Sample records for activated ras alters

  1. Activated Ras alters lens and corneal development through induction of distinct downstream targets

    Reneker Lixing


    Full Text Available Abstract Background Mammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas. Results Ras transgenic lenses and corneal epithelial cells showed increased proliferation with concomitant increases in cyclin D1 and D2 expression. This initial increase in proliferation is sustained in the cornea but not in the lens epithelial cells. Coincidentally, cdk inhibitors p27Kip1 and p57Kip2 were upregulated in the Ras transgenic lenses but not in the corneas. Phospho-Erk1 and Erk2 levels were elevated in the lens but not in the cornea and Spry 1 and Spry 2, negative regulators of Ras-Raf-Erk signaling, were upregulated more in the corneal than in the lens epithelial cells. Both lens and corneal differentiation programs were sensitive to Ras activation. Ras transgenic embryos showed a distinctive alteration in the architecture of the lens pit. Ras activation, though sufficient for upregulation of Prox1, a transcription factor critical for cell cycle exit and initiation of fiber differentiation, is not sufficient for induction of terminal fiber differentiation. Expression of Keratin 12, a marker of corneal epithelial differentiation, was reduced in the Ras transgenic corneas. Conclusions Collectively, these

  2. Ras activation by SOS

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen;


    SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by...... Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather...

  3. Radiation sensitization by inhibition of activated ras

    Background and purpose: Ras has been identified as a significant contributor to radiation resistance. This article reviews preclinical and phase I clinical studies that reported on combining inhibition of activated Ras and downstream effectors of Ras with radiotherapy. Material and methods: transfection studies and RNA interference were used to check the role of the Ras isoforms for intrinsic radiation sensibility. Western blotting was used to control for prenylation inhibition of the respective Ras isoforms and for changes in activity of downstream proteins. Clonogenic assays with human and rodent tumor cell lines served for testing radiosensitivity. In vivo, farnesyltransferase inhibitors (FTIs) and irradiation were used to treat xenograft tumors. Ex vivo plating efficiency measurements, regrowth of tumors, and EF5 staining for detection of hypoxia were endpoints in these studies. Simultaneous treatment with L-778,123 and irradiation was performed in non-small cell lung cancer, head and neck cancer, and pancreatic cancer patients. Results: radiation sensitization was achieved in vitro and in vivo blocking the prenylation of Ras proteins in cell lines with Ras activated by mutations or receptor signaling. Among the many Ras downstream pathways the phosphoinositide 3 (PI3) kinase-Akt pathway was identified as a contributor to Ras-mediated radiation resistance. Furthermore, increased oxygenation was observed in xenograft tumors after FTI treatment. Combined treatment in a phase I study was safe and effective. Conclusion: the rational combination of FTIs with radiotherapy may improve the clinical results of patients with tumors who bear mutant or receptor-signaling activated Ras. (orig.)

  4. Anti-Microbial activity of Talakeshwara Ras

    Prasanna Kumar Tirupati; Srilakshmi Dasari; Ragamala K C; Geeta Balakrishna; Shwetha Seshagiri


    Rasa Shastra, one of the Pharmaco-therapeutic branches of Ayurveda where metals, minerals, poisonous plants and animal products are used after proper processing for internal administration. Talakeshwara Ras is one of Khalvi rasayanas where Emblica officinalis (Dhatri) and minerals Arsenic tri sulphide (Haratala) & Borax (Tankana) are the ingredients. It is indicated for Sarva Kushta at one Masha (1 gm) dose. Anti -Microbial activity of Talakeshwara Ras was done with an intention to evalua...

  5. Amphetamine alters Ras-guanine nucleotide-releasing factor expression in the rat striatum in vivo

    Parelkar, Nikhil K.; Jiang, Qian; Chu, Xiang-Ping; Guo, Ming-Lei; Mao, Li-Min; WANG, John Q.


    Ras-guanine nucleotide-releasing factors (Ras-GRFs) are densely expressed in neurons of the mammalian brain. As a Ras-specific activator predominantly concentrated at synaptic sites, Ras-GRFs activate the Ras-mitogen-activated protein kinase (Ras-MAPK) cascade in response to changing synaptic inputs, thereby modifying a variety of cellular and synaptic activities. While the Ras-MAPK cascade in the limbic reward circuit is well-known to be sensitive to dopamine inputs, the sensitivity of its u...

  6. Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

    Hurwitz, N; Segal, M; Marbach, I; Levitzki, A


    Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements. PMID:7479926

  7. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

    Jesse E. Jun


    Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

  8. A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

    Paweletz Cloud


    Full Text Available Abstract Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90% sensitivity but relatively low (50% specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical

  9. Gamma band activity in the reticular activating system (RAS

    Francisco J Urbano


    Full Text Available This review considers recent evidence showing that cells in three regions of the reticular activating system (RAS exhibit gamma band activity, and describes the mechanisms behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN, intralaminar parafascicular nucleus (Pf, and pontine Subcoeruleus nucleus dorsalis (SubCD all fire in the beta/gamma band range when maximally activated, but no higher. The mechanisms behind this ceiling effect have been recently elucidated. We describe recent findings showing that every cell in the PPN have high threshold, voltage-dependent P/Q-type calcium channels that are essential, while N-type calcium channels are permissive, to gamma band activity. Every cell in the Pf also showed that P/Q-type and N-type calcium channels are responsible for this activity. On the other hand, every SubCD cell exhibited sodium-dependent subthreshold oscillations. A novel mechanism for sleep-wake control based on well-known transmitter interactions, electrical coupling, and gamma band activity is described. The data presented here on inherent gamma band activity demonstrates the global nature of sleep-wake oscillation that is orchestrated by brainstem-thalamic mechanism, and questions the undue importance given to the hypothalamus for regulation of sleep-wakefulness. The discovery of gamma band activity in the RAS follows recent reports of such activity in other subcortical regions like the hippocampus and cerebellum. We hypothesize that, rather than participating in the temporal binding of sensory events as seen in the cortex, gamma band activity manifested in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking and paradoxical sleep. Most of our thoughts and actions are driven by preconscious processes. We speculate that continuous sensory input will induce gamma band activity in the RAS that could participate in the

  10. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu


    R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  11. Alteration of glycolipids in ras-transfected NIH 3T3 cells

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by 125I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition

  12. ras activation in human tumors and in animal model systems.

    Corominas, M; Sloan, S R; Leon, J.; Kamino, H; Newcomb, E W; Pellicer, A


    Environmental agents such as radiation and chemicals are known to cause genetic damage. Alterations in a limited set of cellular genes called proto-oncogenes lead to unregulated proliferation and differentiation. We have studied the role of the ras gene family in carcinogenesis using two different animal models. In one case, thymic lymphomas were induced in mice by either gamma or neutron radiation, and in the other, keratoacanthomas were induced in rabbit skin with dimethylbezanthracene. Hum...

  13. Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes

    Chronic myelomonocytic leukemia (CMML) is a hematological disease close to, but separate from both myeloproliferative disorders (MPD) and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML) or myelodysplastic (MD-CMML) features. Not much is known about the molecular biology of this disease. We studied a series of 30 CMML samples (13 MP- and 11 MD-CMMLs, and 6 acutely transformed cases) from 29 patients by using Agilent high density array-comparative genomic hybridization (aCGH) and sequencing of 12 candidate genes. Two-thirds of samples did not show any obvious alteration of aCGH profiles. In one-third we observed chromosome abnormalities (e.g. trisomy 8, del20q) and gain or loss of genes (e.g. NF1, RB1 and CDK6). RAS mutations were detected in 4 cases (including an uncommon codon 146 mutation in KRAS) and PTPN11 mutations in 3 cases. We detected 11 RUNX1 alterations (9 mutations and 2 rearrangements). The rearrangements were a new, cryptic inversion of chromosomal region 21q21-22 leading to break and fusion of RUNX1 to USP16. RAS and RUNX1 alterations were not mutually exclusive. RAS pathway mutations occurred in MP-CMMLs (~46%) but not in MD-CMMLs. RUNX1 alterations (mutations and cryptic rearrangement) occurred in both MP and MD classes (~38%). We detected RAS pathway mutations and RUNX1 alterations. The latter included a new cryptic USP16-RUNX1 fusion. In some samples, two alterations coexisted already at this early chronic stage

  14. Gamma Band Activity in the RAS-intracellular mechanisms

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.


    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range...

  15. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

    Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))


    Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

  16. Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity.

    Manne, V; Bekesi, E; Kung, H F


    Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits approximately equal to 10 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of ...

  17. An Active RFID Accountability System (RAS) for Constrained Wireless Environments

    Barker, Alan M [ORNL; Hanson, Gregory R [ORNL; Sexton, Angela Kay [ORNL; Jones Jr, J P [ORNL; Freer, Eva B [ORNL; Sjoreen, Andrea L [ORNL


    A team from Oak Ridge National Laboratory (ORNL) has developed an RFID Accountability System (RAS) that allows items with active RFID tags to be tracked in environments where tags may not be able to transmit their location continuously. The system uses activators that transmit a short range signal. Active RFID tags are in a sleep state until they encounter an activator. Then they transmit a signal that is picked up by the antennas installed throughout the building. This paper presents the theory of operation, application areas, lessons learned, and key features developed over the course of seven years of development and use.

  18. Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

    Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34+ cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G0/G1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells

  19. Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung—importance of mouse strain

    Calvert, Richard J.; Tepper, Shirley; Kammouni, Wafa; Anderson, Lucy M.; Kritchevsky, David


    Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For three weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25 mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) gr...

  20. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27Kip Cdk-complexes

    Groth, Anja; Willumsen, Berthe Marie


    and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent......-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal...

  1. Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance | Poster

    Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

  2. RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

    Dominic P Golec

    Full Text Available T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-CD8(- 'double negative' (DN thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+CD8(+ 'double positive' (DP thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

  3. Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

    Hofer, F.; Fields, S; Schneider, C; Martin, G S


    The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The...

  4. CARD9 mediates Dectin-1-induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity.

    Jia, Xin-Ming; Tang, Bing; Zhu, Le-Le; Liu, Yan-Hui; Zhao, Xue-Qiang; Gorjestani, Sara; Hsu, Yen-Michael S; Yang, Long; Guan, Jian-Hong; Xu, Guo-Tong; Lin, Xin


    Dectin-1 functions as a pattern recognition receptor for sensing fungal infection. It has been well-established that Dectin-1 induces innate immune responses through caspase recruitment domain-containing protein 9 (CARD9)-mediated NF-κB activation. In this study, we find that CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands, such as curdlan or Candida albicans yeast. In contrast, we find that CARD9 regulates H-Ras activation by linking Ras-GRF1 to H-Ras, which mediates Dectin-1-induced extracellular signal-regulated protein kinase (ERK) activation and proinflammatory responses when stimulated by their ligands. Mechanistically, Dectin-1 engagement initiates spleen tyrosine kinase (Syk)-dependent Ras-GRF1 phosphorylation, and the phosphorylated Ras-GRF1 recruits and activates H-Ras through forming a complex with CARD9, which leads to activation of ERK downstream. Finally, we show that inhibiting ERK activation significantly accelerates the death of C. albicans-infected mice, and this inhibitory effect is dependent on CARD9. Together, our studies reveal a molecular mechanism by which Dectin-1 induces H-Ras activation that leads to ERK activation for host innate immune responses against fungal infection. PMID:25267792

  5. CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity

    Jia, Xin-ming; Tang, Bing; Zhu, Le-Le; Liu, Yan-Hui; Zhao, Xue-Qiang; Gorjestani, Sara; Hsu, Yen-Michael S.; Yang, Long; Guan, Jian-Hong; Xu, Guo-Tong; Lin, Xin


    Dectin-1 functions as a pattern recognition receptor for sensing fungal infection. It has been well-established that Dectin-1 induces innate immune responses through caspase recruitment domain-containing protein 9 (CARD9)–mediated NF-κB activation. In this study, we find that CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands, such as curdlan or Candida albicans yeast. In contrast, we find that CARD9 regulates H-Ras activation by linking Ras-GRF1 to H-Ras, which mediates De...

  6. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    Schümann, Michael; Dobbelstein, Matthias


    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses to...... grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning the...

  7. Modulation of Ras signaling alters the toxicity of hydroquinone, a benzene metabolite and component of cigarette smoke

    Benzene is an established human leukemogen, with a ubiquitous environmental presence leading to significant population exposure. In a genome-wide functional screen in the yeast Saccharomyces cerevisiae, inactivation of IRA2, a yeast ortholog of the human tumor suppressor gene NF1 (Neurofibromin), enhanced sensitivity to hydroquinone, an important benzene metabolite. Increased Ras signaling is implicated as a causal factor in the increased pre-disposition to leukemia of individuals with mutations in NF1. Growth inhibition of yeast by hydroquinone was assessed in mutant strains exhibiting varying levels of Ras activity. Subsequently, effects of hydroquinone on both genotoxicity (measured by micronucleus formation) and proliferation of WT and Nf1 null murine hematopoietic precursors were assessed. Here we show that the Ras status of both yeast and mammalian cells modulates hydroquinone toxicity, indicating potential synergy between Ras signaling and benzene toxicity. Specifically, enhanced Ras signaling increases both hydroquinone-mediated growth inhibition in yeast and genotoxicity in mammalian hematopoetic precursors as measured by an in vitro erythroid micronucleus assay. Hydroquinone also increases proliferation of CFU-GM progenitor cells in mice with Nf1 null bone marrow relative to WT, the same cell type associated with benzene-associated leukemia. Together our findings show that hydroquinone toxicity is modulated by Ras signaling. Individuals with abnormal Ras signaling could be more vulnerable to developing myeloid diseases after exposure to benzene. We note that hydroquinone is used cosmetically as a skin-bleaching agent, including by individuals with cafe-au-lait spots (which may be present in individuals with neurofibromatosis who have a mutation in NF1), which could be unadvisable given our findings

  8. Immune response to bacteria induces dissemination of Ras-activated Drosophila hindgut cells

    Bangi, Erdem; Pitsouli, Chrysoula; Rahme, Laurence G.; Cagan, Ross; Apidianakis, Yiorgos


    Drosophila hindgut cells exposed to bacterial infection activate the innate immune response. Concomitant expression of the Ras1V12 oncogene leads to extracellular matrix degradation, basal cell invasion and dissemination in the body cavity.

  9. The higher level of complexity of K-Ras4B activation at the membrane.

    Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth


    Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. PMID:26718888

  10. Ki-ras point mutations and proliferation activity in biliary tract carcinomas.

    Ohashi, K; Tstsumi, M.; Nakajima, Y.; Nakano, H; Konishi, Y


    The association between Ki-ras mutations and proliferation activity was investigated in a comprehensive series of biliary tract carcinomas (BTCs). We precisely microdissected samples of tissue from paraffin-embedded sections of 77 BTCs including 22 intrahepatic cholangiocarcinomas (ICCs), 36 extrahepatic cholangiocarcinomas (ECCs), and 19 gall bladder carcinomas (GBCs). Ki-ras mutations at exons 1 and 2 were determined by the polymerase chain reaction-single strand conformation polymorphism (...

  11. Active Erk Regulates Microtubule Stability in H-ras-Transformed Cells

    Rene E. Harrison


    Full Text Available Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

  12. Antroquinonol blocks Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells.

    Ho, Ching-Liang; Wang, Jui-Ling; Lee, Cheng-Chung; Cheng, Hsiu-Yi; Wen, Wu-Che; Cheng, Howard Hao-Yu; Chen, Miles Chih-Ming


    Antroquinonol is the smallest anticancer molecule isolated from Antrodia camphorata thus far. The ubiquinone-like structure of Antroquinonol exhibits a broad spectrum of activity against malignancies in vivo and in vitro. However, the mechanism of action of Antroquinonol remains unclear. Here, we provide evidence that Antroquinonol plays a role in the inhibition of Ras and Ras-related small GTP-binding protein functions through the inhibition of protein isoprenyl transferase activity in cancer cells. Using cell line-based assays, we found that the inactive forms of Ras and Rho proteins were significantly elevated after treatment with Antroquinonol. We also demonstrated that Antroquinonol binds directly to farnesyltransferase and geranylgeranyltransferase-I, which are key enzymes involved in activation of Ras-related proteins, and inhibits enzymes activities in vitro. Furthermore, a molecular docking analysis illustrated that the isoprenoid moiety of Antroquinonol binds along the hydrophobic cavity of farnesyltransferase similar to its natural substrate, farnesyl pyrophosphate. In contrast, the ring structure of Antroquinonol lies adjacent to the Ras-CAAX motif-binding site on farnesyltransferase. The molecular docking study also showed a reasonable correlation with the IC50 values of Antroquinonol analogues. We also found that the levels of LC3B-II and the autophagosome-associated LC3 form were also significantly increased in H838 after Antroquinonol administration. In conclusion, Antroquinonol inhibited Ras and Ras-related GTP-binding protein activation through inhibition of protein isoprenyl transferase activity, leading to activation of autophagy and associated mode of cell death in cancer cells. PMID:25312820

  13. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

    Jennifer S. Liu


    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  14. Role of neuronal Ras activity in adult hippocampal neurogenesis and cognition

    Martina eManns


    Full Text Available Hippocampal neurogenesis in the adult mammalian brain is modulated by various signals like growth factors, hormones, neuropeptides, and neurotransmitters. All of these factors can (but not necessarily do converge on the activation of the G protein p21Ras. We used a transgenic mouse model (synRas mice expressing constitutively activated G12V-Harvey Ras selectively in differentiated neurons to investigate the possible effects onto neurogenesis. Ras activation in neurons attenuates hippocampal precursor cell generation at an early stage of the proliferative cascade before neuronal lineage determination occurs. Therefore it is unlikely that the transgenically activated Ras in neurons mediates this effect by a direct, intracellular signaling mechanism. Voluntary exercise restores neurogenesis up to wild type level presumably mediated by brain derived neurotrophic factor. Reduced neurogenesis is linked to impairments in spatial short-term memory and object recognition, the latter can be rescued by voluntary exercise, as well. These data support the view that new cells significantly increase complexity that can be processed by the hippocampal network when experience requires high demands to associate stimuli over time and/or space.

