Dominant Negative H-ras Mutant Research Articles

JNK signaling pathway is required for bFGF-mediated surface cadherin downregulation on HUVEC

Angiogenesis, the process of new blood vessel formation, is important in wound healing, inflammation, tumorigenesis and metastases. During this process, it is a critical step of the loosening of cellular interactions between endothelial cells, which are dependent on the architecture of adherens junction constructed by homophilic interactions of cell surface cadherins. Several studies suggested that the dynamic changes of cadherins are necessary during angiogenesis. However, the mechanism of cadherins regulation on endothelial cells requires further delineation. Here, we showed that basic fibroblast growth factor (bFGF), a pivotal pro-angiogenic factor, can downregulate typical cadherins (E-, N-, P- and VE-cadherin) expression on the surface of human umbilical vein endothelial cells (HUVECs) via FGF receptor 1 (FGFR1) signaling. The bFGF-mediated surface cadherin downregulation was significantly reversed only when the HUVECs were treated with JNK inhibitor (SP600125), but not ERK (PD98059) or p38 inhibitor (SB203580). Infecting HUVECs with a dominant negative H-Ras mutant (Ras S17N) interferes bFGF-mediated cadherin downregulation, and the result suggests that bFGF attenuates surface cadherin expression on HUVECs via FGFR1 and intracellular Ras-JNK signaling. However, after growth factors withdrawal, FGFR1 blockade or JNK inhibition for 16 h, cadherins were re-expressed on cell surface of HUVECs. But the mRNA or total protein of cadherins had no significant change, suggesting that the effect of bFGF on cadherin expression may work through a post-translational control. Our data first suggest that JNK participates in bFGF-mediated surface cadherin downregulation. Loss of surface cadherins may affect the cell–cell interaction between endothelial cells and facilitate angiogenesis.

Growth suppression of hepatocellular carcinoma by ad-enovirus-mediated transduction of dominant negative H-ras mutant: Kazuhiro Nouso, Tatsuya Fujikawa, Toru Onishi, Mayumi Taniyama, Yoshiyuki Kobayashi, Junko Yokoyama, Yoshitaka Takuma, Harushige Nakatsukasa, Tadashi Hanafusa, Yasuhiro Yumoto, Okayama Univ Grad Sch of Medicine and Dentistry, Okayama-city Japan; Hikaru Ueno, Univ of Occupational and Environmental Health Sch of Medicine, Kitakyushu-city Japan; Takao Tsuji, Okayama Univ

Growth suppression of hepatocellular carcinoma by ad-enovirus-mediated transduction of dominant negative H-ras mutant: Kazuhiro Nouso, Tatsuya Fujikawa, Toru Onishi, Mayumi Taniyama, Yoshiyuki Kobayashi, Junko Yokoyama, Yoshitaka Takuma, Harushige Nakatsukasa, Tadashi Hanafusa, Yasuhiro Yumoto, Okayama Univ Grad Sch of Medicine and Dentistry, Okayama-city Japan; Hikaru Ueno, Univ of Occupational and Environmental Health Sch of Medicine, Kitakyushu-city Japan; Takao Tsuji, Okayama Univ

Effects of ras and rap1 on electrical excitability of differentiated ng108-15 cells

Effects of two small G-proteins, Rap1 and Ras, on the sodium channel activity in NG108-15 cells were studied using sindbis virus-mediated gene transfer. When an activated Rap1A mutant (Rap1-12V, the activated mutant of Rap1 carrying glycine to valine substitution at codon 12) or a dominant-negative H-Ras mutant (Ras-17N, carrying serine to asparagine substitution at codon 17) was expressed in differentiated NG108-15 cells, the proportion of cells generating action potential decreased and the amplitudes of sodium current diminished. This effect was sensitive to an inhibitor of protein kinase A. The effects of a cyclic AMP (cAMP) analog (dibutyl cAMP) on sodium current in these cells were biphasic: inhibitory at lower concentrations (<100 μM) and enhancing at higher concentrations (200–500 μM). The inhibitory phase of cAMP effect was suppressed by an activated Ras mutant (Ras-12V) while the enhancing phase was suppressed by Rap1-12V. These data are consistent with the model that Rap1 and Ras function as counteracting regulators of voltage-gated sodium current through cAMP-dependent mechanisms.

Possible involvement of Rap1 and Ras in glutamatergic synaptic transmission.

