Adhesion States Greatly Affect Cellular Susceptibility to Graphene Oxide: Therapeutic Implications for Cancer Metastasis.

Graphene oxide (GO) has received increasing attention in the life sciences because of its potential for various applications. Although GO is generally considered biocompatible, it can negatively impact cell physiology under some circumstances. Here, we demonstrate that the cytotoxicity of GO greatly varies depending on the cell adhesion states. Human HCT-116 cells in a non-adhered state were more susceptible to GO than those in an adherent state. Apoptosis was partially induced by GO in both adhered and non-adhered cells to a similar extent, suggesting that apoptosis induction does not account for the selective effects of GO on non-adhered cells. GO treatment rapidly decreased intracellular ATP levels in non-adhered cells but not in adhered ones, suggesting ATP depletion as the primary cause of GO-induced cell death. Concurrently, autophagy induction, a cellular response for energy homeostasis, was more evident in non-adhered cells than in adhered cells. Collectively, our observations provide novel insights into GO's action with regard to cell adhesion states. Because the elimination of non-adhered cells is important in preventing cancer metastasis, the selective detrimental effects of GO on non-adhered cells suggest its therapeutic potential for use in cancer metastasis.

Disease-associated H58Y mutation affects the nuclear dynamics of human DNA topoisomerase IIß.

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems and plays critical roles in various nuclear processes. Recently, a heterozygous H58Y substitution in the ATPase domain of human TOP2B was identified from patients with autism spectrum disorder, but its biological significance remains unclear. In this study, we analyzed the nuclear dynamics of TOP2B with H58Y (TOP2B H58Y). Although wild-type TOP2B was highly mobile in the nucleus of a living cell, the nuclear mobility of TOP2B H58Y was markedly reduced, suggesting that the impact of H58Y manifests as low protein mobility. We found that TOP2B H58Y is insensitive to ICRF-187, a TOP2 inhibitor that halts TOP2 as a closed clamp on DNA. When the ATPase activity of TOP2B was compromised, the nuclear mobility of TOP2B H58Y was restored to wild-type levels, indicating the contribution of the ATPase activity to the low nuclear mobility. Analysis of genome-edited cells harboring TOP2B H58Y showed that TOP2B H58Y retains sensitivity to the TOP2 poison etoposide, implying that TOP2B H58Y can undergo at least a part of its catalytic reactions. Collectively, TOP2 H58Y represents a unique example of the relationship between a disease-associated mutation and perturbed protein dynamics.

Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21.

DNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

Crescentic poststreptococcal acute glomerulonephritis accompanied by small vessel vasculitis: case report of an elderly male.

BACKGROUND: Poststreptococcal acute glomerulonephritis (PSAGN) in the elderly tends to have a severe clinical course and often presents with crescentic necrotizing glomerulonephritis in the renal biopsy. However, vasculitis lesions are unusual. CASE PRESENTATION: We present a 71-year-old man who was admitted to our hospital for a recurrent gout attack with a rapid decline of renal function. Low C3 levels and a high anti-streptolysin O titer were observed, while myeloperoxidase- and proteinase 3- antineutrophil cytoplasmic antibody (ANCA) were negative. In addition to cellular crescent and necrosis lesions, diffuse peritubular capillaritis and venulitis as well as small arteriole vasculitis in the glomerular hilus were also apparent. Although granular C3c deposits in the capillary wall and hump lesions were not found, immunofluorescent staining for nephritis-associated plasmin receptor (NAPlr) and in situ zymography for plasmin activity were both positive. We thus diagnosed PSAGN accompanied by small vessel vasculitis. Steroid therapy gradually improved the patient's renal function, and hemodialysis was discontinued after 1 month. CONCLUSIONS: In our case, streptococcus infection might have concurrently provoked vasculitis, and NAPlr staining was useful for confirming diagnosis.

Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells.

Nanosecond pulsed electric fields (nsPEFs) have gained attention as a novel physical stimulus for life sciences. Although cancer therapy is currently their promising application, nsPEFs have further potential owing to their ability to elicit various cellular responses. This study aimed to explore stimulatory actions of nsPEFs, and we used HL-60 cells that were differentiated into neutrophils under cultured conditions. Exposure of neutrophil-differentiated HL-60 cells to nsPEFs led to the extracellular release of chromosomal DNA, which appears to be equivalent to neutrophil extracellular traps (NETs) that serve as a host defense mechanism against pathogens. Fluorometric measurement of extracellular DNA showed that DNA extrusion was rapidly induced after nsPEF exposure and increased over time. Western blot analysis demonstrated that nsPEFs induced histone citrullination that is the hydrolytic conversion of arginine to citrulline on histones and facilitates chromatin decondensation. DNA extrusion and histone citrullination by nsPEFs were cell type-specific and Ca2+-dependent events. Taken together, these observations suggest that nsPEFs drive the mechanism for neutrophil-specific immune response without infection, highlighting a novel aspect of nsPEFs as a physical stimulus.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells.