  15. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

    Hong, Xin; Nguyen, Hung Thanh; Chen, Qingfeng; Zhang, Rui; Hagman, Zandra; Voorhoeve, P Mathijs; Cohen, Stephen M


    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the βTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1. PMID:25180228

  16. Dual Activation of Phospholipase C-ε by Rho and Ras GTPases*

    Seifert, Jason P.; Zhou, Yixing; Hicks, Stephanie N.; Sondek, John; Harden, T. Kendall


    Phospholipase C-ε (PLC-ε) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-ε to stimulate its phospholipase activity. Purified PLC-ε was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P2-containing ph...

  17. Ras pathway activation in gliomas: a strategic target for intranasal administration of perillyl alcohol

    Targeted therapy directed at specific molecular alterations is already creating a shift in the treatment of cancer patients. Malignant gliomas commonly overexpress the oncogenes EGFR and PDGFR and contain mutations and deletions of the tumor suppressor genes PTEN and TP53. Some of these alterations lead to activation of the P13K/Akt and Ras/MAPK pathways, which provide targets for therapy. Perillyl alcohol (POH), the isoprenoid of greatest clinical interest, was initially considered to inhibit farnesyl protein transferase. Follow-up studies revealed that POH suppresses the synthesis of small G proteins, including Ras. Intranasal delivery allows drugs that do not cross the blood-brain barrier to enter the central nervous system. Moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. Applying this method, a phase I/II clinical trial of POH was performed in patients with relapsed malignant gliomas after standard treatment: surgery, radiotherapy, and chemotherapy. POH was administrated in a concentration of 0.3% volume/volume (55 mg) four times daily in an interrupted administration schedule. The objective was to evaluate toxicity and progression-free survival (PFS) after six months of treatment. The cohort consisted of 37 patients, including 29 with glioblastoma multiforme (GBM), 5 with grade III astrocytoma (AA), and 3 with anaplastic oligodendroglioma (AO). Neurological examination and suitable image analysis (computed tomography (CT), magnetic resonance imaging (MRI)) established disease progression. Complete response was defined as neurological stability or improvement of conditions, disappearance of CT/MRI tumor image, and corticosteroid withdraw; partial response (PR) as .50% reduction of CT/MRI tumor image, neurological stability, or improvement of conditions and corticosteroid requirement; progressive course (PC) as .25% increase in CT/MRI tumor image or the appearance of a new lesion; and stable disease as a

  18. Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

    Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection

  19. Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

    D Abaco, G. M.; Whitehead, R. H.; Burgess, A W


    Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are nor...

  20. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T


    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis. PMID:27501536

  1. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie


    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  2. Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells.

    Damian J Junk

    Full Text Available Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1 occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

  3. Analysis of mammalian ras effector functions

    Over the last 8 years or so, a great deal of effort has been put into understanding the biochemical function of the three mammalian p21ras proteins. Single-amino-acid alterations in these three proteins have been detected in 25-50% of some types of human cancers, and it is believed that the somatic mutational event that generated these amino acid substitutions is an important step in the development of these malignancies. The authors report here some of their efforts to look for a possible target for p21ras regulation. In a series of experiments, they have looked for ras-induced changes in another known second-messenger system, namely, the breakdown of phosphatidylinositol (PtdIns) lipids by a phospholipase C. The results lead to the conclusion that NIH-3T3 cells transformed by oncogenic p21ras have an increased basal phospholipase C activity. In a different approach to identify a target for ras, they have analyzed its interaction with the recently described cellular protein, GAP. They have shown that this protein appears to bind ras at a site previously identified as the effector site, strongly implicating GAP as the target protein for p21ras regulation

  4. Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

    Telang Sucheta


    Full Text Available Abstract Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX. Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

  5. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Jianhua Gong

    Full Text Available Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency, hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.



    @@ The monoterpene d-limonene inhibit the plasma-membrane associated P21ras expression and the posttranslational isoprenylation of P21ras, a mechanism that may contribute to its efficacy in the chemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study,the relative abilities of d-limonene to inhibit membrane associated P21ras expression in pancreas tumor cell(PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FTPase) activity during the Ras protein isoprenylation and cell proliferation were determined.Concomitantly,the effects of d-limonene on P21ras localization by immunohistochemistry and H-ras oncogene expression in PaCa tumor cell line by Northern blotting were observed. The results showed that d-limonene inhibited FPTase activity, thus to reduce P21H-ras isoprenylation. d-limonene could decrease P21ras membrane association and increase cytosolic accumulation of P21ras. This phenomenon was also noted when d-limonene-treated PaCa cells were stained immunohistochemically with anti-P21ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21ras membrane association and the alteration of P21ras localization. Inhibition of farnesylation of P21ras altered their intracellular localization and, hence, disrupted their biological activity,but no relationship with H-ras oncogene expression was found.

  7. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation

    Ze-Yi Zheng


    Full Text Available Basal-like breast cancers (BLBCs are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

  8. Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

    Wang, Ai-Guo, E-mail:; Song, Ya-Nan; Chen, Jun; Li, Hui-Ling; Dong, Jian-Yi; Cui, Hai-Peng; Yao, Liang; Li, Xue-Feng; Gao, Wen-Ting; Qiu, Ze-Wen; Wang, Fu-Jin; Wang, Jing-Yu, E-mail:


    Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

  9. Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

    Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

  10. ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

    Bonini Chiara


    Full Text Available Abstract Background The mitogen-activated protein (MAP kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. Results Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. Conclusion These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity.


    陈晓光; YoshihisaYano; TadayoshiHasuma; ToshikoYoshimata; WangYinna; ShuzoOtani


    The monoterpene d-limonene inhibit the plasma membrane associated P21ras expresion and the post-translational isoprenylatlon of P21ras, a mechanism that may contribute to its efficacy in the ehemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study, the relative abilities of d-limonene to inhibit membrane associated P21ras expression in Imncreas tumorcell (PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FT-Pase ) activity during the Rns p~otebi isoprenylation and cell proliferation were determined. Concomitantly,the effects of d-limonene on P21ras localization hy immunohimcchemistry and H-ras oncogene expression in PaCe tutor cell line by Northern blotting were observed. The results showed that ddimonene inhibited FPTase activity, thus to reduce P21H-ras isoprenylation, d limonene couM decrease P21ras meanhrane asso-ciation and increase cytosolie accumulation of P21ras This phenomenon was also noted when d-llmmaene-treated PaCe cells were stained immunohistcchemieally with anti-P21ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21ras membrane assoclaticon aad the alteration of P21ras localization. Inhibition ot farnesylation of P21ras altered their intraeellulsr localization and, hence, disrupted their biological activity, hut no relationship with H ras oneogene expression was found.

  12. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail:


    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular


    WANG Wei; WANG Chun-you; DONG Ji-hua; CHEN Xiong; ZHANG Min; ZHAO Gang


    Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

  14. Activated ras Prevents Downregulation of Bcl-XL Triggered by Detachment from the Extracellular Matrix

    Rosen, Kirill; RAK, Janusz; Leung, Thomas; Dean, Nicholas M.; Kerbel, Robert S.; Filmus, Jorge


    Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptoti...

  15. Detection of K-ras point mutation and telomerase activity during endoscopic retrograde cholangiopancreatography in diagnosis of pancreatic cancer

    Guo-Xiong Zhou; Jie-Fei Huang; Zhao-Shen Li; Guo-Ming Xu; Feng Liu; Hong Zhang


    AIM: To study the value of monitoring K-ras point mutation at codon 12 and telomerase activity in exfoliated cells obtained from pancreatic duct brushings during endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis of pancreatic cancer.METHODS: Exfoliated cells obtained from pancreatic duct brushings during ERCP were examined in 27 patients: 23with pancreatic cancers, 4 with chronic pancreatitis. K-fas point mutation was detected with the polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP). Telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAPELISA).RESULTS: The telomerase activities in 27 patients were measured in 21 exfoliated cell samples obtained from pancreatic duct brushings. D450 value of telomerase activities in pancreatic cancer and chronic pancreatitis were 0.446±0.27and 0.041±0.0111, respectively. Seventy-seven point eight percent (14/18) of patients with pancreatic cancer and none of the patients with chronic pancreatitis showed telomerase activity in cells collected from pancreatic duct brushings when cutoff value of telomerase activity was set at 2.0. The K-ras gene mutation rate (72.2%) in pancreatic cancer was higher than that in chronic pancreatitis (33.3%)(P<0.05). In considering of both telomerase activities and K-ras point mutation, the total positive rate was 83.3%(15/18), and the specificity was 100%.CONCLUSION: Changes of telomerase activities and K-ras point mutation at codon 12 may be an early event of malignant progression in pancreatic cancer. Detection of telomerase activity and K-ras point mutation at codon 12may be complementary to each other, and is useful in diagnosis of pancreatic cancer.

  16. Dietary turmeric modulates DMBA-induced p21ras, MAP kinases and AP-1/NF-κB pathway to alter cellular responses during hamster buccal pouch carcinogenesis

    The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-κB, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-κB DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-κB, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements

  17. The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

    Chiara Sandri; Guido Serini; Francesca Caccavari; Donatella Valdembri; Chiara Camillo; Stefan Veltel; Martina Santambrogio; Letizia Lanzetti; Fedenco Bussolino; Johanna Ivaska


    During developmental and tumor angiogenesis,semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases.R-Ras is mainly expressed in vascular cells,where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms.We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and-angiogenic activity of R-Ras.Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia.Upon binding,GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF)to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes.Here,the R-Ras/RIN2/Rab5 signaling module activates Racl-dependent cell adhesion via TIAM1,a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate.In conclusion,the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Racl.

  18. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)–transfected cells enhanced activation of the phosphatidylinositol-3-kinase–Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)–neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor α was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR–phosphatidylinositol-3-kinase–Akt cascade.

  19. Role of 239Pu-induced chromosome alterations and mutated Ki-v-ras oncogene during liver-cancer induction in Chinese hamsters and mice

    Chromosome aberrations and mutated oncogenes can cause important changes during carcinogenesis. Model systems are being studied in which defined cellular and molecular changes can be quantitated and altered, and tumor frequency, type, and time of appearance can be evaluated. Dose-response relationships for Pu Citrate-induced chromosome aberrations and liver cancer were measured in Chinese hamsters. Chromosome aberrations increased linearly according to dose, with a slope of 4.8 x 10-1 aberrations/cell/Gy; liver-tumor incidence was 1.1 x 10-1 tumors/animal/Gy. The dose was calculated at the 50% survival time. The interaction between Pu and Ki-v-ras, an altered, dominant-acting oncogene, on the induction of liver cancer was measured in B6C3F1 mice. The neo oncogene was used as a negative control in these studies. The Ki-v-ras oncogene was inserted into a viral vector and incorporated into the livers of mice either 30 days before or after the incorporation of 239Pu. Compared with both the controls and the mice injected with a single insult, mortality increased in groups of animals that received combined exposure to oncogenes, CCl4, and 239Pu. The relationships between molecular and cellular damage and the induction of cancer is being defined in both mice and Chinese hamsters

  20. Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

    Nadja Chevalier

    Full Text Available Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes. Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin. All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

  1. The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

    Jaiswal, R. K.; Moodie, S A; Wolfman, A; Landreth, G E


    Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity whi...

  2. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc’h, Nathalie


    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the del...

  3. Activation of Wnt signaling bypasses the requirement for RTK/Ras signaling during C. elegans vulval induction

    Gleason, Julie E.; Korswagen, Hendrik C.; Eisenmann, David M


    During Caenorhabditis elegans vulval development, activation of receptor tyrosine kinase/Ras and Notch signaling pathways causes three vulval precursor cells (VPCs) to adopt induced cell fates. A Wnt signaling pathway also acts in cell fate specification by the VPCs, via regulation of the Hox gene lin-39. We show here that either mutation of pry-1 or expression of an activated BAR-1 β-catenin protein causes an Overinduced phenotype, in which greater than three VPCs adopt induced cell fates. T...

  4. Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line

    Matsumura, Itaru; Nakajima, Koichi; Wakao, Hiroshi; Hattori, Seisuke; Hashimoto, Koji; Sugahara, Hiroyuki; Kato, Takashi; Miyazaki, Hiroshi; Hirano, Toshio; Kanakura, Yuzuru


    Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to cla...

  5. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    Kolkova, K; Novitskaya, V; Pedersen, N;


    ), protein kinase C (PKC), and the Ras-mitogen-activated protein (MAP) kinase pathway. This was done using a coculture system consisting of PC12-E2 cells grown on fibroblasts, with or without NCAM expression, allowing NCAM-NCAM interactions resulting in neurite outgrowth. PC12-E2 cells were transiently......The signal transduction pathways associated with neural cell adhesion molecule (NCAM)-induced neuritogenesis are only partially characterized. We here demonstrate that NCAM-induced neurite outgrowth depends on activation of p59(fyn), focal adhesion kinase (FAK), phospholipase Cgamma (PLCgamma...... transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor...

  6. Activation of the H-ras oncogene in hepatocellular carcinomas initiated with diethylnitrosamine and promoted by a dietary methyl deficiency

    High molecular weight DNA was isolated from control livers and from hepatocellular carcinomas produced in F344 rats initiated with diethylnitrosamine (20 mg/kg body weight) then fed a methyl-deficient diet. The DNAs were used to transfect NIH 3T3 cells by the calcium phosphate precipitation technique. Cultures were scored for foci of morphologically transformed cells after 21 days. Three of 24 DNAs isolated from tumors initiated with diethylnitrosamine were able to transform NIH 3T3 cells (frequency = 0.01 to 0.04 foci/μg DNA). Secondary transformants were produced when DNA isolated from transformed foci were used in the transfection assay. The presence of rat DNA sequences in the primary transformants was demonstrated using a probe specific for rat repetitive sequences. High molecular weight DNA isolated from each of the 3 primary transformants was digested with BamH1 and was subjected to Southern blot analysis using a 32P-labelled probe for the H-ras gene. Each transformant tested exhibited a fragment not present in 3T3 DNA that hybridized with the H-ras probe. Twelve control DNAs isolated from livers of tumor-free animals were negative in the transfection assay. The results are consistent with the involvement of the activated H-ras gene in the development of these chemically-initiated carcinomas in methyl-deficient animals

  7. Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.

    Owen, R D; Ostrowski, M C


    Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-r...

  8. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis; the role of p53 and p21

    Escandell, José M.; Kaler, Pawan; Recio, M. Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija


    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke3 cells. However, the presence of oncogenic k-Ras significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransfromed intestinal epithelial cells with inducible expression of k-RasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  9. cDNA cloning and chromosomal mapping of a novel human GAP (GAP1M), GTPase-activating protein of Ras

    Li, Shaowei; Nakamura, Shun; Hattori, Seisuke [National Center of Neurology and Psychiatry, Kodaira, Tokyo (Japan)] [and others


    We have previously isolated a novel Ras GTPase-activating protein (Ras GAP), Gapl{sup m}, from rat brain. Gap1{sup m} is considered to be a negative regulator of the Ras signaling pathways, like other Ras GAPs, neurofibromin, which is a gene product of the neurofibromatosis type I gene, and p120GAP. In this study we have isolated a human cDNA of this Gap and mapped the gene. The gene encodes a protein of 853 amino acids that shows 89% sequence identity to rat Gapl{sup m}. The human gene was mapped to chromosome 3 by PCR analysis on a panel of human-mouse hybrid cells. FISH analysis refined the location of the gene further to 3q22-q23. 11 refs., 2 figs.

  10. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita


    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  11. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Gennaro Altamura


    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  12. T24 human bladder carcinoma cells with activated Ha-ras protooncogene: nontumorigenic cells susceptible to malignant transformation with carcinogen.

    Senger, D. R.; Perruzzi, C A; Ali, I U


    A comparative analysis of T24 human bladder carcinoma cells and N-methyl-N'-nitro-N-nitrosoguanidine (MeNNG)-transformed derivatives (MeNNG-T24 cells) revealed the following: (i) The presence of an activated c-Ha-ras gene (in the absence of the normal allele) is insufficient to confer upon T24 cells a tumor-associated phenotype. (ii) MeNNG-transformed T24 cells not only acquire tumor-associated (in vitro) traits (growth in soft agar and rhodamine retention) but, are highly tumorigenic in nude...

  13. Grb2 Is a Negative Modulator of the Intrinsic Ras-GEF Activity of hSos1

    Zarich, Natasha; Oliva, José Luis; Martínez, Natalia; Jorge, Rocío; Ballester, Alicia; Gutiérrez-Eisman, Silvia; García-Vargas, Susana; Rojas, José M


    hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modu...

  14. A New View of Ras Isoforms in Cancers.

    Nussinov, Ruth; Tsai, Chung-Jung; Chakrabarti, Mayukh; Jang, Hyunbum


    Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. PMID:26659836

  15. Constitutive K-RasG12D activation of ERK2 specifically regulates 3D invasion of human pancreatic cancer cells via MMP-1.

    Botta, Gregory P; Reginato, Mauricio J; Reichert, Maximilian; Rustgi, Anil K; Lelkes, Peter I


    Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-Ras(G12D) that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of extracellular signal-regulated kinase (ERK) 1/2 and proinvasive matrix metalloproteinases (MMP), indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras/ERK1/2 signaling and its influence on MMP-directed stromal invasion in primary human pancreatic ductal epithelial cells (PDEC) have yet to be elucidated in three-dimensions. Expression of oncogenic K-Ras(G12D) alone in genetically defined PDECs reveals increased invadopodia and epithelial-to-mesenchymal transition markers, but only when cultured in a three-dimensional model incorporating a basement membrane analog. Activation of ERK2, but not ERK1, also occurs only in K-Ras(G12D)-mutated PDECs cultured in three-dimensions and is a necessary intracellular signaling event for invasion based upon pharmacologic and short hairpin RNA (shRNA) inhibition. Increased active invasion of K-Ras(G12D) PDECs through the basement membrane model is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 RNA, its secreted protein, and its proteolytic cleavage activity are amplified in K-Ras(G12D) PDECs when assayed by real-time quantitative PCR, ELISA, and fluorescence resonance energy transfer (FRET). Importantly, shRNA silencing of MMP-1 mimics ERK2 inhibition and disrupts active, vertical PDEC invasion. ERK2 isoform and MMP-1 targeting are shown to be viable strategies to attenuate invasion of K-Ras(G12D)-mutated human pancreatic cancer cells in a three-dimensional tumor microenvironment. PMID:22160930

  16. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis; the role of p53 and p21

    Escandell, José M.; Kaler, Pawan; Recio, M. Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija


    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We com...

