| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cell and Tumor Biology |
1 Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford and 2 Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut
Requests for reprints: Fernando Ferrer, Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, CT. Phone: 860-545-9658; Fax: 860-545-9545; E-mail: fferrrer{at}ccmckids.org/.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
protein they couple to intracellularly (5). Unlike S1P1 that only couples to Rac via Gi, S1P2 and to a lesser extent S1P3 also couple and stimulate Rho via G12/13 (6). S1P4, which is highly expressed in the lymphoid system, couples to Gi and even more effectively to G12/13 but not to Gq (7). G protein–coupling properties of S1P5, which is almost exclusively expressed in neuronal tissue, is not well characterized at present. Thus, the ability of S1P to bind to multiple S1P receptors, which in turn activate multiple G protein–coupled pathways, creates a complex signaling system downstream of S1P receptors. Pathways controlling the activation of small GTPases Rac and Rho are of particular interest for their roles in cell migration during angiogenesis and cancer metastasis. The dichotomous effects of S1P on cell migration are cell type specific. S1P stimulates chemotaxis in vascular endothelial cells and embryonic fibroblasts that primarily express S1P1 but inhibits cell migration in vascular smooth muscle cells, neutrophils, and B16 melanoma cells that predominantly express S1P2 (8–12). Therefore, the balance of S1P receptor expression in any given cell seems to dictate its migration response to S1P. The model based on these observations ascribes Rac-dependent, chemoattractive responses primarily to Gi-coupled receptors S1P1 and S1P3 and chemorepellant activities via S1P2/G12/13-mediated activation of Rho. Recent evidence suggests an additional mechanism of Rac and Rho regulation, such that S1P-mediated activation of Rho by S1P2 leads to the inhibition of Rac (13). Studies by Takuwa et al. have supported the notion that S1P2-mediated inhibition of tumor cell migration and metastasis occurs as a consequence of down-regulation of cellular Rac. However, these results are largely based on work done in B16 mouse melanoma cells that are unique in that they constitutively express only S1P2 (14). Data derived from human tumor cell lines and tissues show that multiple S1P receptors are typically present in a cell. Thus, the net result of tumor cell response to S1P stimulation represents the aggregate effect of the various S1P receptors and their downstream effectors.
Glioblastoma multiforme is a locally aggressive tumor associated with a poor prognosis despite aggressive therapy. Recently, Van Brocklyn et al. established that glioma cells express S1P1, S1P2, and S1P3 and that S1P is a mitogen for glioblastoma multiforme cells (15). The authors also report that S1P enhances motility and invasion of glioblastoma cells independent of metalloproteinase secretion (16). Since these initial observations, we have more comprehensively quantitated the expression level of all of the S1P receptors and metabolizing enzymes in several human glioblastoma cell lines and studied the effects of S1P on glioblastoma multiforme proliferation and migration. Our data indicate that S1P has a profound and differential effect on glioblastoma cell migration dependent on S1P receptor, specifically S1P2, expression, and contrary to prior murine tumor studies, on Rho kinase activity without concomitant inhibition of Rac activation. These findings shed new light on our understanding of S1P regulation of tumor cell migration.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Quantitative reverse transcription-PCR. Total RNA was prepared as previously described using the RNA STAT-60 single-step isolation procedure as previously described (17). Total RNA was treated with DNase I before reverse transcription to ensure removal of all contaminating DNA. PCR primers were designed with PrimerExpress software developed by Applied Biosystems (Foster City, CA) for optimal product length, GC content, and melting temperature for quantitative reverse transcription-PCR (RT-PCR) using SYBR Green I DNA binding dye technology (18). Primers span at least one intron whenever possible to ensure exclusive amplification of cDNA. Duplicate SYBR Green PCR reactions for each sample were done using an ABI 7900HT instrument. All reactions underwent a final dissociation curve determination to ensure a single PCR product at the correct melting temperature. Fluorescence data were exported and quantitated using a statistical model that corrects for PCR efficiency for each reaction (19). Results are expressed relative to the internal control gene glyceraldehyde-3-phosphate dehydrogenase. RT-PCR analysis of minus RT reactions for all genes were negative demonstrating that the observed products detected were indeed cDNA and not amplified from contaminating genomic DNA (data not shown).
