Distinguish response of low-dose radiation with different dose-rate on gene expression of human coronary artery endothelial cells: a bioinformatic study based on transcriptomic sequencing.

PURPOSE: People are exposed to low-dose radiation in medical diagnosis, occupational, or life circumstances, but the effect of low-dose radiation on human health is still controversial. The biological effects of radiation below 100 mGy are still unproven. In this study, we observed the effects of low-dose radiation (100 mGy) on gene expression in human coronary artery endothelial cells (HCAECs) and its effect on molecular signaling. MATERIALS AND METHODS: HCAECs were exposed to 100 mGy ionizing radiation at 6 mGy/h (low-dose-rate) or 288 mGy/h (high-dose-rate). After 72 h, total RNA was extracted from sham or irradiated cells for Quant-Seq 3'mRNA-Seq, and bioinformatic analyses were performed using Metascape. Gene profiling was validated using qPCR. RESULTS: Compared to the non-irradiated control group, 100 mGy of ionizing radiation at 6 mGy/h altered the expression of 194 genes involved in signaling pathways related to heart contraction, blood circulation, and cardiac myofibril assembly differentially. However, 100 mGy at 288 mGy/h altered expression of 450 genes involved in cell cycle-related signaling pathways, including cell division, nuclear division, and mitosis differentially. Additionally, gene signatures responding to low-dose radiation, including radiation dose-specific gene profiles (HIST1H2AI, RAVER1, and POTEI) and dose-rate-specific gene profiles (MYL2 for the low-dose-rate and DHRS9 and CA14 for the high-dose-rate) were also identified. CONCLUSIONS: We demonstrated that 100 mGy low-dose radiation could alter gene expression and molecular signaling pathways at the low-dose-rate and the high-dose-rate differently. Our findings provide evidence for further research on the potential impact of low-dose radiation on cardiovascular function.

An 18.6-µW/Ch TDM-Based 8-Channel Noncontact ECG Recording IC with Common-Mode Interference Suppression.

This paper presents an 8-channel electrocardiogram (ECG) monitoring integrated circuit (IC) controlled by time-division multiplexing (TDM). The proposed TDM compensates the electrode DC offsets by forming an individual discrete-time feedback loop per channel while sharing an analog frontend. This enables a chopping-free open-loop amplification, achieving a high input impedance suitable for a noncontact ECG monitoring. In addition, a common-mode interference (CMI) cancellation scheme is also introduced in the proposed TDM schedule for the realization of a pseudo-driven-right leg (DRL) in a multichannel environment. The designed system is implemented in 180 nm CMOS. The chip dissipates 18.6 µW/channel including the power consumption by ADC. It shows the total-CMRR of 100 dB against CMI voltage swing up to 20 VPP. The chip is verified in noncontact 8-channel ECG using conventional passive electrodes.

An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage.

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.

Endothelial-to-mesenchymal transition in anticancer therapy and normal tissue damage.

Endothelial-to-mesenchymal transition (EndMT) involves the phenotypic conversion of endothelial-to-mesenchymal cells, and was first discovered in association with embryonic heart development. EndMT can regulate various processes, such as tissue fibrosis and cancer. Recent findings have shown that EndMT is related to resistance to cancer therapy, such as chemotherapy, antiangiogenic therapy, and radiation therapy. Based on the known effects of EndMT on the cardiac toxicity of anticancer therapy and tissue damage of radiation therapy, we propose that EndMT can be targeted as a strategy for overcoming tumor resistance while reducing complications, such as tissue damage. In this review, we discuss EndMT and its roles in damaging cardiac and lung tissues, as well as EndMT-related effects on tumor vasculature and resistance in anticancer therapy. Modulating EndMT in radioresistant tumors and radiation-induced tissue fibrosis can especially increase the efficacy of radiation therapy. In addition, we review the role of hypoxia and reactive oxygen species as the main stimulating factors of tissue damage due to vascular damage and EndMT. We consider drugs that may be clinically useful for regulating EndMT in various diseases. Finally, we argue the importance of EndMT as a therapeutic target in anticancer therapy for reducing tissue damage.

Stromal Cell-Derived Factor 1 Protects Brain Vascular Endothelial Cells from Radiation-Induced Brain Damage.