  17. RAS Laboratory Groups

    The RAS Initiative uses multiple technologies to attack RAS-driven cancers. The resources of the Frederick National Lab allocated to the RAS Hub are organized into seven laboratory groups, each contributing to the collaborative effort.

  18. The RAS Initiative

    NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

  19. Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype

    Pluk Helma


    Full Text Available Abstract Background The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. Results Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. Conclusion The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

  20. Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

    Yu Jing


    Full Text Available Abstract Background Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma. Methods We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation. Results Promoter methylation of RASSF1A could be detected in 71.05% (27/38 of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p p p p Conclusion Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated

  1. The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence.

    Thompson, Keyata N; Whipple, Rebecca A; Yoon, Jennifer R; Lipsky, Michael; Charpentier, Monica S; Boggs, Amanda E; Chakrabarti, Kristi R; Bhandary, Lekhana; Hessler, Lindsay K; Martin, Stuart S; Vitolo, Michele I


    A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common. PMID:26497685

  2. Activating Mutations and/or Expression Levels of Tyrosine Kinase Receptors GRB7, RAS, and BRAF in Testicular Germ Cell Tumors

    Alan McIntyre


    Full Text Available Amplification and/or overexpression of genes encoding tyrosine kinase receptors KIT and ERBB2 have been reported in testicular germ cell tumors (TGCTs. These receptors can bind the adaptor molecule GRB7 encoded by a gene adjacent to ERBB2 at 17q12, a region also frequently gained in TGCTs. GRB7 binding may be involved in the activation of RAS signaling and KRAS2 maps to 12p, which is constitutively gained in TGCT and lies within a minimum overlapping region of amplification at 12pl1.2–12.1, a region we have previously defined. RAS proteins activate BRAF, and activating mutations of genes encoding these proteins have been described in various tumors. Here we determine the relationships between expression levels and activating mutations of these genes in a series of 65 primary TGCTs and 4 TCGT cell lines. High levels of expression and activating mutations in RAS were mutually exclusive events, and activating mutations in RAS were only identified in the seminoma subtype. Mutations in BRAF were not identified. Increased ERBB2 expression was associated with differentiated nonseminoma histology excised from lymph nodes postchemotherapy. Mutation, elevated expression, and correlations between expression levels of KRAS2, GRB7, and KIT are consistent with their involvement in the development of TGCTs.

  3. Ras does not contribute to the facilitation of hippocampal synaptic plasticity enabled by environmental enrichment.

    Novkovic, T; Heumann, R; Manahan-Vaughan, D


    Environmental enrichment (EE), which mimics the wealth of sensory, motor and cognitive stimuli that arise through intense interactions with the ambient environment, results in enhanced hippocampal long-term potentiation (LTP) and spatial learning. A key molecular factor in the mediation of these changes is the brain-derived neurotrophic factor (BDNF). One of the downstream cascades that is activated by BDNF is the cascade linked to the small GTPase, Ras, that triggers mitogen-activated protein kinase (MAPK) activity and is part of the cAMP response element-binding protein (CREB) pathway that can lead to synaptic restructuring to support LTP. Here, we explored whether persistent activation of Ras in neurons further enhances LTP following EE of rodents. Immediately following weaning, transgenic mice that expressed constitutively activated neuronal Ras, or their wildtype (Wt) littermates, underwent 3weeks of constant EE. In the absence of EE, theta burst stimulation (TBS) evoked LTP in the CA1 region of transgenic mice that was not significantly different from LTP in Wts. After 3weeks of EE, hippocampal LTP was improved in Wt mice. Enriched transgenic mice showed an equivalent level of LTP to enriched Wts, but it was not significantly different from non-enriched synRas controls. Western blot analysis performed after a pull-down assay showed that non-enriched transgenic mice expressed higher Ras activity compared to non-enriched Wts. Following EE, Ras activity was reduced in transgenics to levels detected in Wts. These results show that constitutive activation of Ras does not mimic the effects of EE on LTP. In addition, EE results in an equivalent enhancement of LTP transgenics and Wts, coupled with a decrease in Ras activity to Wt levels. This suggests that permanent activation of Ras in neurons of synRas animals following EE results in an altered feedback regulation of endogenous Ras activity that is not a key factor in LTP enhancements. The maintenance of Ras within

  4. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

    Fiorella Guadagni


    Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733


    Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor on Tyrosine 845 Is Required for Zinc-induced Ras ActivationWeidong Wu 1 , Lee M. Graves 2 , Gordon N. Gill 3 , Sarah J. Parsons 4 , and James M. Samet 51 Center for Environmental Medicine and Lung Biolo...

  6. Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells.

    Baba, I; Shirasawa, S; Iwamoto, R; Okumura, K; Tsunoda, T; Nishioka, M; Fukuyama, K; Yamamoto, K; Mekada, E; Sasazuki, T


    To identify the genes located downstream of the activated Ki-Ras signaling pathways in human colon cancer cells, a PCR-based cDNA subtraction library was constructed between HCT116 cells and HCT116-derived activated Ki-ras-disrupted cells (HKe3). One of the genes in HCT116 that was evidently up-regulated was epiregulin, a member of the epidermal growth factor family that is expressed in many kinds of human cancer cells. HKe3-stable transfectants expressing activated Ki-Ras regained over-expression of epiregulin. To further elucidate the biochemical structure and significance of epiregulin expression in tumorigenesis, HKe3-stable transfectants expressing epiregulin (e3-pSE cells) were established. Epiregulin existed as highly glycosylated membrane-bound forms, and TPA rapidly induced ectodomain shedding of epiregulin. Furthermore, the conditioned medium of e3-pSE cells showed more DNA synthesis for 32D cells expressing epidermal growth factor receptor (DER) cells than that of HKe3. Although anchorage-independent growth in soft agar was not observed for e3-pSE cells, tumorigenicity in nude mice was observed evidently, and their growth rate was correlated with each amount of exogenous epiregulin expression. These results suggested that activated Ki-Ras will be one of the factors contributing to the overexpression of epiregulin in human colon cancer cells, and that epiregulin will play a critical role in human tumorigenesis in vivo. PMID:11156386

  7. The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization.

    Licata, Stephanie C; Pierce, R Christopher


    Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. PMID:12641723

  8. New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution

    Klink Björn U


    Full Text Available Abstract Background In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes. Results X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido-triphosphate, a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom. Conclusion For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.

  9. Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

    Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y


    Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity. PMID:24853419

  10. A novel quinoline, MT477: suppresses cell signaling through Ras molecular pathway, inhibits PKC activity, and demonstrates in vivo anti-tumor activity against human carcinoma cell lines.

    Jasinski, Piotr; Welsh, Brandon; Galvez, Jorge; Land, David; Zwolak, Pawel; Ghandi, Lori; Terai, Kaoru; Dudek, Arkadiusz Z


    MT477 is a novel thiopyrano[2,3-c]quinoline that has been identified using molecular topology screening as a potential anticancer drug with a high activity against protein kinase C (PKC) isoforms. The objective of the present study was to determine the mechanism of action of MT477 and its activity against human cancer cell lines. MT477 interfered with PKC activity as well as phosphorylation of Ras and ERK1/2 in H226 human lung carcinoma cells. It also induced poly-caspase-dependent apoptosis. MT477 had a dose-dependent (0.006 to 0.2 mM) inhibitory effect on cellular proliferation of H226, MCF-7, U87, LNCaP, A431 and A549 cancer cell lines as determined by in vitro proliferation assays. Two murine xenograft models of human A431 and H226 lung carcinoma were used to evaluate tumor response to intraperitoneal administration of MT477 (33 microg/kg, 100 microg/kg, and 1 mg/kg). Tumor growth was inhibited by 24.5% in A431 and 43.67% in H226 xenografts following MT477 treatment, compared to vehicle controls (p < 0.05). In conclusion, our empirical findings are consistent with molecular modeling of MT477's activity against PKC. We also found, however, that its mechanism of action occurs through suppressing Ras signaling, indicating that its effects on apoptosis and tumor growth in vivo may be mediated by Ras as well as PKC. We propose, therefore, that MT477 warrants further development as an anticancer drug. PMID:17957339

  11. The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose Cytarabine

    Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML). However there is still controversy about its clinical relevance on the treatment outcome of this leukemia. Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C) used in post induction consolidation chemotherapy in adult AML patients. Patients and Methods: The study comprised 71de novo AML patients with a male: Female ratio of 1.4: 1; their ages ranged from 21-59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G banding and K-RAS mutation detection using realtime PCR. The patients were randomized into 2 groups (gps) according to the ara-C dose used in consolidation treatment, HDAC gp receiving 400 mg ara-C and LDAC gp receiving 100 mg ara-C. They were followed over a period of 5 years. Results: Mutations in the K-RAS gene (mutRAS) were detected in 23 patients (32%) with the remaining 48 patients (68%) having wild type RAS (wtRAS). Blast cell percentage was significantly lower in mutRAS compared to wtRAS patients (p=<0.001). The M4 subtype of AML and cases with Inv 16 showed significantly higher frequencies in mutRAS compared to wtRAS patients, (p=0.015, 0.003, respectively). The patients were followed up for a median of 43 months (range 11-57 months). There was no significant difference in overall survival (OS) between mutRAS and wtRAS patients (p=0.326). Within the mutRAS patients treated with HDAC, cumulative OS was significantly higher than those treated with LDAC (p=0.001). This was not the case in the wtRAS group (p=0.285). There was no significant difference in disease The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose

  12. The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine

    Ahmad EI


    Full Text Available Ebtesam I Ahmad, Heba H Gawish, Nashwa MA Al Azizi, Ashraf M ElhefniClinical Pathology Department, Hematology and Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University, Sharkia, EgyptBackground: Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML. However, little is known about its clinical relevance in the treatment outcome for this leukemia.Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C used in postinduction consolidation chemotherapy in adult AML patients.Patients and methods: The study comprised of 71 de novo AML patients with male/female ratio 1.4:1; their ages ranged from 21–59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G-banding (Giemsa staining, and K-RAS mutation detection using real-time polymerase chain reaction. The patients were randomized into two groups according to the ara-C dose used in consolidation treatment, the high the dose ara-C (HDAC group receiving 400 mg ara-C and-low-dose ara-C (LDAC group receiving 100 mg ara-C; they were followed over a period of five years.Results: Mutations in the K-RAS gene (mutRAS were detected in 23 patients (32% with the remaining 48 patients (68% having wild-type RAS (wtRAS. The percent of blast cells was significantly lower in mutRAS compared to wtRAS patients (P ≤ 0.001 while M4 subtype of AML and Inv(16 frequencies were significantly higher in mutRAS compared to wtRAS patients (P = 0.015 and (P = 0.003, respectively. The patients were followed up for a median of 43 months (range 11–57 months. There was no significant difference in overall survival (OS between mutRAS and wtRAS (P = 0.326. Within the mutRAS

  13. Past, Present, and Future of Targeting Ras for Cancer Therapies.

    Tan, Zhi; Zhang, Shuxing


    For decades, mutant Ras (mut-Ras) proteins have been identified as drivers of multiple cancers including pancreatic, lung, and colon cancers. However, targeting this oncogene has been challenging and no Ras inhibitors are on the market to date. Lately several candidates targeting the downstream pathways of Ras signaling, including PI3K and Raf, were approved for cancer treatment. However, they do not present promising therapeutic effects on patients harboring Ras mutations. Recently, a variety of compounds have been reported to impair the activity of Ras, and these exciting discoveries reignite the hope for development of novel drugs targeting mut-Ras. In this article, we will review the progress made in this field and the current state-of-the-art technologies to develop Ras inhibitors. Also we will discuss the future direction of targeting Ras. PMID:26423695

  14. Activated Ras signaling pathways and reovirus oncolysis: an update on the mechanism of preferential reovirus replication in cancer cells



    Full Text Available The development of wild-type, unmodified Type 3 Dearing (T3D strain reovirus as an anticancer agent has currently expanded to 32 clinical trials (both completed and ongoing involving reovirus in the treatment of cancer. It has been more than 30 years since the potential of reovirus as an anticancer agent was first identified in studies that demonstrated the preferential replication of reovirus in transformed cell lines but not in normal cells. Later investigations have revealed the involvement of activated Ras signaling pathways (both upstream and downstream and key steps of the reovirus infectious cycle in promoting preferential replication in cancer cells with reovirus-induced cancer cell death occurring through necrotic, apoptotic, and autophagic pathways. There is increasing evidence that reovirus-induced antitumor immunity involving both innate and adaptive responses also contributes to therapeutic efficacy though this discussion is beyond the scope of this article. Here we review our current understanding of the mechanism of oncolysis contributing to the broad anticancer activity of reovirus. Further understanding of reovirus oncolysis is critical in enhancing the clinical development and efficacy of reovirus.

  15. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

    Cheng, Yougan; Othmer, Hans


    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

  16. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network.

    Cheng, Yougan; Othmer, Hans


    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that [Formula: see text] cycling modulated by Ric8, a nonreceptor guanine exchange factor for [Formula: see text] in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both [Formula: see text] and Gβγ are essential for direction sensing, in that membrane-localized [Formula: see text], the activated GTP-bearing form of [Formula: see text], leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient 'memory' to eliminate the 'back-of-the wave' problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since

  17. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover

    Hong, Xin; Nguyen, Thanh Hung; Chen, Qingfeng;


    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of...

  18. RalA, a GTPase targeted by miR-181a, promotes transformation and progression by activating the Ras-related signaling pathway in chronic myelogenous leukemia

    Luo, Xiaochuang; Yang, Juhua; Li, Yumin; Li, Tianfu; Wang, Ruirui; Fei, Jia


    BCR/ABL is a well-known activator of multiple signaling pathways. RalA, a Ras downstream signaling molecule and a small GTPase, plays an important role in Bcr-Abl-induced leukemogenesis but the exact mechanism remains elusive. Here, we show that RalA GTPase activity is commonly high in chronic myelogenous leukemia (CML) cell lines and patient samples. Overexpression of RalA results in malignant transformation and progression, and induces resistance to imatinib (IM) in BaF3 and K562 cell lines. RalA reduced survival and led to IM resistance in a xenografted mouse model. Ablation of RalA by either siRNA or miR-181a, a RalA targeting microRNA, attenuated the malignant phenotypes in K562 cells. RBC8, a selective Ral inhibitor, enhanced the inhibitory effects of IM in K562, KCL22 and BaF3-P210 cells. Interestingly, the phospho-specific protein microarray assay revealed that multiple phosphorylation signal proteins were decreased by RalA inhibition, including SAPK, JNK, SRC, VEGFR2, P38 MAPK, c-Kit, JunB, and Keratin18. Among them, P38 MAPK and SAPK/JNK are Ras downstream signaling kinases. Taken together, RalA GTPase might be an important oncogene activating the Ras-related signaling pathway in CML. PMID:26967392

  19. Absolute Quantification of Endogenous Ras Isoform Abundance.

    Craig J Mageean

    Full Text Available Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.

  20. Activation of RAS family members confers resistance to ROS1 targeting drugs.

    Cargnelutti, Marilisa; Corso, Simona; Pergolizzi, Margherita; Mévellec, Laurence; Aisner, Dara L.; Dziadziuszko, Rafal; Varella-Garcia, Marileila; Comoglio, Paolo M.; Doebele, Robert C.; Vialard, Jorge; Giordano, Silvia


    The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse. To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activatio...

  1. Cardiac remodelling and RAS inhibition.

    Ferrario, Carlos M


    Risk factors such as hypertension and diabetes are known to augment the activity and tissue expression of angiotensin II (Ang II), the major effector peptide of the renin-angiotensin system (RAS). Overstimulation of the RAS has been implicated in a chain of events that contribute to the pathogenesis of cardiovascular (CV) disease, including the development of cardiac remodelling. This chain of events has been termed the CV continuum. The concept of CV disease existing as a continuum was first proposed in 1991 and it is believed that intervention at any point within the continuum can modify disease progression. Treatment with antihypertensive agents may result in regression of left ventricular hypertrophy, with different drug classes exhibiting different degrees of efficacy. The greatest decrease in left ventricular mass is observed following treatment with angiotensin converting enzyme inhibitors (ACE-Is), which inhibit Ang II formation. Although ACE-Is and angiotensin receptor blockers (ARBs) provide significant benefits in terms of CV events and stroke, mortality remains high. This is partly due to a failure to completely suppress the RAS, and, as our knowledge has increased, an escape phenomenon has been proposed whereby the human sequence of the 12 amino acid substrate angiotensin-(1-12) is converted to Ang II by the mast cell protease, chymase. Angiotensin-(1-12) is abundant in a wide range of organs and has been shown to increase blood pressure in animal models, an effect abolished by the presence of ACE-Is or ARBs. This review explores the CV continuum, in addition to examining the influence of the RAS. We also consider novel pathways within the RAS and how new therapeutic approaches that target this are required to further reduce Ang II formation, and so provide patients with additional benefits from a more complete blockade of the RAS. PMID:27105891

  2. A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

    Smida, M.; Posevitz-Fejfar, A.; Hořejší, Václav; Schraven, B.; Lindquist, J.A.


    Roč. 110, č. 2 (2007), s. 596-605. ISSN 0006-4971 R&D Projects: GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : PAG * Ras * lipid rafts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.896, year: 2007

  3. ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

    Posada, Itziar M. D.; Serulla, Marc; Zhou, Yong; Oetken-Lindholm, Christina


    Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events. PMID:27437940

  4. Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS-p110 crosstalk.

