Ken ichi Yano

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.

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.

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 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.

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.

Dynamic behavior of DNA topoisomerase IIβ 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 IIβ 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 IIβ to the DSB site. Detergent extraction followed by immunofluorescence showed the tight association of endogenous Topo IIβ with DSB sites. Photobleaching analysis revealed that Topo IIβ is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIβ mobility and thereby prevented Topo IIβ recruitment to DSBs. Furthermore, Topo IIβ knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo IIβ in HR-mediated DSB repair. Taken together, these results highlight a novel aspect of Topo IIβ functions in the cellular response to DSBs.

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.

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.