Rap1A, first identified as a suppressor of transformed phenotype induced by an activated ras oncogene, is abundantly expressed in the brain. Its neurophysiological function, however, is poorly understood. When an activated Rap1A mutant (Rap1-12V) or a dominant negative H-Ras mutant (Ras-17N) was expressed in CA1 neurons in cultured hippocampal slices using the sindbis virus-mediated gene transfer technique, NMDA receptor current in response to Schaffer collateral stimulation was suppressed. Expression of activated H-Ras mutant (Ras-12V) resulted in the elevation of both NMDA receptor current and AMPA receptor current. These results implicate counteracting functions of Ras and Rap1 in the regulation of NMDA receptor-mediated synaptic transmission and a positive regulatory role of Ras in AMPA receptor-mediated synaptic transmission.

V-Crk activates the phosphoinositide 3-kinase/AKT pathway by utilizing focal adhesion kinase and H-Ras.

v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.

Adenovirus-mediated gene therapy for bladder cancer in an orthotopic model using a dominant negative H-ras mutant.

It has been suggested that abnormal Ras function is important in the carcinogenesis and progression of bladder cancer. Our aim was to investigate the efficacy of transurethral inoculation of an adenovirus expressing the dominant negative H-ras mutant N116Y against orthotopically implanted human bladder-cancer cells in nude mice. We used a replication-defective adenovirus vector containing the beta-galactosidase gene (AdCMV-LacZ) as a control and the N116Y gene (AdCMV-N116Y) as the therapeutic vector under the transcriptional control of the cytomegalovirus promoter. We initially investigated the in vitro growth-suppressive effects of AdCMV-N116Y on 2 human bladder-cancer cell lines, KU-7 and UMUC-2. Thereafter, we examined the inhibitory effects of AdCMV-N116Y on the 2 orthotopically implanted cell lines in nude mice. Intravesically created, orthotopic human bladder cancers were established in female KSN athymic nude mice with 1x 10(7) cancer cells. Then, 2, 3 and 4 days following implantation, 1 x 10(9) pfu of AdCMV-LacZ or AdCMV-N116Y were administered transurethrally. In vitro growth assays revealed significant growth suppression (>95%) with apoptosis of target cells treated with AdCMV-N116Y compared to AdCMV-LacZ. Transurethral inoculation of AdCMV-N116Y into the bladder brought about a significant reduction in size (73% to 90%) and number (47% to 78%) of orthotopically implanted human bladder tumors compared to AdCMV-LacZ or PBS. Normal mucosa in nude mice had minor inflammation with the infiltration of mononuclear cells. Our results suggest that gene therapy via transurethral inoculation of AdCMV-N116Y holds promise for the treatment of human bladder cancer.

The dominant negative H-ras mutant, N116Y, suppresses growth of metastatic human pancreatic cancer cells in the liver of nude mice

In pancreatic cancer, the mutation of c-K-ras is a critical event of tumor growth and metastasis. We have previously demonstrated a dominant negative effect of N116Y on the growth of pancreatic cancer cells. To evaluate the potential of N116Y for suppressing the metastatic growth of pancreatic tumor cells, we made a replication-deficient recombinant N116Y adenovirus driven by the carcinoembryonic antigen (CEA) promoter (Ad CEA-N116Y). We demonstrated that the expression of N116Y, growth inhibition, and apoptotic death induction were all specific to pancreatic cancer cell lines (PCI-35 and PCI-43) that were promoter positive, whereas no growth retardation was observed in human embryonic pancreas-derived cell line 1C3D3 after Ad CEA-N116Y infection. We examined the effect of Ad CEA-N116Y on the metastatic growth of PCI-43 colonies in liver, which was generated by tumor injection into the spleen of nude mice. The results showed that Ad CEA-N116Y effectively reduced the number of metastatic colonies without any complication by injecting intrasplenically 5 days after tumor cell inoculation. Thus, N116Y can selectively suppress the metastatic growth of pancreatic tumor cell by using the CEA promoter-driven adenovirus vector indicating that N116Y gene therapy may be potentially useful for the treatment of pancreatic cancer patients with liver micrometastasis.

Endothelin-mediated vascular growth requires p42/p44 mitogen-activated protein kinase and p70 S6 kinase cascades via transactivation of epidermal growth factor receptor.

Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, exerts a growth-promoting effect on vascular smooth muscle cells, implicating its pathogenic role in vascular remodeling. To gain insight into the cellular and molecular mechanism whereby ET-1 induces vascular growth, we studied whether transactivation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, are required for activation of p42/p44 mitogen-activated protein (MAP) kinase and p70 S6 kinase (p70S6K), and subsequent growth-promotion by ET-1 in cultured rat vascular smooth muscle cells. Immunoblotting with antiphosphotyrosine antibody revealed that ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR. ET-1 rapidly increased association of EGFR and Shc with glutathione-S-transferase-Grb2 fusion protein. The ET-1-induced activation of MAP kinase was reduced by an EGFR kinase inhibitor (AG1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG1296). AG1478 dose-dependently decreased ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake. The ET-1-induced tyrosine phosphorylation of EGFR, as well as MAP kinase activation, was inhibited by an ETA receptor antagonist and intracellular Ca2+ antagonists but not by an ETB receptor antagonist, pertussis toxin, or protein kinase C inhibitors. In addition, dominant negative mutant of H-Ras and a MAP kinase kinase (MEK-1) inhibitor (PD98059) completely blocked ET-1-induced MAP kinase activation as well as [3H]leucine and [3H]thymidine uptake. Both AG1478 and PD98059 inhibited ET-1-induced phosphorylation and activation of p70S6K. Furthermore, rapamycin, a selective inhibitor of mammalian target of rapamycin, completely blocked ET-1-stimulated [3H]leucine and [3H]thymidine uptake. These results suggest that ETA receptor-mediated vascular growth by ET-1 requires both MAP kinase and p70S6K cascades mediated partly via Ca2+-dependent EGFR transactivation.

CrkL Activates Integrin-Mediated Hematopoietic Cell Adhesion Through the Guanine Nucleotide Exchange Factor C3G

CrkL is a member of the Crk family of adapter proteins consisting mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in hematopoietic cells and has been implicated in pathogenesis of chronic myelogenous leukemia. However, its function has not been precisely defined. Here, we show that overexpression of CrkL enhances the adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced increase in cell adhesion was blocked by antibodies against VLA-4 (4β1) and VLA-5 (5β1) but was observed without changes in surface expression levels of these integrins. Studies using CrkL mutants demonstrated that the SH2 domain is partially required for enhancing cell adhesion, whereas the C-terminal SH3 domain as well as the tyrosine phosphorylation site (Y207) is dispensable. In contrast, the N-terminal SH3 domain, involved in binding C3G and other signaling molecules, was showed to play a crucial role, because a mutant defective of this domain showed an inhibitory effect on the cell adhesion to fibronectin. Furthermore, overexpression of C3G also increased the adhesion of hematopoietic cells to fibronectin, whereas a C3G mutant lacking the guanine nucleotide exchange domain abrogated the CrkL-induced increase in cell adhesion. On the other hand, a dominant negative mutant of H-Ras or that of Raf-1 enhanced the basal and CrkL-induced cell adhesion and that of R-Ras modestly decreased the adhesion. Taken together, these results indicate that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the guanine nucleotide exchange activity of C3G.

CrkL Activates Integrin-Mediated Hematopoietic Cell Adhesion Through the Guanine Nucleotide Exchange Factor C3G

AbstractCrkL is a member of the Crk family of adapter proteins consisting mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in hematopoietic cells and has been implicated in pathogenesis of chronic myelogenous leukemia. However, its function has not been precisely defined. Here, we show that overexpression of CrkL enhances the adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced increase in cell adhesion was blocked by antibodies against VLA-4 (α4β1) and VLA-5 (α5β1) but was observed without changes in surface expression levels of these integrins. Studies using CrkL mutants demonstrated that the SH2 domain is partially required for enhancing cell adhesion, whereas the C-terminal SH3 domain as well as the tyrosine phosphorylation site (Y207) is dispensable. In contrast, the N-terminal SH3 domain, involved in binding C3G and other signaling molecules, was showed to play a crucial role, because a mutant defective of this domain showed an inhibitory effect on the cell adhesion to fibronectin. Furthermore, overexpression of C3G also increased the adhesion of hematopoietic cells to fibronectin, whereas a C3G mutant lacking the guanine nucleotide exchange domain abrogated the CrkL-induced increase in cell adhesion. On the other hand, a dominant negative mutant of H-Ras or that of Raf-1 enhanced the basal and CrkL-induced cell adhesion and that of R-Ras modestly decreased the adhesion. Taken together, these results indicate that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the guanine nucleotide exchange activity of C3G.

Suppression of Erk activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y.