    Tamaskovic, Rastislav; Schwill, Martin; Nagy-Davidescu, Gabriela; Jost, Christian; Schaefer, Dagmar C; Verdurmen, Wouter P R; Schaefer, Jonas V; Honegger, Annemarie; Plückthun, Andreas


    Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS-p110α interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerization-incompetent state, which entail pan-ErbB inhibition and a permanent OFF state in the oncogenic signalling, thereby triggering extensive apoptosis in ErbB2-addicted tumours. Thus, these novel insights into mechanisms underlying network robustness provide a guide for overcoming adaptation response to ErbB2/ErbB3-targeted therapy. PMID:27255951

  5. RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines.

    Ksionda, O; Melton, A A; Bache, J; Tenhagen, M; Bakker, J; Harvey, R; Winter, S S; Rubio, I; Roose, J P


    Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRAS(G12D), are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRAS(G12D) T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRAS(G12D) signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity. PMID:26549032

  6. Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.

    Samid, D; Flessate, D M; Friedman, R M


    Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion. Images PMID:2439904

  7. MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy

    Neupane, Manish; Clark, Allison P.; Landini, Serena;


    activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRAS(G12C)-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required...... for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action. MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45-58. ©2015 AACR.This article...

  8. Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

    Nielsen-Preiss, Sheila M; Allen, Melissa P; Xu, Mei; Linseman, Daniel A; Pawlowski, John E; Bouchard, R J; Varnum, Brian C; Heidenreich, Kim A; Wierman, Margaret E


    GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration. PMID:17332061

  9. NSD2 contributes to oncogenic RAS-driven transcription in lung cancer cells through long-range epigenetic activation.

    García-Carpizo, Verónica; Sarmentero, Jacinto; Han, Bomie; Graña, Osvaldo; Ruiz-Llorente, Sergio; Pisano, David G; Serrano, Manuel; Brooks, Harold B; Campbell, Robert M; Barrero, Maria J


    The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression. PMID:27604143

  10. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht


    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  11. Ha-ras oncogene activation in mammary glands of N-methyl-N-nitrosourea-treated rats genetically resistant to mammary adenocarcinogenesis.

    Lu, S J; Archer, M.C.


    A single dose of N-methyl-N-nitrosourea given to sexually immature female Buf/N rats produces a high incidence of mammary adenocarcinomas. A large percentage of these tumors contain the Ha-ras oncogene, activated by a G----A transition at the second nucleotide of codon 12. Copenhagen rats, on the other hand, are completely resistant to mammary tumor induction by a number of carcinogens, including N-methyl-N-nitrosourea. Here we show, using a sensitive method involving PCR, that codon 12 Ha-ra...

  12. K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

    Hernández-Porras, Isabel; Schuhmacher, Alberto J; Garcia-Medina, Raquel; Jiménez, Beatriz; Cañamero, Marta; de Martino, Alba; Guerra, Carmen


    The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27174785

  13. XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice.

    Semenchenko, Kostyantyn; Wasylyk, Christine; Cheung, Henry; Tourrette, Yves; Maas, Peter; Schalken, Jack A; van der Pluijm, Gabri; Wasylyk, Bohdan


    Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy. PMID:27427904

  14. Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

    Yu Jing; Liu Wei; Chen Yeshan; Liu Hongli; Wang Tao; Wu Gang


    Abstract Background Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking...

  15. Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf

    Bergo, Martin O.; Gavino, Bryant J.; Hong, Christine; Beigneux, Anne P.; McMahon, Martin; Casey, Patrick J.; Young, Stephen G.


    Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcysteine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methylation is important for targeting of the proteins to the plasma membrane. We hypothesized that a knockout of Icmt would reduce the ability of cells to be transformed by K-Ras. Fibroblasts harboring a floxed Icmt allele and expressing activated K-Ras (K-Ras-Icmtflx/flx) were treated with Cre-adenovir...

  16. The role of non-ras transforming genes in chemical carcinogenesis

    Cooper, C.S. (Inst. of Cancer Research, Surrey (England))


    DNA transfection experiments using the NIH 3T3 mouse fibroblast cell line have demonstrated that chemically induced tumors and chemically transformed cell lines frequently contain dominant transforming genes. Although many of the genes detected using the NIH 3T3 transfection-transformation assay are activated versions of H-ras, K-ras, and N-ras, in some experimental systems activated forms of genes such as met and neu that are unrelated to ras have been observed. The activated met gene was originally detected in a human cell line that had been transformed by exposure to N-methyl-N{prime}-nitro-N-nitrosoguanidine. Subsequent studies demonstrated that the met proto-oncogene encodes a novel growth factor receptor and that gene activation involves the production of a chimeric gene in which the regions of met encoding the extracellular and transmembrane domains of the receptor are replaced by the 5{prime}-region of an unrelated gene called trp. The presence of genetic alterations in chemically induced malignancies has also been assessed in cytogenetic studies and by Southern analysis of DNA from neoplastic cells. These studies have demonstrated the presence of altered versions the c-myc and mos genes in plasmocytomas induced in mice following exposure to pristane or mineral oil and of activated pim-1 and c-myc genes in thymomas that arise in AKR mice following treatment with N-methyl-N-nitrosourea. Analyses of the mechanisms of activation of these non-ras genes has provided important insights into the different ways in which genes may become activated following chemical exposure.

  17. Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis?

    Repasky, Gretchen A; Chenette, Emily J; Der, Channing J


    Ras proteins function as signal transducers and are mutationally activated in many human cancers. In 1993, Raf was identified as a key downstream effector of Ras signaling, and it was believed then that the primary function of Ras was simply to facilitate Raf activation. However, the subsequent discovery of other proteins that are effectors of Ras function suggested that oncogenic activities of Ras are mediated by both Raf-dependent and Raf-independent signaling. Further complexity arose with the identification of Ras effectors with putative tumor suppressor, rather than oncogenic, functions. However, the recent identification of B-raf mutations in human cancers has renewed the debate regarding whether Raf activation alone promotes Ras-mediated oncogenesis. In this article, we summarize the current knowledge of the contribution of Ras effectors in Ras-mediated oncogenesis. PMID:15519853

  18. K-Ras-Independent Effects of the Farnesyl Transferase Inhibitor L-744,832 on Cyclin B1/Cdc2 Kinase Activity, G2/M Cell Cycle Progression and Apoptosis in Human Pancreatic Ductal Adenocarcinoma Cell

    Si Young Song


    Full Text Available Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs. However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage -inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 µM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.

  19. Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain.

    Bradford, A P; Conrad, K E; Wasylyk, C; Wasylyk, B; Gutierrez-Hartmann, A


    The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enh...

  20. Altered myoelectric activity in the experimental blind loop syndrome.

    Justus, P G; Fernandez, A; Martin, J.L.; King, C E; Toskes, P P; Mathias, J R


    Nutrient malabsorption and diarrhea are characteristic of the blind loop syndrome. Alterations in motility have been implicated as a cause of bacterial overgrowth, but the possibility that altered motility may result from alterations in the flora has not been explored. The purpose of this study was to characterize the myoelectric activity of the small intestine in the blind loop rat model. Eight groups of rats were studied: rats with self-filling blind loops, which develop bacterial overgrowt...

  1. Radiosensitivity and ras oncogene expression in preneoplastic rat tracheal epithelial cells

    The sensitivity of preneoplastic rat tracheal epithelial (RTE) cells to the cytotoxic effects of high- and low-LET radiation, and the modulating effect of the viral ras oncogene on this sensitivity were determined. Two lines of preneoplastic RTE cells have the same responsiveness to high-LET radiation, but differ in their responsiveness to a transfected ras oncogene and in their sensitivities to low-LET radiation. Cells that respond to ras by becoming neoplastic are more resistant to the cytotoxic effects of low-LET radiation than cells that are not transformable by ras. The radiosensitivity of ras-responsive cells was not altered by transfection with ras. However, transfection of ras-non responsive cells with ras decreased their sensitivity to low-LET radiation. These data suggest that the ability of cells to repair radiation damage changes as they progress to neoplasia. (author)

  2. Mammalian target of rapamycin complex I (mTORC1 activity in ras homologue enriched in brain (Rheb-deficient mouse embryonic fibroblasts.

    Marlous J Groenewoud

    Full Text Available The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs, we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.

  3. Ras Conformational Ensembles, Allostery, and Signaling.

    Lu, Shaoyong; Jang, Hyunbum; Muratcioglu, Serena; Gursoy, Attila; Keskin, Ozlem; Nussinov, Ruth; Zhang, Jian


    Ras proteins are classical members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. In this review, we focus on data that have accumulated over the past few years pertaining to the conformational ensembles and the allosteric regulation of Ras proteins and their interpretation from our conformational landscape standpoint. The Ras ensemble embodies all states, including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states serving as a reservoir for emerging functions. The ensemble is shifted by distinct mutational events, cofactors, post-translational modifications, and different membrane compositions. A better understanding of Ras biology can contribute to therapeutic strategies. PMID:26815308

  4. The role of p19 C-H-Ras protein in metastasis and proliferative pathways

    García Cruz, Roseli Marlen


    Ras is an evolutionary and conserved family of genes present in many organisms, in humans, Ras is conformed by three members called N-Ras, K-Ras and H-Ras located on chromosomes 1, 11 and 12 respectively (1, 2). Ras proteins act as a molecular switch, activating many signalling pathways through phosphorylation of GTPases; so their punctual mutations promote a constitutive activation in their GTPase function that fosters carcinogenesis, loss of adhesion and invasion of malignant cells (3-9). I...

  5. Deletion of Pim kinases elevates the cellular levels of reactive oxygen species and sensitizes to K-Ras-induced cell killing.

    Song, J H; An, N; Chatterjee, S; Kistner-Griffin, E; Mahajan, S; Mehrotra, S; Kraft, A S


    The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-Ras(G12V)) and undergo cell death. Transduction of K-Ras(G12V) into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuated ROS production and reversed the cytotoxic effects of K-Ras(G12V) in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-Ras(G12V)-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-Ras(G12V)-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases have an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth. PMID:25241892

  6. Pharmacological modulation of oncogenic Ras by natural products and their derivatives: Renewed hope in the discovery of novel anti-Ras drugs.

    Quah, Shun Ying; Tan, Michelle Siying; Teh, Yuan Han; Stanslas, Johnson


    Oncogenic rat sarcoma (Ras) is linked to the most fatal cancers such as those of the pancreas, colon, and lung. Decades of research to discover an efficacious drug that can block oncogenic Ras signaling have yielded disappointing results; thus, Ras was considered "undruggable" until recently. Inhibitors that directly target Ras by binding to previously undiscovered pockets have been recently identified. Some of these molecules are either isolated from natural products or derived from natural compounds. In this review, we described the potential of these compounds and other inhibitors of Ras signaling in drugging Ras. We highlighted the modes of action of these compounds in suppressing signaling pathways activated by oncogenic Ras, such as mitogen-activated protein kinase (MAPK) signaling and the phosphoinositide-3-kinase (PI3K) pathways. The anti-Ras strategy of these compounds can be categorized into four main types: inhibition of Ras-effector interaction, interference of Ras membrane association, prevention of Ras-guanosine triphosphate (GTP) formation, and downregulation of Ras proteins. Another promising strategy that must be validated experimentally is enhancement of the intrinsic Ras-guanosine triphosphatase (GTPase) activity by small chemical entities. Among the inhibitors of Ras signaling that were reported thus far, salirasib and TLN-4601 have been tested for their clinical efficacy. Although both compounds passed phase I trials, they failed in their respective phase II trials. Therefore, new compounds of natural origin with relevant clinical activity against Ras-driven malignancies are urgently needed. Apart from salirasib and TLN-4601, some other compounds with a proven inhibitory effect on Ras signaling include derivatives of salirasib, sulindac, polyamine, andrographolide, lipstatin, levoglucosenone, rasfonin, and quercetin. PMID:27016467

  7. Does mental exertion alter maximal muscle activation?

    Vianney Rozand; Benjamin Pageaux


    Mental exertion is known to impair endurance performance, but its effects on neuromuscular function remain unclear. The purpose of this study was to test the hypothesis that mental exertion reduces torque and muscle activation during intermittent maximal voluntary contractions of the knee extensors. Ten subjects performed in a randomized order three separate mental exertion conditions lasting 27 minutes each: i) high mental exertion (incongruent Stroop task), ii) moderate mental exertion (con...

  8. Does mental exertion alter maximal muscle activation?

    Rozand, Vianney; Pageaux, Benjamin; Marcora, Samuele M.; Papaxanthis, Charalambos; Lepers, Romuald


    Mental exertion is known to impair endurance performance, but its effects on neuromuscular function remain unclear. The purpose of this study was to test the hypothesis that mental exertion reduces torque and muscle activation during intermittent maximal voluntary contractions of the knee extensors. Ten subjects performed in a randomized order three separate mental exertion conditions lasting 27 min each: (i) high mental exertion (incongruent Stroop task), (ii) moderate mental exertion (congr...

  9. Ras and Rheb Signaling in Survival and Cell Death

    One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively

  10. The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.

    Kozma, S C; Bogaard, M E; Buser, K; Saurer, S M; Bos, J. L.; Groner, B; Hynes, N E


    We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is a...

  11. The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH

    Willumsen, B M; Vass, W C; Velu, T J;


    We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass......-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype....

  12. Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1

    Botta, Gregory P; Reginato, Mauricio J.; Reichert, Maximillian; RUSTGI, ANIL K.; Lelkes, Peter I.


    Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-RasG12D that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of ERK1/2 and pro-invasive matrix metalloproteinases (MMPs); indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras – ERK1/2 ...

  13. Myelin alters the inflammatory phenotype of macrophages by activating PPARs

    Bogie, Jeroen; Jorissen, Winde; Mailleux, Jo; Vanmierlo, Tim; van Horssen, Jack; Hellings, Niels; Stinissen, Piet; Hendriks, J. J. A.; Nijland, Philip G.; Zelcer, Noam


    Background Foamy macrophages, containing myelin degradation products, are abundantly found in active multiple sclerosis (MS) lesions. Recent studies have described an altered phenotype of macrophages after myelin internalization. However, mechanisms by which myelin affects the phenotype of macrophages and how this phenotype influences lesion progression remain unclear. Results We demonstrate that myelin as well as phosphatidylserine (PS), a phospholipid found in myelin, reduce nitri...

  14. Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin

    Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradyknin to activate phospholipase C-mediated phosphoinositide hydrolysis. In [3H]inositol-labeled rat-1 fibroblasts, PDGF resulted in a 2-fold increase in the level of [3H]inositol trisphosphate (InsP3) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of [3H]inositol monophosphate (InsP1) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either [3H]InsP3 or [3H]InsP1. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21/sup ras/ and the loss of PDGF-stimulated [3H]InsP1 accumulation. The authors propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors

  15. Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors

    The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, the authors have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 104 foci per μg of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, they found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which the authors had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment

  16. Alterations in HIV-1 LTR promoter activity during AIDS progression

    HIV-1 variants evolving in AIDS patients frequently show increased replicative capacity compared to those present during early asymptomatic infection. It is known that late stage HIV-1 variants often show an expanded coreceptor tropism and altered Nef function. In the present study we investigated whether enhanced HIV-1 LTR promoter activity might also evolve during disease progression. Our results demonstrate increased LTR promoter activity after AIDS progression in 3 of 12 HIV-1-infected individuals studied. Further analysis revealed that multiple alterations in the U3 core-enhancer and in the transactivation-response (TAR) region seem to be responsible for the enhanced functional activity. Our findings show that in a subset of HIV-1-infected individuals enhanced LTR transcription contributes to the increased replicative potential of late stage virus isolates and might accelerate disease progression

  17. Human activities change marine ecosystems by altering predation risk.

    Madin, Elizabeth M P; Dill, Lawrence M; Ridlon, April D; Heithaus, Michael R; Warner, Robert R


    In ocean ecosystems, many of the changes in predation risk - both increases and decreases - are human-induced. These changes are occurring at scales ranging from global to local and across variable temporal scales. Indirect, risk-based effects of human activity are known to be important in structuring some terrestrial ecosystems, but these impacts have largely been neglected in oceans. Here, we synthesize existing literature and data to explore multiple lines of evidence that collectively suggest diverse human activities are changing marine ecosystems, including carbon storage capacity, in myriad ways by altering predation risk. We provide novel, compelling evidence that at least one key human activity, overfishing, can lead to distinct, cascading risk effects in natural ecosystems whose magnitude exceeds that of presumed lethal effects and may account for previously unexplained findings. We further discuss the conservation implications of human-caused indirect risk effects. Finally, we provide a predictive framework for when human alterations of risk in oceans should lead to cascading effects and outline a prospectus for future research. Given the speed and extent with which human activities are altering marine risk landscapes, it is crucial that conservation and management policy considers the indirect effects of these activities in order to increase the likelihood of success and avoid unfortunate surprises. PMID:26448058

  18. Intrinsic Brain Activity in Altered States of Consciousness

    Boly, M.; Phillips, C.; Tshibanda, L.; Vanhaudenhuyse, A.; Schabus, M.; Dang-Vu, T.T.; Moonen, G.; Hustinx, R.; Maquet, P.; Laureys, S.


