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PubMed2005

Resveratrol modulates gene expression associated with apoptosis, proliferation and cell cycle in cells with mutated human c-Ha-Ras, but does not alter c-Ha-Ras mRNA or protein expression.

An accumulating body of evidence suggests that resveratrol can inhibit carcinogenesis through antiproliferative and apoptotic effects. One proposed mechanism for this is the modulation of genes, for example, Ras and p53, frequently associated with human cancer. To test the effect of resveratrol on gene expression, we used the WR-21 cell line because it contains a mutated human c-Ha-ras gene. Cells at > or =70% confluency were incubated with media alone or with increasing concentrations of trans-resveratrol (0.1-1000 microM) for 24 h. Resveratrol (30-100 microM) decreased cellular proliferation by 80% (bromodeoxyuridine incorporation) and increased apoptosis by 60% (TUNEL). Cells were then treated with media alone or with 50-microM resveratrol for 24 h. RNA was isolated for nylon-based macroarray analyses and protein for immunoblotting. Resveratrol increased (+) and decreased (-) gene expression associated with apoptosis (Birc5+, Cash+, Mcl-1+, Mdm2+, Rpa-like+), cellular proliferation (Ctsd+, Mdm2+, Egr1+, ODC+) and cell cycle (cyclin D+, cyclin g+, Gadd45a-, Mad2l-, Mdm2+). Resveratrol consistently increased by > or =6-fold Mdm2 expression and other downstream p53 effectors, but not p53 itself at 24 h. Subsequent cell cycle analysis indicated a significant accumulation of cells in G2/M, and a decrease in G1/G0 suggesting a G2/M blockade. Further RT-PCR and Western blot analyses indicated no differential changes in Ras mRNA expression or p21(ras) protein levels, respectively. These results suggest that resveratrol potently inhibits cellular proliferation, increases apoptosis, alters cell cycle dynamics and modulates associated gene expression. Furthermore, these effects appear mediated, in part, by p53 without direct modulation of mutant c-Ha-ras expression.

Animals↗

PubMed2000

Identification, using molecular dynamics, of an effector domain of the ras-binding domain of the raf-p74 protein that is uniquely involved in oncogenic ras-p21 signaling.

By comparing the average structures, computed using molecular dynamics, of the ras-binding domain of raf (RBD) bound to activated wild-type ras-p21 and its homologous inhibitory protein, rap-1A, we formerly identified three domains of the RBD that changed conformation between the two complexes, residues 62-76, 97-110, and 111-121. We found that one synthetic peptide, corresponding to RBD residues 97-110, selectively inhibited oncogenic ras-p21-induced oocyte maturation. In this study, we performed molecular dynamics on the Val 12-ras-p21-RBD complex and compared its average structure with that for the wild-type protein. We find that there is a large displacement of a loop involving these residues when the structures of the two complexes are compared. This result corroborates our former finding that the RBD 97-110 peptide inhibits only signal transduction by oncogenic ras-p21 and suggests that oncogenic p21 uses this loop to interact with raf in a unique manner.

Amino Acid Substitution↗

PubMed2002

Inhibition of ras-induced oocyte maturation by peptides from ras-p21 and GTPase activating protein (GAP) identified as being effector domains from molecular dynamics calculations.

In the accompanying article, using molecular dynamics calculations, we found that the 66-77 and 122-138 domains in ras-p21 and the 821-827, 832-845, 917-924, 943-953, and 1003-1020 domains in GAP have different conformations in complexes of GAP with wild-type and oncogenic ras-p21. We have now synthesized peptides corresponding to each of these domains and coinjected them into oocytes with oncogenic p21, which induces oocyte maturation, or injected them into oocytes incubated with insulin that induces maturation by activating wild-type cellular ras-p21. We find that all of these peptides inhibit both agents but do not inhibit progesterone-induced maturation that occurs by a ras-independent pathway. The p21 66-77 and 122-138 peptides cause greater inhibition of oncogenic p21. On the other hand, the GAP 832-845 and 1003-1021 peptides inhibit insulin-induced maturation to a significantly greater extent. Since we have found that activated wild-type and oncogenic p21 activate downstream targets like raf differently, these GAP peptides may be useful probes for identifying elements unique to the wild-type ras-p21 pathway.