Stromal cell-derived factor 1 (SDF-1) and its main receptor, CXC chemokine receptor 4 (CXCR4), play a critical role in endothelial cell function regulation during cardiogenesis, angiogenesis, and reendothelialization after injury. The expression of CXCR4 and SDF-1 in brain endothelial cells decreases due to ionizing radiation treatment and aging. SDF-1 protein treatment in the senescent and radiation-damaged cells reduced several senescence phenotypes, such as decreased cell proliferation, upregulated p53 and p21 expression, and increased senescence-associated beta-galactosidase (SA-ß-gal) activity, through CXCR4-dependent signaling. By inhibiting extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription protein 3 (STAT3), we confirmed that activation of both is important in recovery by SDF-1-related mechanisms. A CXCR4 agonist, ATI2341, protected brain endothelial cells from radiation-induced damage. In irradiation-damaged tissue, ATI2341 treatment inhibited cell death in the villi of the small intestine and decreased SA-ß-gal activity in arterial tissue. An ischemic injury experiment revealed no decrease in blood flow by irradiation in ATI2341-administrated mice. ATI2341 treatment specifically affected CXCR4 action in mouse brain vessels and partially restored normal cognitive ability in irradiated mice. These results demonstrate that SDF-1 and ATI2341 may offer potential therapeutic approaches to recover tissues damaged during chemotherapy or radiotherapy, particularly by protecting vascular endothelial cells.

Development and Validation of a Deep Learning System for Staging Liver Fibrosis by Using Contrast Agent-enhanced CT Images in the Liver.

Purpose To develop and validate a deep learning system (DLS) for staging liver fibrosis by using CT images in the liver. Materials and Methods DLS for CT-based staging of liver fibrosis was created by using a development data set that included portal venous phase CT images in 7461 patients with pathologically confirmed liver fibrosis. The diagnostic performance of the DLS was evaluated in separate test data sets for 891 patients. The influence of patient characteristics and CT techniques on the staging accuracy of the DLS was evaluated by logistic regression analysis. In a subset of 421 patients, the diagnostic performance of the DLS was compared with that of the radiologist's assessment, aminotransferase-to-platelet ratio index (APRI), and fibrosis-4 index by using the area under the receiver operating characteristic curve (AUROC) and Obuchowski index. Results In the test data sets, the DLS had a staging accuracy of 79.4% (707 of 891) and an AUROC of 0.96, 0.97, and 0.95 for diagnosing significant fibrosis (F2-4), advanced fibrosis (F3-4), and cirrhosis (F4), respectively. At multivariable analysis, only pathologic fibrosis stage significantly affected the staging accuracy of the DLS (P = .016 and .013 for F1 and F2, respectively, compared with F4), whereas etiology of liver disease and CT technique did not. The DLS (Obuchowski index, 0.94) outperformed the radiologist's interpretation, APRI, and fibrosis-4 index (Obuchowski index range, 0.71-0.81; P ˂ .001) for staging liver fibrosis. Conclusion The deep learning system allows for accurate staging of liver fibrosis by using CT images. © RSNA, 2018 Online supplemental material is available for this article.

Neuroprotective effect of triflusal and its main metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB), in the postischemic brain.

2-Hydroxy-4-trifluoromethylbenzoic acid (HTB) is a metabolite of triflusal (TF), and has been reported to exert anti-inflammatory effect. In this study, the authors investigated whether HTB has a neuroprotective effect against ischemic brain injuries. We showed that intravenous administration of HTB (5mg/kg) 30min before or 1, 3, or 6h after middle cerebral artery occlusion (MCAO) reduced brain infarct to 10.4±3.3%, 16.9±2.3%, 22.2±1.5% and 40.7±7.5%, respectively, of that of treatment-naive MCAO controls, and the therapeutic time window extended to 9h after MCAO (40.7±7.5%). Furthermore, HTB suppressed infarct formation, protected motor activities, and ameliorated neurological deficits more effectively than by TF or salicylic acid (SA). HTB markedly suppressed microglial activation and proinflammatory cytokines expressions in the postischemic brain and in BV2 cells and suppressed LPS-induced nitrite production by inhibiting IkB degradation. In addition, HTB suppressed NMDA-induced neuronal cell death more effectively than TF or SA in primary cortical neuron cultures. Together, these results indicate that HTB has multi-modal protective effects against ischemic brain damage that encompass anti-inflammatory, anti-excitotoxicity, and anti-Zn2+-toxicity effects.

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1.

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1­NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1­NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1­NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1­NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1­NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents.