Our previous studies demonstrated that introduction of a dominant negative H-ras mutant, N116Y, inhibits the growth of various types of cancer cells in vitro. In this study, we tested the efficacy of N116Y in blocking the growth of esophageal cancer cells using an adenoviral vector. Infection with N116Y adenovirus, (AdCMV-N116Y), in which N116Y expression is driven by the cytomegalovirus promoter, significantly reduced the in vitro growth of all esophageal cancer cell lines studied. Esophageal cancer cells that contained wild-type K-ras and H-ras (TE8, SGF3, SGF7) were more sensitive to AdCMV-N116Y than HEC46 cells that expressed mutant K-ras protein. Most importantly, direct injection of AdCMV-N116Y into TE8- or SGF3-induced tumors in nude mice suppressed their growth significantly. To examine the suppressive mechanism of N116Y, cell cycle profile and the activation of extracellular signal-regulated kinase 2 (Erk2) were examined by flow cytometry and Western blot analysis, respectively. In TE8 cells, progression into S phase was clearly blocked after infection with AdCMV-N116Y. Infection with AdCMV-N116Y did not strongly suppress the activation of Erk2 after EGF stimulation in serum-starved HEC46 cells, whereas it completely suppressed activation in TE8, SGF3 and SGF7 cells. Our observations suggest that N116Y reduces growth of human esophageal cancer cells and suppresses the activation of Erk2; they also indicate that N116Y is a potential candidate gene for human esophageal cancer gene therapy.

The Significance of RAS Guanine Nucleotide Exchange Factor, Son of Sevenless Protein, in Renal Cell Carcinoma Cell Lines

Purpose The aim of the present study is to clarify the significance of the Ras guanine-nucleotide exchange reaction in the proliferation of human renal cell carcinoma cell lines. Materials and Methods We examined the expression of human son of sevenless-1 (hSos-1) protein and the epidermal growth factor (EGF) receptor in human cell carcinoma cell lines by Western blot analysis. Additionally, a dominant negative H-ras mutant, N116Y, which is known to inhibit the Ras guanine-nucleotide exchange reaction, was transfected into these cell lines by lipofection. Results Human renal cell carcinoma cell lines expressed much higher amounts of the EGF receptor and hSos-1 protein than normal kidney tissue. Moreover, the N116Y ras mutant could strongly suppress cellular proliferation in these cell lines. Conclusions Augmentation of the Ras guanine-nucleotide exchange reaction might be essential to the proliferation of human renal cell carcinoma cells.

The significance of Ras guanine nucleotide exchange factor, son of sevenless protein, in renal cell carcinoma cell lines.

The aim of the present study is to clarify the significance of the Ras guanine-nucleotide exchange reaction in the proliferation of human renal cell carcinoma cell lines. We examined the expression of human son of sevenless-1 (hSos-1) protein and the epidermal growth factor (EGF) receptor in human renal cell carcinoma cell lines by Western blot analysis. Additionally, a dominant negative H-ras mutant, N116Y, which is known to inhibit the Ras guanine-nucleotide exchange reaction, was transfected into these cell lines by lipofection. Human renal cell carcinoma cell lines expressed much higher amounts of the EGF receptor and hSos-1 protein than normal kidney tissue. Moreover, the N116Y ras mutant could strongly suppress cellular proliferation in these cell lines. Augmentation of the Ras guanine-nucleotide exchange reaction might be essential to the proliferation of human renal cell carcinoma cells.

A Human Protein Selected for Interference with Ras Function Interacts Directly with Ras and Competes with Raf1

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

Induction of Apoptosis by a Dominant Negative H-RAS Mutant (116Y) in K562 Cells

Recent extensive work on apoptosis has begun to reveal its molecular mechanisms. Several genes that regulate apoptosis have been identified. Among them, the BCL2 gene is considered to be an important gene that inhibits apoptosis. However, there must be other genes, yet to he identified, which suppress apoptosis. It has been suggested that the activation of RAS function by BCR-ABL fusion protein in chronic myelogenous leukemia may be an important mechanism in the BCR-ABL mediated transformation. Therefore, in this study we have investigated whether the suppression of endogenous H-RAS function inhibits the BCR-ABL mediated transforming activity in a K562 human chronic myelogenous leukemia cell line. The induced expression of a dominant negative v-H-RAS mutant (116Y) in K562 cells has resulted in cell death. The morphological characteristics and the detection of fragmented DNA by gel electrophoresis in the dead cells have revealed that this cell death is apoptosis. These results directly indicate that the RAS gene as well as the BCL2 gene has an ability to suppress apoptosis.