with 25 mM glucose and AQP1 adenovirus. Data are eight independent experiments in duplicate ± SEM. (B) Effect of AQP1 overexpression on endothelin-1 mRNA expression. Cells were incubated under indicated conditions for 24 h. The expression levels of endothelin-1 mRNA were measured by quantitative RT-PCR analysis. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus. Data are five independent experiments in duplicate ± SEM. (C) Effect of AQP1 overexpression on endothelin-1 secretion. Cells were incubated under indicated conditions for 24 h, and endothelin-1 secretion was measured by ELISA assay. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus. Data are six independent experiments in duplicate ± SEM. (D) Effect of AQP1 overexpression on fibronectin mRNA expression. Cells were incubated under indicated conditions for 24 h. The expression levels of fibronectin mRNA were measured by quantitative RT-PCR analysis. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus. Data are five independent experiments in duplicate ± SEM. (E) Effect of AQP1 overexpression on cell apoptosis. Cells were incubated under indicated conditions for 168 hours. Data are expressed as the mean number of positive cells/section of 10 independent sections ± SEM. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus.</p

Cellular hypoxia was attenuated by inhibiting mitochondrial respiration and MnSOD overexpression <i>in vitro and vivo</i>.

<p>(A) Effect of mitochondrial respiratory inhibitors on cellular hypoxia. Cells were incubated for 3 h with indicated reagents (5 μM rotenone, 10 μM antimycin A) and 10 μM pimonidazole. Relative intensity of pimonidazole staining was measured. *P < 0.05 compared with 21% O₂ and 5.5 mM glucose; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and 5 μM rotenone; †P < 0.05 compared with 21% O₂, 5.5 mM glucose and 10 μM antimycin A. Rote, rotenone; anti, antimycin A. Data are 10 independent experiments in duplicate ± SEM. (B) Effect of manganese superoxide dismutase (MnSOD) overexpression on pimonidazole staining. Cells transduced with MnSOD or control adenovirus were incubated for 3 h under indicated conditions and 10 μM pimonidazole, and relative intensity of pimonidazole staining was measured. Black bars = control adenovirus; white bars = MnSOD adenovirus. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and MnSOD adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and MnSOD adenovirus. Data from B and C are eight independent experiments in duplicate ± SEM. (C) Effect of MnSOD overexpression on LOX-1 phosphorescence. Cells transduced with MnSOD or control adenovirus were incubated for 24 h under indicated conditions and 2 μM LOX-1, and the relative intensity of LOX-1 phosphorescence was measured. Black bars = control adenovirus; white bars = MnSOD adenovirus. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 5.5 mM glucose, and MnSOD adenovirus; †P < 0.05 compared with 21% O₂, 25 mM glucose, and MnSOD adenovirus. (D-G) Immunofluorescence for 8-OHdG (8-hydroxy-2'-deoxyguanosine, D and E) and pimonidazole (F and G) in mice glomeruli (<i>blue</i>: 4′,6-diamidino-2-phenylindole; <i>red</i>: 8-OHdG; <i>green</i>: pimonidazole). Diabetes mellitus (DM) was induced in C57Bl/6 mice (8–10 weeks old) by streptozotocin injection. Immunohistochemistry was performed at 4 weeks after the onset of DM. Scale bars represent 20 μm. *P < 0.05 compared with non-DM control mice; #P < 0.05 compared with non-DM MnSOD-Tg mice; †P < 0.05 compared with diabetic MnSOD-Tg mice. n = 6/group</p

Proposed model of the pathogenesis of diabetic complications.

<p>High glucose increases mitochondrial reactive oxygen species (mtROS) generation. High glucose also induces cellular hypoxia through increased O₂ consumption in mitochondria. Cellular hypoxia may also be affected through suppressed aquaporin-1 (AQP1) expression induced by mtROS generation. Hyperglycemia-induced cellular hypoxia and mtROS generation may simultaneously promote hyperglycemic damage including overproduction of endothelin-1 and fibronectin, and induction of apoptosis, which leading to diabetic vascular complications.</p

Metagenomic analyses of the gut microbiota associated with colorectal adenoma.

Recent studies have suggested an association between certain members of the Fusobacterium genus, especially F. nucleatum, and the progression of advanced colorectal carcinoma (CRC). We assessed such an association of the gut microbiota in Japanese patients with colorectal adenoma (CRA) or intramucosal CRC using colonoscopy aspirates. We analyzed samples from 81 Japanese patients, including 47 CRA and 24 intramucosal CRC patients, and 10 healthy subjects. Metagenomic analysis of the V3-V4 region of the 16S ribosomal RNA gene was performed. The linear discriminant analysis (LDA) effect size (LEfSe) method was used to examine microbial dysbiosis, revealing significant differences in bacterial abundances between the healthy controls and CRA or intramucosal CRC patients. In particular, F. varium was statistically more abundant in patients with CRA and intramucosal CRC than in healthy subjects. Here, we present the metagenomic profile of CRA and intramucosal CRC and demonstrate that F. varium is at least partially involved in the pathogenesis of CRA and intramucosal CRC

AQP1 is involved in high glucose-induced cellular hypoxia and is affected by mitochondrial ROS generation.

<p>(A) Expression levels of membrane aquaporin-1 (AQP1) protein. Cells transduced with manganese superoxide dismutase (MnSOD) or control adenovirus were incubated under indicated conditions for 24 h, and membrane proteins were examined by western blotting for detection of membrane AQP1 expression. The experiments were repeated at least four times. *P < 0.05 compared with 5.5 mM glucose and control adenovirus; #P < 0.05 compared with 5.5 mM glucose, 19.5 mM L-glucose, and control adenovirus; †P < 0.05 compared with 5.5 mM glucose, MnSOD adenovirus. Data from A and B are four independent experiments in duplicate ± SEM. (B) Hydrogen peroxide attenuated membrane AQP1 expression. Cells were incubated under indicated conditions for 30 min, and membrane proteins were subjected to western blotting. The experiments were repeated at least four times. *P < 0.05 compared with no agent. (C and D) Immunofluorescence for AQP1 in mouse glomeruli (<i>red</i>: AQP1). C57Bl/6 mice (8 weeks old) were made diabetes mellitus (DM) by streptozotocin injection. Immunohistochemistry was performed at 4 weeks after the onset of DM. Scale bars represent 20 μm. *P < 0.05 compared with non-DM control mice. n = 5/group. (E and F) Effect of AQP1 overexpression on pimonidazole staining. Cells infected with AQP1 or control adenovirus were incubated for 3 h, and relative intensity of pimonidazole staining (<i>green</i>) was measured. Black bars = control adenovirus; white bars = AQP1 adenovirus. *P < 0.05 compared with 21% O₂, 5.5 mM glucose, and control adenovirus; #P < 0.05 compared with 21% O₂, 25 mM glucose, and AQP1 adenovirus; †P < 0.05 compared with 21% O₂, 5.5 mM glucose, and AQP1 adenovirus. Data are eight independent experiments in duplicate ± SEM.</p