[The Japanese Colonial Medicine as Seen Through the Life of Fujita Tsuguakira].

Fujita Tsuguakira was a man who established Jahyeuiwon, a governmental medical facility, during the Residency-General Period and took over the presidency of a committee in the Japanese Government-General of Chosun after Chosun was annexed to Japanese. In addition, he is a man well qualified to be placed on the top of the list when discussing the Japanese colonial medicine in Chosun, considering his personal history of getting evolved in the colonial rule of Taiwan for seven years as an army surgeon. He led the colonial medicine in Chosun for nine years before and after the Japanese annexation of Korea. He was engaged in almost all the areas related to the colonial medicine such as anti-cholera projects, Hansung Sanitation Union, Deahan Hospital, Chosun Chongdokbu Hospital, Jahyeuiwon, medical schools affiliated to the Japanese Government-General of Chosun. In all respects, his life was in sync with the expansionist strategies of Imperial Japan. Especially, his deeds in Chosun was an "active aid to the instructions" from Army Minister Terauchi Masatake " as Sato Kozo testifies. Fujita was chosen by the military, and so he faithfully served the role given from it. The rewards that he received form the military attest to this fact. He took the position of Surgeon General in Army Medical Service on September, 1912, the top place that an army surgeon could hold. The position was first given to the officer who worked outside Japan proper, and he was the only army surgeon with no doctorial degree to receive such title except for Ishiguro Tadanori who was the first army surgeon in Japan. To sum up, Fujita was not a "doctor" but a "military officer". His walk of life mainly lay in the role of an aider adjusted to the ups and downs and the speeds of the plans of Imperial Japan to invade the continent. Therefore, the Japanese colonial medicine controlled by such man as Fujita in Chosun was inevitably studded with the military things. As a chief in the army medicine, what was important to him was the hospitals for managing the armed troops and projects for preventing infectious disease that could threaten the military sanitation. As a result, the medical service for those under the colonial rule was naturally put on the back burner. This study was conducted mainly based on Fujita's memoirs called Army Surgeon General Fujita Tsuguakira (1943), and accordingly it would be not without limitations. However, as he is a man who cannot be put aside when discussing the Japanese colonial medicine in Chosun, the records by this study of his life and past activities are expected to give no small amount of contribution to these discussions.

XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is well known as an antagonist of XIAP-mediated caspase inhibition. Although XAF1 serves as a tumor-suppressor gene, the role of XAF1 in cellular senescence remains unclear. We found that XAF1 expression was increased by genotoxic agents, such as doxorubicin and ionizing radiation in pulmonary microvascular endothelial cells, consequently leading to premature senescence. Conversely, downregulation of XAF1 in premature senescent cells partially overcame endothelial cell senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by XAF1 induction. XAF1 expression was transcriptionally regulated by Bromodomain 7 (BRD7). XAF1 induction with interferon-gamma (IFN-γ) treatment was abrogated by BRD7 knockdown, which resulted in blocking interferon-induced senescence. In lung cancer cells, XAF1 tumor suppressor activity was decreased by BRD7 knockdown, and inhibition of tumor growth by IFN-γ did not appear in BRD7-depleted xenograft tumors. These data suggest that XAF1 is involved in BRD7-associated senescence and plays an important role in the regulation of endothelial senescence through a p53-dependent pathway. Furthermore, regulation of the BRD7/XAF1 system might contribute to tissue or organismal aging and protection against cellular transformation.

Enhancement of cellular radiation sensitivity through degradation of Chk1 by the XIAP-XAF1 complex.

X-linked inhibitor of apoptosis (XIAP) and Chk1 are potential molecular targets in radiotherapy. However, their molecular association in the regulation of radiation sensitivity has been rarely studied. Here, we show that XIAP modulates radiation sensitivity by regulating stability of Chk1 in lung cancer cells. Both Chk1 and XIAP are highly expressed in various lung cancer cells. Overexpression of XIAP increased cell survival following genotoxic treatments by preventing downregulation of Chk1. However, XIAP reversed Chk1-protective activity in the presence of XIAP-associated factor 1 (XAF1) by degrading Chk1 via ubiquitination-dependent proteasomal proteolysis. The XIAP-XAF1 complex-mediated Chk1 degradation also required CUL4A and DDB1. Chk1 or XIAP was associated with DDB1 and CUL4A. Depletion of CUL4A or DDB1 prevented the XIAP-XAF1-mediated Chk1 degradation suggesting involvement of a CUL4A/DDB1-based E3 ubiquitin ligase in the process or its collaboration with XIAP E3 ligase activity. Taken together, our findings show that XIAP plays a dual role in modulation of Chk1 stability and cell viability following IR. In the absence of XAF1, XIAP stabilizes Chk1 under IR with corresponding increase of cell viability. By contrast, when XAF1 is overexpressed, XIAP facilitates Chk1 degradation, which leads to enhancement of radiation sensitivity. This selective regulation of Chk1 stability by XIAP and XAF1 could be harnessed to devise a strategy to modulate radiation sensitivity in lung cancer cells.

