Electrotaxis of lung cancer cells in a multiple-electric-field chip

We report a microfluidic cell culture chip that was used for long-term electrotaxis study on a microscope. The cellular response under three different electric field strengths was studied in a single channel microfluidic chip. Electric field (EF) inside the microchamber was numerically simulated and compared to the measured value. Lung cancer cell lines with high and weak metastasis potential, CL1–5 and CL1–0, respectively, were used to demonstrate the function of the multi-field chip (MFC). The two cell lines exhibited greatly different response under the applied EF of E = 74–375 mV/mm. CL1–5 cells migrated toward the anode while CL1–0 cells did not show obvious response. Under the applied EF, cell orientation was observed accompanying the cell migration. Judging from the different temporal responses of the orientation and the migration, it is proposed that the two EF-induced responses may involve different signaling pathways.     

Computational exploration of the activated pathways associated with DNA damage response in breast cancer

Molecular signaling events regulate cellular activity. Cancer stimulating signals trigger cellular responses that evade the regulatory control of cell development. To understand the mechanism of signaling regulation in cancer, it is necessary to identify the activated pathways in cancer. We have developed RepairPATH, a computational algorithm that explores the activated signaling pathways in cancer. The RepairPATH integrates RepairNET, an assembled protein interaction network associated with DNA damage response, with the gene expression profiles derived from the microarray data. Based on the observation that cofunctional proteins often exhibit correlated gene expression profiles, it identifies the activated signaling pathways in cancer by systematically searching the RepairNET for proteins with significantly correlated gene expression profiles. Analyzing the gene expression profiles of breast cancer, we found distinct similarities and differences in the activated signaling pathways between the samples from the patients who developed metastases and the samples from the patients who were disease free within 5 years. The cellular pathways associated with the various DNA repair mechanisms and the cell-cycle checkpoint controls are found to be activated in both sample groups. One of the most intriguing findings is that the pathways associated with different cellular processes are functionally coordinated through BRCA1 in the disease-free sample group, whereas such functional coordination is absent in the samples from patients who developed metastases. Our analysis revealed the potential cellular pathways that regulate the signaling events in breast cancer.     

Steered migration and changed morphology of human astrocytes by an applied electric field

Direct current electric field (DC EF) plays a role in influencing the biological behaviors and functions of cells. We hypothesize that human astrocytes (HAs) could also be influenced in EF. Astrocytes, an important type of nerve cells with a high proportion quantitatively, are generally activated and largely decide the brain repair results after brain injury. So far, no electrotaxis study on HAs has been performed. We here obtained HAs derived from brain trauma patients. After purification and identification, HAs were seeded in the EF chamber and recorded in a time-lapse image system. LY294002 and U0126 were then used to probe the role of PI3K or ERK signaling pathway on cellular behaviors. The results showed that HAs could be guided to migrate to the anode in DC EFs, in a voltage-dependent manner. The HAs displayed elongated cell bodies and reoriented perpendicularly to the EF in morphology. When treated with LY294002 or U0126, alternation of parameters such as cellular verticality, track speed, displacement speed, long axis, vertical length and circularity were inhibited partly as expected, while the EF-induced directedness was not terminated even at a high drug dosage which was not consistent with previous electrotaxis studies. In conclusion, applied EFs steered the patient-derived HAs directional migration and changed morphology, in which PI3K and ERK pathways at least partially participate. The characteristics of HAs to EF stimulation may be involved in wound healing and neural regeneration, which could be utilized as a novel treatment strategy in brain injury.     

Beyond ATM: the protein kinase landscape of the DNA damage response

The DNA of all organisms is constantly subjected to damaging agents, both exogenous and endogenous. One extremely harmful lesion is the double-strand break (DSB), which activates a massive signaling network - the DNA damage response (DDR). The chief activator of the DSB response is the ATM protein kinase, which phosphorylates numerous key players in its various branches. Recent phosphoproteomic screens have extended the scope of damage-induced phosphorylations beyond the direct ATM substrates. We review the evidence for the involvement of numerous other protein kinases in the DDR, obtained from documentation of specific pathways as well as high-throughput screens. The emerging picture of the protein phosphorylation landscape in the DDR broadens the current view on the role of this protein modification in the maintenance of genomic stability. Extensive cross-talk between many of these protein kinases forms an interlaced signaling network that spans numerous cellular processes. Versatile protein kinases in this network affect pathways that are different from those they have been identified with to date. The DDR appears to be one of the most extensive signaling responses to cellular stimuli.     

Evaluation of EGFR and RTK Signaling in the Electrotaxis of Lung Adenocarcinoma Cells under Direct-Current Electric Field Stimulation

Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells’ response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.     

LIRs/ILTs/MIRs, inhibitory and stimulatory Ig-superfamily receptors expressed in myeloid and lymphoid cells

Cells exhibit a complex network of inhibitory and stimulatory signaling pathways, which interact with each other to maintain an homeostatic balance and modulate cellular responses to external stimuli. During most of the 1980s, a great effort was put into the characterization of stimulatory cell surface receptors for cytokines and growth factors. In the last decade, a large number of inhibitory receptors have been identified and it has become apparent that inhibitory signaling pathways are subject to intricate regulatory mechanisms. Inhibitory and stimulatory signaling pathways work in concert with each other to establish activation thresholds and provide sensitive tuning mechanisms that help control cellular responses. LIRs/ILTs/MIRs are a novel family of inhibitory and stimulatory receptors expressed both in myeloid and lymphoid cells. They contain two or four immunoglobulin-like domains in the extracellular region and their cytoplasmic domains are either very short and without any signaling motifs or are long and contain a variable number of immunoreceptor tyrosine-based inhibition motifs (ITIMs). LIRs within the first group send stimulatory signals by association with the FcR common gamma chain and LIRs within the second group deliver inhibitory signals by association with the protein tyrosine phosphatase SHP-1. This review summarizes our current knowledge on the LIRs, their ligands, and biological functions.     