Amino Acid Sequence↗

PubMed1990

Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins.

We have previously identified a membrane factor capable of stimulating guanine nucleotide exchange activity for ras p21 proteins. The ras guanine nucleotide exchange factor (rGEF) was purified from bovine brain to near homogeneity by successive chromatographies on DE52 DEAE-cellulose, Sepharose 6B, hydroxylapatite, and FPLC phenyl-Superose resins. SDS/polyacrylamide gel electrophoresis of the purified rGEF showed a single major protein with a molecular mass of 35 kDa. rGEF increased the exchange rate of GDP in normal [Gly12]p21 or oncogenic [Val12]p21 up to 30- to 40-fold under physiological concentrations of Mg2+. Since the factor was free from GDP/GTP binding activity and nonspecific GDP hydrolytic activity, we propose that rGEF may regulate GDP/GTP exchange reaction of ras proteins in response to external growth signals. Moreover, rGEF enhanced the dissociation of bound GDP from some of ras-like G proteins, R-ras, rap1-A, rab1-B, and rho proteins, raising the possibility that rGEF may affect the activities of these proteins.

Animals↗

PubMed1991

ras isoprenylation is required for ras-induced but not for NGF-induced neuronal differentiation of PC12 cells.

We have used compactin, an inhibitor of mevalonate biosynthesis, to block p21ras posttranslational modification and membrane association in PC12 cells. Previous studies have demonstrated a requirement for isoprenylation for mitogenic effects of activated p21ras in mammalian cells and for function of RAS gene products in yeast. Immunoprecipitation of [35S]methionine-labeled p21ras from PC12 cell homogenates confirmed that the processed p21ras species is missing from compactin-treated PC12 cells. Immunoprecipitation from particulate and cytosolic fractions of PC12 cells confirmed that compactin blocks p21ras membrane association: p21ras is confined to the cytosol fraction. Induction of neuronal differentiation and ornithine decarboxylase (ODCase) transcription by oncogenic p21N-ras does not occur in compactin-treated cells indicating that activity of oncogenic p21N-ras expressed in PC12 cells is abolished by compactin treatment. Thus, p21ras isoprenylation or association with the membrane appears to be required for early responses and neuronal differentiation attributable to p21ras activation. In contrast, blockade of p21ras isoprenylation and membrane association by compactin treatment did not significantly reduce PC12 cell responses to NGF. Responses examined included rapid phosphorylation of tyrosine hydroxylase, rapid induction of ODCase expression, survival in serum-free medium and neuronal differentiation. Compactin blocked growth factor-induced rapid changes in cell surface morphology but did so whether this response was induced by NGF or by EGF. These results indicate that functional p21ras is not necessary for responses to NGF which in turn implies that if a ras-dependent NGF signal transduction pathway exists, as has been previously suggested, at least one additional ras-independent pathway must also be present.

Animals↗

PubMed1998

Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos.

The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells, the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain of Sos (DH-PH-Sos) was defective in Rac activation but regained Rac stimulating activity when it was coexpressed with activated Ras. Ras-mediated activation of DH-PH-Sos did not require activation of mitogen-activated protein kinase but it was dependent on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling pathways.

Actins↗

PubMed1993

Expression of ras-P21 and ras gene alteration in pleomorphic adenomas.

The expression of ras-P21 protein and gene alteration of the K-ras gene were examined in 12 cases of pleomorphic adenoma of the salivary gland. The tumor cells in all 12 cases were strongly stained for ras-P21 protein by the indirect immunoperoxidase method, suggesting the enhanced expression of ras-P21 protein in pleomorphic adenomas. Analysis of the PCR products for the K-ras gene by temperature-gradient gel electrophoresis (TGGE) revealed that gene alterations such as point mutations occurred in 4 out of 12 pleomorphic adenoma tissues examined.