Characterization of DNA damage-induced cellular senescence by ionizing radiation in endothelial cells.

PURPOSE: Inhibition of growth in mammalian cells in response to damage or stress is known as cellular senescence. Increasing evidence suggests that double-strand breaks (DSB) commonly mediate cellular senescence. Recently, radiation exposure has been reported to induce premature senescence. MATERIALS AND METHODS: We investigated whether ionizing radiation (IR) at 4 Gy induces cellular senescence with DNA damage response in human umbilical vein endothelial cells (HUVEC). To determine alterations in gene expression on IR exposure, we have developed a DNA microarray analysis system that contains genes known to be involved in replicative senescence. RESULTS: The damage by IR exposure is shown to result in a variety of senescence-like phenotypes such as changes in cell morphology, decrease in cell proliferation, increase in senescence- associated ß-galactosidase (SA-ß-gal) staining, and suppression of angiogenic activity. Moreover, the expression levels of several genes associated with cell cycle regulation are remarkably increased in IR-exposed endothelial cells. We found that IGFBP5 (insulin-like growth factor binding protein 5), PLAT (plasminogen activator), SNAI2 (snail homolog 2), JAG1 (jagged 1), SPRY4 (Sprouty homolog 4), and CD44 were upregulated, whereas CFB (complement factor B), VCAM1 (vascular cell adhesion molecule 1), AQP1 (aquaporin 1), LOXL1 (lysyl oxidase-like 1), and RBPMS (RNA-binding protein with multiple splicing) were down- regulated in both radiation-damaged and old cells. CONCLUSIONS: These results imply that the IR-induced phenotype may be enhanced by alterations in genes associated with senescence.

An international survey of physicians regarding clinical trials: a comparison between Kyoto University Hospital and Seoul National University Hospital.

BACKGROUND: International clinical trials are now rapidly expanding into Asia. However, the proportion of global trials is higher in South Korea compared to Japan despite implementation of similar governmental support in both countries. The difference in clinical trial environment might influence the respective physicians' attitudes and experience towards clinical trials. Therefore, we designed a questionnaire to explore how physicians conceive the issues surrounding clinical trials in both countries. METHODS: A questionnaire survey was conducted at Kyoto University Hospital (KUHP) and Seoul National University Hospital (SNUH) in 2008. The questionnaire consisted of 15 questions and 2 open-ended questions on broad key issues relating to clinical trials. RESULTS: The number of responders was 301 at KUHP and 398 at SNUH. Doctors with trial experience were 196 at KUHP and 150 at SNUH. Among them, 12% (24/196) at KUHP and 41% (61/150) at SUNH had global trial experience. Most respondents at both institutions viewed clinical trials favorably and thought that conducting clinical trials contributed to medical advances, which would ultimately lead to new and better treatments. The main reason raised as a hindrance to conducting clinical trials was the lack of personnel support and time. Doctors at both university hospitals thought that more clinical research coordinators were required to conduct clinical trials more efficiently. KUHP doctors were driven mainly by pure academic interest or for their desire to find new treatments, while obtaining credits for board certification and co-authorship on manuscripts also served as motivation factors for doctors at SNUH. CONCLUSIONS: Our results revealed that there might be two different approaches to increase clinical trial activity. One is a social level approach to establish clinical trial infrastructure providing sufficient clinical research professionals. The other is an individual level approach that would provide incentives to encourage doctors to participate in and conduct clinical trials.

Interferon-gamma enhances radiation-induced cell death via downregulation of Chk1.

