EGF Induces Cell Motility and Multi-Drug Resistance Gene Expression in Breast Cancer Cells

The epidermal growth factor receptor (EGFR) is important for normal development, differentiation, and cell proliferation. Deregulation of EGFR has been observed in breast cancer. EGFR and signal pathways activated by these receptors have been associated with an advanced tumor stage and a poor clinical prognosis in breast cancer, however, the precise mechanisms responsible for this process are still not known. Here we show that treatment of MCF-7 breast cancer cells with EGF activated Akt and ERK, induced morphological changes, and increased cell motility. In addition, the constitutive expression of Raf-1 and the use of a MEK inhibitor demonstrated the participation of the Raf/MEK/ERK pathway in these processes. Importantly we detected that EGF induced MRP-1, 3, 5 and 7 gene expression and an increase in MRP1 promoter activity. In conclusion, treatment of MCF-7 breast cancer cells with EGF, in the absence of other growth factors, resulted in activation of EGFR signal transduction pathways; which were related with cell motility and drug resistance.     

PAK1 Kinase Promotes Cell Motility and Invasiveness through CRK-II Serine Phosphorylation in Non-Small Cell Lung Cancer Cells

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.     

Roles of the Mitochondria and Endoplasmic Reticulum in Calcium Elevation during Osmotic Shock in Adrenocortical Cells

Steroid hormones participate in various metabolic processes, and dysfunction of the adrenocortical system leads to numerous pathologies in humans. One of the factors that can influence the secretory properties of adrenocorticocytes is changes in the cell volume observed during osmotic shock. In our study, we tested the hypothesis that osmotic stress modifies intracellular Ca²⁺ signalling and in such a way can influence the secretion of steroids by adrenocorticocytes. The effects of hyperosmotic stress on the cytosolic Ca²⁺ concentration ([Ca] _i ) in cultured adrenocortical cells from the zona fasciculata of the rat adrenals were investigated using the indicator fura-2 technique. Our experiments have shown that exposure of the cells to a hyperosmotic solution caused a decrease in the cell volume, as well as a reversible rise in the [Ca] _i . Calcium-free media partly eliminated [Ca] _i responses. Pretreatment of the cells with thapsigargin or CCCP (blockers of internal calcium stores) significantly decreased the magnitude of responses induced by osmotic stress. These findings indicate that osmotic shock causes an increase in the [Ca] _i in adrenocortical cells, mostly due to depletion of the intracellular stores, and may in such a way stimulate steroidogenesis.     

Decreased Plakophilin-1 Expression Promotes Increased Motility in Head and Neck Squamous Cell Carcinoma Cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

2,4-D和NAA在拟南芥细胞分裂和伸长中的作用分析

以拟南芥悬浮细胞体系为实验材料,研究了人工合成生长素NAA和2,4-D外源处理对细胞形态、细胞鲜重、细胞分裂指数等指标的影响.结果表明:2μmol/L 2,4-D可有效促进细胞分裂但不影响细胞伸长;同样浓度的NAA主要诱导细胞的伸长;细胞伸长和细胞分裂是2个不相偶联的过程;在2,4-D所诱导的细胞分裂过程中异三聚体G-蛋白α-亚基GPA1强表达.     

Role of Cell Wall Calcium in Red Light- inhibited Elongation of Hypocotyls of Etiolated Phaseolus radiatus

When the hypocotyl segments of Phaseolus radiatus L. were incubated in CaC12 (1 mmol/L) medium, the cell wall calcium was increased over threefold more than those incubated in a Ca2+ -free medium. However, red light inhibited elongation of the hypocotyl was 20% to 25% both in the medium with or without Ca2 + . The amount of calcium removed from the wall by ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) ( 1 to 10 mmol/L) was 58.13 % to 75.33%, which offset the red light-inhibited elongation of the hypocotyl by 61.29% to 87.1%. Moreover, treatment with the channel blocker, verapamil ( 10 to 100 μ mol/L), wall calcium was the same as that of the darkness control, by which the red light-inhibited growth was also offset. La3 + ( 100 to 1 000 μmol/L) had no effect on wall calcium as compared to hypocotyl segments treated with red light alone, but eliminated the inhibitory effect of red light. Treatment with the calcium ionophore, A23187 (10 to 100 μmol/L), red light-inhibited elongation was abolished by 66.67% to 142.45% while wall calcium was reduced by 24.53% to 42.81%. In addition, calmodulin antagonist chlorpromazine (1 to 10 μmol/L) also counter acted the red light-induced elongation inhibition. These data indicated that exogenous Ca2+ was involved in the red light-inhibition effect, but that' did not mean that Ca2 + was not required. Perhaps Ca2 + in the wall itself was sufficient for red light-induced inhibition of hypocotyl elongation. The role of wall calcium might be quite complex, it not only acted as a signal of influx Ca2 + from the Ca2 + pool, but also played a regulatory role in the cell wall.     

Calcium Ionophore-Induced Degradation of Neurofilament and Cell Death in MSN Neuroblastoma Cells

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca²⁺-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EQTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.     

MicroRNA-31 Is Overexpressed in Cutaneous Squamous Cell Carcinoma and Regulates Cell Motility and Colony Formation Ability of Tumor Cells

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.     

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.     