    Spontaneous brain activity has recently received increasing interest in the neuroimaging community. However, the value of resting-state studies to a better understanding of brain–behavior relationships has been challenged. That altered states of consciousness are a privileged way to study the relationships between spontaneous brain activity and behavior is proposed, and common resting-state brain activity features observed in various states of altered consciousness are reviewed. Early positron emission tomography studies showed that states of extremely low or high brain activity are often associated with unconsciousness. However, this relationship is not absolute, and the precise link between global brain metabolism and awareness remains yet difficult to assert. In contrast, voxel-based analyses identified a systematic impairment of associative frontoparieto–cingulate areas in altered states of consciousness, such as sleep, anesthesia, coma, vegetative state, epileptic loss of consciousness, and somnambulism. In parallel, recent functional magnetic resonance imaging studies have identified structured patterns of slow neuronal oscillations in the resting human brain. Similar coherent blood oxygen level–dependent (BOLD) systemwide patterns can also be found, in particular in the default-mode network, in several states of unconsciousness, such as coma, anesthesia, and slow-wave sleep. The latter results suggest that slow coherent spontaneous BOLD fluctuations cannot be exclusively a reflection of conscious mental activity, but may reflect default brain connectivity shaping brain areas of most likely interactions in a way that transcends levels of consciousness, and whose functional significance remains largely in the dark. PMID:18591474

  19. EMT-induced stemness and tumorigenicity are fueled by the EGFR/Ras pathway.

    Dominic Chih-Cheng Voon

    Full Text Available Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT. However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 (-/- p53 (-/- murine gastric epithelial (GIF-14 cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-β1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-β1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.

  20. Ras signaling enhances the activity of C/EBPalpha to induce granulocytic differentiation by phosphorylation of serine 248

    Singh, Sheo Mohan


    The transcription factor C/EBPa regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBPa gene do not express detectable levels of the G-CSF receptor and produce no neutrophils. We have recently shown that C/EBPa function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBPa is regulated both in myelopoiesis and leukemogenesis, is not fully understood. The current study demonstrates th...

  1. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    O. Kauko; T.D. Laajala; M. Jumppanen; P. Hintsanen; V. Suni; P. Haapaniemi; G. Corthals; T. Aittokallio; J. Westermarck; S.Y. Imanishi


    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how

  2. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    Kauko, O.; Laajala, T. D.; Jumppanen, M.; Hintsanen, P; Suni, V.; Haapaniemi, P.; Corthals, G.; Aittokallio, T.; Westermarck, J; Imanishi, S.Y.


    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantificat...

  3. Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats

    Sørensen, Nanna Møller; Kobaek-Larsen, Morten; Bonne, Anita;


    The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer...... result is in accordance with those of similar studies in other rat models and with most human colorectal cancers. Immunohistochemical analyses of beta-catenin did not reveal loss of gene activity, nor did the sequencing of Ki-ras reveal mutations. These results are in contrast to most findings in...... comparable rat studies. The deviations may be due to differences in exposure to the carcinogen or difference in strain and/or age. The lack of beta-catenin and Ki-ras alterations in this colon cancer model is unlike human sporadic colorectal cancers where these genetic changes are common findings....

  4. Environmental noise alters gastric myoelectrical activity: Effect of age

    James S Castle; Jin-Hong Xing; Mark R Warner; Mark A Korsten


    AIM: To evaluate the effect of age and acoustic stress on gastric myoelectrical activity (GMA) and autonomic nervous system function,METHODS: Twenty-one male subjects (age range 22-71years, mean 44 years) were recruited and exposed, in random order, to three auditory stimuli (Hospital noise,conversation babble and traffic noise) after a 20-min baseline. All periods lasted 20 min and were interspersed with a 10 min of recovery. GMA was obtained using a Synectics Microdigitrapper. Autonomic nerve function was assessed by monitoring blood pressure and heart rate using an automatic recording device.RESULTS: Dominant power tended to decrease with increase of age (P<0.05). The overall percentage of three cycle per minute (CPM) activity decreased during exposure to hospital noise (12.0%, P < 0.05), traffic noise (13.9%, P < 0.05), and conversation babble(7.1%). The subjects in the younger group (< 50 years)showed a consistent reduction in the percentage of 3CPM activity during hospital noise (22.9%, P < 0.05),traffic noise (19.0%, P < 0.05), and conversation babble(15.5%). These observations were accompanied by a significant increase in bradygastria: hospital noise (P< 0.05) and traffic noise (P < 0.05). In contrast, the subjects over 50 years of age did not exhibit a significant decrease in 3 CPM activity. Regardless of age, noise did not alter blood pressure or heart rate.CONCLUSION: GMA changes with age. Loud noise can alter GMA, especially in younger individuals. Our data indicate that even short-term exposure to noise may alter the contractility of the stomach.

  5. The oncoprotein H-RasV12 increases mitochondrial metabolism

    Arumugam Sengodagounder


    Full Text Available Abstract Background Neoplastic cells increase glycolysis in order to produce anabolic precursors and energy within the hypoxic environment of a tumor. Ras signaling is activated in several cancers and has been found to regulate metabolism by enhancing glycolytic flux to lactate. We examined the effects of sequential immortalization and H-RasV12-transformation of human bronchial epithelial cells on the anabolic fate of fully-labeled 13C-glucose-derived carbons using two-dimensional total correlated spectroscopic analysis-nuclear magnetic resonance spectroscopy (2D TOCSY-NMR. Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells unexpectedly increased tricarboxylic acid cycle activity as measured by the direct conversion of 13C-glucose carbons into the anabolic substrates glutamate/glutamine, aspartate and uridine. We then observed that immortalization and H-RasV12-transformation of bronchial epithelial cells caused a stepwise increase in oxygen consumption, a global measure of electron transport chain activity. Importantly, ectopic expression of H-RasV12 sensitized immortalized cells to the ATP-depleting and cytotoxic effects of electron transport perturbation using the complex I inhibitor rotenone. Conclusion Taken together, these data indicate that the oncoprotein H-RasV12 increases mitochondrial metabolism and provide new rationale for the targeting of the tricarboxylic acid cycle and electron transport chain as anti-neoplastic strategies.

  6. An Orthosteric Inhibitor of the Ras-Sos Interaction

    Patgiri, Anupam; Yadav, Kamlesh K.; Arora, Paramjit S.; Bar-Sagi, Dafna


    Mimics of α-helices on protein surfaces have emerged as powerful reagents for antagonizing protein-protein interactions, which are difficult to target with small molecules. Herein we describe the design of a cell-permeable synthetic α-helix based on the guanine nucleotide exchange factor Sos that interferes with Ras-Sos interaction and downregulates Ras signaling in response to receptor tyrosine kinase activation.

  7. Altered brain activity for phonological manipulation in dyslexic Japanese children

    Yamamoto, Hisako; Oba, Kentaro; Terasawa, Yuri; Moriguchi, Yoshiya; Uchiyama, Hitoshi; Seki, Ayumi; Koeda, Tatsuya; Inagaki, Masumi


    Because of unique linguistic characteristics, the prevalence rate of developmental dyslexia is relatively low in the Japanese language. Paradoxically, Japanese children have serious difficulty analysing phonological processes when they have dyslexia. Neurobiological deficits in Japanese dyslexia remain unclear and need to be identified, and may lead to better understanding of the commonality and diversity in the disorder among different linguistic systems. The present study investigated brain activity that underlies deficits in phonological awareness in Japanese dyslexic children using functional magnetic resonance imaging. We developed and conducted a phonological manipulation task to extract phonological processing skills and to minimize the influence of auditory working memory on healthy adults, typically developing children, and dyslexic children. Current experiments revealed that several brain regions participated in manipulating the phonological information including left inferior and middle frontal gyrus, left superior temporal gyrus, and bilateral basal ganglia. Moreover, dyslexic children showed altered activity in two brain regions. They showed hyperactivity in the basal ganglia compared with the two other groups, which reflects inefficient phonological processing. Hypoactivity in the left superior temporal gyrus was also found, suggesting difficulty in composing and processing phonological information. The altered brain activity shares similarity with those of dyslexic children in countries speaking alphabetical languages, but disparity also occurs between these two populations. These are initial findings concerning the neurobiological impairments in dyslexic Japanese children. PMID:24052613

  8. Comparison of liver oncogenic potential among human RAS isoforms

    Chung, Sook In; Moon, Hyuk; Ju, Hye-Lim; Kim, Dae Yeong; Cho, Kyung Joo; Ribback, Silvia; Dombrowski, Frank; Calvisi, Diego F.; Ro, Simon Weonsang


    Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p mice lived significantly longer than KRRAS4BG12V mice (p mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene. PMID:26799184

  9. Society News: PhD theses could win prizes; Last chance for IYA2009 grants; New Fellows; RAS Fellows win prizes; Need a job? Need staff? RAS Library Saturdays


    Fellows who are PhD student supervisors should be on the lookout for exceptionally good work from research students submitting their theses this year, for nomination for the RAS Michael Penston Astronomy Prize and the RAS Keith Runcorn Prize. The RAS is offering one last chance to apply for grants towards International Year of Astronomy activities, but you'll have to apply soon. The Society sends congratulations to Fellows of the RAS who have recently received prestigious awards for their work.

  10. Mice lacking Ras-GRF1 show contextual fear conditioning but not spatial memory impairments: convergent evidence from two independently generated mouse mutant lines

    Raffaele ed'Isa


    Full Text Available Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning the Brambilla’s mice with a third mouse line (GENA53 in which a nonsense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in the Brambilla’s mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdalar functions but possibly to some distinct hippocampal connections specific to

  11. A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

    Li Sheng-Fowler

    Full Text Available As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM, DNA (100 µg was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.

  12. Alteration In Bones Metabolism In Active Rheumatoid Arthritis

    The strength and integrity of the human skeleton depends on a delicate equilibrium between bone resorption and bone formation. Osteocalcin (OC) is synthesized by osteoblasts and is considered to be a marker of bone formation and helps in corporating calcium into bone tissue. Rheumatoid arthritis (RA) is an autoimmune inflammatory joint disease characterized by bone complication including bone pain, erosion and osteoporosis. The aim of the present study is to evaluate some factors responsible in bone metabolism termed OC, vitamin D (vit. D), oncostatin M (OSM), ionized calcium and alkaline phosphatase. Fifty pre-menopausal female patients with active RA and twenty healthy controls of the same age were included in the present study. Radioimmunoassay (RIA) was used to estimate serum OC and active vitamin D. The quantitative determination of ionized calcium and alkaline phosphatase were carried out colorimetrically. OSM was measured by ELISA and serum levels of OC and active vitamin D were significantly decreased in RA patients as compared to those of the control group. On the other hand, the levels of serum OSM, ionized calcium and alkaline phosphatase were significantly increased in the RA patients as compared to their healthy control subjects. The results of this study indicated that early investigation and therapy of disturbances of bone metabolism in active RA are necessary for better prognosis and exhibited the importance of OC as a diagnostic tool of alterations of bone metabolism in RA patients.

  13. Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats.

    Yin, Zong-Zhu; Jin, Hai-Ling; Yin, Xue-Zhe; Li, Tian-Zhu; Quan, Ji-Shu; Jin, Zeng-Nan


    AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, P<0.05) and the expression of mutant p53 and p21(ras) protein was lower than that of hepatic preneoplastic lesions (33% and 22% positive respectively in DEN+AAF group vs negative in DEN+AAF+BR group). Both CH(2)Cl(2) and H(2)O extracts from BR had anti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice. PMID:11819701

  14. N-ras mutations in human cutaneous melanoma from sun-exposed body sites.

    Van 't Veer, L.J.; Burgering, B M; Versteeg, R.; Boot, A J; Ruiter, D J; Osanto, S; Schrier, P. I.; Bos, J L


    In 7 of 37 patients with cutaneous melanoma, mutations in the N-ras gene were found. The primary tumors of these seven patients were exclusively localized on body sites continuously exposed to sunlight. Moreover, the ras mutations were all at or near dipyrimidine sites known to be targets of UV damage. Two primary tumors were biclonal with respect to ras mutation. An active role for UV irradiation in induction of the mutations is suggested.

  15. Alteration of biophysical activity of pulmonary surfactant by aluminosilicate nanoparticles.

    Kondej, Dorota; Sosnowski, Tomasz R


    The influence of five different types of aluminosilicate nanoparticles (NPs) on the dynamic surface activity of model pulmonary surfactant (PS) (Survanta) was studied experimentally using oscillating bubble tensiometry. Bentonite, halloysite and montmorillonite (MM) NPs, which are used as fillers of polymer composites, were characterized regarding the size distribution, morphology and surface area. Particle doses applied in the studies were estimated based on the inhalation rate and duration, taking into account the expected aerosol concentration and deposition efficiency after penetration of NPs into the alveolar region. The results indicate that aluminosilicate NPs at concentrations in the pulmonary liquid above 0.1 mg cm(-3) are capable of promoting alterations of the original dynamic biophysical activity of the PS. This effect is indicated by deviation of the minimum surface tension, stability index and the size of surface tension hysteresis. Such response is dependent on the type of NPs present in the system and is stronger when particle concentration increases. It is suggested that interactions between NPs and the PS must be related to the surfactant adsorption on the suspended particles, while in the case of surface-modified clay NPs the additional washout of surface-active components may be expected. It is speculated that observed changes in surface properties of the surfactant may be associated with undesired health effects following extensive inhalation of aluminosilicate NPs in the workplace. PMID:23363039

  16. Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

    Putty-Reddy Sudhir

    Full Text Available BACKGROUND: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. METHODOLOGY/PRINCIPAL FINDINGS: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77 using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60% of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. CONCLUSIONS/SIGNIFICANCE: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

  17. Pseudorabies virus infection alters neuronal activity and connectivity in vitro.

    Kelly M McCarthy


    Full Text Available Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV, infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural

  18. Radiosensitivity of small-cell lung cancer xenografts compared with activity of c-myc, N-myc, L-myc, c-raf-1 and K-ras proto-oncogenes

    Rygaard, K; Slebos, R J; Spang-Thomsen, M


    Oncogenes of the myc family c-raf-1 and K-ras have been reported to modulate radiosensitivity. We examined the possible relationship between in vivo radiosensitivity to single-dose irradiation with 3-10 Gy, and activity of these proto-oncogenes in 2 sets of small-cell lung cancer (SCLC) xenografts......-19 expressed identical amounts of c-raf-1 and high levels of c-myc mRNA, but neither expressed N-myc or L-myc. None of the tumours was mutated at codon 12 or K-ras. Our results show that SCLC xenografts with different radiosensitivity may express identical amounts of some of the proto-oncogenes...... reported to modulate radiosensitivity. Thus, factors other than activation of the examined proto-oncogenes must be involved in causing the differences in radiosensitivity found in the SCLC xenografts. Possible long-term effects of irradiation on proto-oncogene expression was examined in xenografts of GLC...

  19. Evidence implicating the Ras pathway in multiple CD28 costimulatory functions in CD4+ T cells.

    Sujit V Janardhan

    Full Text Available CD28 costimulation is a critical event in the full activation of CD4(+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L to transduce normal, Coxsackie-Adenovirus Receptor (CAR transgenic CD4(+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.

  20. Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

    The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

  1. Disrupting the oncogenic synergism between nucleolin and Ras results in cell growth inhibition and cell death.

    Sari Schokoroy

    Full Text Available BACKGROUND: The ErbB receptors, Ras proteins and nucleolin are major contributors to malignant transformation. The pleiotropic protein nucleolin can bind to both Ras protein and ErbB receptors. Previously, we have demonstrated a crosstalk between Ras, nucleolin and the ErbB1 receptor. Activated Ras facilitates nucleolin interaction with ErbB1 and stabilizes ErbB1 levels. The three oncogenes synergistically facilitate anchorage independent growth and tumor growth in nude mice. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we used several cancer cell lines. The effect of Ras and nucleolin inhibition was determined using cell growth, cell death and cell motility assays. Protein expression was determined by immunohistochemistry. We found that inhibition of Ras and nucleolin reduces tumor cell growth, enhances cell death and inhibits anchorage independent growth. Our results reveal that the combined treatment affects Ras and nucleolin levels and localization. Our study also indicates that Salirasib (FTS, Ras inhibitor reduces cell motility, which is not affected by the nucleolin inhibitor. CONCLUSIONS/SIGNIFICANCE: These results suggest that targeting both nucleolin and Ras may represent an additional avenue for inhibiting cancers driven by these oncogenes.


    Ni Putu Sriwidyani


    Full Text Available Karsinogenesis kanker kolorektal merupakan proses multi-step, melibatkan berbagai abnormalitasgenetik. Mutasi gen K RAS sering ditemukan pada tumor ini. K RAS adalah gen yang menyandi proteinK ras, suatu produk proto-onkogen yang merupakan komponen penting pada jalur pensignalan darireseptor permukaan sel untuk mengontrol proliferasi, diferensiasi, dan kematian sel. Kebanyakanmutasi terjadi pada kodon 12 dan 13 dari ekson 1. Protein K ras mutan akan menyebabkan aktivasipersisten dari banyak signal downstream dari pertumbuhan dan survival sel. Pemeriksaan adanyamutasi pada gen K RAS memegang peranan penting pada prognosis dan terapi dari kanker kolorektal.[MEDICINA 2013;44:97-100].

  3. PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

    Ting Jin; Weizhong Liu; Yixuan Zhang; Yi Pan; Zhenzhen Wang; Walter G Thomas; Yan Chen; Qiurong Ding; Heng Huang; Daqian Xu; Yuhui Jiang; Ben Zhou; Zhenghu Li; Xiaomeng Jiang; Jing He


    Ras plays a pivotal role in many cellular activities,and its subcellular compartmentalization provides spatial and temporal selectivity.Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family.PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus.Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner.Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas,NRas and KRas4A,but not KRas4B.PAQR10 and PAQR11 can also interact with HRas,NRas and KRas4A,but not KRas4B.The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state.Consistently,knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus.Intriguingly,PAQR10 and PAQR11 are able to interact with RasGRP1,a guanine nucleotide exchange protein of Ras,and increase Golgi localization of RasGRP1.The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11.The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1.Furthermore,differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11.Collectively,this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.

  4. Coexistence of K-ras mutations and HPV infection in colon cancer

    Tezol Ayda


    Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

  5. Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo.

    Jasinski, Piotr; Zwolak, Pawel; Terai, Kaoru; Dudek, Arkadiusz Z


    MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind. PMID:19010291

  6. The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

    Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmΔ4A/tmΔ4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras -/- (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmΔ4A/tmΔ4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmΔ4A/tmΔ4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmΔ4A/tmΔ4A and K-ras -/- ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras -/- cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmΔ4A/tmΔ4A ES cells were more resistant to etoposide-induced apoptosis than K-ras -/- cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice

  7. Retraction: "Activated K-Ras and INK4a/Arf Deficiency Promote Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With Cancer Stem Cell Phenotype" by Wang et al.