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

Dynamic behavior of DNA topoisomerase IIß in response to DNA double-strand breaks.

DNA topoisomerase II (Topo II) is crucial for resolving topological problems of DNA and plays important roles in various cellular processes, such as replication, transcription, and chromosome segregation. Although DNA topology problems may also occur during DNA repair, the possible involvement of Topo II in this process remains to be fully investigated. Here, we show the dynamic behavior of human Topo IIß in response to DNA double-strand breaks (DSBs), which is the most harmful form of DNA damage. Live cell imaging coupled with site-directed DSB induction by laser microirradiation demonstrated rapid recruitment of EGFP-tagged Topo IIß to the DSB site. Detergent extraction followed by immunofluorescence showed the tight association of endogenous Topo IIß with DSB sites. Photobleaching analysis revealed that Topo IIß is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIß mobility and thereby prevented Topo IIß recruitment to DSBs. Furthermore, Topo IIß knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo IIß in HR-mediated DSB repair. Taken together, these results highlight a novel aspect of Topo IIß functions in the cellular response to DSBs.

Calcium-dependent activation of transglutaminase 2 by nanosecond pulsed electric fields.

Exposure of cultured human cells to nanosecond pulsed electric fields (nsPEFs) elicits various cellular events, including Ca2+ influx and cell death. Recently, nsPEFs have been regarded as a novel physical treatment useful for biology and medicine, but the underlying mechanism of action remains to be fully elucidated. In this study, we investigated the effect of nsPEFs on transglutaminases (TGs), enzymes that catalyze covalent protein modifications such as protein-protein crosslinking. Cellular TG activity was monitored by conjugation of cellular proteins with biotin-cadaverine, a cell-permeable pseudosubstrate for TGs. We applied nsPEFs to HeLa S3 cells and found that overall catalytic activity of cellular TGs was greatly increased in a Ca2+-dependent manner. The Ca2+ ionophore ionomycin significantly augmented nsPEF-induced TG activation, further supporting the importance of Ca2+. Among human TG family members, TG2 is known to be the most ubiquitously expressed, and its catalytic activity requires elevated intracellular Ca2+. Given the requirement of Ca2+ for TG activation by nsPEFs, we performed depletion of TG2 by RNA interference (RNAi). We observed that TG2 RNAi suppressed the nsPEF-induced TG activation and partially alleviated the cytotoxic effects of nsPEFs. These findings demonstrate that TG2 activation is a Ca2+-dependent event in nsPEF-exposed cells and exerts negative effects on cell physiology.

Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin-induced neutrophil extracellular trap-osis (NETosis)-like cell death in cultured human leukemia cells.

We previously reported that the nucleoside antibiotic tunicamycin (TN), a protein glycosylation inhibitor triggering unfolded protein response (UPR), induced neutrophil extracellular trap-osis (NETosis)-like cellular suicide and, thus, discharged genomic DNA fibers to extracellular spaces in a range of human myeloid cell lines under serum-free conditions. In this study, we further evaluated the effect of TN on human promyelocytic leukemia HL-60 cells using time-lapse microscopy. Our assay revealed a previously unappreciated early event induced by TN-exposure, in which, at 30-60 min after TN addition, the cells extruded their nuclei into the extracellular space, followed by discharge of DNA fibers to form NET-like structures. Intriguingly, neither nuclear extrusion nor DNA discharge was observed when cells were exposed to inducers of UPR, such as brefeldin A, thapsigargin, or dithiothreitol. Our findings revealed novel nuclear dynamics during TN-induced NETosis-like cellular suicide in HL-60 cells and suggested that the toxicological effect of TN on nuclear extrusion and DNA discharge was not a simple UPR.

The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

Different involvement of extracellular calcium in two modes of cell death induced by nanosecond pulsed electric fields.

