Biomedical subjects

M Symons

Publications and source records attributed to M Symons.

At least 37 records · Page 2Linked to original sources

PubMed1998

Mas oncogene signaling and transformation require the small GTP-binding protein Rac.

The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.

3T3 Cells↗

PubMed1997

Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular signal-regulated kinase pathways.

Directed cell migration is essential for a variety of important biological processes ranging from development and angiogenesis to metastasis. Ras plays a pivotal role in the signaling cascade that governs chemotaxis of fibroblasts toward platelet-derived growth factor-BB (PDGF-BB). Ras activates multiple downstream pathways, which include the extracellular signal-regulated kinase (ERK), Rac, and Ral signaling cascades. We therefore investigated the role of the Rac and ERK pathways in cell migration. We showed that migration of fibroblasts toward PDGF-BB is inhibited by expression of dominant negative Asn-17 Rac1. Blocking of the ERK pathway by either expression of dominant negative Ala-218/Ala-222-mitogen-activated protein kinase kinase (A218/A222-MEK1) or by a MEK-specific inhibitor did not inhibit migration toward PDGF-BB. In contrast, migration toward soluble fibronectin was suppressed by inhibition of the ERK pathway but not by Asn-17 Rac1 expression. These results indicate that directed cell migration mediated by different receptor classes in response to different ligands differentially utilizes the Rac and ERK pathways and suggest that Rac might play a critical role in pathological processes such as angiogenesis and metastasis.

Animals↗

PubMed1997

Role of Rho family proteins in phospholipase D activation by growth factors.

Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD). In order to determine the role of the Rho family of small GTPases in growth factor-mediated PLD activation, we used cells transfected with wild type and mutant Rac1. In response to epidermal growth factor (EGF), PLD activity was greatly increased in Rat1 fibroblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1, consistent with Rac1 mediating the action of this growth factor. In contrast, in cells treated with platelet-derived growth factor (PDGF) or phorbol ester, the wtRac1 cells showed little or no enhancement of PLD activity, and the response was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C. Both growth factors produced an attenuated PLD response in cells expressing constitutively active V12Rac1, but these cells showed other changes, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation. The effects of EGF and PDGF on phosphoinositide phospholipase C activity were not enhanced in cells expressing wtRac1 or inhibited in those expressing N17Rac1. In cells expressing constitutively active V12Rac1, basal phosphoinositide phospholipase C was elevated, but there were no significant effects of EGF or PDGF. We used C3 transferase of Clostridium botulinum, which ADP-ribosylates and inactivates RhoA, to investigate the involvement of RhoA in the activation of PLD by PDGF. Cells expressing wtRac1 and N17Rac1 showed a decreased PLD in response to PDGF when treated with C3 transferase, indicating a role for RhoA. In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGF or protein kinase C.

Animals↗

PubMed1997

Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.

Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.

3T3 Cells↗

PubMed1997

Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation.

The Rho family members Cdc42, Rac, and Rho play a central role in the organization of the actin cytoskeleton and regulate transcription. Whereas Rac and Rho have been implicated in transformation by oncogenic Ras, the role of Cdc42 in this process remains unknown. In this study, we found that Rat1 fibroblasts expressing constitutively active V12-Cdc42 were anchorage independent and proliferated in nude mice but failed to show enhanced growth in low serum. Similar to V12-Rac1-expressing Rat1 fibroblasts, V12-Cdc42 lines displayed a high frequency of multinucleated cells. Interestingly, coexpression of dominant negative N17-Rac1 blocked the V12-Cdc42-induced multinucleated phenotype but not growth in soft agar, indicating that Cdc42 controls anchorage independence in a Rac-independent fashion. We also showed that dominant negative N17-Cdc42 inhibited Ras focus formation and anchorage-independent growth and caused reversion of the transformed morphology, indicating that Cdc42 is necessary for Ras transformation. N17-Cdc42 caused only partial inhibition of Ras-induced low-serum growth, however. In contrast, whereas N17-Rac1 also effectively inhibited Ras-induced anchorage independence, it did not revert the morphology of Ras-transformed cells. N17-Rac1 strongly inhibited low-serum growth of Ras-transformed cells, however. Together, these data provide a novel function for Cdc42 in cell proliferation and indicate that Cdc42 and Rac play distinct roles in growth control and Ras transformation.

3T3 Cells↗

PubMed1997

A search for the intermediate radical, ONOO, in the reaction between oxygen and nitric oxide in solution.

The most probable initial reaction between .NO and O2 is direct addition to give the peroxyl radical ONOO.. In view of the potential importance of this radical in biology, we have searched extensively for its formation, using EPR spectroscopy and rapid freezing techniques. At best, only extremely low concentrations were detected, and in most systems, no signals were detectable. We conclude that this radical is unlikely to be of major importance per se in biological systems, in contrast with its one electron adduct, the peroxynitrite anion.