    The above article, published online on November 23, 2012 in Wiley Online Library (, has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 4B and C to be inappropriately manipulated and re-labeled. Literature Cited Wang Z, Ali S, Banerjee S, Bao B, Li Y, Azmi AS, Korc M, Sarkar FH. 2013. Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype. J Cell Physiol 228:556-562; doi: 10.1002/jcp.24162. PMID:27315162

  8. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos


    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc. PMID:26873620

  9. Dexras1 a unique ras-GTPase interacts with NMDA receptor activity and provides a novel dissociation between anxiety, working memory and sensory gating.

    Carlson, G C; Lin, R E; Chen, Y; Brookshire, B R; White, R S; Lucki, I; Siegel, S J; Kim, S F


    Dexras1 is a novel GTPase that acts at a confluence of signaling mechanisms associated with psychiatric and neurological disease including NMDA receptors, NOS1AP and nNOS. Recent work has shown that Dexras1 mediates iron trafficking and NMDA-dependent neurodegeneration but a role for Dexras1 in normal brain function or psychiatric disease has not been studied. To test for such a role, mice with germline knockout (KO) of Dexras1 were assayed for behavioral abnormalities as well as changes in NMDA receptor subunit protein expression. Because Dexras1 is up-regulated during stress or by dexamethasone treatment, we included measures associated with emotion including anxiety and depression. Baseline anxiety-like measures (open field and zero maze) were not altered, nor were depression-like behavior (tail suspension). Measures of memory function yielded mixed results, with no changes in episodic memory (novel object recognition) but a significant decrement on working memory (T-maze). Alternatively, there was an increase in pre-pulse inhibition (PPI), without concomitant changes in either startle amplitude or locomotor activity. PPI data are consistent with the direction of change seen following exposure to dopamine D2 antagonists. An examination of NMDA subunit expression levels revealed an increased expression of the NR2A subunit, contrary to previous studies demonstrating down-regulation of the receptor following antipsychotic exposure (Schmitt et al., 2003) and up-regulation after exposure to isolation rearing (Turnock-Jones et al., 2009). These findings suggest a potential role for Dexras1 in modulating a selective subset of psychiatric symptoms, possibly via its interaction with NMDARs and/or other disease-related binding-partners. Furthermore, data suggest that modulating Dexras1 activity has contrasting effects on emotional, sensory and cognitive domains. PMID:26946266

  10. Alteration of swelling clay minerals by acid activation

    Steudel, A.; Batenburg, L.F.; Fischer, H.R.; Weidler, P.G.; Emmerich, K.


    The bulk material of six dioctahedral and two trioctahedral swellable clay minerals was leached in H2SO4 and HCl at concentrations of 1.0, 5.0 and 10.0 M at 80 °C for several hours. Alteration of the clay mineral structures was dependent on the individual character of each mineral (chemical composit

  11. Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

    Buhrman, Greg; O; #8242; Connor, Casey; Zerbe, Brandon; Kearney, Bradley M.; Napoleon, Raeanne; Kovrigina, Elizaveta A.; Vajda, Sandor; Kozakov, Dima; Kovrigin, Evgenii L.; Mattos, Carla (NCSU); (MCW); (BU)


    We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

  12. Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

    Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y


    The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. PMID:26811072

  13. Altered expression of the IQGAP1 gene in human lung cancer cell lines

    Mitchell, C.E.; Palmisano, W.A.; Lechner, J.F. [and others


    IQGAP1 is a GTPase activation protein that accelerates GTP hydrolysis by normal p21 ras proteins. Therefore, IQGAP1 could act as an upstream affector of p21 ras activity by convert in excess amounts of active GTP-21 ras to inactive GDP-21 ras. IQGAP1 displays extensive sequence similarity to the catalytic domain of all previously reported ras GAPs, including the tumor suppressor gene protein neurofibromatosis type 1 (NF1). It has been shown that abnormal NF1 protein cannot negatively regulate the activity of ras proteins in neuroblast cells. This observation supports the hypothesis that NF1 is a tumor suppressor gene whose product acts upstream of ras. IQGAP1 is primarily expressed in lung, where it may play a role similar to NF1 in regulating the activity of H-ras or K-ras proteins. IQGAP1 functions as other GAPs by controlling the activity of ras.

  14. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith


    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  15. High physical activity in young children suggests positive effects by altering autoantigen-induced immune activity.

    Carlsson, E; Ludvigsson, J; Huus, K; Faresjö, M


    Physical activity in children is associated with several positive health outcomes such as decreased cardiovascular risk factors, improved lung function, enhanced motor skill development, healthier body composition, and also improved defense against inflammatory diseases. We examined how high physical activity vs a sedentary lifestyle in young children influences the immune response with focus on autoimmunity. Peripheral blood mononuclear cells, collected from 55 5-year-old children with either high physical activity (n = 14), average physical activity (n = 27), or low physical activity (n = 14), from the All Babies In Southeast Sweden (ABIS) cohort, were stimulated with antigens (tetanus toxoid and beta-lactoglobulin) and autoantigens (GAD65 , insulin, HSP60, and IA-2). Immune markers (cytokines and chemokines), C-peptide and proinsulin were analyzed. Children with high physical activity showed decreased immune activity toward the autoantigens GAD65 (IL-5, P < 0.05), HSP60 and IA-2 (IL-10, P < 0.05) and also low spontaneous pro-inflammatory immune activity (IL-6, IL-13, IFN-γ, TNF-α, and CCL2 (P < 0.05)) compared with children with an average or low physical activity. High physical activity in young children seems to have positive effects on the immune system by altering autoantigen-induced immune activity. PMID:25892449

  16. Reduced Expression of the Extracellular Calcium-Sensing Receptor (CaSR) Is Associated with Activation of the Renin-Angiotensin System (RAS) to Promote Vascular Remodeling in the Pathogenesis of Essential Hypertension

    Wang, La-mei; Tang, Na; Zhong, Hua; Liu, Yong-min; Li, Zhen; Feng, Qian; He, Fang


    The proliferation of vascular smooth muscle cells (VSMCs), remodeling of the vasculature, and the renin-angiotensin system (RAS) play important roles in the development of essential hypertension (EH), which is defined as high blood pressure (BP) in which secondary causes, such as renovascular disease, are absent. The calcium-sensing receptor (CaSR) is involved in the regulation of BP. However, the underlying mechanisms by which the CaSR regulates BP are poorly understood. In the present study, the role of the CaSR in EH was investigated using male spontaneously hypertensive rats (SHRs) and rat and human plasma samples. The percentages of medial wall thickness to external diameter (WT%), total vessel wall cross-sectional area to the total area (WA%) of thoracic arteries, as well as the percentage of wall area occupied by collagen to total vessel wall area (CA%) were determined. Tissue protein expression and plasma concentrations of the CaSR, cyclic adenosine monophosphate (cAMP), renin, and angiotensin II (Ang II) were additionally assessed. WT%, WA%, and CA% were found to increase with increasing BP, whereas the plasma concentration of CaSR was found to decrease. With increasing BP, the levels of smooth muscle actin and calponin decreased, whereas those of osteopontin and proliferating cell nuclear antigen increased. The CaSR level negatively correlated with the levels of cAMP and Ang II, but positively correlated with those of renin. Our data suggest that reduced expression of the CaSR is correlated with activation of the RAS, which induces increased vascular remodeling and VSMC proliferation, and thereby associated with EH in the SHR model and in the Han Chinese population. Our findings provide new insights into the pathogenesis of EH. PMID:27391973

  17. Phosphorylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

    Cobitz, A.R.; Yim, E.H.; Brown, W.R.; Perou, C.M.; Tamanoi, Fuyuhiko (Univ. of Chicago, IL (USA))


    RAS1 and RAS2 proteins of Saccharomyces cerevisiae are guanine nucleotide-binding proteins involved in the regulation of adenylate cyclase. In this paper, the authors report that these proteins are phosphorylated. The phosphorylation of RAS1 protein is demonstrated by treating with alkaline phosphatase as well as by labeling with ({sup 32}P)orthophosphate. The phosphorylation occurs exclusively on serine residues and phosphorylated RAS1 protein is predominantly membrane localized. The phosphorylation of RAS2 protein is demonstrated by similar {sup 32}P-labeling experiments. The phosphorylation occurs exclusively on serine residues and phosphopeptide analyses suggest that only two major phosphorylated tryptic peptides are generated from the RAS2 protein. These results provide evidence for the phosphorylation of RAS proteins in vivo. Furthermore, the demonstration that the phosphorylation occurs exclusively on serine residues and that the RAS2 protein contains only two major phosphorylated tryptic peptides argues that the phosphorylation may be physiologically significant.

  18. Phosphorylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

    RAS1 and RAS2 proteins of Saccharomyces cerevisiae are guanine nucleotide-binding proteins involved in the regulation of adenylate cyclase. In this paper, the authors report that these proteins are phosphorylated. The phosphorylation of RAS1 protein is demonstrated by treating with alkaline phosphatase as well as by labeling with [32P]orthophosphate. The phosphorylation occurs exclusively on serine residues and phosphorylated RAS1 protein is predominantly membrane localized. The phosphorylation of RAS2 protein is demonstrated by similar 32P-labeling experiments. The phosphorylation occurs exclusively on serine residues and phosphopeptide analyses suggest that only two major phosphorylated tryptic peptides are generated from the RAS2 protein. These results provide evidence for the phosphorylation of RAS proteins in vivo. Furthermore, the demonstration that the phosphorylation occurs exclusively on serine residues and that the RAS2 protein contains only two major phosphorylated tryptic peptides argues that the phosphorylation may be physiologically significant

  19. Using the capital markets in Ras Gas

    In December 1996, Ras Laffan Liquefied Natural Gas Company Ltd (Ras Gas) closed a multi-source financing that included an offering of US$1.2bn of bonds. The sponsors of the Ras Gas project overcame a number of obstacles on the road to closing the capital markets offering. This article provides a general overview of capital markets offerings in international project financings and discusses how Ras Gas was able to successfully integrate a capital markets offering into a financing plan which included a commercial bank facility and several export-credit agency facilities. (Author)

  20. Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells.

    Allegra, Maryline; Zaragkoulias, Andreas; Vorgia, Elena; Ioannou, Marina; Litos, Gabriele; Beug, Hartmut; Mavrothalassitis, George


    Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-β (TGF-β) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-β-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-β-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-β signaling through Smads was mostly unaffected, and ERF suppressed the TGF-β-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-β-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-β-signaling pathways. PMID:22875994

  1. Loss of RASSF1A Expression in Colorectal Cancer and Its Association with K-ras Status

    Dan Cao


    Full Text Available Background. The RAS-association domain family 1 A (RASSF1A is a classical member of RAS effectors regulating cell proliferation and apoptosis. Loss of RASSF1A expression may shift the balance towards a growth-promoting effect without the necessity of activating K-ras mutations. Its potential association with K-ras mutations in colorectal cancer (CRC is unclear. Methods. RASSF1A expression was examined in normal mucosa, adenoma, and tumor tissues of colon and rectum, respectively. We examined the association of RASSF1A expression, mutations of K-ras, and EGFR status in 76 primary CRCs. The relationship between clinicopathological characteristics and RASSF1A expression was also analyzed. Results. RASSF1A expression level decreased progressively in normal mucosa, adenoma and, tumor tissues, and the loss of RASSF1A expression occurred more frequently in tumor tissues. Of 76 primary CRCs, loss of RASSF1A expression and/or K-ras mutations were detected in 77% cases. Loss of RASSF1A expression was more frequent in K-ras wild-type than in mutation cases (63% versus 32%, . Conclusions. Our study indicates that loss of RASSF1A may be involved in pathogenesis of CRC, its expression was found predominantly in K-ras wild-type CRCs, suggesting that it may be another way of affecting RAS signaling, in addition to K-ras mutations.

  2. The research progress on function of Ras guanyl nucleotide releasing protein family%Ras 鸟苷酸释放蛋白家族的功能研究新进展

    杨柳; 陶娟; 涂亚庭


    Ras鸟苷酸释放蛋白家族(Ras guanyl nucleotide releasing proteins ,RasGRPs)是鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factors ,GEFs)中的一员,由4种类型的蛋白质组成。 RasGRPs能通过交换鸟嘌呤核苷酸将Ras蛋白从无活性的GDP形式变成有活性的GTP形式,从而活化Ras。4种不同类型的RasGRP蛋白拥有共同的分子结构。其结构包括一个由Ras 交换中心和CDC25区域组成的催化中心,同时还拥有一对非典型的EF臂及C1端。异常表达的不同RasGRP蛋白在不同的疾病发病过程中发挥作用。%Ras guanyl nucleotide releasing proteins ( RasGRPs) were one of guanine nucleotide exchange factors .They consist of four types of proteins .RasGRPs can activate Ras from GDP form to GTP form through the exchange of guanine nucleotide .Four differ-ent types of RasGRP proteins share a common molecular structure .The structure consists of a catalytic center including Ras exchange centers and CDC25 region.They also have a pair of atypical EF arms and C 1 side.The abnormal expressions of different RasGRP pro-teins play a role in different diseases .

  3. Chronic hyperammonemia alters the circadian rhythms of corticosteroid hormone levels and of motor activity in rats.

    Ahabrach, Hanan; Piedrafita, Blanca; Ayad, Abdelmalik; El Mlili, Nisrin; Errami, Mohammed; Felipo, Vicente; Llansola, Marta


    Patients with liver cirrhosis may present hepatic encephalopathy with a wide range of neurological disturbances and alterations in sleep quality and in the sleep-wake circadian rhythm. Hyperammonemia is a main contributor to the neurological alterations in hepatic encephalopathy. We have assessed, in an animal model of chronic hyperammonemia without liver failure, the effects of hyperammonemia per se on the circadian rhythms of motor activity, temperature, and plasma levels of adrenal corticosteroid hormones. Chronic hyperammonemia alters the circadian rhythms of locomotor activity and of cortisol and corticosterone levels in blood. Different types of motor activity are affected differentially. Hyperammonemia significantly alters the rhythm of spontaneous ambulatory activity, reducing strongly ambulatory counts and slightly average velocity during the night (the active phase) but not during the day, resulting in altered circadian rhythms. In contrast, hyperammonemia did not affect wheel running at all, indicating that it affects spontaneous but not voluntary activity. Vertical activity was affected only very slightly, indicating that hyperammonemia does not induce anxiety. Hyperammonemia abolished completely the circadian rhythm of corticosteroid hormones in plasma, completely eliminating the peaks of cortisol and corticosterone present in control rats at the start of the dark period. The data reported show that chronic hyperammonemia, similar to that present in patients with liver cirrhosis, alters the circadian rhythms of corticosteroid hormones and of motor activity. This suggests that hyperammonemia would be a relevant contributor to the alterations in corticosteroid hormones and in circadian rhythms in patients with liver cirrhosis. PMID:19998493

  4. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort.

    Ines, Chaar; Donia, Ounissi; Rahma, Boughriba; Ben Ammar, Azza; Sameh, Amara; Khalfallah, Taher; Abdelmajid, Ben Hmida; Sabeh, Mzabi; Saadia, Bouraoui


    According to the multistep route of genetic alterations in the colorectal adenoma-carcinoma sequence, the complex K-ras/p53 mutation is one of the first alterations to occur and represent an important genetic event in colorectal cancer (CRC). An evaluation of the mutation spectra in K-ras and p53 gene was effected in 167 Tunisian patients with sporadic CRC to determine whether our populations have similar pattern of genetic alteration as in Maghrebin's population. Mutation patterns of codon 12-13 of K-ras and exon 5-8 of p53 were analyzed by immunohistochemistry and PCR-SSCP and confirmed by sequencing. Mutations in the K-ras gene were detected in 31.13 % and affect the women more than the men (p = 0.008). Immunostaining showed that expression of p21 ras was correlated with the advanced age (p = 0.004), whereas loss of signal was associated with mucinous histotype (p = 0.003). Kaplan-Meier survival curve found that patients with the K-ras mutation had a shorter survival compared with patients without mutation (p = 0.005). Alteration in p53 was seen in 17.4 % of patients and affects three hot spot codons such as 175, 245, and 248. Overexpression of p53 was seen in 34.1 % and correlated with tumor node metastasis (TNM) advanced stage (p = 0.037) and mucinous histotype (p = 0.001). A high concordance between p53 expression and alteration (p<0.005) was shown. Concomitant mutations in K-ras and p53 gene were detected in only 4 % of tumors. K-ras and p53 undergo separate pathways in colorectal tumorogenesis. Interestingly, mutations in the K-ras gene might be considered a valuable prognostic factor correlated to poor outcome. p53 gene alterations were rather low in our set, and methylation pattern of p53 is required to elucidate the molecular basis of this protein in CRC. PMID:24763823

  5. 8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells.

    Mohammed, Idrees; Hampton, Shahienaz E; Ashall, Louise; Hildebrandt, Emily R; Kutlik, Robert A; Manandhar, Surya P; Floyd, Brandon J; Smith, Haley E; Dozier, Jonathan K; Distefano, Mark D; Schmidt, Walter K; Dore, Timothy M


    Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure-activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer. PMID:26706114

  6. RasGRPs are targets of the anti-cancer agent ingenol-3-angelate.

    Xiaohua Song

    Full Text Available Ingenol-3-angelate (I3A is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A's effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP's C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin's lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.

  7. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

    Jones, S; Vignais, M L; Broach, J R


    The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also o...

  8. Characterization of p21Ras-mediated apoptosis induced by protein kinase C inhibition and application to human tumor cell lines.