Cell migration assay. Migration assays were done in 96-well transwell chambers with 8-µm polycarbonate membrane filters (Neuroprobe, Gaithersburg, MD) separating the lower and upper culture compartments. Cells were serum starved in DMEM containing 0.1% fatty acid–free BSA for 2 hours before trypsinizing and plated in triplicate into the top chamber at 5 x 104 per well in 0.39 mL serum-free medium containing 0.1% fatty acid–free BSA (Sigma, St. Louis, MO). The bottom chamber contained various concentrations of S1P in serum-free medium containing 0.1% fatty acid–free BSA or medium alone or complete growth medium. Cells were allowed to migrate for 5 hours in a humidified chamber at 37°C with 5% CO2. After the incubation period, the filter was removed and nonmigrated cells on the upper side of the filter were removed with a cotton swab. The filters were fixed overnight with 4% formaldehyde in PBS at 4°C. Attached cells were stained with 0.1% crystal violet and quantitated by densitometric analysis using ImageQuant software (Molecular Dynamics). Absorbance in pixel values for equivalent areas of triplicate wells were averaged and used to calculate fold migration relative to control.
Rac and Rho activation assays. The GTPase-binding domain of p21-activated kinase (PAK; amino acids, 67-150) or rhotekin (C21; amino acids, 7-89; a kind gift of Dr. Shuh Narumiya, Kyoto University, Kyoto, Japan) were obtained in a bacterial expression vector and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST; refs. 20, 21). All steps were done on ice or at 4°C. Bacteria from 500 mL of 2x LB culture containing 50 µg/mL Ampicillin and 0.1 mmol/L isopropyl-L-thio-B-D-galactopyranoside were pelleted and lysed with bacteria lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 100 mmol/L NaCl, 5% glycerol, 0.1% Triton X-100, 1 mmol/L DTT, and 0.1 mmol/L phenylmethylsulfonyl fluoride]. GST-fusion proteins were purified from cleared bacterial cell lysates by adsorption to glutathione-Sepharose beads for 1 hour. Beads were washed with bacterial lysis buffer and aliquoted to 50 µL bead volume per reaction before adding 500 µg of 100 nmol/L S1P-treated cell extract in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.5), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mol/L NaCl, 10 mmol/L MgCl2, 1x protease inhibitors] and brought to a total reaction volume of 800 µL with PBS. Extracts loaded with GTP-
-S were included as a positive control in all experiments. Affinity precipitation of GST-C21-RhoAGTP or GST-PAK-Rac1GTP was carried out for 1 hour at 4°C before washing thrice with Sander's bead wash [50 mmol/L Tris-HCl (pH 7.4), 0.1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L MgCl2, 10% glycerol, 1x protease inhibitors] and resuspending final bead pellet in 50 µL 4x Laemmli sample buffer. Proteins were separated on 12% SDS-PAGE gels, transferred to nitrocellulose membrane, and blotted with specific antibodies for RhoA (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or Rac1 (1:2,000, BD Biosciences, San Jose, CA). Equal loading was confirmed by blotting total extracts against RhoA or Rac1. Results were quantitated by densitometric analysis and expressed as fold above background.
Fluorescence microscopy. Cells cultured in fibronectin-coated, glass bottom 35-mm dishes were fixed in 4% formaldehyde, permeabilized with 0.2% Triton X-100, and stained with tetramethyl rhodamine B isothiocyanate–labeled phalloidin (Sigma) to visualize filamentous actin and counter stained with 4',6-diamidino-2-phenylindole to visualize nuclei. The cells were observed under on a Zeiss LSM 510 confocal microscope. The percentage of cells exhibiting stress fibers, cell rounding, or lamellipodia and membrane-ruffling structures were determined by counting the number of cells with each phenotype in five fields using standard fluorescence microscopy.