Adenoma, Pleomorphic↗

PubMed1991

[A comparison study of immunohistochemical detection of ras P21 and N-ras mRNA in situ hybridization in hepatocellular carcinoma and pericancerous nontumor liver tissue].

30 cases of hepatocellular carcinoma and pericancerous nontumor samples were studied by examining the serial sections for immunohistochemical detection of P21 product accompanying identification of N-ras mRNA by in situ hybridization with biotinylated N-ras cDNA probe and a significant correlation was noticed between the results obtained (P less than 0.02). It was suggested that immunohistochemical detection of ras oncogene P21 product may indicate the expression of ras oncogene. The higher expression of N-ras mRNA of the carcinoma and the non-tumor tissue peripheral to the cancer by in situ hybridization were 53.33% and 28.57% respectively. The positive ratio of P21 were 96.67% and 90.48%.

Carcinoma, Hepatocellular↗

PubMed1991

Co-localization of ras gene products and cathepsin L in cytoplasmic vesicles in v-Ha-ras-transformed NIH3T3 mouse fibroblasts.

Subcellular localisation of Ha-ras gene products (p21) and cathepsin L was investigated by immunocytochemical studies. Western blotting analysis by using v-Ha-ras p21 deletion-insertion mutants indicated that NCC-RAS-001 and -004 monoclonal antibodies recognize epitopes within amino acids 126-140 and 146-165, respectively, of Ha-ras p21. Indirect immunofluorescent studies using these antibodies showed that ras p21 is distributed in patch-shaped structures in the cytoplasm. Cathepsin L whose activity is effectively inhibited by p21s in vitro was found in the regions where p21s are concentrated. These p21-concentrated regions may be derived from endosome-like vesicles because they are also labeled with Lucifer Yellow. The co-localization of p21 and cathepsin L in cytoplasmic vesicles was also confirmed by electron microscopic immunocytochemistry.

3T3 Cells↗

PubMed1989

Characterization of monoclonal antibody R256, specific for activated ras p21 with arginine at 12, and analysis of breast carcinoma of v-Harvey-ras transgenic mouse.

The ras family of proto-oncogenes can be activated into transforming genes by single point mutations resulting in p21 products with amino acid substitutions at positions 12, 13 and 61. Here we describe a monoclonal antibody designated R256 which reacts specifically with a synthetic peptide corresponding to amino acids 5 to 16 of ras p21 activated by the substitution of arginine for glycine at position 12. It does not react with peptides representing other activating substitutions at position 12. Western blot studies showed that R256 reacts specifically with recombinant ras p21 containing an arginine-12 substitution but is unreactive with the normal glycine-12 recombinant p21. R256 binds the activated Kirsten p21 in human lung tumor cell line A2182, which contains an activated arginine-12 p21. R256 is also specific for arginine-12 p21 extracted from NIH3T3 cells transformed by the viral Harvey-ras p21, containing arginine at 12, and is not reactive with transformants containing activated p21s with valine, serine, aspartic acid, glutamic acid, cysteine or normal glycine at position 12. When R256 was used to test for expression of arginine-12 p21 in tissues of transgenic mice containing an MMTV/v-Harvey-ras transgene, the monoclonal antibody was able to determine that the arginine-12 p21 transgene product was present in breast tumors but not in normal tissues. The ability of R256 to react specifically with arginine-12 p21 may prove to be valuable in the study of ras oncogenesis in model systems and in determining the presence of arginine-12 p21 in human tumors.

Animals↗

PubMed1995

Intracellular expression of the monoclonal anti-ras antibody Y13-259 blocks the transforming activity of ras oncogenes.

