SEARCH · PubMed Health

Results for “RAS”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources

PubMed2001

Basis for signaling specificity difference between Sos and Ras-GRF guanine nucleotide exchange factors.

Sos and Ras-GRF are two families of guanine nucleotide exchange factors that activate Ras proteins in cells. Sos proteins are ubiquitously expressed and are activated in response to cell-surface tyrosine kinase stimulation. In contrast, Ras-GRF proteins are expressed primarily in central nervous system neurons and are activated by calcium/calmodulin binding and by phosphorylation. Although both Sos1 and Ras-GRF1 activate the Ras proteins Ha-Ras, N-Ras, and Ki-Ras, only Ras-GRF1 also activates the functionally distinct R-Ras GTPase. In this study, we determined which amino acid sequences in these exchange factors and their target GTPases are responsible for this signaling specificity difference. Analysis of chimeras and individual amino acid exchanges between Sos1 and Ras-GRF1 revealed that the critical amino acids reside within an 11-amino acid segment of their catalytic domains between the second and third structurally conserved regions (amino acids (aa) 828-838 in Sos1 and 1057-1067 in Ras-GRF1) of Ras guanine nucleotide exchange factors. In Sos1, this segment is in helix B, which is known to interact with the switch 2 region of Ha-Ras. Interestingly, a similar analysis of Ha-Ras and R-Ras chimeras did not identify the switch 2 region of Ha-Ras as encoding specificity. Instead, we found a more distal protein segment, helix 3 (aa 91-103 in Ha-Ras and 117-129 in R-Ras), which interacts instead primarily with helix K (aa 1002-1016) of Sos1. These findings suggest that specificity derives from the fact that R-Ras-specific amino acids in the region analogous to Ha-Ras helix 3 prevent a functional interaction with Sos1 indirectly, possibly by preventing an appropriate association of its switch 2 region with helix B of Sos1. Although previous studies have shown that helix B of Sos1 and helix 3 of Ha-Ras are involved in promoting nucleotide exchange on Ras proteins, this study highlights the importance of these regions in establishing signaling specificity.

Amino Acid Sequence↗

PubMed2003

Expression of Ras GTPases in normal kidney and in glomerulonephritis.

BACKGROUND: Small monomeric Ras GTPases play critical and specific roles in the control of cellular proliferation and apoptosis but the expression of the three Ras isoforms (Ha-Ras, Ki-Ras and N-Ras) in human renal tissue is unknown. This work is an immunohistochemical study of Ras expression in normal renal tissue and in membranous glomerulonephritis (MGN), IgA nephropathy (IgAN) and IgA-negative mesangioproliferative glomerulonephritis (MPGN). METHODS: Formalin-fixed, paraffin-embedded tissue was stained using pan-Ras monoclonal antibody (mAb) and Ras isoform-specific mAb. Detection employed a (DAKO Envision) modified polymer system. RESULTS: The expression of Ras isoforms in normal human kidney was cell-specific. For example, N-Ras was detected in tubule epithelial cells but not in glomerular or interstitial cells. Ki-Ras was expressed in mesangial cells, interstitial cells and in proximal convoluted tubule cells (PCT) (particularly localized at brush borders) and in collecting duct cells (CD) (localized to cell membranes) but not in podocytes. Cytoplasmic Ha-Ras was detected in all the above cell types except podocytes. MGN was associated with podocyte expression of all three Ras isoforms and with reduced mesangial cell expression of Ha-Ras and Ki-Ras. IgAN was characterized by podocyte expression of Ha-Ras (but not Ki-Ras) and reduced mesangial cell expression of Ki-Ras without alterations in mesangial Ha-Ras expression. MPGN was associated with reduced mesangial cell Ha-Ras and Ki-Ras expression without significant podocyte Ras expression. CONCLUSION: These disease-specific and isoform-specific alterations in Ras expression may be of significance in pathogenesis and warrant further functional investigation.

Fibroblasts↗

PubMed1997

Dominant transformation by mutated human ras genes in vitro requires more than 100 times higher expression than is observed in cancers.