Statistical analysis. Data are presented as the mean ± SE and statistical differences between two groups were analyzed using Student's t test. For multiple comparisons one-way ANOVA with post hoc Dunnett's multiple comparisons correction where appropriate. P < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Enzymes that metabolize S1P may alter signaling through this pathway. We next measured the mRNA expression level of SK1, SK2, SPP, and SGPL in the same cell lines. As shown in Supplemental Fig. 1B, SK1, SK2, SPP, and SGPL were expressed at different levels. Transcripts for SK1 or SK2 were highest of the four genes examined in U87, U118, and U138 (Supplemental Fig. S1B). These data indicate that the level of gene expression for the S1P receptors and metabolizing enzymes is highly variable, and suggests that sphingolipid metabolism and S1P signaling may contribute to biological responses.
Differential effect of S1P on glioblastoma cell migration. S1P has been shown to either promote or inhibit cellular migration depending on the cell type examined (4). We therefore studied the role of S1P on glioblastoma migration in vitro and determined that S1P has a profound and differential effect on migration as shown in Fig. 1. U87 cells treated with 1 µmol/L S1P showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited (Fig. 1A). The effects of S1P on migration were dose-dependent and observed at 10 to 100 nmol/L concentrations, which is in the range of receptor activation (data not shown).
|
S1P2 mediates inhibition of migration in U118 cells. Because pertussis toxin was unable to block S1P-mediated inhibition of migration in U118 cells, a mechanism other than Gi is responsible. Interestingly, expression of S1P2/EDG-5, which couples to G12/13, correlated with the inhibitory effect on migration. For example, in U87 cells that express low levels of S1P2 mRNA, S1P is a potent inducer of migration (Fig. 2A). Conversely, in cell lines that express high levels of S1P2, S1P strongly inhibits migration, particularly in U118 and U138 cell lines (Fig. 2A). Indeed, in previous findings from Takuwa's laboratory, S1P2 was shown to inhibit migration of Chinese hamster ovary (CHO) cells and melanoma cells (22). To substantiate the notion that S1P2 is involved in the inhibition of glioblastoma cell migration, we tested the effect of the S1P analogue FTY720-phosphate (FTY720-P), which is an agonist for all S1P receptors except S1P2 (23). In U118 cells that express high levels of S1P2, 100 nmol/L FTY720-P was unable to significantly inhibit migration in U118 cells, whereas the same concentration of S1P was inhibitory (Fig. 2B). Concentrations of FTY720-P ranging from 10 nmol/L to 1 µmol/L were unable to inhibit U118 cell migration, suggesting that S1P-mediated inhibition occurs via S1P2 signaling pathways (Fig. 2C).
|
|
S1P2 siRNA treatment of U118 cells completely abrogated S1P inhibition of cell migration, whereas a control siRNA was without an effect (Fig. 3D). The specificity of S1P2 siRNA was further confirmed in a recovery experiment using S1P2 siRNA with the rat S1P2 adenovirus expression vector, which is not targeted by the human S1P2 siRNA. These cells were inhibited by S1P similarly to cells in Fig. 3A that overexpressed S1P2 (Fig. 3D). These data corroborate overexpression studies establishing that S1P2 mediates inhibition of migration of glioblastoma cells.