Exposure of cultured cells to nanosecond pulsed electric fields (nsPEFs) induces various cellular responses, including the influx of extracellular Ca2+ and cell death. Recently, nsPEFs have been regarded as a novel means of cancer therapy, but their molecular mechanism of action remains to be fully elucidated. Here, we demonstrate the involvement of extracellular Ca2+ in nsPEF-induced cell death. Extracellular Ca2+ was essential for necrosis and consequent poly(ADP-ribose) (PAR) formation in HeLa S3 cells. Treatment with a Ca2+ ionophore enhanced necrosis as well as PAR formation in nsPEF-exposed HeLa S3 cells. In the absence of extracellular Ca2+, HeLa S3 cells were less susceptible to nsPEFs and exhibited apoptotic proteolysis of caspase 3 and PARP-1. HeLa S3 cells retained the ability to undergo apoptosis even after nsPEF exposure but instead underwent necrosis, suggesting that necrosis is the preferential mode of cell death. In K562 and HEK293 cells, exposure to nsPEFs resulted in the formation of necrosis-associated PAR, whereas Jurkat cells exclusively underwent apoptosis independently of extracellular Ca2+. These observations demonstrate that the mode of cell death induced by nsPEFs is cell-type dependent and that extracellular Ca2+ is a critical factor for nsPEF-induced necrosis.

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells.

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

[Effect of magnesium supplementation on early-stage hypomagnesemia in patients treated with cetuximab].

Cetuximab is a monoclonal antibody that binds to the EGFR, and is used in patients with colorectal cancer. The most common toxicities associated with the use of cetuximab are rash and hypomagnesemia. Hypomagnesemia is a major adverse event, but it has often been ignored in past studies and its management has not been characterized. The aim of this study was to obtain a better understanding of the overall incidence of hypomagnesemia and to evaluate the usefulness of our original treatment guidelines for hypomagnesemia in patients receiving cetuximab-based therapy. We investigated 15 patients who were treated with cetuximab(with or without combined chemotherapy)between October 2008 and November 2010. Thirteen patients developed hypomagnesemia: 11 patients had Grade 1, one patient had Grade 2, and one patient had severe hypomagnesemia(Grade 3). Grade 1 hypomagnesemia was observed after an average of 7. 5±4. 8 weeks of treatment. None of the patients developed Grade 2 or higher hypomagnesemia after the implementation of our treatment guidelines. In conclusion, cetuximab treatment is associated with a significant risk of hypomagnesemia. The early monitoring and effective management of hypomagnesemia are important for patients receiving cetuximab-based therapy.

Vancomycin Ophthalmic Ointment 1% for methicillin-resistant Staphylococcus aureus or methicillin-resistant Staphylococcus epidermidis infections: a case series.

OBJECTIVES: To investigate the efficacy and safety of Vancomycin Ophthalmic Ointment 1% (Toa Pharmaceutical Co., Ltd, Toyama, Japan) in patients with external ocular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant Staphylococcus epidermidis (MRSE). DESIGN: A case series. SETTING: This study was a multicentre, open-label, uncontrolled study in Japan approved as orphan drug status. PARTICIPANTS: Patients with MRSA or MRSE external ocular infections unresponsive to the treatment of fluoroquinolone eye drops. INTERVENTIONS: Vancomycin Ophthalmic Ointment 1% was administered four times daily. PRIMARY AND SECONDARY OUTCOME MEASURES: The subjective and objective clinical scores and bacterial cultures were collected at days 0 (baseline), 3, 7 and 14. The primary outcome was clinical response evaluation (efficacy rate) determined as complete response, partial response, no response and worsening. Secondary outcome was the eradication of the bacteria. Safety was assessed by adverse events including cases in which neither MRSA nor MRSE was detected. RESULTS: Twenty-five cases with MRSA (20) or MRSE (5) infections were enrolled. Of these 25 cases, 4 discontinued the treatment due to the negative results for bacterial culture during screening or at baseline. Of the 21 cases with conjunctivitis (14), blepharitis (3), meibomitis (1), dacryocystitis (2) or keratitis (1), 14 (66.7%) cases were evaluated as being excellently (complete response, 2 cases) or well (partial response, 12 cases) treated. The eradication rates were 68.4% in MRSA (13 of 19 cases) and 100% in MRSE (2 of 2 cases). Ten adverse events occurred in 7 (28.0%) of 25 cases at the local administration site. CONCLUSIONS: Vancomycin Ophthalmic Ointment 1% was considered to be useful for the treatment of intractable ocular MRSA/MRSE infections.

Nanosecond pulsed electric fields activate AMP-activated protein kinase: implications for calcium-mediated activation of cellular signaling.

Nanosecond pulsed electric fields (nsPEFs) are increasingly being recognized as a potential tool for use in the life sciences. Exposure of human cells to nsPEFs elicits the formation of small membrane pores, intracellular Ca(2+) mobilization, signaling pathway activation, and apoptosis. Here we report the activation of AMP-activated protein kinase (AMPK) by nsPEFs. AMPK activation is generally achieved by the phosphorylation of AMPK in response to changes in cellular energy status and is mediated by two protein kinases, LKB1 and CaMKK. Exposure to nsPEFs rapidly induced phosphorylation of AMPK and its downstream target ACC in both LKB1-proficient and LKB1-deficient cells. In LKB1-deficient cells, AMPK activation by nsPEFs was mediated by CaMKK and required extracellular Ca(2+), which suggested the occurrence of Ca(2+) mobilization and its participation in AMPK activation by nsPEFs. Our results provide experimental evidence for a direct link between activated cellular signaling and Ca(2+) mobilization in nsPEF-exposed cells.