The gene-mutation-cancer hypothesis holds that mutated cellular protooncogenes, such as point-mutated proto-ras, "play a dominant part in cancer," because they are sufficient to transform transfected mouse cell lines in vitro [Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994) Molecular Biology of the Cell (Garland, New York)]. However, in cells transformed in vitro mutated human ras genes are expressed more than 100-fold than in the cancers from which they are isolated. In view of the discrepancy between the very low levels of ras transcription in cancers and the very high levels in cells transformed in vitro, we have investigated the minimal level of human ras expression for transformation in vitro. Using point-mutated human ras genes recombined with different promoters from either human metallothionein-IIA or human fibronectin or from retroviruses we found dominant in vitro transformation of the mouse C3H cell line only with ras genes linked to viral promoters. These ras genes were expressed more than 120-fold higher than are native ras genes of C3H cells. The copy number of transfected ras genes ranged from 2-6 in our system. In addition, nondominant transformation was observed in a small percentage (2-7%) of C3H cells transfected with ras genes that are expressed less than 20 times higher than native C3H ras genes. Because over 90% of cells expressing ras at this moderately enhanced level were untransformed, transformation must follow either a nondominant ras mechanism or a non-ras mechanism. We conclude that the mutated, but normally expressed, ras genes found in human and animal cancers are not likely to "play a dominant part in cancer." The conclusion that mutated ras genes are not sufficient or dominant for cancer is directly supported by recent discoveries of mutated ras in normal animals, and in benign human tissue, "which has little potential to progress" [Jen, J., Powell, S. M., Papadopoulos, N., Smith, K. J., Hamilton, S. R., Vogelstein, B. & Kinzler, K. W. (1994) Cancer Res. 54, 5523-5526]. Even the view that mutated ras is necessary for cancer is hard to reconcile with (i) otherwise indistinguishable cancers with and without ras mutations, (ii) metastases of the same human cancers with and without ras mutations, (iii) retroviral ras genes that are oncogenic without point mutations, and (iv) human tumor cells having spontaneously lost ras mutation but not tumorigencity.

Animals↗

PubMed2005

Activation of the RAS pathway is predictive for a chemosensitive phenotype of acute myelogenous leukemia blasts.

PURPOSE: Activation of the RAS pathway plays a major role in cancer cells. In acute myeloid leukemia (AML), mutations of the RAS genes cause an intrinsic activation of this pathway. Until now, clinical studies could not find clear association of RAS mutations with the clinical outcome after AML therapy. This could be due to alternative initiating events for activation of the RAS pathway like constitutive tyrosine kinase activation or mutations in Ras-regulating genes. EXPERIMENTAL DESIGN: In total, 191 AML patients (126 as training population and 65 as test population) were studied for Ras activity with a glutathione S-transferase pull-down assay using Raf binding of activated Ras. RESULTS: AML samples showed a wide range of Ras activity values, which was in contrast to normal bone marrow donors who showed no or very limited Ras activity. Using a Ras binding score based on semiquantitative Western blotting, we defined patients with strong Ras activity and compared Ras activity with RAS mutation. Surprisingly, only a minority of RAS mutated AML samples (22.2%) showed strong Ras activity, whereas 25 patients presented strong Ras activity in the absence of RAS mutations. Clinical outcome did not show differences according to RAS mutations. In contrast, Ras activity predicted for a high response rate (P <0.05) and proved to be an independent factor for overall survival rate (P <0.05) in younger AML patients receiving high-dose 1-beta-D-arabinofuranosylcytosine as induction therapy. CONCLUSION: The data highlight the role for alternative pathways of Ras activation without RAS mutations. Intrinsically activated Ras seems to increase sensitivity of the AML blast to high-dose 1-beta-D-arabinofuranosylcytosine therapy.

Antimetabolites, Antineoplastic↗

PubMed2003

p38 kinase is a key signaling molecule for H-Ras-induced cell motility and invasive phenotype in human breast epithelial cells.