Lack of inhibition of the Rac GTPase in S1P-mediated inhibition of glioblastoma cell migration. Having established the requirement for S1P2 on S1P-mediated inhibition of migration in U118 cells, we investigated whether small GTPases such as Rho and Rac are involved. In particular, we checked for Rho-dependent inhibition of Rac, which was shown recently in melanoma and CHO cells (24). As shown in Supplemental Fig. S2, treatment of U118 cells with 100 nmol/L S1P led to a robust activation of Rho by 1 minute that was returned to baseline after for 30 minutes. Interestingly, Rac was also activated nearly 2-fold and did not return to baseline within 30 minutes of stimulation, despite the fact that S1P inhibits migration in these cells (Supplemental Fig. S2). To confirm these findings we did a similar time course in U118 cells overexpressing S1P2 or ß-galactosidase as a control (Fig. 4A). As shown in Fig. 4A, stimulation of U118 cells overexpressing S1P2 with 100 nmol/L S1P resulted in a robust activation of Rho and Rac. In fact, overexpression of S1P2 led to a more potent activation of Rac compared with ß-galactosidase transduced cells. To rule out cell type–specific effects on Rac and Rho, we also did overexpression experiments with the U87 cell line. As shown in Fig. 4B, a 1-minute stimulation of U87 cells overexpressing S1P2 led to a dramatic induction of activated Rho with a concomitant modest increase in Rac activity. Taken together, these results do not reveal any evidence for inhibition of Rac GTPase during S1P-mediated inhibition of migration in glioblastoma cells, further confirming that modulation of S1P2 receptor expression alone inhibits migratory responses.
|
|
|
Because U87 and U138 cell lines expressed relatively high levels of SK1 mRNA (Supplemental Fig. S1B), we investigated whether the SK inhibitor dimethyl-sphingosine (DMS) could influence the growth of these cells in vitro. DMS potently inhibited proliferation of U87 (high SK1) and U138 (lower SK1) at concentrations >1 µM (Supplemental Fig. S3B). DMS-treated cells rapidly died as evidenced by the cells rounding up and detaching from the dish. U138 cells behaved similarly (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We found that S1P has a profound and differential effect on glioblastoma motility. Only one cell line, U87, of three migrated in a pertussis toxin sensitive manner in response to S1P (Fig. 1). U118 and U138 cell lines were strongly inhibited by S1P (Fig. 1A). Inhibition of migration by S1P was insensitive to pertussis toxin, thereby implicating non-Gi linked signaling pathways (Fig. 1B). Our data support several reports in the literature that define Gi-linked S1P1 and S1P3 as chemoattractant receptors and G12/13-linked S1P2 as a chemorepellant receptor (24).
A major finding of the present study is the correlation between the level of S1P2 mRNA expression and degree of S1P-mediated inhibition of migration (Fig. 2). This correlation was confirmed in migration experiments using FTY720-P, an S1P analogue that binds to all S1P receptors except S1P2 (23), which was ineffective at inhibiting migration (Fig. 2B). FTY720-P does not significantly induce migration of U118 cells despite their expression of S1P3. This may be due to the fact that the affinity of FTY720-P for S1P receptors varies and is reported to be greatest for S1P1 (23). Consequently, a different effect between responses mediated via S1P1 versus S1P3 could be expected. Furthermore, unlike S1P1, S1P3 can also signal through G12/13 thereby inhibiting migration. Finally, despite having similar receptor profiles, tumor cells may exhibit varied responses to FTY720-P due to other signal transduction abnormalities. Our data agree with that obtained by Osada et al., who used a recently developed S1P2 antagonist, JTE-013, to reverse the inhibitory actions of S1P on migration in vascular endothelial cells and smooth muscles cells (27).
We proved that S1P2 mediates inhibition of migration in response to S1P by over expression of S1P2, as well as S1P2 gene silencing via siRNA (Fig. 3). Importantly, U118 cells are capable of S1P-stimulated migration as shown by adenoviral overexpression of S1P1 (Fig. 3A), so the effects of S1P are not due to the lack of downstream signaling machinery. Recent findings by Yamaguchi et al. show that S1P treatment of mouse B16 melanoma cells that express only S1P2 significantly reduced lung metastasis after tail vein injection, and these effects could be potentiated or aggravated by overexpression of S1P2 or S1P1, respectively (28). Because S1P2 expression varied in the three glioblastoma lines we tested, more comprehensive studies on primary tumors are warranted to determine whether S1P2 expression changes during tumor progression, and whether changes have value in predicting subsequent invasion, tumor sequelae development after surgery, and poor prognosis.