Nanosecond pulsed electric fields act as a novel cellular stress that induces translational suppression accompanied by eIF2α phosphorylation and 4E-BP1 dephosphorylation.

Recent advances in electrical engineering enable the generation of ultrashort electric fields, namely nanosecond pulsed electric fields (nsPEFs). Contrary to conventional electric fields used for DNA electroporation, nsPEFs can directly reach intracellular components without membrane destruction. Although nsPEFs are now recognized as a unique tool in life sciences, the molecular mechanism of nsPEF action remains largely unclear. Here, we present evidence that nsPEFs act as a novel cellular stress. Exposure of HeLa S3 cells to nsPEFs quickly induced phosphorylation of eIF2α, activation of its upstream stress-responsive kinases, PERK and GCN2, and translational suppression. Experiments using PERK- and GCN2-knockout cells demonstrated dual contribution of PERK and GCN2 to nsPEF-induced eIF2α phosphorylation. Moreover, nsPEF exposure yielded the elevated GADD34 expression, which is known to downregulate the phosphorylated eIF2α. In addition, nsPEF exposure caused a rapid decrease in 4E-BP1 phosphorylation irrespective of the PERK/GCN2 status, suggesting participation of both eIF2α and 4E-BP1 in nsPEF-induced translational suppression. RT-PCR analysis of stress-inducible genes demonstrated that cellular responses to nsPEFs are distinct from those induced by previously known forms of cellular stress. These results provide new mechanistic insights into nsPEF action and implicate the therapeutic potential of nsPEFs for stress response-associated diseases.

Nanosecond pulsed electric fields activate MAPK pathways in human cells.

Application of nanosecond pulsed electric fields (nsPEFs) has attracted attention as a unique tool in life sciences, especially for cancer therapy, but the molecular mechanism of its action on living organisms is yet to be fully elucidated. Here, we report a transient activation of signaling pathways involving mitogen-activated protein kinases (MAPKs) by nsPEFs. Application of nsPEFs to HeLa S3 cells induced phosphorylation of MAPKs, including p38, JNK and ERK, and their upstream kinases. The application of nsPEFs also elicited elevated phosphorylation of downstream factors including MSK1, Hsp27, ATF2, p90RSK, and c-Jun. In addition, the application of nsPEFs led to the transcriptional activation of immediate early genes in the MAPK pathways. Treatment with inhibitors of the MAPK pathways suppressed nsPEF-induced protein phosphorylation and gene expression downstream of MAPKs, confirming the functional connection between the nsPEF-activated MAPKs and the observed induction of the downstream events. Taken together, these results provide important clues to the action of nsPEFs on human cells and demonstrate a new possibility for the utilization of nsPEFs in the control of various biological phenomena involving activation of the MAPK pathways.

Activation of the JNK pathway by nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.

First synthesis of dodecasubstituted porphycenes.

The synthesis of dodecasubstituted porphycenes has not been reported, to date. Herein, the preparation of tetramethyloctaethylporphycene by a McMurry-type coupling of 3,3',4,4'-tetraethyl-5,5'-diformyl-2,2'-bipyrrole was attempted at first, but dodecasubstituted porphycene was not successfully obtained and only pyrrolocyclophene was obtained. The structure of the pyrrolocyclophene was determined by (1)H NMR spectroscopy, FAB MS, and X-ray crystal-structure analysis. The pyrrolocyclophene was not successfully oxidized to porphycene. Then, the McMurry-type coupling of bicyclo[2.2.2]octadiene (BCOD)-fused 5,5'-diacyl-2,2'-bipyrroles was performed and tetra-meso-octa-ß-substituted (dodecasubstituted) porphycenes were successfully obtained for the first time. The structures were determined by (1)H NMR spectroscopy and X-ray crystal-structure analysis. The crystal structures and NMR spectra were compared carefully with octasubstituted porphycenes, and there was a good correlation between the position of the substituents, the N1−−N2 and N1−−N4 distances of the porphycene inner nitrogen atoms, and NMR chemical shifts of the inner NH protons, which expressed the strength of N−−H⋅⋅⋅N hydrogen bonding between N1 and N2. These results suggested that the BCOD structure was relatively compact compared with common alkyl groups and that was why the dodecasubstituted porphycenes were available this time. UV/Vis absorption and fluorescence properties are also discussed.