Ras expression has been suggested as a marker for tumor aggressiveness of breast cancer,including the degrees of invasion and tumor recurrence.We showed previously that H-ras, but not N-ras, up-regulates matrix metalloproteinase 2 expression and induces invasive phenotype in MCF10A human breast epithelial cells (A. Moon, et al. Int. J. Cancer, 85: 176-181, 2000). In this study, we show that H-ras also promotes cell motility more effectively than N-ras in MCF10A cells. We have investigated H-ras-specific signaling pathway(s) critical for H-ras-mediated cell motility and invasive phenotype. Whereas neither H-ras nor N-ras activated c-Jun NH(2)-terminal kinase 1, both H-ras and N-ras effectively activated extracellular signal-regulated protein kinase (ERK) -1,2. Importantly, prominent activation of p38 mitogen-activated protein kinase was shown only in H-ras-activated cells but not in N-ras-activated MCF10A cells. Functional significance of H-ras-activated p38 in invasiveness and cell motility was evidenced by studies using SB203580, a chemical inhibitor of p38, and a dominant-negative construct of p38. Whereas inhibition of c-Jun NH(2)-terminal kinase 1 activity had no effect on H-ras-induced MCF10A cell invasion and motility, the inhibition of the ERK pathway using a chemical inhibitor PD98059 or dominant-negative mutant of mitogen-activated protein/ERK kinase 1, an activator of ERKs, significantly reduced H-ras-induced invasion and migration. We also provide evidence that p38 and, to a lesser degree, ERKs, are critical for H-ras-mediated up-regulation of matrix metalloproteinase 2. Taken together, the present study shows that H-ras activation of both p38 and ERKs induces cell invasion and motility, whereas N-ras activation of ERKs alone is not sufficient. This study reveals the p38 kinase as a key signaling molecule differentially regulated by H-ras and N-ras, leading to H-ras-specific cell invasive and migrative phenotypes in human breast epithelial cells.

Breast↗

PubMed1997

N-ras protein: frequent quantitative and qualitative changes occur in human colorectal carcinomas.

Point mutation and overexpression are recognized mechanisms for ras activation in malignancy. However, little information is available on overexpression of N-ras protein compared with H- or K-ras proteins, as N-ras-specific antibodies have only recently become available. For comparative analyses of ras protein levels, we have probed Western blots of extracts from 9 normal human tissues and 55 pairs of colorectal carcinoma and matched control mucosa, using monoclonal antibodies (MAbs) specific for H-, K- or N-ras proteins. On multi-tissue blots, N-ras protein was more highly expressed in colon than in the other human tissues analyzed, suggesting a role for N-ras in colorectal function. N-ras protein levels in multiple independent extracts of normal colon mucosa were consistently higher than either K-ras protein or H-ras protein levels. In 69% of colon carcinomas, N-ras protein levels were increased an average of 4.8-fold over normal mucosa. Overexpression of K-ras protein was also observed in colon cancers but less frequently (13% of cases) than N-ras protein. H-ras protein levels were too low for comparative studies. Alterations in N-ras protein mobility, possibly reflecting increased post-translational processing, were also detected in 42% of colon carcinomas. N-ras protein, typically present as a single 23 kDa band in normal mucosa, was expressed in some cancers as a 22 kDa band or as multiple bands of 20-23 kDa. Sequencing of N-ras DNA from 6 carcinomas with these variations in protein mobility did not reveal mutations in codons 12, 13, 59 or 61. Thus, frequent quantitative and qualitative changes in N-ras protein expression, which do not appear to correlate with the presence of typical N-ras point mutations, result in abnormal N-ras protein patterns in human colorectal carcinomas.

Antibodies, Monoclonal↗

PubMed2000

How does GAP catalyze the GTPase reaction of Ras? A computer simulation study.

The formation of a complex between p21(ras) and GAP accelerates the GTPase reaction of p21(ras) and terminates the signal for cell proliferation. The understanding of this rate acceleration is important for the elucidation of the role of Ras mutants in tumor formation. In principle there are two main options for the origin of the effect of GAP. One is a direct electrostatic interaction between the residues of GAP and the transition state of the Ras-GAP complex and the other is a GAP-induced shift of the structure of Ras to a configuration that increases the stabilization of the transition state. This work examines the relative importance of these options by computer simulations of the catalytic effect of Ras. The simulations use the empirical valence bond (EVB) method to study the GTPase reaction along the alternative associative and dissociative paths. This approach reproduces the trend in the overall experimentally observed catalytic effect of GAP: the calculated effect is 7 +/- 3 kcal/mol as compared to the observed effect of approximately 6.6 kcal/mol. Furthermore, the calculated effect of mutating Arg789 to a nonpolar residue is 3-4 kcal/mol as compared to the observed effect of 4.5 kcal/mol for the Arg789Ala mutation. It is concluded, in agreement with previous proposals, that the effect of Arg789 is associated with its direct interaction with the transition state charge distribution. However, calculations that use the coordinates of Ras from the Ras-GAP complex (referred to here as Ras') reproduce a significant catalytic effect relative to the Ras coordinates. This indicates that part of the effect of GAP involves a stabilization of a catalytic configuration of Ras. This configuration increases the positive electrostatic potential on the beta-phosphate (relative to the corresponding situation in the free Ras). In other words, GAP stabilizes the GDP bound configuration of Ras relative to that of the GTP-bound conformation. The elusive oncogenic effect of mutating Gln61 is also explored. The calculated effect of such mutations in the Ras-GAP complex are found to be small, while the observed effect is very large (8.7 kcal/mol). Since the Ras is locked in its Ras-GAP configuration in our simulations, we conclude that the oncogenic effect of mutation of Gln61 is indirect and is associated most probably with the structural changes of Ras upon forming the Ras-GAP complex. In view of these and the results for the Ras' we conclude that GAP activates Ras by both direct electrostatic stabilization of the transition state and an indirect allosteric effect that stabilizes the GDP-bound form. The present study also explored the feasibility of the associative and dissociative mechanism in the GTPase reaction of Ras. It is concluded that the reaction is most likely to involve an associative mechanism.