In the present study, we explored the mechanism of S1P2-mediated inhibition of migration in glioblastoma cells by examining downstream effectors of S1P2. Functional data for S1P2 indicates that this receptor couples primarily to G12 or G13, which activate the small GTPase Rho (5). Whereas it has been shown that Rac and Rho are both required for establishing cell polarity in terms of "frontness" and "backness," respectively, excessive Rho activity seems to inhibit polarization and motility (29). Rho mediates stress fiber formation and focal adhesion, processes associated with stationary cells, through a phosphorylation cascade beginning with p160 Rho kinase (ROCK), and ultimately impeding activity of cofilin and myosin light-chain phosphatase thereby stabilizing actin filaments (26). Not surprisingly, U118 cells stimulated with 100 nmol/L S1P resulted in a 5-fold induction of RhoA activity and 2-fold activation of Rac after 1 minute (Supplemental Fig. S2). Contrary to the model proposed by Takuwa et al., Rac was also activated in both cell lines even when S1P2 was overexpressed (Fig. 4). These data clearly indicate that S1P2-specific inhibition of Rac activity is not involved in S1P-mediated inhibition of migration in the human glioblastoma cell lines examined.
To gain further insight into events following Rho activation, we examined the effect of the specific ROCK inhibitor Y-27632 on S1P-mediated migration (Fig. 5). ROCK activity was found required for S1P-mediated inhibition of U118 cells but independent of S1P-induced migration of U87 (Fig. 5). Our data contradicts a recent report by Sugimoto et al. who did not observe any prevention of S1P-S1P2–mediated inhibition of migration by Y-27632 or a structurally unrelated Rho kinase inhibitor HA-1077 in CHO cells stably transfected with S1P2 (13). These disparate observations suggest that there exist both Rho-kinase–dependent and –independent mechanisms for Rho regulation of migration in different cell types.
The ability of cells to coordinately reorganize the actin cytoskeleton is a hallmark of motile cells. Confocal microscopy of actin-stained S1P-stimulated U118 cells that are inhibited to migrate in response to S1P resulted in dramatic stress fiber formation, as expected (Fig. 6A, e). U87 cells, on the other hand, are potently stimulated to migrate in response to S1P and showed lamellipodia and membrane ruffling structures in response to S1P, as anticipated (Fig. 6B, e). Our findings support results by Okamoto et al. who used CHO cells expressing S1P1 or S1P2 mediated lamellipodia and membrane ruffling or stress fibers, respectively, in response to S1P (22). Stress fiber formation was blocked in U118 cells pretreated with the Rho kinase inhibitor Y-27632, which agrees with previous findings by Sugimoto et al. using CHO cells overexpressing S1P2 (13). Confocal microscopy data correlates with migration assays with U118 and U87 cells showing that S1P-S1P2 receptor signaling results in ROCK-dependent inhibition of migration and stress fiber formation and, conversely, activation of S1P-S1P1 signaling pathways leads to stimulation of migration through Rac activation and lamellipodia formation.
Having established the requirement for S1P2 in S1P-mediated inhibition of migration, we were interested in knowing whether S1P2 also influenced the proliferative capacity of glioblastoma cells in vitro. Interestingly, a recent finding showed that S1P inhibits proliferation of rat hepatocytes by activating Rho via S1P2 (30). In proliferation experiments we consistently found S1P to be a weak mitogen for glioblastoma cell lines tested at doses ranging from 10 nmol/L to 1 µmol/L (Supplemental Fig. S3A). Our data on the proliferative capacity of U87 and U118 glioblastoma cells in response to S1P agrees with that observed by Van Brocklyn et al. who showed that U87 and U118 cells treated with 100 nmol/L S1P resulted in a 1.0- and 1.02-fold increase, respectively (15). We conclude that whereas S1P2 has an important role in glioblastoma cell migration, it does not correlate with proliferation.