Electron Spin Resonance Spectroscopy↗

PubMed1996

The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades.

PEA3, a member of the Ets family of transcriptional regulatory proteins, binds to the PEA3 promoter element and stimulates transcription through this site. The activity of the PEA3 element is regulated by mitogens, activated receptor tyrosine kinases, and oncogenic members of the Ras signal transduction pathway. However, it is not clear whether PEA3 mediates transcriptional regulation by these agents because a number of different Ets proteins can functionally interact with the PEA3 element. To specifically learn whether the activity of PEA3 is regulated, we investigated the ability of constitutively-activated Ras (Ha-RasV12) and signaling proteins downstream of Ras to alter PEA3-dependent reporter gene expression in COS cells. Ha-RasV12 and activated proteins in both the extra-cellular regulated kinase (ERK) and the stress-activated protein kinase (SAPK) or Jun N-terminal kinase (JNK) cascades independently stimulated PEA3-mediated gene expression. Ha-RasV12 stimulation of PEA3 activity was reduced by dominant-negative mutants in each of these protein kinase cascades, suggesting that Ras activates PEA3 through both pathways. Furthermore, the ability of unique activators of each kinase cascade to stimulate PEA3-dependent gene expression was selectively reduced by dominant-negative mutants within the homologous but not the heterologous pathway. Hence two distinct mitogen-activated protein kinase (MAPK) cascades regulate PEA3 activity. PEA3 was phosphorylated in vivo at serine residues consistent with the possibility that it may be a direct target of MAPKs.

Animals↗

PubMed1996

Activation of Na+-H+ exchange is necessary for RhoA-induced stress fiber formation.

The ubiquitously expressed Na+-H+ exchanger isoform, NHE1, functions in regulating intracellular pH and cell volume. We recently determined that the GTPase Galpha13 stimulates NHE1 activity through a RhoA-dependent mechanism (Hooley, R., Yu, C.-Y., Symons, M., and Barber, D. L. (1996) J. Biol. Chem. 271, 6152-6158). RhoA belongs to the Ras superfamily of GTPases and is a key regulator of actin stress fiber formation. We therefore investigated the relationship between RhoA, NHE1 activity, and the regulation of stress fiber assembly. Using two independent approaches, pharmacological inhibition of NHE1 and NHE1-deficient cells, we determined that the induction of stress fibers by lysophosphatidic acid and RhoA is dependent on increased NHE1 activity. These results indicate that stimulation of NHE1 acts downstream of RhoA in a pathway that controls stress fiber formation.

Actins↗

PubMed1996

G alpha 13 stimulates Na+-H+ exchange through distinct Cdc42-dependent and RhoA-dependent pathways.

Activity of the ubiquitously expressed Na+-H+ exchanger subtype NHE1 is stimulated upon activation of receptor tyrosine kinases and G protein-coupled receptors. The intracellular signaling pathways mediating receptor regulation of the exchanger, however, are poorly understood. Using transient expression of dominant interfering and constitutively active alleles in CCL39 fibroblasts, we determined that the GTPases Ha-Ras and Galpha 13 stimulate NHE1 through distinct signaling cascades. Exchange activity stimulated by constitutively active RasV12 occurs through a Rafl- and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase (MEK)-dependent mechanism. Constitutively active Galpha 13QL, recently shown to stimulate the Jun kinase cascade, activates NHE1 through a Cdc42- and MEK kinase (MEKK1)-dependent mechanism that is independent of Rac1. Constitutively active Rac1V12 does stimulate NHE1 through a MEKK1-dependent mechanism, but dominant interfering Rac1N17 does not inhibit Galpha 13QL-mediated or constitutively active Cdc42V12-mediated stimulation of the exchanger. Conversely, Cdc42NI7 does not inhibit Rac1V12 activation of NHE1, suggesting that Rae I and Cdc42 independently regulate a MEKK1-dependent activation of the exchanger. Rapid (<10 min) stimulation of NHE1 with a Ga13/Gaz chimera also was inhibited by a kinase-inactive MEKK. Galpha 13QL, but not RasV12, also stimulates NHE1 through a RhoA-dependent pathway that is independent of MEKK, and microinjection of mutationally active Galpha 13 results in a Rho phenotype of increased stress fiber formation. These findings indicate a new target for Rho-like proteins: the regulation of H+ ex- change and intracellular pH. Our findings also suggest that a MEKK cascade diverges to regulate effectors other than transcription factors.

Animals↗

PubMed1996

Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization.

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

Actins↗

PubMed1996

Rho family GTPases: the cytoskeleton and beyond.