Computer Simulation↗

PubMed1986

Harvey ras genes transform without mutant codons, apparently activated by truncation of a 5' exon (exon -1).

The hypothesis is tested that the ras gene of Harvey sarcoma virus (Ha-SV) and the proto-ras DNAs from certain tumor cells derive transforming function from specific codons in which they differ from normal proto-ras genes. Molecularly cloned Harvey proviral vectors carrying viral ras, normal rat proto-ras, and recombinant ras genes in which the virus-specific ras codons 12 and 59 were replaced by proto-ras equivalents each transformed aneuploid mouse 3T3 cells after latent periods that ranged from 4 to 10 days. Viruses with or without virus-specific ras codons all transformed diploid rat cells in 3-5 days equally well. However, in the absence of virus replication, mutant codons were beneficial for transforming function. Deletion of non-ras regions of Ha-SV did not affect transforming function. We conclude that specific ras codons are not necessary for transforming function. Comparisons of the ras sequences of Ha-SV, BALB SV, and Rasheed SV with sequences of proto-ras genes from rat and man revealed an upstream proto-ras exon, termed exon -1. The 3' end of this exon is present in all three viruses and in a ras pseudogene of the rat. Since ras genes transform without mutation and since exon -1 is truncated in viral ras genes and all transforming proto-ras DNAs of the Harvey and the Kirsten ras family, we propose that ras genes are activated by truncation of exon -1 either via viral transduction or artificially via cloning and transfection. The proposal implies that untruncated proto-ras genes with point mutations may not be cellular cancer genes.

Animals↗

PubMed1996

Antisense oligonucleotides demonstrate a dominant role of c-Ki-RAS proteins in regulating the proliferation of diploid human fibroblasts.

Although members of the RAS protein family (Ha-, Ki-, and N-RAS) are known to play a key role in normal cell proliferation and to be frequently mutated in naturally occurring tumors, it remains unclear which of these proteins functions to regulate growth in normal cells. Gene-specific oligonucleotides (oligos) against c-Ki-RAS (ISIS 6957), c-Ha-RAS (ISIS 2503), and oncogenic Ha-RAS (ISIS 2570) were used to analyze the requirement for individual RAS proteins in the proliferation of diploid human lung fibroblasts (MRC-5), and human bladder carcinoma cell lines with (T24) or without (J-82) a RAS mutation. The oncogenic Ha-RAS oligo substantially inhibited T24 cell proliferation, whereas the c-Ki-RAS and control (ISIS 1966) oligos had little effect. Interestingly, in MRC-5 cells the c-Ki-RAS but not c-Ha-RAS oligo was effective in inhibiting cell proliferation. No inhibition was seen in the J-82 cells with either oligo. In Western analysis, p21 RAS protein was decreased following treatment with the oncogenic Ha-RAS oligo in T24 cells or the c-Ki-RAS oligo in MRC-5 cells, whereas no reductions were observed in J-82 cells with either oligo. The specificity of these oligos was demonstrated in Northern analyses in which both Ha-RAS and Ki-RAS oligo treatment resulted in reduced levels of their respective mRNAs in all three cell lines, whereas the mutant Ha-RAS mRNA in T24 cells was most effectively reduced with the oncogenic Ha-RAS oligo. These results demonstrate that oncogenic Ha-RAS plays an important role in the proliferation of T24 cells, whereas c-Ki-RAS contributes predominantly to the proliferation of normal MRC-5 cells.