Zoledronic Acid,Bevacizumab and Dexamethasone-Induced Apoptosis,Mitochondrial Oxidative Stress,and Calcium Signaling Are Decreased in Human Osteoblast-Like Cell Line by Selenium Treatment

Increased intracellular free calcium ion (Ca²⁺) concentration induces excessive oxidative stress and apoptosis. Medical procedures such as zoledronic acid (Zol), bevacizumab (Bev), and dexamethasone (Dex) are usually used in the treatment of bone diseases (osteoporosis, Paget’s disease, etc.) and to prevent metastasis in the bone although the procedures induce osteonecrosis of the jaw through excessive production of reactive oxygen species (ROS). Recently, we observed regulator roles of selenium (Se) on apoptosis and Ca²⁺ entry through transient receptor potential vanilloid 1 (TRPV1) channels in the cancer cell lines. Therefore, Se may modulate Zol, Bev, and Dex-induced oxidative stress and apoptosis through regulation of TRPV1 channel. In the current study, we investigated the protective effects of Se on apoptosis and oxidative stress through TRPV1 in Zol, Bev, and Dex-induced osteoblast-like cell line. We used human osteoblast-like cell line (Saos-2), and the cells were divided into 12 groups as control, Zol, Bev, Dex, Se, Zol+Se, Bev+Se, Dex+Se, Zol+Dex, Zol+Dex+Se, Zol+Bev, and Zol+Bev+Se which were incubated with drugs (Zol, Bev, Dex, and Se) for 24 h. The cytosolic free Ca²⁺ concentration was increased by Zol, Bev, Dex, Zol+Bev, and Zol+Dex, although it was reduced by Se treatment. However, Zol, Bev, and Dex-induced increase in apoptosis, caspase 3, caspase 9, poly (ADP-ribose) polymerase 1 expression levels, and intracellular ROS production values in the cells were decreased by Se treatments. In conclusion, we observed that Zol, Bev, and Dex-induced apoptosis, mitochondrial oxidative stress, and calcium signaling are decreased in human osteoblast-like cell line by the Se treatment. Our findings may be relevant to the etiology and treatment of Zol, Bev, and Dex-induced osteonecrosis by Se.     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

Stimulated and Unstimulated Saliva Levels of Calcium and Magnesium in Giardiasis

Giardia lamblia causes malabsorption. The aim of this study was to evaluate serum and saliva calcium and magnesium levels in patients with giardiasis. Thirty patients with giardiasis as a case and 30 person without giardiasis as a control group were enrolled. The stimulated and unstimulated whole saliva and serum calcium and magnesium levels were assayed by Arsenazo reaction and xylidyl blue complex methods, respectively. Mean calcium and magnesium level was low in serum and stimulated saliva of case group than that of controls. However, they were higher in the unstimulated saliva of the case group. It is suggested that patients suffering from giardiasis have low calcium and magnesium levels, and they lose the most of calcium and magnesium by saliva during unstimulated condition.     

Stem Elongation and Cell Wall Proteins in Flowering Plants

Abstract: The growth of stems (hypocotyls, epicotyls) and stem-like organs (coleoptiles) in developing seedlings is largely due to the elongation of cells in the sub-apical region of the corresponding organ. According to the organismal concept of plant development, the thick outer epidermal wall, which can be traced back to the peripheral cell wall of the zygote, creates a sturdy organ sheath that determines the rate of stem elongation. The cells of the inner tissues are the products of secondary partitioning of one large protoplast; these turgid, thin-walled cells provide the driving force for organ growth. The structural differences between these types of cell walls are described (outer walls: thick, sturdy, helicoidal cellulose architecture; inner walls: thin, extensible, transversely-oriented cellulose microfibrils). On the basis of these facts, current models of cell wall loosening (and wall stiffening) are discussed with special reference to the expansin, enzymatic polymer remodelling and osmiophilic particle hypothesis. It is concluded that the exact biochemical mechanism(s) responsible for the coordinated yielding of the growth-controlling peripheral organ wall(s) have not yet been identified.     

The Inhibition of KCa3.1 Channels Activity Reduces Cell Motility in Glioblastoma Derived Cancer Stem Cells

In the present study we evaluated the expression of the intermediate conductance calcium-activated potassium (KCa3.1) channel in human glioblastoma stem-like cells (CSCs) and investigated its role in cell motility. While the KCa3.1 channel is not expressed in neuronal- and glial-derived tissues of healthy individuals, both the KCa3.1 mRNA and protein are present in the glioblastoma tumor population, and are significantly enhanced in CSCs derived from both established cell line U87MG and a primary cell line, FCN9. Consistent with these data, voltage-independent and TRAM-34 sensitive potassium currents imputable to the KCa3.1 channel were recorded in the murine GL261 cell line and several primary human glioblastoma cells lines. Moreover, a significantly higher KCa3.1 current was recorded in U87MG-CD133 positive cells as compared to the U87MG-CD133 negative subpopulation. Further, we found that the tumor cell motility is strongly associated with KCa3.1 channel expression. Blockade of the KCa3.1 channel with the specific inhibitor TRAM-34 has in fact a greater impact on the motility of CSCs (reduction of 75%), which express a high level of KCa3.1 channel, than on the FCN9 parental population (reduction of 32%), where the KCa3.1 channel is expressed at lower level. Similar results were also observed with the CSCs derived from U87MG. Because invasion of surrounding tissues is one of the main causes of treatment failure in glioblastoma, these findings can be relevant for future development of novel cancer therapeutic drugs.     

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.     

Shear Stress-induced Redistribution of Vascular Endothelial-Protein-tyrosine Phosphatase (VE-PTP) in Endothelial Cells and Its Role in Cell Elongation

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.