Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain

Background: The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated. Results: We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms. Conclusion: The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.

An analysis of the cooperative mechano-sensitive feedback between intracellular signaling, focal adhesion development, and stress fiber contractility

Cells communicate with their external environment via focal adhesions and generate activation signals that in turn trigger the activity of the intracellular contractile machinery. These signals can be triggered by mechanical loading that gives rise to a cooperative feedback loop among signaling, focal adhesion formation, and cytoskeletal contractility, which in turn equilibrates with the applied mechanical loads. We devise a signaling model that couples stress fiber contractility and mechano-sensitive focal adhesion models to complete this above mentioned feedback loop. The signaling model is based on a biochemical pathway where IP3 molecules are generated when focal adhesions grow. These IP3 molecules diffuse through the cytosol leading to the opening of ion channels that disgorge Ca2+ from the endoplasmic reticulum leading to the activation of the actin/myosin contractile machinery. A simple numerical example is presented where a one-dimensional cell adhered to a rigid substrate is pulled at one end, and the evolution of the stress fiber activation signal, stress fiber concentrations, and focal adhesion distributions are investigated. We demonstrate that while it is sufficient to approximate the activation signal as spatially uniform due to the rapid diffusion of the IP3 through the cytosol, the level of the activation signal is sensitive to the rate of application of the mechanical loads. This suggests that ad hoc signaling models may not be able to capture the mechanical response of cells to a wide range of mechanical loading events. © 2011 American Society of Mechanical Engineers.

Aniracetam restores the effects of amyloid-beta protein or ageing on membrane fluidity and intracellular calcium concentration in mice synaptosomes

In the present study, we observed the in vitro effect of aniracetam on membrane fluidity and free calcium concentrations (Ca(2+)i) of frontal cortical (FC) and hippocampal (HP) synaptosomes of aged mice and young mice treated with amyloid-beta protein (A

Relationship between trichosanthin cytotoxicity and its intracellular concentration

Trichosanthin (TCS) is a type I ribosome-inactivating protein with board spectrum of biological activity. Toxicity of this compound differs in different cell lines and this study examined the cause of such difference. It is generally believed that TCS toxicity is mediated through intracellular ribosome inactivation. Therefore, TCS toxicity should be determined by the amount inside cells rather than outside. Three different cell types IC21, JAR and Vero cell lines were chosen with high, medium and low sensitivity to TCS. Intracellular concentrations of fluorescein isothiocyanate labeled TCS were determined by laser scanning confocal microscopy. A good relationship was demonstrated between intracellular TCS concentration and toxicity. Highest intracellular concentration was found in IC21, followed by JAR, and lowest in Vero cells. When the intracellular TCS concentrations in these cells were reduced by using a competitive inhibitor to block cell entry, cytotoxicity was not observed. In conclusion, there is strong evidence to indicate that cytotoxicity of TCS is dependent on its intracellular concentration. Variation of cytotoxicity in different cells may be related to the mechanisms affecting its internalization. (C) 2002 Published by Elsevier Science Ireland Ltd.

Quantification of the effects of antibodies on the extra- and intracellular dynamics of Salmonella enterica.

Antibodies are known to be essential in controlling Salmonella infection, but their exact role remains elusive. We recently developed an in vitro model to investigate the relative efficiency of four different human immunoglobulin G (IgG) subclasses in modulating the interaction of the bacteria with human phagocytes. Our results indicated that different IgG subclasses affect the efficacy of Salmonella uptake by human phagocytes. In this study, we aim to quantify the effects of IgG on intracellular dynamics of infection by combining distributions of bacterial numbers per phagocyte observed by fluorescence microscopy with a mathematical model that simulates the in vitro dynamics. We then use maximum likelihood to estimate the model parameters and compare them across IgG subclasses. The analysis reveals heterogeneity in the division rates of the bacteria, strongly suggesting that a subpopulation of intracellular Salmonella, while visible under the microscope, is not dividing. Clear differences in the observed distributions among the four IgG subclasses are best explained by variations in phagocytosis and intracellular dynamics. We propose and compare potential factors affecting the replication and death of bacteria within phagocytes, and we discuss these results in the light of recent findings on dormancy of Salmonella.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

One-Step Synthesis of Folic Acid Protected Gold Nanoparticles and Their Receptor-Mediated Intracellular Uptake

We report here a facile method to obtain folic acid (FA)-protected gold nanoparticles (Au NPs) by heating an aqueous solution of HAuCl4/FA in which FA acts as both the reducing and stabilizing agent. The successful formation of FA-protected Au NPs is demonstrated by UV/Vis spectroscopy, transmission electron microscopy (TEM), selected-area electron diffraction (SAED), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). ne intracellular uptake of these nanoparticles is facilitated by HeLa cells overexpressing the folate reporter, which itself is significantly inhibited by free FA in a competitive assay as quantified by inductively coupled plasma mass spectroscopy (ICP-MS). This simple one-step approach affords a new perspective for creating functional nanomaterials, and the resulting biocompatible, functional Au NPs may find some prospective applications in various biomedical fields.