Interferon-gamma (IFNγ) is a cytokine with roles in immune responses as well as in tumor control. Interferon is often used in cancer treatment together with other therapies. Here we report a novel approach to enhancement of cancer cell killing by combined treatment of IFNγ with ionizing radiation. We found that IFNγ treatment alone in HeLa cells induced phosphorylation of Chk1 in a time- and dose-dependent manner, and resulted in cell arrest. Moreover IFNγ treatment was correlated with attenuation of Chk1 as the treatment shortened protein half-life of Chk1. As Chk1 is an essential cell cycle regulator for viability after DNA damage, attenuation of Chk1 by IFNγ pre-treatment in HeLa cells resulted in increased cell death following ionizing radiation about 2-folds than ionizing radiation treatment alone whereas IFNγ treatment alone had little effect on cell death. X-linked inhibitor of apoptosis-associated factor 1 (XAF1), an IFN-induced gene, seems to partly regulate IFNγ-induced Chk1 destabilization and radiation sensitivity because transient depletion of XAF1 by siRNA prevented IFNγ-induced Chk1 attenuation and partly protected cells from IFNγ-enhanced radiation cell killing. Therefore the results provide a novel rationale to combine IFNγ pretreatment and DNA-damaging anti-cancer drugs such as ionizing radiation to enhance cancer cell killing.

Identification of ELAVL4 as a modulator of radiation sensitivity in A549 non-small cell lung cancer cells.

Identification of genes that modulate radiation sensitivity provides important tools to study cellular responses to ionizing radiation. We combined DNA microarrays and viability assays to identify modulators of radiation sensitivity in A549 lung cancer cells. Up-regulated genes were selected from microarray experiments and RNA expression levels were confirmed by real-time RT-PCR analysis. Cell viability assays such as clonogenic assay, MTT and FACS analysis of cell death, identified the ELAVL4 gene as a novel modulator of radiation sensitivity. ELAVL4 expression was induced following ionizing irradiation. Depletion of the ELAVL4 gene increased radiation sensitivity of A549 cells as shown by decreased surviving cell fraction following irradiation in clonogenic assay. Enhanced radiation sensitivity of ELAVL4-depleted cells was attributable to decreased cell proliferation as well as increased apoptotic cell death following irradiation. Thus the endogenous function of ELAVL4 in relation to radiation sensitivity might be the regulation of cell proliferation and death. This approach to identification of modulators for radiation sensitivity has several advantages in terms of functional selectivity, stringency and time. Further analysis of the modulators should find potential use in the application of radiation biomarkers as well as modulators of cellular radiation responses.

Enhancement of radiation sensitivity in lung cancer cells by celastrol is mediated by inhibition of Hsp90.

The radiosensitizing activity of celastrol, a quinone methide triterpene was examined. We found that celastrol treatment of the NCI-H460 lung cancer cell line increased radiation-induced cell killing. The increased radiosensitivity was correlated with decreased levels of Hsp90 clients, such as EGFR, ErbB2 and survivin as well as with increased p53 expression. Celastrol inhibited the ATP-binding activity of Hsp90. Furthermore, celastrol treatment dissociated an Hsp90 client protein, EGFR, and this in turn resulted in degradation of the client protein. These results were not observed with another structurally similar triterpenoid, 6ß-acetonyl-22ß-hydroxytingenol (TG), suggesting that a specific structural feature of the triterpenoid is required for radiosensitization. Moreover celastrol treatment increased p53 levels by phosphorylating Ser15 and Ser20 residues as well as by inhibiting its proteasomal degradation. Celastrol may be considered an effective radiosensitizer acting as an inhibitor of Hsp90 and a p53 activator. The two activities could be applicable to a broad range of cancer cells with either wild-type or mutant p53 because either activity could be effective for the enhancement of radiation cell killing. Further analysis with other triterpenoids should identify the functional moiety of the structure and additional candidates for effective radiosensitizers, which can be used in combined radiotherapy.

Proto-oncogenic H-Ras, K-Ras, and N-Ras are involved in muscle differentiation via phosphatidylinositol 3-kinase.

Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras (c-Ras) in muscle differentiation remains unclear. The active GTP-bound form of Ras has been known to associate with diverse effectors including Raf, phosphatidylinositol 3-kinase (PI3K), Ral-GDS, and other molecules to transmit downstream signals. We hypothesize that c-Ras may stimulate muscle differentiation by selectively activating PI3K, an important mediator for muscle differentiation. In our experiments, inhibition of c-Ras by farnesyltransferase inhibitors and a dominant negative form of H-Ras (Ras S17N) suppressed muscle differentiation. Consistently, individual knockdown of H-Ras, K-Ras, and N-Ras by siRNAs all blocked muscle differentiation. Interestingly, we found that c-Ras preferentially interacts with PI3K rather than its major binding partner c-Raf, during myogenic differentiation, with total c-Ras activity remaining unchanged. PI3K and its downstream myogenic pathway, the Nox2/NF-kappaB/inducible nitric oxide synthase (iNOS) pathway, were found to be suppressed by inhibition of c-Ras activity during differentiation. Furthermore, expression of a constitutively active form of PI3K completely rescued the differentiation block and reactivated the Nox2/NF-kappaB/iNOS pathway in c-Ras-inhibited cells. On the basis of our results, we conclude that contrary to oncogenic Ras, proto-oncogenic H-Ras, K-Ras, and N-Ras are directly involved in the promotion of muscle differentiation via PI3K and its downstream signaling pathways. In addition, PI3K pathway activation is associated with a concurrent suppression of the otherwise predominantly activated Raf/Mek/Erk pathway.

Endogenous hydrogen peroxide regulates glutathione redox via nuclear factor erythroid 2-related factor 2 downstream of phosphatidylinositol 3-kinase during muscle differentiation.

We reported previously that endogenous reactive oxygen species (ROS) function as myogenic signaling molecules. It has also been determined that excess ROS induce electrophile-response element (EpRE)-driven gene expression via activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Nonetheless, the relationship between the metabolism of ROS (eg, H(2)O(2)) through glutathione (GSH) up-regulation, GSH-dependent reduction of H(2)O(2), and Nrf2-dependent gene regulation is not well established. Therefore, we attempted to determine whether H(2)O(2) controls the intracellular GSH redox state via the Nrf2-glutamate-cysteine ligase (GCL)/glutathione reductase (GR)-GSH signaling pathway. In our experiments, enhanced H(2)O(2) generation was accompanied by an increase in both total GSH levels and the GSH/GSSG ratio during muscle differentiation. Both GCL and GR transcriptional expression levels were markedly increased during muscle differentiation but reduced by catalase treatment. Nrf2 protein expression and nuclear translocation increased during myogenesis. The inhibition of GCL, GR, and Nrf2 both by inhibitors and by RNA interference blocked muscle differentiation. Phosphatidylinositol 3-kinase regulated the expression of the GCL C (a catalytic subunit) and GR genes via the induction of Nrf2 nuclear translocation and expression. In conclusion, endogenous H(2)O(2) generated during muscle differentiation not only functions as a signaling molecule, but also regulates the GSH redox state via activation of the Nrf2-GCL/GR-GSH signaling pathway downstream of phosphatidylinositol 3-kinase.

A new method for purification of functional recombinant GST-cyclophilin A protein from E. coli.

The expression of glutathione-S-transferase (GST) fusion protein is extensively utilized in the study of protein-protein interactions. In the commonly used purification method, the overexpressed GST fusion protein is bound to the glutathione (GSH)-coupled resins via affinity chromatography, and then eluted by an excessive quantity of reduced GSH. However, this technique has certain limitations, such as low product purity, retention of GSH in the sample, as well as relatively high cost. To overcome these limitations, in this study, elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer. By using this method, highly purified GST-cyclophilin A (CypA) fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities. Moreover, the procedure is also cost-effective, due to the low cost of formic acid as compared with GSH.

Suppression of c-Src activity stimulates muscle differentiation via p38 MAPK activation.

Role of c-Src in muscle differentiation has been controversial. Here, we investigated if c-Src positively or negatively regulates muscle differentiation, using H9c2 and C2C12 cell lines. Inhibition of c-Src by treatment with PP1 and SU6656, pharmacologic inhibitors of Src family kinases, or by expression of a dominant negative c-Src, all induced muscle differentiation in proliferation medium (PM). In differentiating cells in differentiation medium (DM), c-Src activity gradually decreased and reached basal level 3 days after induction of differentiation. Inhibition of c-Src suppressed Raf/MEK/ERK pathway but activated p38 MAPK. Inhibition of p38 MAPK did not affect c-Src activity in PM. However, it reactivated Raf/MEK/ERK pathway in c-Src-inhibited cells regardless of PM or DM. Concomitant inhibition of c-Src and p38 MAPK activities blocked muscle differentiation in both media. In conclusion, suppression of c-Src activity stimulates muscle differentiation by activating p38 MAPK uni-directionally.