    Liou, James S; Chen, James S; Faller, Douglas V


    Suppression of PKC activity can selectively induce apoptosis in cells expressing a constitutively activated p21Ras protein. We demonstrate that continued expression of p21Ras activity is required in PKC-mediated apoptosis because farnesyltransferase inhibitors abrogated the loss of viability in p21Ras-transformed cells occurring following PKC inhibition. Studies utilizing gene transfer or viral vectors demonstrate that transient expression of oncogenic p21Ras activity is sufficient for induction of apoptosis by PKC inhibition, whereas physiologic activation of p21Ras by growth factor is not sufficient to induce apoptosis. Mechanistically, the p21Ras-mediated apoptosis induced by PKC inhibition is dependent upon mitochondrial dysregulation, with a concurrent loss of mitochondrial membrane potential (psim). Cyclosporine A, which prevented the loss of psim, also inhibited HMG-induced DNA fragmentation in cells expressing an activated p21Ras. Induction of apoptosis by PKC inhibition in human tumors with oncogenic p21Ras mutations was demonstrated. Inhibition of PKC caused increased apoptosis in MIA-PaCa-2, a human pancreatic tumor line containing a mutated Ki-ras allele, when compared to HS766T, a human pancreatic tumor line with normal Ki-ras alleles. Furthermore, PKC inhibition induced apoptosis in HCT116, a human colorectal tumor line containing an oncogenic Ki-ras allele but not in a subline (Hke3) in which the mutated Ki-ras allele had been disrupted. The PKC inhibitor 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG), significantly reduced p21Ras-mediated tumor growth in vivo in a nude mouse MIA-PaCa-2 xenograft model. Collectively these studies suggest the therapeutic feasibility of targeting PKC activity in tumors expressing an activated p21Ras oncoprotein. PMID:14603530

  9. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    Pathan, Akbar Ali Khan; Panthi, Bhavana; Khan, Zahid; Koppula, Purushotham Reddy; Alanazi, Mohammed Saud; Sachchidanand; Parine, Narasimha Reddy; Chourasia, Mukesh


    Objective Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein.

  10. Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

    Mali, Aniket V; Bhise, Sunita S; Hegde, Mahabaleshwar V; Katyare, Surendra S


    The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50 % while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64 % respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85 % increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose. PMID:27382204

  11. Sensitization of human pancreatic cancer cells harboring mutated K-ras to apoptosis.

    Ling Shen

    Full Text Available Pancreatic cancer is a devastating human malignancy and gain of functional mutations in K-ras oncogene is observed in 75%-90% of the patients. Studies have shown that oncogenic ras is not only able to promote cell growth or survival, but also apoptosis, depending upon circumstances. Using pancreatic cancer cell lines with or without expressing mutated K-ras, we demonstrated that the inhibition of endogenous PKC activity sensitized human pancreatic cancer cells (MIA and PANC-1 expressing mutated K-ras to apoptosis, which had no apoptotic effect on BxPC-3 pancreatic cancer cells that contain a normal Ras as well as human lung epithelial BAES-2B cells. In this apoptotic process, the level of ROS was increased and PUMA was upregulated in a p73-dependent fashion in MIA and PANC-1 cells. Subsequently, caspase-3 was cleaved. A full induction of apoptosis required the activation of both ROS- and p73-mediated pathways. The data suggest that PKC is a crucial factor that copes with aberrant K-ras to maintain the homeostasis of the pancreatic cancer cells harboring mutated K-ras. However, the suppression or loss of PKC disrupts the balance and initiates an apoptotic crisis, in which ROS and p73 appear the potential, key targets.

  12. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21RasC118S) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG

  13. Analysis of RAS oncogene mutations in human lymphoid malignancies.

    Neri, A.; Knowles, D M; Greco, A.; McCormick, F; Dalla-Favera, R


    We investigated the frequency of mutations activating RAS oncogenes in human lymphoid malignancies, including B- and T-cell-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. By the polymerase chain reaction/oligonucleotide hybridization method, DNA from 178 cases was analyzed for activating mutations involving codons 12 and 61 of the HRAS, KRAS and NRAS genes and codon 13 of the NRAS gene. Mutations involving codons 12 or 13 of the NRAS gene were de...

  14. Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

    Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms

  15. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M


    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  16. Ras1CA overexpression in the posterior silk gland improves silk yield

    Li Ma; Hanfu Xu; Jinqi Zhu; Sanyuan Ma; Yan Liu; Rong-Jing Jiang; Qingyou Xia; Sheng Li


    Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm,Bombyx mori,but has reached a plateau during the last decades. For the first time,we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1CA oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%,while increasing food consumption by only 20%. Ras activation by Ras1CA overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins,up-regulated fibroin mRNA levels,increased total DNA content,and stimulated endoreplication. Moreover,Rasl activation increased cell and nuclei sizes,enriched subcellular organelles related to protein synthesis,and stimulated ribosome biogenesis for mRNA translation. We conclude that Rasl activation increases cell size and protein synthesis in the posterior silk gland,leading to silk yield improvement.

  17. RAS and RHO Families of GTPases Directly Regulate Distinct Phosphoinositide 3-Kinase Isoforms

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S.; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian


    Summary RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind an...

  18. Analysis of ras genes and linked viral sequences in rat hepatocarcinogenesis.

    Chandar, N.; Lombardi, B; Schulz, W.; Locker, J


    After long-term feeding of a choline-devoid diet to rats, the authors analyzed rasK, rasH, and rasN transcripts and gene structure in livers and liver tumors. They controlled their analysis by studying cell lines derived from chemically induced hepatomas. Transcripts from all three genes were elevated in all tumors, but not in the livers from which they arose. The transcript elevations may represent an effect of active cell proliferation in the tumors. Clone HiHi-3, derived from the Kirsten m...

  19. Silver and Gold Nanoparticles Alter Cathepsin Activity In vitro

    Speshock, Janice L.; Braydich-Stolle, Laura K.; Szymanski, Eric R.; Hussain, Saber M.


    Nanomaterials are being incorporated into many biological applications for use as therapeutics, sensors, or labels. Silver nanomaterials are being utilized for biological implants and wound dressings as an antiviral material, whereas gold nanomaterials are being used as biological labels or sensors due to their surface properties and biocompatibility. Cytotoxicity data of these materials are becoming more prevalent; however, little research has been performed to understand how the introduction of these materials into cells affects cellular processes. Here, we demonstrate the impact that silver and gold nanoparticles have on cathepsin activity in vitro. Cathepsins are important cellular proteases that are imperative for proper immune system function. We have selected to examine gold and silver nanoparticles due to the increased use of these materials in biological applications. This manuscript depicts how both of these types of nanomaterials affect cathepsin activity, which could impact the host's immune system and its ability to respond to pathogens. Cathepsin B activity decreases in a dose-dependent manner with all nanoparticles tested. Alternatively, the impact of nanoparticles on cathepsin L activity depends greatly on the type and size of the material.

  20. Altered Error-Related Activity in Patients with Schizophrenia

    Koch, Kathrin; Wagner, Gerd; Schultz, Christoph; Schachtzabel, Claudia; Nenadic, Igor; Axer, Martina; Reichenbach, Jurgen R.; Sauer, Heinrich; Schlosser, Ralf G. M.


    Deficits in working memory (WM) and executive cognitive control are core features of schizophrenia. However, findings regarding functional activation strengths are heterogeneous, partly due to differences in task demands and behavioral performance. Previous investigators proposed integrating these heterogeneous findings into a comprehensive model…

  1. Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF.

    Baljuls, Angela; Dobrzyński, Maciej; Rauch, Jens; Rauch, Nora; Kolch, Walter


    RAF family kinases are central components of the Ras-RAF-MEK-ERK cascade. Dimerization is a key mechanism of RAF activation in response to physiological, pathological and pharmacological signals. It is mediated by a dimer interface region in the RAF kinase domain that is also conserved in KSR, a scaffolding protein that binds RAF, MEK and ERK. The regulation of RAF dimerization is incompletely understood. Especially little is known about the molecular mechanism involved in the selection of the dimerization partner. Previously, we reported that Ras-dependent binding of the tumour suppressor DiRas3 to C-RAF inhibits the C-RAF:B-RAF heterodimerization. Here we show that DiRas3 binds to KSR1 independently of its interaction with activated Ras and RAF. Our data also suggest that depending on the local stoichiometry between DiRas3 and oncogenic Ras, DiRas3 can either enhance homodimerization of KSR1 or recruit KSR1 to the Ras:C-RAF complex and thereby reduce the availability of C-RAF for binding to B-RAF. This mechanism, which is shared between A-RAF and C-RAF, may be involved in the regulation of Ras12V-induced cell transformation by DiRas3. PMID:27368419

  2. Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

    Overexpression of Aurora-A and mutant Ras (RasV12) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either RasV12and wild-type Aurora-A (designated WT) or RasV12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. Overexpression of wild-type Aurora-A and mutation of RasV12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the RasV12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the RasV12 transformants. Wild-type-Aurora-A enhances focus formation and aggregation of the RasV12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway

  3. Genetic Transferability of Anomalous Irradiation Alterations of Antibiotic Activity

    Bass, George E.


    It previously has been discovered that visible light irradiation of crystalline substrates can lead to enhancement of subsequent enzymatic reaction rates as sharply peaked oscillatory functions of irradiation time. The particular activating irradiation times can vary with source of a given enzyme and thus, presumably, its molecular structure. The experiments reported here demonstrate that the potential for this anomalous enzyme reaction rate enhancement can be transferred from one bacterial s...


    Hong-wei Wang; Lin Yuan; Tie-liang Zhao; He Huang; Hong Chen; Di Wu


    The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure.It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan (6S-chitosan) and 3,6-O-sulfated chitosan (3,6S-chitosan),but decreased greatly after being bound to 2-N-6-O-sulfated chitosan (2,6S-chitosan).Meanwhile,among these sulfated chitosans,2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra.These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme,lysozyme undergoes conformational change of different magnitudes,which results in corresponding levels of lysozyme activity.Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance (SPR) suggested that their affinities might be determined by their molecular structures.

  5. Alterations in electrodermal activity and cardiac parasympathetic tone during hypnosis.

    Kekecs, Zoltán; Szekely, Anna; Varga, Katalin


    Exploring autonomic nervous system (ANS) changes during hypnosis is critical for understanding the nature and extent of the hypnotic phenomenon and for identifying the mechanisms underlying the effects of hypnosis in different medical conditions. To assess ANS changes during hypnosis, electrodermal activity and pulse rate variability (PRV) were measured in 121 young adults. Participants either received hypnotic induction (hypnosis condition) or listened to music (control condition), and both groups were exposed to test suggestions. Blocks of silence and experimental sound stimuli were presented at baseline, after induction, and after de-induction. Skin conductance level (SCL) and high frequency (HF) power of PRV measured at each phase were compared between groups. Hypnosis decreased SCL compared to the control condition; however, there were no group differences in HF power. Furthermore, hypnotic suggestibility did not moderate ANS changes in the hypnosis group. These findings indicate that hypnosis reduces tonic sympathetic nervous system activity, which might explain why hypnosis is effective in the treatment of disorders with strong sympathetic nervous system involvement, such as rheumatoid arthritis, hot flashes, hypertension, and chronic pain. Further studies with different control conditions are required to examine the specificity of the sympathetic effects of hypnosis. PMID:26488759

  6. Postnatal foraging demands alter adrenocortical activity and psychosocial development.

    Lyons, D M; Kim, S; Schatzberg, A F; Levine, S


    Mother squirrel monkeys stop carrying infants at earlier ages in high-demand (HD) conditions where food is difficult to find relative to low-demand (LD) conditions. To characterize these transitions in psychosocial development, from 10- to 21-weeks postpartum we collected measures of behavior, adrenocortical activity, and social transactions coded for initiator (mother or infant), goal (make-contact or break-contact), and outcome (success or failure). Make-contact attempts were most often initiated by HD infants, but mothers often opposed these attempts and less than 50% were successful. Break-contact attempts were most often initiated by LD infants, but mothers often opposed these attempts and fewer LD than HD infant break-contact attempts were successful. Plasma levels of cortisol were significantly higher in HD than LD mothers, but differences in adrenocortical activity were less consistent in their infants. HD and LD infants also spent similar amounts of time nursing on their mothers and feeding on solid foods. By rescheduling some transitions in development (carry-->self-transport), and not others (nursing-->self-feeding), mothers may have partially protected infants from the immediate impact of an otherwise stressful foraging task. PMID:9589217

  7. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  8. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)


    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  9. Refeeding alters superoxide dismutase activity in Chinese hamster ovary cells

    The authors previously showed superoxide dismutase (SOD) activity is increased in heat shocked Chinese hamster ovary (CHO) and ovarian carcinoma (OvCa) cells during the time period when thermotolerance (TT) is observed (Ca Res 45,3029). SOD is also increased in OvCa cells following transient exposure to ethanol, carbonyl cyanide-N-chlorophenyl-hydrazone, or hypoxia; all treatments which induce TT (1986 Rad Res Abstr Co-2). As these experiments involved refeeding of cell cultures, the authors examined the effect of refeeding on SOD in CHO cells. Refeeding confluent CHO cells with fresh McCoy's 5A medium containing 10% FCS decreased SOD 0 to 6 hours after refeeding, which was due to loss of the mitochondrial or Mn SOD. Addition of glucose to the medium at the concentration originally found in the medium caused a similar decline in SOD. At 6-24 hours after refeeding or the addition of glucose an increase in Mn SOD was observed. These results suggest metabolic status can affect Mn SOD in the cell. The possible role of metabolic regulation of SOD in heat sensitivity is being investigated

  10. Altered behaviour in spotted hyenas associated with increased human activity

    Boydston, E.E.; Kapheim, K.M.; Watts, H.E.; Szykman, M.; Holekamp, K.E.


    To investigate how anthropogenic activity might affect large carnivores, we studied the behaviour of spotted hyenas (Crocuta crocuta) during two time periods. From 1996 to 1998, we documented the ecological correlates of space utilization patterns exhibited by adult female hyenas defending a territory at the edge of a wildlife reserve in Kenya. Hyenas preferred areas near dense vegetation but appeared to avoid areas containing the greatest abundance of prey, perhaps because these were also the areas of most intensive livestock grazing. We then compared hyena behaviour observed in 1996-98 with that observed several years earlier and found many differences. Female hyenas in 1996-98 were found farther from dens, but closer to dense vegetation and to the edges of their territory, than in 1988-90. Recent females also had larger home ranges, travelled farther between consecutive sightings, and were more nocturnal than in 1988-90. Finally, hyenas occurred in smaller groups in 1996-98 than in 1988-90. We also found several changes in hyena demography between periods. We next attempted to explain differences observed between time periods by testing predictions of hypotheses invoking prey abundance, climate, interactions with lions, tourism and livestock grazing. Our data were consistent with the hypothesis that increased reliance on the reserve for livestock grazing was responsible for observed changes. That behavioural changes were not associated with decreased hyena population density suggests the behavioural plasticity typical of this species may protect it from extinction. ?? 2003 The Zoological Society of London.

  11. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21RasC118S). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway

  12. NAM: The 2004 RAS National Astronomy Meeting

    Jones, Barrie; Norton, Andrew


    This year's RAS National Astronomy Meeting was held at the Open University's Milton Keynes campus from 29 March to 2 April. The event was organized by members of the OU Physics & Astronomy Department and Planetary & Space Science Research Institute. Around 450 people attended the meeting, at which more than 220 talks were presented, along with around 90 posters. Co-chairs of RAS NAM04, Barrie Jones and Andrew Norton, summarize.

  13. Low concentrations of hydrogen peroxide or nitrite induced of Paracoccidioides brasiliensis cell proliferation in a Ras-dependent manner.

    Ana Eliza Coronel Janu Haniu

    Full Text Available Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM, should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS and nitrogen (RNS species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM or NO2 (0.1-0.25 µM stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD fused with GST (RBD-Byr2-GST to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.

  14. Expression of ras oncogene and major histocompatibility complex (MHC) antigen in carcinomas of the uterine cervix

    Consecutive 50 cases of squamous cell carcinomas of the uterine cervix diagnosed in 1992 were subjected to immunohistochemical study for ras oncogene product (p21) and MHC class II (DR) antigen using a microprobe immunostainer. Activated ras and aberrant DR expression were noted in 26 cases (52%) and 11 cases (22%) of cervical squamous cell carcinomas, respectively, without difference among histologic types. The reaction was mainly intracytoplasmic, with granular staining pattern and diffuse distribution. No direct histologic correlation between ras and DR expression was found. Four cases with HPV 16/18 DNA in superficial koilocytotic cells, revealed by in situ hybridization, showed various expression of ras and DR, and these 3 factors histologically did not seem to be affected one another. (Author)

  15. Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development

    Dagorn Jean


    Full Text Available Abstract Background In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from rasV12/E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development. Results Among the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors. Conclusions Some of the genes identified in this work, whose expression is altered upon rasV12/E1A transformation of MEFs, could be new cancer therapeutic targets.

  16. Altered sensorimotor activation patterns in idiopathic dystonia-an activation likelihood estimation meta-analysis of functional brain imaging studies

    Løkkegaard, Annemette; Herz, Damian M; Haagensen, Brian N;


    . Further, study size was usually small including different types of dystonia. Here we performed an activation likelihood estimation (ALE) meta-analysis of functional neuroimaging studies in patients with primary dystonia to test for convergence of dystonia-related alterations in task-related activity....... Hum Brain Mapp 37:547-557, 2016. © 2015 Wiley Periodicals, Inc....

  17. Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

    Berrier, Allison L.; Mastrangelo, Anthony M.; Downward, Julian; Ginsberg, Mark; LaFlamme, Susan E.


    Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact β cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spread...

  18. Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling.

    Diaz, B.; Barnard, D.; Filson, A; MacDonald, S; King, A.; Marshall, M.


    Activation of the Raf serine/threonine protein kinases is tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a lesser extent, serine 339 is essential for the biological and enzymatic activit...