Until recently, members of the Rho family of GTPases were considered primarily to be regulators of the distinct structures making up the actin cytoskeleton. Several Rho GTPases have now also been shown to play an important role in cell transformation. In addition, Cdc42, Rac and Rho activate transcription, providing a possible mechanism for their role in growth control.

Actins↗

PubMed1995

A role for Rho in Ras transformation.

The small GTP-binding proteins Rac and Rho are key elements in the signal-transduction pathways respectively controlling the formation of lamellipodia and stress fibers induced by growth factors or oncogenic Ras. We recently reported that Rac function is necessary for Ras transformation and that expression of constitutively activated Rac1 is sufficient to cause malignant transformation. We now show that, although expression of constitutively activated V14-RhoA in Rat 1 fibroblasts does not cause transformation on its own, it strongly cooperates with constitutively active RafCAAX in focus-formation assays in NIH 3T3 cells. Furthermore, dominant-negative N19-RhoA inhibits focus formation by V12-H-Ras and RafCAAX in NIH 3T3 cells, and stable coexpression of N19-RhoA and V12-H-Ras in Rat1 fibroblasts reverts Ras transformation. Interestingly, stress fiber formation is inhibited in V12-H-Ras lines and restored by coexpression of N19-RhoA. We conclude that Rho drives at least two separate pathways, one that induces stress fiber formation and another one that is important for transformation by oncogenic Ras.

Alkyl and Aryl Transferases↗

PubMed1995

The GTP-binding protein Rac does not couple PI 3-kinase to insulin-stimulated glucose transport in adipocytes.

BACKGROUND: In insulin-sensitive cells, such as adipocytes and skeletal muscle, the activation of phosphoinositide 3-kinase (PI 3-kinase) is thought to be critical in allowing insulin to stimulate both the uptake of glucose and the translocation of a specialized glucose transporter, GLUT4, to the plasma membrane. However, the downstream mediators that couple PI 3-kinase to GLUT4 translocation are still not known. Recent studies have shown that the GTP-binding protein Rac mediates some of the biological effects of PI 3-kinase, and these findings have led to the suggestion that Rac may be a common mediator for a variety of responses mediated by PI 3-kinase. To determine whether Rac couples PI 3-kinase to glucose uptake in adipocytes, we produced 3T3-L1 cells expressing either a constitutively active Rac1 (V12 Rac1, containing a valine residue at position 12) or a dominant-inhibitory Rac1 (N17 Rac1, containing an asparagine residue at position 17). RESULTS: The stable expression of both V12 Rac1 and N17 Rac1 led to observable phenotypes in 3T3-L1 cells; expression of V12 Rac1 resulted in constitutive formation of lamellipodia and constitutive activation of the cJun-N-terminal kinase (JNK), whereas expression of N17 Rac1 inhibited the insulin-stimulated formation of lamellipodia. However, neither basal glucose uptake nor insulin-stimulated glucose uptake was affected by the expression of either mutant Rac protein. In addition, expression of V12 Rac1 did not reverse the inhibition of insulin-stimulated glucose uptake caused by the PI 3-kinase inhibitor wortmannin. CONCLUSIONS: These findings provide direct evidence that PI 3-kinase does not use Rac to couple the insulin receptor to glucose uptake in adipocytes. Furthermore, the finding that Rac does not mediate glucose uptake in response to insulin is consistent with the idea that PI 3-kinase couples to a variety of different effector molecules in cells, and suggests that some of the specificity in the biological responses elicited by PI 3-kinase may be mediated by the activation of different effector molecules.

3T3 Cells↗

PubMed1995

A multileaf collimator field prescription preparation system for conventional radiotherapy.

PURPOSE: The purpose of this work is to develop a prescription preparation system for efficient field shaping using a multileaf collimator that can be used in community settings as well as research institutions. The efficiency advantage of the computer-controlled multileaf collimator, over cerrobend blocks, to shape radiation fields has been shown in conformal treatments, which typically require complete volumetric computerized tomographic data for three-dimensional radiation treatment planning--a utility not readily available to the general community. As a result, most patients today are treated with conventional radiation therapy. Therefore, we believe that it is very important to fully use the same efficiency advantage of multileaf collimator as a block replacement in conventional practice. METHODS AND MATERIAL: The multileaf collimator prescription preparation system developed by us acquires prescription images from different sources, including film scanner and radiation treatment planning systems. The multileaf collimator angle and leaf positions are set from the desired field contour defined on the prescription image, by minimizing the area discrepancies. Interactive graphical tools include manual adjustment of collimator angle and leaf positions, and definition of portions of the field edges that require maximal conformation. Data files of the final leaf positions are transferred to the multileaf collimator controller via a dedicated communication link. RESULTS: We have implemented the field prescription preparation system and a network model for integrating the multileaf collimator and other radiotherapy modalities for routine treatments. For routine plan evaluation, isodose contours measured with film in solid water phantom at prescription depth are overlaid on the prescription image. Preliminary study indicates that the efficiency advantage of the MLC over cerrobend blocks in conformal therapy also holds true for conventional treatments. CONCLUSION: Our model of computer-controlled prescription, evaluation, and treatment using multileaf collimators can be effectively implemented in both community settings and research institutions. The resultant increase in treatment efficiency and accuracy is now available for conventional radiotherapy.