Cell Division↗

PubMed1991

Clonal dominance of select subsets of viral Kirsten ras(+)-transformed 3T3 cells during tumor progression.

Long-term culturing of viral Kirsten-ras-oncogene (v-Ki-ras)-transformed BALB/c 3T3 cells (KiMSV) selectively enriches for cells which have deleted the viral oncogene. In contrast, v-Ki-ras in vivo is amplified and expression increased in all late-stage tumors and lung metastases relative to late-passage KiMSV cells being injected. The nature and significance of these selection processes, in terms of the v-Ki-ras gene, have been explored using genetically-tagged cells, as have the properties of v-Ki-ras- revertant subclones. Inoculation of KiMSV late-passage cells (containing less than 5% v-Ki-ras+ cells) into nude mice, generated primary and lung metastatic tumors with the v-Ki-ras gene at increased dosage in all tumors and their single-cell clones, isolated at both early and late stages of tumor development; this demonstrates early and specific in vivo selection for v-Ki-ras+ cells in both induction and progression of tumors in this system. v-Ki-ras- revertant subclones, isolated from late-passage KiMSV cells and inoculated individually into athymic nude mice, yielded tumors for only 1 of the 4 revertants, with no evidence for v-Ki-ras sequences in these tumor cells, thereby revealing a v-Ki-ras-independent mechanism for tumor formation in a small subset of revertant cells. Mixtures of the 4 subclones yielded tumors in all animals, although at a much longer latent period than observed with v-Ki-ras+ cells. Experiments with mixtures of v-Ki-ras- revertant cells and pSV2neo0tagged/v-Ki-ras+ cells (both complex NeoR cell mixtures and individual NeoR clones tested) at various cell ratios revealed clonal variability among v-Ki-ras+ cells for dominance during tumor formation. Moreover, the complex NeoR cell mixtures yielded both primary and metastatic tumors with simplified patterns of pSV2neo integration sites, suggesting that secondary genetic or epigenetic events, in addition to v-Ki-ras, contribute to the tumor-progressing phenotype. These experiments taken together demonstrate (a) clone-specific early selection of distinct v-Ki-ras+ cells amongst themselves and over v-Ki-ras- cells in both induction and progression of tumors, (b) reduced tumorigenic competence of v-Ki-ras- revertant cells, with a small subset displaying a v-Ki-ras-independent mechanism for tumor formation in this BALB/c 3T3 system, and (c) the significance of additional genetic or epigenetic events for tumor-progressing competence in unique subsets of v-Ki-ras+ cells.

Animals↗

PubMed1999

Concepts in Ras-directed therapy.

Ras proteins are key transducers of growth signals regulated by cell surface receptors. They are anchored to the inner surface of the cell membrane where receptor-mediated signalling induces Ras activation (GDP/GTP exchange) and inactivation (stimulation of Ras GTPase activity). Ras-GTP in turn activates a multitude of signalling cascades controlling cell growth and differentiation. Aberrant Ras function (mostly constitutive activation) contributes to the development of many types of neoplastic human diseases. Activating mutations in ras genes, leading to the expression of Ras proteins insensitive to Ras-GTPase activating proteins, are found in as many as 30% of all human tumours. This suggests that Ras is an appropriate target for drug design. Remarkable improvements in the understanding of post-translational modifications in Ras that promote Ras-membrane anchorage, in the mechanisms of activation and inactivation of Ras, and in the interactions of Ras with a plethora of effector molecules have led to the development of new concepts for Ras-directed therapy. The most advanced approach has been that of farnesyltransferase inhibitors (FTIs) designed to inhibit the farnesylation of Ras required for membrane anchorage and transforming activity. FTIs now in clinical trials have been extensively reviewed. Here we review the progress in the development of FTIs and in the development of other promising concepts for Ras-directed therapy. These include compounds such as S-farnesylthiosalicylic acid (FTS), which disrupt the proper anchorage of Ras with the cell membrane and inhibit human tumour growth in animal models, and compounds that interfere with interactions of Ras with its downstream effectors. We conclude with a description of a recently described novel drug concept that could restore the defective GTPase activity of oncogenic Ras and with the interesting results of reovirus-induced tumour regression observed in animal models of human tumours containing an intact Ras signalling pathway.