  19. Epidermal Growth Factor Receptor (EGFR gene copy number (GCN correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH and chromogenic in situ hybridization (CISH analysis

    Scartozzi Mario


    Full Text Available Abstract Background K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Methods Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Results Forty-four patients were available for analysis. We observed a partial remission in 9 (60% and 2 (9% cases with a FISH EGFR GCN ≥ 2.6 and Conclusion FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

  20. Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

    Bitencourt, C.S. [Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Duarte, C.G.; Azzolini, A.E.C.S.; Assis-Pandochi, A.I. [Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)


    Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

  1. Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells

    Medullary thyroid carcinoma (MTC) is an endocrine tumor of the thyroid C cells that expresses high levels of the neuroendocrine peptide hormone calcitonin. During tumor progression in the host, there is an apparent loss of differentiation in MTC cells that involves a consistent decrease in calcitonin content of the tumor cells associated with decreased expression of the calcitonin gene and/or changes in a mRNA alternative-processing pattern away from that characteristic of the parent thyroid C cell. The authors now report that introduction of the viral Harvey ras (v-Ha-ras) oncogene into cultured human MTC cells can reverse such changes in gene expression and can induce endocrine differentiation of the tumor cells. The expression of v-Ha-ras is associated with decreased cellular proliferation and DNA synthesis. There is a marked increase in the number of cytoplasmic secretory granules that are a classic feature of differentiated thyroid C cells. v-Ha-ras expression induces increased expression of the calcitonin gene and the processing of the primary gene transcript is shifted to favor calcitonin mRNA rather than calcitonin-gene-related peptide (CGRP) mRNA production. These studies with cultured human MTC cells provide a model system to study the role of Ha-ras and related genes in neuroendocrine differentiation. The findings suggest an important approach for identifying genes in solid tumors whose altered expression may play a role in the impaired maturational capacity characteristic of cancer cells during tumor progression

  2. Post-transcriptional regulation of connexin43 in H-Ras-transformed cells.

    Mustapha Kandouz

    Full Text Available Connexin43 (Cx43 expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3' and 5'Untranslated regions (UTRs and found an opposing effect; a 5'UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3(Ras, while the 3'UTR is active only in normal NIH-3T3(Neo cells and completely silenced in NIH-3T3(Ras cells. Most importantly, we identified a previously unknown regulatory element within the 3'UTR, named S1516, which accounts for this 3'UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3(Neo compared to NIH-3T3(Ras cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.

  3. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    Pathan AAK


    Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the

  4. Genomic heterogeneity and instability in colorectal cancer: spectral karyotyping, glutathione transferase-Ml and ras.

    Bartos, Jeremy D; Stoler, Daniel L; Matsui, Sei-ichi; Swede, Helen; Willmott, Lyndsay J; Sait, Sheila N; Petrelli, Nicholas J; Anderson, Garth R


    Genomic instability in cancer is frequently described as being either chromosomal instability or microsatellite instability, although when events within chromosomes are monitored, extensive intrachromosomal instability is also found. Spectral karyotyping was used to visualize how extensively genomic instability gives rise to intratumor genomic heterogeneity in sporadic colorectal carcinomas. Two factors were then examined which might relate to intrachromosomal instability in colorectal cancers: the presence of the glutathione transferase-Ml gene to detoxify potential carcinogens, and the presence of activated ras which has been associated with chromosomal instability when first expressed. Intrachromosomal genomic instability was previously determined by inter-(simple sequence repeat) PCR (inter-SSR PCR) and by fractional allelic loss rate for 348 markers. GSTM1 status was determined for each of 49 tumors through use of specific PCR, and 28 of the tumors showed the GSTM1 null genotype. A significant association was found between GSTMl-null status and elevated inter-(simple sequence repeat) PCR instability. In contrast, no association was found with fractional allelic loss rate. The first exons of the K-ras and H-ras oncogenes were sequenced in 72 colorectal cancers; 19 of the tumors had a mutation in codon 12 of the K-ras gene (24.5%), but no H-ras mutations were found. A weak correlation (p=0.10) was observed between mutant K-ras and inter-(simple sequence repeat) PCR genomic instability, and no association existed with fractional allelic loss rate. PMID:15542115

  5. Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.

    Uno, I.; Mitsuzawa, H.; Matsumoto, K.; Tanaka, K; Oshima, T.; Ishikawa, T


    Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 pr...

  6. Basal but not luminal mammary epithelial cells require PI3K/mTOR signaling for Ras-driven overgrowth.

    Plichta, Kristin A; Mathers, Jessica L; Gestl, Shelley A; Glick, Adam B; Gunther, Edward J


    The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment of origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic three-dimensional culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted coexpression of oncogenic H-RAS(G12V) together with a nuclear fluorescent reporter. Monitoring of H-RAS(G12V)-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RAS(G12V) drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiologic modes used during growth factor-initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase or the mammalian target of rapamycin pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia. PMID:23010075

  7. Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

    Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU → GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.

  8. Altered lower leg muscle activation patterns in patients with cerebral palsy during cycling on an ergometer

    Alves-Pinto, Ana; Blumenstein, Tobias; Turova, Varvara; Lampe, Renée


    Objective Cycling on a recumbent ergometer constitutes one of the most popular rehabilitation exercises in cerebral palsy (CP). However, no control is performed on how muscles are being used during training. Given that patients with CP present altered muscular activity patterns during cycling or walking, it is possible that an incorrect pattern of muscle activation is being promoted during rehabilitation cycling. This study investigated patterns of muscular activation during cycling on a recumbent ergometer in patients with CP and whether those patterns are determined by the degree of spasticity and of mobility. Methods Electromyographic (EMG) recordings of lower leg muscle activation during cycling on a recumbent ergometer were performed in 14 adult patients diagnosed with CP and five adult healthy participants. EMG recordings were done with an eight-channel EMG system built in the laboratory. The activity of the following muscles was recorded: Musculus rectus femoris, Musculus biceps femoris, Musculus tibialis anterior, and Musculus gastrocnemius. The degree of muscle spasticity and mobility was assessed using the Modified Ashworth Scale and the Gross Motor Function Classification System, respectively. Muscle activation patterns were described in terms of onset and duration of activation as well as duration of cocontractions. Results Muscle activation in CP was characterized by earlier onsets, longer periods of activation, a higher occurrence of agonist–antagonist cocontractions, and a more variable cycling tempo in comparison to healthy participants. The degree of altered muscle activation pattern correlated significantly with the degree of spasticity. Conclusion This study confirmed the occurrence of altered lower leg muscle activation patterns in patients with CP during cycling on a recumbent ergometer. There is a need to develop feedback systems that can inform patients and therapists of an incorrect muscle activation during cycling and support the training

  9. Galectin-3 mediates cross-talk between K-Ras and Let-7c tumor suppressor microRNA.

    Ran Levy

    Full Text Available BACKGROUND: Galectin-3 (Gal-3 and active (GTP-bound K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60% of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50% of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1 induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.

  10. K-Ras and mitochondria: Dangerous liaisons

    Jiri Neuzil; Jakub Rohlena; Lan-Feng Dong


    It is well documented that the KRAS oncogene efficiently transforms non-malignant cells,and there is some evidence for the role of mitochondria in this process.Now Peng Huang and colleagues show that K-Ras induction results early on in mitochondria assuming the phenotype consistent with the so-called Warburg effect,i.e.,increased glycolysis and attenuated oxidative phosphorylation.Thus the K-Ras protein capable of swift induction of phenotypic changes typical of cancer cells,yet these changes are reversible,and for cells to irreversibly reach their full malignant potential a much longer K-Ras expression is required,implicating mitochondria in the longer-term effects of the oncogene.

  11. CD8(+) T cells mediate RAS-induced psoriasis-like skin inflammation through IFN-γ.

    Gunderson, Andrew J; Mohammed, Javed; Horvath, Frank J; Podolsky, Michael A; Anderson, Cherie R; Glick, Adam B


    The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RAS(V12G) in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses, and increased T helper type 1 (Th1)/Th17 and T cell type 1 (Tc1)/Tc17 skin infiltration. The majority of skin-infiltrating CD8(+) T cells coexpressed IFN-γ and IL-17A. When RAS was expressed on a Rag1-/- background, microabscess formation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation were suppressed. Depletion of CD8(+), but not CD4(+), T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17(+) γδ T cells, and epidermal hyperproliferation to levels similar to a Rag1-/- background. Reconstitution of Rag1-/- inducible RAS mice with purified CD8(+) T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ, but not of IL-17A, in CD8(+) T-cell-reconstituted Rag1-/- mice expressing RAS blocked CD8-mediated skin inflammation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation. These results show that CD8(+) T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ. PMID:23151849

  12. Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling

    Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients

  13. Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

    Baldwin, Kenneth M.; Haddad, Fadia


    The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

  14. Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

    Soon-Jeong JEONG; Su-Gwan KIM; Jiyun YOO; Mi-Young HAN; Joo-Cheol PARK; Heung-Joong KIM; Seong Soo KANG; Baik-Dong CHOI; Moon-Jin JEONG


    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells.Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins.The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ interacted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway,was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  15. Alterations in the activity and structure of pectin methylesterase treated by high pressure carbon dioxide.

    Zhou, Linyan; Wu, Jihong; Hu, Xiaosong; Zhi, Xian; Liao, Xiaojun


    The influence of high pressure carbon dioxide (HPCD) on the activity and structure of pectin methylesterase (PME) from orange was investigated. The pressures were 8-30 MPa, temperature 55 degrees C and time 10 min. HPCD caused significant inactivation on PME, the lowest residual activity was about 9.3% at 30 MPa. The SDS-PAGE electrophoretic behavior of HPCD-treated PME was not altered, while changes in the secondary and tertiary structures were found. The beta-structure fraction in the secondary structure decreased and the fluorescence intensity increased as HPCD pressures were elevated. After 7-day storage at 4 degrees C, no alteration of its activity and no reversion of its beta-structure fraction were observed, while its fluorescence intensity further decreased. PMID:19256556

  16. Byzantine seals from the Ras fortress

    Ivanišević Vujadin


    Full Text Available In this paper, seals found at the location of the Ras fortress (Tvrđava Ras have been published. Inscriptions on these seals show that they used to belong to persons which could be identified with certain military commanders who served under Alexios I Komnenos. The seals in question are: the seals of protonobelissimos Eustathios Kamytzes, Constantine Dalassenos Doukas, protoproedros and doux Constantine Kekaumenos and a certain person called Alexios. [Projekat Ministarstva nauke Republike Srbije, br. 177021 i br. 177032

  17. RAS Symposium Draws Hundreds of Attendees | Poster

    They call themselves “rasologists”: scientists who study the RAS family of genes and the cancers that can arise due to mutations within them. This field of research is at the heart of some sobering numbers. Almost a third of all human cancers, including 95 percent of pancreatic cancers, are driven by mutated RAS genes. The American Cancer Society estimates there were 48,960 new cases of pancreatic cancer in the United States in 2015 and 40,560 deaths from the disease.

  18. RAS in Pregnancy and Preeclampsia and Eclampsia

    M. Rodriguez


    Full Text Available Preeclampsia is a common disease of pregnancy characterized by the presence of hypertension and commitment of many organs, including the brain, secondary to generalized endothelial dysfunction. Its etiology is not known precisely, but it involved several factors, highlighting the renin angiotensin system (RAS, which would have an important role in the origin of multisystem involvement. This paper reviews the evidence supporting the involvement of RAS in triggering the disease, in addition to the components of this system that would be involved and how it eventually produces brain engagement.

  19. Prenatal immune activation alters hippocampal place cell firing characteristics in adult animals.

    Wolff, Amy R; Bilkey, David K


    Prenatal maternal immune activation (MIA) is a risk factor for several developmental neuropsychiatric disorders, including autism, bipolar disorder and schizophrenia. Adults with these disorders display alterations in memory function that may result from changes in the structure and function of the hippocampus. In the present study we use an animal model to investigate the effect that a transient prenatal maternal immune activation episode has on the spatially-modulated firing activity of hippocampal neurons in adult animals. MIA was induced in pregnant rat dams with a single injection of the synthetic cytokine inducer polyinosinic:polycytidylic acid (poly I:C) on gestational day 15. Control dams were given a saline equivalent. Firing activity and local field potentials (LFPs) were recorded from the CA1 region of the adult male offspring of these dams as they moved freely in an open arena. Most neurons displayed characteristic spatially-modulated 'place cell' firing activity and while there was no between-group difference in mean firing rate between groups, place cells had smaller place fields in MIA-exposed animals when compared to control-group cells. Cells recorded in MIA-group animals also displayed an altered firing-phase synchrony relationship to simultaneously recorded LFPs. When the floor of the arena was rotated, the place fields of MIA-group cells were more likely to shift in the same direction as the floor rotation, suggesting that local cues may have been more salient for these animals. In contrast, place fields in control group cells were more likely to shift firing position to novel spatial locations suggesting an altered response to contextual cues. These findings show that a single MIA intervention is sufficient to change several important characteristics of hippocampal place cell activity in adult offspring. These changes could contribute to the memory dysfunction that is associated with MIA, by altering the encoding of spatial context and by

  20. The RAS Problem: Turning Off a Broken Switch

    The RAS gene is commonly mutated in cancer and researchers are working to better understand how to develop drugs that can target the RAS protein, which for many years has been considered to be “undruggable.”

  1. RAS Projects at the NCI Frederick National Lab

    The RAS Initiative involves a number of projects focusing on ways to inhibit the proliferation of cancer cells by mutant RAS proteins. Projects are conducted at the FNLCR hub, with collaboration from the research community nationwide.

  2. Society News: Monica Grady awarded CBE; Grubb Parsons Lecture 2012; Join the RAS; Astronomy on radio for kids; New Fellows; Peter D Hingley


    RAS Fellow Prof. Monica Grady has been made a Commander of the Most Excellent Order of the British Empire (CBE), in recognition of her services to space science. The RAS sponsors the annual Grubb Parsons Lecture, which this year took place on 6 June at the University of Durham. If you are a professional astronomer, geophysicist, or similar, a student studying these disciplines, or simply someone with a serious interest in them, we urge you to apply for membership of the RAS. Outreach is an important activity for the RAS. We recently supported an astronomy series called Deep Space High on the digital radio channel Fun Kids.

  3. Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis

    K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN ≥ 2.6 and < 2.6 respectively (p = 0.002) and in 10 (36%) and 1 (6%) cases with a CISH EGFR GCN ≥ 2.12 and < 2.12 respectively (p = 0.03). Median TTP was 7.7 and 6.4 months in patients showing increased FISH and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively). FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab

  4. CD8+ T cells Mediate RAS-induced Psoriasis-like Skin Inflammation Through IFN-γ

    Gunderson, Andrew J.; Mohammed, Javed; Horvath, Frank; Podolsky, Michael, J.; Anderson, Cherie; Glick, Adam B.


    The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RASV12G in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses and increased Th1/Th17 and Tc1/Tc17 skin infiltration. The majority of skin infiltrating CD8+ T cells co-expressed IFN-γ and IL-17A. When RAS was exp...

  5. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents.

    Melville B Vaughan

    Full Text Available BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent. METHODOLOGY/PRINCIPAL FINDINGS: Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras, were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63, basement-membrane formation (collagen IV, laminin-5, and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes. CONCLUSIONS/SIGNIFICANCE: The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

  6. Copy number variants including RAS pathway genes-How much RASopathy is in the phenotype?

    Lissewski, Christina; Kant, Sarina G; Stark, Zornitza; Schanze, Ina; Zenker, Martin


    The RASopathies comprise a group of clinically overlapping developmental syndromes the common pathogenetic basis of which is dysregulated signal flow through the RAS-MAPK pathway. Mutations in several components or modifiers of the pathway have been identified in Noonan syndrome and related disorders. Over the past years copy number variants (CNVs) encompassing RAS pathway genes (PTPN11, RAF1, MEK2, or SHOC2) have been reported in children with developmental syndromes. These observations raised speculations that the associated phenotypes represent RASopathies, implying that the increased or reduced expression of the respective RAS pathway component and a consecutive dysregulation of RAS pathway signalling is responsible for the clinical picture. Herein, we present two individuals and three of their relatives harboring duplications of either 3p25.2 including the RAF1 locus or 19p13.3 including the MEK2 locus. Duplication carriers exhibited variable clinical phenotypes including non-specific facial dysmorphism, short stature, and learning difficulties. A careful review of the literature supported the impression that phenotypes associated with CNVs including RAS pathway genes commonly share non-specific symptoms with RASopathies, while the characteristic "gestalt" is lacking. Considering the known molecular pathogenesis of RASopathies, it is questionable that a modest increase in the expression of a functionally normal signaling component can mimic the effects of a qualitatively abnormal (hyperactive) mutant protein. We thus argue that current empirical and biological evidence is still insufficient to allow the conclusion that an altered copy number of a RAS pathway component is indeed the mechanism that is critical for the phenotype associated with CNVs including RASopathy genes. PMID:25974318

  7. Status of neutron complex of INR RAS

    The neutron complex of INR RAS consists of two sources of neutrons, beam stop, lead slowing down spectrometer and solid state spectrometers. The description of objects and their condition, the program of planned researches, co-operation with other institutes of the Moscow Region, progress reached for last two years are introduced in the article. (author)

  8. Reducing Cost: RAP and RAS Considerations

    Huber, Gerry


    This session will discuss of how local government decision-makers can achieve greater value without sacrificing quality or service life. The session will touch upon use of Higher Recycled Asphalt Pavement (RAP), Recycled Asphalt Shingles (RAS), Asphalt Mix Design for Heavy Traffic vs. Light Traffic, Speed of Construction, Perpetual Pavement and Pavement Rehabilitation Strategies.

  9. Alteration and modulation of protein activity by varying post-translational modification

    Thompson, David N.; Reed, David W.; Thompson, Vicki S.; Lacey, Jeffrey A.; Apel, William A.


    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

  10. Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

    Carroll, Ian M.; Ringel-Kulka, Tamar; Ferrier, Laurent; Wu, Michael C.; Siddle, Jennica P.; Bueno, Lionel; Ringel, Yehuda


    Objective: Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear. Aim: The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples. Design: In order to capture a wide range of fec...