CBL‐mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores

During adaptation and developmental processes cells respond through nonlinear calcium‐decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B‐like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL‐interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N‐terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII‐independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra‐ and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.     

Ca<Superscript>2+</Superscript>-Signaling,Alternative Splicing and Endoplasmic Reticulum Stress Responses

Ca²⁺-signaling, alternative splicing, and stress responses by the endoplasmic reticulum are three important cellular activities which can be strongly interconnected to alter the expression of protein isoforms in a tissue dependent manner or during development depending on the environmental conditions. This integrated network of signaling pathways permits a high degree of versatility and adaptation to metabolic, developmental and stress processes. Defects in its regulation may lead to cellular malfunction.     

Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo

It has long been known that cells can be induced to migrate by the application of small d.c. electric fields (EFs), a phenomenon referred to as galvanotaxis. We recently reported some significant effects of electric signals of physiological strength in guiding cell migration and wound healing. We present here protocols to apply an EF to cells or tissues cultured in an electrotactic chamber. The chamber can be built to allow controlled medium flow to prevent the potential development of chemical gradients generated by the EFs. It can accommodate cells on planar culture or tissues in 3D gels. Mounted on an inverted microscope, this setup allows close and well-controlled observation of cellular responses to electric signals. As similar EFs are widely present during development and wound healing, this experimental system can be used to simulate and study cellular and molecular responses to electric signals in these events.     

Systems biology of virus-host signaling network interactions

Viruses have evolved to manipulate the host cell machinery for virus propagation, in part by interfering with the host cellular signaling network. Molecular studies of individual pathways have uncovered many viral host-protein targets; however, it is difficult to predict how viral perturbations will affect the signaling network as a whole. Systems biology approaches rely on multivariate, context-dependent measurements and computational analysis to elucidate how viral infection alters host cell signaling at a network level. Here we describe recent advances in systems analyses of signaling networks in both viral and non-viral biological contexts. These approaches have the potential to uncover virus- mediated changes to host signaling networks, suggest new therapeutic strategies, and assess how cell-to-cell variability affects host responses to infection. We argue that systems approaches will both improve understanding of how individual virus-host protein interactions fit into the progression of viral pathogenesis and help to identify novel therapeutic targets.     

Dynamics of TGF-β/Smad signaling

The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the non-canonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.     

PKC and PLA2: Probing the complexities of the calcium network

Lipid signaling and phosphorylation cascades are fundamental to calcium signaling networks. In this review, we will discuss the recent laboratory findings for the phospholipase A₂ (PLA₂)/protein kinase C (PKC) pathway within cellular calcium networks. The complexity and connectivity of these ubiquitous cellular signals make interpretation of experimental results extremely challenging. We present here computational methods which have been developed to conquer such complex data, and how they can be used to make models capable of accurately predicting cellular responses within multiple calcium signaling pathways. We propose that information obtained from network analysis and computational techniques provides a rich source of knowledge which can be directly translated to the laboratory benchtop.     

Cellular response to bioactive lipid peroxidation products

Reactive aldehydes, such as 4-hydroxy-2-nonenal, have been implicated as inducers in generating intracellular reactive oxygen species and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli. Here, I briefly summarize a novel signaling pathway in cellular response, in which aldehyde-stimulated detoxification response is mediated by cyclooxygenase metabolites. These findings argue that lipid mediators could induce a cellular process that represents a cellular defense program against toxic compounds.     

Stomatal movements and long-distance signaling in plants

As the nerve-mediated signaling in animals, long-distance signaling in plants is a prerequisite for plants to be able to perceive environmental stimuli and initiate adaptive responses. While intracellular signal transduction has been attracting considerable attentions, studies on long-distance signaling in plants has been relatively overlooked. Stomatal movements are well recognized as a model system for studies on cellular signal transduction. It has been demonstrated that the stomatal movements may be frequently tuned by long-distance signaling under various environmental stimuli. Stomatal movements can not only respond to persistent stress stimuli but also respond to shock stress stimuli. Stomatal responses to drought stress situations may be best characterized in terms of interwoven networks of chemical signaling pathways playing predominant roles in these adaptive processes. In cases of shock stress stimuli, stomatal movements can be more sensitively regulated through the long-distance signaling but with distinctive patterns not observed for drought or other persistent stresses. Here, the fundamental characteristics of stomatal movements and associated long-distance signaling are reviewed and the implications for plant responses to environmental stresses are discussed.Key words: stomatal movement, long-distance signaling, environmental stresses, abscisic aci, pH signaling, hydraulic signaling, cytokinins, acetylcholine, heat-shock, electric signal     

Cellular response to bioactive lipid peroxidation products

Reactive aldehydes, such as 4-hydroxy-2-nonenal, have been implicated as inducers in generating intracellular reactive oxygen species and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli. Here, I briefly summarize a novel signaling pathway in cellular response, in which aldehyde-stimulated detoxification response is mediated by cyclooxygenase metabolites. These findings argue that lipid mediators could induce a cellular process that represents a cellular defense program against toxic compounds.     

Proteome profiling reveals potential toxicity and detoxification pathways following exposure of BEAS-2B cells to engineered nanoparticle titanium dioxide

Oxidative stress is known to play important roles in engineered nanomaterial‐induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial‐mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS‐2B and nanoscale titanium dioxide. Utilizing 2‐DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2‐DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses^? canonical pathways and Ingenuity Pathway Analyses tox lists to create protein‐interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS‐2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide‐treated BEAS‐2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein‐interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.