Journal Article↗

PubMed1998

Presence of activated ras correlates with increased cysteine proteinase activities in human colorectal carcinomas.

The metastatic potential of ras-transfected cells has been attributed in part to significant ras induction of proteinase expression. To determine whether primary cancers also demonstrate higher cysteine proteinase activities in the presence of activated ras genes or altered ras protein expression, we have analyzed 60 primary human colorectal carcinomas for ras gene or protein changes together with the expression of cathepsins B and L. Cancers containing K-ras mutations (47% of 60 carcinomas) demonstrated greater increases in cathepsin L activity than cancers without K-ras mutations (p = 0.029), with particularly significant correlations for earlier stage cancers (Dukes' A and B carcinomas, p = 0.006). Western blots used to characterize ras protein patterns in the same cancer/normal pairs have demonstrated that N-ras protein is more highly expressed in colon tissues than H- or K-ras proteins and that N-ras overexpression occurs in almost 70% of colorectal cancers, with or without a concurrent change in electrophoretic mobility of N-ras protein. Our current study has now shown that N-ras protein overexpression alone does not significantly induce cathepsin B or L activity levels in colon cancers. However, carcinomas demonstrating altered N-ras protein forms, in the absence of any K- or N-ras mutations, expressed significantly higher levels of cathepsin B and L activities compared with carcinomas with normal N-ras protein banding patterns. Our data suggest that colorectal carcinomas with either K-ras mutations or altered forms of N-ras protein may increase their tumorigenic potential via the induction of cathepsin L or B expression levels. Our results also confirm that ras oncogene up-regulation of cathepsin B and L activities, previously reported in cultured cells, is a frequent event in primary human colorectal carcinomas.

Blotting, Western↗

PubMed2001

Disrupting the transforming activity of shrimp ras(Q(61)K) by deleting the CAAX box at the C-terminus.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus. Ras transcription and protein levels had increased significantly in the cells transfected with the S-ras plasmid, compared to cells transfected with a control plasmid pcDNA3.1. The bacterially expressed GTP-locked S-Ras(Q(61)K) is successfully prenylated by rat protein geranylgeranyltransferase I (PGGTase I) and then polymerized with tubulin, in agreement with findings for GTP-locked mammalian K(B)-Ras(Q(61)K) in vitro. Shrimp protein farnesyltransferase (PFTase) of shrimp did not prenylate the GTP-locked shrimp S-Ras(Q(61)K) (Lin and Chuang. 1998. J Exp Zool 281:565-573), whereas rat PFTase efficiently catalyzed the farnesylation of GTP-locked S-Ras(Q(61)K). To investigate the effect of geranylgeranylation on cellular transformation, we generated S-ras(Q(61)K) mutants with deletion of the CAAX box [S-ras(Q(61)K)(-caax)] or replacement of the CAAX box [S-ras(Q(61)K)(Kcaax)] or replacement of the arginine-rich domain [S-ras(Q(61)K)(K-Lys)] with corresponding sequences from rat K(B)-ras(Q(61)K). BALB/3T3 cells transfected with DNA encoding S-ras(Q(61)K), S-ras(Q(61)K)(KCAAX), S-ras(Q(61)K)(K-Lys) were transformed successfully, but S-ras(Q(61)K)(-CAAX) was defective in its ability to transform. Thus, prenylation at CAAX is required for transformation. Either the geranylgeranylated or the farnesylated S-Ras(Q(61)K) was endowed with abilities to transform. The arginine-rich region in S-Ras or the lysine-rich clusters from the rat K(B)-Ras appear not essential for activity to transform.

3T3 Cells↗

PubMed1990

Suppression of c-ras transformation by GTPase-activating protein.

The ras genes are required for normal cell growth and mediate transformation by oncogenes encoding protein tyrosine kinases. Normal ras can transform cells in vitro and in vivo, but mutationally activated ras does so much more efficiently, and highly transforming mutant versions of ras have been isolated from a variety of human and animal tumours. The ras genes encode membrane-associated, guanine nucleotide-binding proteins that are active when GTP is bound and inactive when GDP is bound. The slow intrinsic GTPase activity of normal mammalian Ras proteins can be greatly accelerated by the GTPase-activating protein (GAP), which is predominantly cytoplasmic. This activity of GAP, which can increase with cell density in contact-inhibited cells, suggests that it functions as a negative, upstream regulator of ras. Other studies, however, show that GAP interacts with a region of ras-encoded protein implicated in ras effector function, which raises the possibility that GAP might also be a downstream target of ras. Mutationally activated ras-encoded proteins also interact with GAP, although they are resistant to its catalytic activity. In an attempt to define the role of GAP in ras-mediated transformation, we examined the effects on transformation of normal or mutant ras when cells overexpress GAP. We found that GAP suppresses transformation of NIH 3T3 cells by normal Ha-ras (c-ras) but does not inhibit transformation by activated Ha-ras (v-ras). These results support the hypothesis that GAP functions as a negative regulator of normal ras and make it unlikely that GAP alone is the ras target.