Microinjection of the anti-ras antibody Mab Y13-259 modifies ras function and can induce temporary reversion of the transformed phenotype in mutant ras-transformed cells. Intracellular production of neutralizing antibodies represents an approach to investigate the regulation of gene function. The genes coding for the heavy and light chains of Mab Y13-259 were isolated from a cDNA library. NIH3T3 cells transfected with heavy and light chain expression vectors produced functional anti-ras antibody. The production of functional antibody did not require glycosylation. To ensure that the antibody entered the cytoplasm and not the secretory pathway, the hydrophobic leader sequences of both chains were removed and replaced with synthetic initiator sequences. The modified heavy chain gene was cloned under the control of the murine sarcoma virus long terminal repeat, and the light chain gene under the control of the mouse mammary tumor virus long terminal repeat, which allows the induction of light chain expression in the presence of dexamethasone. When both heavy and light chain genes were expressed in cells with activated ras (morphologically transformed) in the presence of dexamethasone, we observed phenotypic reversion to characteristics of nontransformed cells. These experiments show that intracellular expression of antibodies can also be used as an alternative to analyze biological functions of a given protein.

3T3 Cells↗

PubMed1993

[Studies on ras P21 expression and mutation at 12th codon of C-Ha-ras oncogene in squamous cell carcinoma of the larynx].

Monoclonal antibody directed against the ras oncogene product P21 was used to evaluate P21 expression in 42 cases of tumors of the larynx. The results showed that there was a higher level of ras P21 expression in squamous cell carcinoma of the larynx (LSCC) than in papilloma of the larynx (LP) and the normal tissues adjacent to LSCC, P < 0.05. We had also detected G-->T mutation at codon 12 of Ha-ras oncogenes in LSCC using polymerase chain reaction (PCR). PCR products were hybridized with mutation-specific oligonucleotide probes. Thirty per cent (6/20) LSCC cases harbored this mutation. But G-->T mutation was absent in 3 LP and 5 normal tissues adjacent to LSCC. A relationship may exist between the presence of G-->T mutation and Jakobsson's malignant grading result. All of 6 LSCC cases containing G-->T mutation were in high malignant grading group, P < 0.05. This study implicates that overexpression of ras P21 and presence of Ha-ras G-->T mutation may be one of the molecular mechanisms of maintenance of malignancy and progression of LSCC.

Base Sequence↗

PubMed1999

Evidence that mutational activation of the ras genes may not be involved in aflatoxin B(1)-induced human hepatocarcinogenesis, based on sequence analysis of the ras and p53 genes.

Exposure to aflatoxin B(1) (AFB(1)) is one of the risk factors for developing hepatoma. In rats, activation of the ras gene is a prevalent event in AFB(1)-induced hepatocarcinogenesis. It is not clear whether a similar event occurs in humans. By analysis of codon 249 of the p53 gene, six of 36 human hepatoma samples were found to show a G-->T transversion, suggesting that AFB(1) may be a risk factor for hepatocarcinogenesis. However, analysis at codons 12, 13, and 61 in the ras family genes revealed a A-->T transversion at codon 61 of the N-ras gene in a single tumor. Apparently, ras activation is rare in human hepatoma, and the mutation detected might not be induced by AFB(1). This suggests that activation of the ras gene may not be a major event in AFB(1)-related human hepatocarcinogenesis.

Aflatoxin B1↗

PubMed1987

Production of chimeric protein coded by the fused viral H-ras and human N-ras genes in Escherichia coli.

A new method for the production of a chimeric protein of two related genes has been developed. The nucleotide sequences of the region from the N terminus to the 86th amino acid (aa) residue of human N-ras and of the Harvey sarcoma virus (Ha-MuSV) H-ras are 80% homologous. We isolated the DNA fragment encoding the N-terminal portion up to the 70th aa residue from plasmid pH-1 which encodes the total genome of Ha-MuSV, and the DNA fragment encoding the C-terminal portion from the 40th aa to the C terminus from plasmid p6a1 which includes the human N-ras cDNA but lacks the N-terminal portion. After partial digestion of both fragments with phage lambda exonuclease, which creates 3'-protruding ends, a hybrid was formed between 73% homologous single-stranded DNA portions at the 3' ends of both fragments. The hybrid was recloned on pBR322 after repairing with Escherichia coli DNA polymerase I and DNA ligase. The chimeric v-H/N-ras gene composed of the N-terminal portion of v-H-ras gene and the remaining region of N-ras gene was inserted into an expression vector containing two tandem trp promoters and a terminator, and expressed in E. coli. The chimeric protein was found to accumulate to approx. 10% of total cellular proteins.