Equipment Design↗

PubMed1995

Regulation of phospholipase D in HL60 cells. Evidence for a cytosolic phospholipase D.

Phospholipase D (PLD) activity that was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was detected in cytosol and membranes of HL60 cells. GTP gamma S-stimulated PLD activity was detected in the membranes when exogenous labeled phosphatidylcholine was used in the presence of phosphatidylethanolamine and phosphatidylinositol 4,5-bisphosphate, but not when [3H]myristic acid-labeled endogenous substrate was used. Cytosolic PLD co-chromatographed with small GTP-binding proteins on anion-exchange columns, but subsequent chromatography separated these. Reconstitution studies demonstrated ADP ribosylation factor (ARF) as a regulator of cytosolic PLD, whereas the Rho proteins RhoA and CDC42Hs were ineffective. The cytosolic enzyme showed very little activity in the absence of GTP gamma S and was stimulated by 2 mM Ca2+, whereas the membrane enzyme had significant basal activity and was inhibited by Ca2+. Rho-specific GDP dissociation inhibitor inhibited GTP gamma S stimulation of membrane PLD activity in the presence and absence of cytosol. The stimulation in GDP dissociation inhibitor-treated membranes could be partially recovered by the addition of recombinant Rho proteins (RhoA, Rac1, CDC42Hs). RhoA and Rac1 were also stimulatory in untreated membranes. However, Western blot analysis of membranes showed the presence of RhoA, but not Rac1 or CDC42Hs, suggesting that RhoA was the endogenous small GTP-binding protein involved in GTP-dependent PLD activity in membranes in the absence of cytosol. ARF also stimulated the membrane PLD in the presence of GTP gamma S, and the combination of RhoA and ARF showed a synergistic effect. These results show the presence of ARF-dependent PLD activity in both cytosol and membranes. The membranes contain another PLD activity for which the endogenous regulator appears to be RhoA. The data suggest the existence of at least two different PLD isozymes in HL60 cells.

Cell Line↗

PubMed1995

PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.

BACKGROUND: Phosphoinositide 3-kinases (PI 3-kinases) are thought to play an important role in coordinating the responses elicited by a variety of growth factors, oncogene products and inflammatory stimuli. These responses include activation of membrane ruffling, chemotaxis, glucose transport, superoxide production, neurite outgrowth and pp70 S6 kinase. Some of these responses are also known to be regulated by Rac, a small GTP-binding protein related to Ras. Neither the transducing elements upstream of Rac, nor those downstream of PI 3-kinase, have been defined. RESULTS: We show here that platelet-derived growth factor (PDGF) can stimulate an increase in the level of GTP-Rac by at least two distinct mechanisms: firstly, by increased guanine nucleotide exchange; and secondly, by inhibition of a Rac GTPase activity. The first of these mechanisms is essential for the activation of Rac, and we show that it is dependent upon PDGR-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphosphate. CONCLUSIONS: These results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.

Cell Line↗

PubMed1995

An essential role for Rac in Ras transformation.

The GTPase Rac1 is a key component in the reorganization of the actin cytoskeleton that is induced by growth factors or oncogenic Ras1. Here we investigate the role of Rac1 in cell transformation and show that Rat1 fibroblasts expressing activated Val-12 Rac1 (Rac1 with valine at residue 12) display all the hallmarks of malignant transformation. In a focus-forming assay in NIH3T3 fibroblasts to measure the efficiency of transformation, we found that dominant-negative Asn-17 Rac1 inhibited focus formation by oncogenic Ras, but not by RafCAAX, a Raf kinase targeted to the plasma membrane by virtue of the addition of a carboxyterminal localization signal from K-Ras. This indicates that Rac is essential for transformation by Ras. In addition, Val-12 Rac1 synergizes strongly with RafCAAX in focus-formation assays, indicating that oncogenic Ras drives both the Rac and MAP-kinase pathways, which cooperate to cause transformation.

3T3 Cells↗

PubMed1995

The Rac and Rho pathways as a source of drug targets for Ras-mediated malignancies.

The small GTP-binding proteins Rac and Rho are key control elements in the reorganization of the actin cytoskeleton induced by growth factors or oncogenic Ras. It has been established recently that Rac and Rho also play a crucial role in Ras transformation. This suggests that the elements in the pathways regulated by Rac and Rho are valid targets for cancer therapy. Several important components of those signaling pathways have now have identified.

Animals↗