Enzymes involved in S1P metabolism can perturb the levels of S1P. Previous reports in the literature have shown that overexpression of sphingosine kinase in NIH 3T3 cells leads to increased proliferation and acquisition of a transformed phenotype (31). We used a competitive inhibitor of sphingosine kinase, DMS, to assess whether high sphingosine kinase mRNA expression identified in U87 cells conferred a growth advantage (Supplemental Fig. S3B). Our results did not support a mechanism of enhanced growth potential in high SK expressing cells, because both U87 and U118 cells were equally inhibited by DMS (Supplemental Fig. S3B).
In conclusion, our results show that S1P2 mediates inhibition of migration in vitro in response to S1P, and this inhibition is dependent on Rho kinase activity but not concomitant inhibition of Rac activity. Manipulating S1P2 expression, receptor activity, or downstream effectors such as Rho-kinase may be a point for intervention in human cancers.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received 6/29/04. Revised 1/27/05. Accepted 2/21/05.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3- and ß1-containing integrins. J Biol Chem 2001;276:11830–7.This article has been cited by other articles:
|
|
 |
|
  G. Cattoretti, J. Mandelbaum, N. Lee, A. H. Chaves, A. M. Mahler, A. Chadburn, R. Dalla-Favera, L. Pasqualucci, and A. J. MacLennan Targeted Disruption of the S1P2 Sphingosine 1-Phosphate Receptor Gene Leads to Diffuse Large B-Cell Lymphoma Formation Cancer Res., November 15, 2009; 69(22): 8686 - 8692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Kapitonov, J. C. Allegood, C. Mitchell, N. C. Hait, J. A. Almenara, J. K. Adams, R. E. Zipkin, P. Dent, T. Kordula, S. Milstien, et al. Targeting Sphingosine Kinase 1 Inhibits Akt Signaling, Induces Apoptosis, and Suppresses Growth of Human Glioblastoma Cells and Xenografts Cancer Res., September 1, 2009; 69(17): 6915 - 6923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Skoura and T. Hla Regulation of vascular physiology and pathology by the S1P2 receptor subtype Cardiovasc Res, May 1, 2009; 82(2): 221 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Komachi, H. Tomura, E. Malchinkhuu, M. Tobo, C. Mogi, T. Yamada, T. Kimura, A. Kuwabara, H. Ohta, D.-S. Im, et al. LPA1 receptors mediate stimulation, whereas LPA2 receptors mediate inhibition, of migration of pancreatic cancer cells in response to lysophosphatidic acid and malignant ascites Carcinogenesis, March 1, 2009; 30(3): 457 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-F. Lee, S. Gordon, R. Estrada, L. Wang, D. L. Siow, B. W. Wattenberg, D. Lominadze, and M.-J. Lee Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H33 - H42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kimura, T. Ohmori, Y. Kashiwakura, R. Ohkawa, S. Madoiwa, J. Mimuro, K. Shimazaki, Y. Hoshino, Y. Yatomi, and Y. Sakata Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction * Supplemental Materials and Methods Stroke, December 1, 2008; 39(12): 3411 - 3417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Estrada, Q. Zeng, H. Lu, H. Sarojini, J.-F. Lee, S. P. Mathis, T. Sanchez, E. Wang, C. D. Kontos, C.-Y. Lin, et al. Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells J. Biol. Chem., October 31, 2008; 283(44): 30363 - 30375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Bartolome, N. Wright, I. Molina-Ortiz, F. J. Sanchez-Luque, and J. Teixido Activated G{alpha}13 Impairs Cell Invasiveness through p190RhoGAP-Mediated Inhibition of RhoA Activity Cancer Res., October 15, 2008; 68(20): 8221 - 8230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-H. Li, T. Sanchez, A. Pappalardo, K. R. Lynch, T. Hla, and F. Ferrer Induction of Antiproliferative Connective Tissue Growth Factor Expression in Wilms' Tumor Cells by Sphingosine-1-Phosphate Receptor 2 Mol. Cancer Res., October 1, 2008; 6(10): 1649 - 1656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Bryan, B. S. Paugh, D. Kapitonov, K. M. Wilczynska, S. M. Alvarez, S. K. Singh, S. Milstien, S. Spiegel, and T. Kordula Sphingosine-1-Phosphate and Interleukin-1 Independently Regulate Plasminogen Activator Inhibitor-1 and Urokinase-Type Plasminogen Activator Receptor Expression in Glioblastoma Cells: Implications for Invasiveness Mol. Cancer Res., September 1, 2008; 6(9): 1469 - 1477. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-i. Takashima, N. Sugimoto, N. Takuwa, Y. Okamoto, K. Yoshioka, M. Takamura, S. Takata, S. Kaneko, and Y. Takuwa G12/13 and Gq mediate S1P2-induced inhibition of Rac and migration in vascular smooth muscle in a manner dependent on Rho but not Rho kinase Cardiovasc Res, September 1, 2008; 79(4): 689 - 697. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tolle, A. Pawlak, M. Schuchardt, A. Kawamura, U. J. Tietge, S. Lorkowski, P. Keul, G. Assmann, J. Chun, B. Levkau, et al. HDL-Associated Lysosphingolipids Inhibit NAD(P)H Oxidase-Dependent Monocyte Chemoattractant Protein-1 Production Arterioscler Thromb Vasc Biol, August 1, 2008; 28(8): 1542 - 1548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Aoki, T. Yokoyama, K. Fujiwara, A. M. Tari, R. Sawaya, D. Suki, K. R. Hess, K. D. Aldape, S. Kondo, R. Kumar, et al. Phosphorylated Pak1 Level in the Cytoplasm Correlates with Shorter Survival Time in Patients with Glioblastoma Clin. Cancer Res., November 15, 2007; 13(22): 6603 - 6609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Novgorodov, M. El-Alwani, J. Bielawski, L. M. Obeid, and T. I. Gudz Activation of sphingosine-1-phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration FASEB J, May 1, 2007; 21(7): 1503 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Damirin, H. Tomura, M. Komachi, J.-P. Liu, C. Mogi, M. Tobo, J.-Q. Wang, T. Kimura, A. Kuwabara, Y. Yamazaki, et al. Role of lipoprotein-associated lysophospholipids in migratory activity of coronary artery smooth muscle cells Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2513 - H2522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wu, H. Chen, B. Parker, E. Rubin, T. Zhu, J. S. Lee, P. Argani, and S. Sukumar HOXB7, a Homeodomain Protein, Is Overexpressed in Breast Cancer and Confers Epithelial-Mesenchymal Transition Cancer Res., October 1, 2006; 66(19): 9527 - 9534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Salhia, F. Rutten, M. Nakada, C. Beaudry, M. Berens, A. Kwan, and J. T. Rutka Inhibition of Rho-Kinase Affects Astrocytoma Morphology, Motility, and Invasion through Activation of Rac1 Cancer Res., October 1, 2005; 65(19): 8792 - 8800. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), 200 nmol/L siRNA S1P2 (
), or a combination of 200 nmol/L siRNA S1P2 and adenovirus rat S1P2 (
). Points, fold over control for triplicate wells; bars, ±SE. Experiments were performed twice with similar results.
) were assayed for their ability to migrate in response to increasing doses of S1P. Points, fold over control for triplicate wells from an experiment that was repeated twice; bars, ±SE.