Amino Acid Sequence↗

PubMed1998

Synergistic action of R-Ras and IGF-1 on Bcl-xL expression and caspase-3 inhibition in BaF3 cells: R-Ras and IGF-1 control distinct anti-apoptotic kinase pathways.

R-Ras and insulin-like growth factor-1 (IGF-1) synergistically inhibit apoptosis of BaF3 cells upon interleukin-3 deprivation. To characterize the mechanism of this synergistic inhibition, we examined the effect of R-Ras and IGF-1 on several apoptosis-related proteins. Extracellular signal-regulated kinase (ERK) was activated by IGF-1, but not by R-Ras. In contrast, Akt was activated strongly by R-Ras, but weakly by IGF-1. It was also found that R-Ras and IGF-1 cooperatively induced Bcl-xL expression and inhibited caspase-3 activation.

Apoptosis↗

PubMed1999

Cell-free synthesis of the Ras-binding domain of c-Raf-1: binding studies to fluorescently labelled H-ras.

It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.

Binding Sites↗

PubMed2001

Malolactomycin D, a potent inhibitor of transcription controlled by the Ras responsive element, inhibits Ras-mediated transformation activity with suppression of MMP-1 and MMP-9 in NIH3T3 cells.

To search for anti-cancer agents, a screening system for Ras signal inhibitors was developed using a NIH3T3 cell line with an introduced reporter gene which is controlled by the Ras-responsive element (RRE). With this screening system, malolactomycin D was identified as a selective inhibitor of transcription from the RRE. This compound was found to preferentially inhibit the anchorage-independent growth rather than the anchorage-dependent growth of Ras-transformed NIH3T3 cells. The expression of matrix metalloproteinases MMP-1 and MMP-9, which have RRE in their promoters, were reduced by treatment with malolactomycin D at the translational and transcriptional levels. Analysis of the activity of mitogen-activated protein (MAP) kinases, which play important roles in transduction of the Ras signal, showed that malolactomycin D inhibits the activation of p38 MAP kinase and Jun N-terminal-kinase (JNK) but not extracellular signal-regulated kinase 1 or 2 (ERK1 or 2). These findings suggest that by inhibiting the pathway that leads to the activation of p38 MAP kinase and JNK, malolactomycin D suppresses the expression of MMPs. Since MMPs play important roles in metastasis and maintenance of the microenvironment of tumor cells, both of which facilitate tumor growth, the inhibition of MMPs by malolactomycin D is believed to contribute to its ability to inhibit Ras-mediated tumorigenesis.

3T3 Cells↗

PubMed1991

IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein.

The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. (i) Extracts of yeast cells overexpressing IRA2 stimulated the GTPase activity of the yeast RAS2 protein. (ii) An epitope for a monoclonal antibody (12CA5) was added to the N terminus of the IRA2 protein. The GAP activity of extracts prepared from cells expressing this fusion protein was shown to be immunoprecipitable by 12CA5. (iii) An IRA2 protein fused to glutathione S-transferase (GST) was produced and partially purified from Escherichia coli cells. GAP activity was detected with this purified GST-IRA2 fusion protein. (iv) The GAP activity of IRA2 proteins described above did not stimulate the GTPase activity of the RAS2Val19 protein, a protein having an amino acid alteration analogous to that found in mammalian oncogenic ras proteins. This result parallels studies showing that mammalian GAP is incapable of stimulating the GTPase activity of mammalian oncogenic proteins. The remarkable conservation between the GAP activity in mammalian and yeast cells supports the idea that the function of GAP is to negatively regulate ras proteins in mammalian cells.

Escherichia coli↗