Involvement of intracellular free Ca(2+ )in enhanced release of herpes simplex virus by hydrogen peroxide

BACKGROUND: It was reported that elevation of the intracellular concentration of free Ca(2+ )([Ca(2+)]i) by a calcium ionophore increased the release of herpes simplex virus type 1 (HSV-1). Freely diffusible hydrogen peroxide (H(2)O(2)) is implied to alter Ca(2+ )homeostasis, which further enhances abnormal cellular activity, causing changes in signal transduction, and cellular dysfunction. Whether H(2)O(2 )could affect [Ca(2+)]i in HSV-1-infected cells had not been investigated. RESULTS: H(2)O(2 )treatment increased the amount of cell-free virus and decreased the proportion of viable cells. After the treatment, an elevation in [Ca(2+)]i was observed and the increase in [Ca(2+)]i was suppressed when intracellular and cytosolic Ca(2+ )were buffered by Ca(2+ )chelators. In the presence of Ca(2+ )chelators, H(2)O(2)-mediated increases of cell-free virus and cell death were also diminished. Electron microscopic analysis revealed enlarged cell junctions and a focal disintegration of the plasma membrane in H(2)O(2)-treated cells. CONCLUSION: These results indicate that H(2)O(2 )can elevate [Ca(2+)]i and induces non-apoptotic cell death with membrane lesions, which is responsible for the increased release of HSV-1 from epithelial cells

Effect of ultrasound on herpes simplex virus infection in cell culture

<p>Abstract</p> <p>Background</p> <p>Ultrasound has been shown to increase the efficiency of gene expression from retroviruses, adenoviruses and adeno-associated viruses. The effect of ultrasound to stimulate cell membrane permeabilization on infection with an oncolytic herpes simplex virus type 1 (HSV-1) was examined.</p> <p>Results</p> <p>Vero monkey kidney cells were infected with HSV-1 and exposed to 1 MHz ultrasound after an adsorption period. The number of plaques was significantly greater than that of the untreated control. A combination of ultrasound and microbubbles further increased the plaque number. Similar results were obtained using a different type of HSV-1 and oral squamous cell carcinoma (SCC) cells. The appropriate intensity, duty cycle and time of ultrasound to increase the plaque number were 0.5 W/cm², 20% duty cycle and 10 sec, respectively. Ultrasound with microbubbles at an intensity of 2.0 W/cm², at 50% duty cycle, or for 40 sec reduced cell viability.</p> <p>Conclusion</p> <p>These results indicate that ultrasound promotes the entry of oncolytic HSV-1 into cells. It may be useful to enhance the efficiency of HSV-1 infection in oncolytic virotherapy.</p

Mechanism Underlying IKK Activation Mediated by the Linear Ubiquitin Chain Assembly Complex (LUBAC)

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex

Role of p53 mutation in the effect of boron neutron capture therapy on oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Boron neutron capture therapy (BNCT) is a selective radiotherapy, being effective for the treatment of even advanced malignancies in head and neck regions as well as brain tumors and skin melanomas. To clarify the role of p53 gene, the effect of BNCT on oral squamous cell carcinoma (SCC) cells showing either wild- (SAS/neo) or mutant-type (SAS/mp53) p53 was examined.</p> <p>Methods</p> <p>Cells were exposed to neutron beams in the presence of boronophenylalanine (BPA) at Kyoto University Research Reactor. Treated cells were monitored for modulations in colony formation, proliferation, cell cycle, and expression of cell cycle-associated proteins.</p> <p>Results</p> <p>When SAS/neo and SAS/mp53 cells were subjected to BNCT, more suppressive effects on colony formation and cell viability were observed in SAS/neo compared with SAS/mp53 cells. Cell cycle arrest at the G1 checkpoint was observed in SAS/neo, but not in SAS/mp53. Apoptotic cells increased from 6 h after BNCT in SAS/neo and 48 h in SAS/mp53 cells. The expression of p21 was induced in SAS/neo only, but G2 arrest-associated proteins including Wee1, cdc2, and cyclin B1 were altered in both cell lines.</p> <p>Conclusion</p> <p>These results indicate that oral SCC cells with mutant-type are more resistant to BNCT than those with wild-type p53, and that the lack of G1 arrest and related apoptosis may contribute to the resistance. At a physical dose affecting the cell cycle, BNCT inhibits oral SCC cells in p53-dependent and -independent manners.</p

Involvement of Hydrogen Peroxide in Safingol-Induced Endonuclease G-Mediated Apoptosis of Squamous Cell Carcinoma Cells

Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a mitochondrial apoptogenic mediator—endonuclease G (endo G)—and apoptosis of human oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown. The levels of hydrogen peroxide (H2O2) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H2O2 levels but not extracellular H2O2 levels, indicating the production of H2O2. The cell killing effect of safingol and H2O2 was diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that apoptotic cell death occurred by treatment with H2O2 and safingol. The number of apoptotic cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the cytoplasm and an association of endo G with mitochondria was observed. After treatment with H2O2 and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic cells and the translocation of endo G. Knock down of endo G diminished the cell killing effect of H2O2 and safingol. These results suggest that H2O2 is involved in the endo G-mediated apoptosis of oral SCC cells by safingol

Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability

Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects

Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound

Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the 134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval