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.

Effect of Nanosecond Pulsed Currents on Directions of Cell Elongation and Migration through Time-Lapse Analysis.

It is generally known that cells elongate perpendicularly to an electric field and move in the direction of the field when an electric field is applied. We have shown that irradiation of plasma-simulated nanosecond pulsed currents elongates cells, but the direction of cell elongation and migration has not been elucidated. In this study, a new time-lapse observation device that can apply nanosecond pulsed currents to cells was constructed, and software to analyze cell migration was created to develop a device that can sequentially observe cell behavior. The results showed nanosecond pulsed currents elongate cells but do not affect the direction of elongation and migration. It was also found the behavior of cells changes depending on the conditions of the current application.

Nanosecond pulsed current under plasma-producing conditions induces morphological alterations and stress fiber formation in human fibrosarcoma HT-1080 cells.

Cold atmospheric plasma (CAP) is a promising means for various biomedical applications, including cancer therapy. Although the biological action of CAP is considered to be brought about by synergistic effects of reactive species and electrical factors of CAP, limited information is currently available on the contribution of electrical factors to CAP-induced cell responses. We have previously demonstrated that nanosecond pulsed current (nsPC) under CAP-producing conditions significantly promoted the motility of human HT-1080 cells. In this study, we explored the effects of nsPC on cell morphology associated with cell motility. We observed that nsPC stimulation caused extended cell shape, membrane protrusion formation, and increased cell surface area, but not cell death induction. nsPC stimulation also caused elevated intracellular ROS and Ca2+. HT-1080 cells can undergo two modes of cell motility, namely mesenchymal and ameboid motility, and we found that morphological features of mesenchymal motility was partly shared with nsPC-stimulated cells. Furthermore, nsPC-stimulated cells had extended stress fibers composed of filamentous actin. Taken together, this study provides a novel insight into the electrical aspect of CAP action, and we speculate that nsPC activates a certain mechanism involving intracellular signaling for stress fiber formation, leading to altered cell morphology and increased cell motility.

A fluorescence method to visualize the nuclear boundary by the lipophilic dye DiI.

Here, we describe a procedure to fluorescently contrast the nuclear boundary using the lipophilic carbocyanine dye DiI in cultured human cells. Our procedure is simple and is applicable to detect nuclear boundary defects, which may be relevant to studies on nuclear envelope dynamics, micronuclei formation and cancer biology. ABBREVIATIONS: DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate; NE: nuclear envelope; RanBP2: Ran-binding protein 2/Nucleoporin 358.

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.

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.

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.

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 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.

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.

The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.

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.

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.

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.

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.

Ankle foot orthosis that prevents slippage for tibial rotation in knee osteoarthritis patients.

Knee osteoarthritis (OA) is a disease caused by age-related muscle weakness, obesity, or sports injury that leads to gait disability due to pain during walking. Knee OA is characterized by abnormal knee joint alignment and rotational dyskinesia, which are believed to worsen the symptoms. We previously developed an ankle orthosis that mechanically induces the rotation of the lower limb in conjunction with that of the ankle joint. This orthosis can effectively correct the alignment of the knee joint. However, slippage between the orthosis and leg can occur during walking, decreasing the corrective force. In this study, we clarify the effect of slippage between the orthosis and body on the correction force of the orthosis, and develop a lower leg tracking mechanism to suppress slippage and minimize reduction of force. The effectiveness of the proposed mechanism was evaluated by three-dimensional motion analysis of gait. Analysis results confirmed that the proposed mechanism was effective in suppressing slippage and improving correction force, demonstrating the effectiveness of the mechanism for knee OA.

System for Operating Electric Wheelchairs Using Only the Remaining Functions of the Thumbs of Muscular Dystrophy Patients.

For patients with muscular dystrophy, a motor dysfunction, who have difficulty operating an electric wheelchair with joysticks, a simplified one-input device is used. However, avoiding obstacles can be time-consuming. In this study, we analyzed the motor functions of the thumb of a patient with severe muscular dystrophy and identified the operations that did not cause physical fatigue. Then, we developed an operation support system to continuously operate. Finally, we conducted experiments comparing the proposed system with the conventional system and verified the effectiveness of the proposed system based on the steering accuracy of the electric wheelchair and the task completion time.

DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks.

Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases mu and lambda and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.

Molecular mechanism of protein assembly on DNA double-strand breaks in the non-homologous end-joining pathway.

Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Upon DSB induction, a living cell quickly activates the NHEJ pathway comprising of multiple molecular events. However, it has been difficult to analyze the initial phase of DSB responses in living cells, primarily due to technical limitations. Recent advances in real-time imaging and site-directed DSB induction using laser microbeam allow us to monitor the spatiotemporal dynamics of NHEJ factors in the immediate-early phase after DSB induction. These new approaches, together with the use of cell lines deficient in each essential NHEJ factor, provide novel mechanistic insights into DSB recognition and protein assembly on DSBs in the NHEJ pathway. In this review, we provide an overview of recent progresses in the imaging analyses of the NHEJ core factors. These studies strongly suggest that the NHEJ core factors are pre-assembled into a large complex on DSBs prior to the progression of the biochemical reactions in the NHEJ pathway. Instead of the traditional step-by-step assembly model from the static view of NHEJ, a novel model for dynamic protein assembly in the NHEJ pathway is proposed. This new model provides important mechanistic insights into the protein assembly at DSBs and the regulation of DSB repair.