Animals↗

PubMed2004

NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation.

The C terminus, also known as the hypervariable region (residues 166-189), of H-, N-, and K-Ras proteins has sequence determinants necessary for full activation of downstream effectors such as Raf kinase and PI-3 kinase as well as for the correct targeting of Ras proteins to lipid rafts and non-raft membranes. There is considerable interest in understanding how residues in the extreme C terminus of the different Ras proteins and farnesylation of the CaaX box cysteine affect Ras membrane localization and allosteric activation of Raf kinase. To provide insights into the structural and dynamic changes that occur in Ras upon farnesylation, we have used NMR spectroscopy to compare the properties of truncated H-Ras (1-166), to non-processed full-length H-Ras (residues 1-185) and full-length (1-189) farnesylated H-Ras. We report that the C-terminal helix alpha-5 extends to residue N172, and the remaining 17 amino acid residues in the C terminus are conformationally averaged in solution. Removal of either 23 or 18 amino acid residues from the C terminus of full length H-Ras generates truncated H-Ras (1-166) and H-Ras (1-171) proteins, respectively, that have been structurally characterized and are biochemical active. Here we report that C-terminal truncation of H-Ras results in minor structural and dynamic perturbations that are propagated throughout the H-Ras protein including increased flexibility of the central beta-sheet and the C-terminal helix alpha-5. Ordering of residues in loop-2, which is involved in Raf CRD binding is also observed. Farnesylation of full-length H-Ras at C186 does not result in detectable conformational changes in H-Ras. Chemical shift mapping studies of farnesylated and non-farnesylated forms of H-Ras with the Raf-CRD show that the farnesyl moiety, the extreme H-Ras C terminus and residues 23-30, contribute to H-Ras:Raf-CRD interactions, thereby increasing the affinity of H-Ras for the Raf-CRD.

Cell Membrane↗

PubMed2005

H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells.

Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.

Breast↗

PubMed2004

Selective occurrence of ras mutations in benign and malignant thyroid follicular neoplasms in Taiwan.

Previous studies have demonstrated that point mutations in all three ras genes (H-ras, K-ras, and N-ras) may occur in thyroid neoplasia. However, the overall incidence of ras mutations in thyroid tumors and their frequency in specific histologic types varies widely in different series. Many earlier studies have chosen allele-specific oligonucleotide hybridization approaches to examine ras mutations without further confirmation of the positive samples by DNA sequencing. In this study, mutational hot spots in exon 1 (codons 12/13) and exon 2 (codon 61) of the H-ras, K-ras, and N-ras were polymerase chain reaction (PCR) amplified and sequenced with an automatic sequencer. ras mutations were detected in 4 of 89 (4.5%) benign and malignant thyroid tumors. Three of 8 follicular carcinomas exhibited mutations in codon 61 of H-ras, K-ras, and N-ras, respectively, and mutation at codon 61 of N-ras was found in 1 of 12 follicular adenomas. No mutations were observed in the other tumors, which included 20 nodular goiters, 5 Hürthle cell adenomas, 42 papillary carcinomas, and 2 undifferentiated carcinomas. Our results, obtained by the direct sequencing technique, indicate a lower overall prevalence of ras oncogenes in thyroid tumors than reports in earlier series. However, the frequency of ras mutations in specific histotype of thyroid tumors and their exclusive involvement of codon 61 in our series are similar to those studies utilizing DNA sequencing to detect or to confirm ras gene alterations. The selective occurrence of ras mutations in benign and malignant follicular neoplasms indicates that ras gene alterations have a specific and early role in the development of follicular type of thyroid tumors in Taiwan.

Adenocarcinoma, Follicular↗

PubMed2006

Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets.

Rats treated with the alkylating agent methylnitrosourea (MNU) develop multiple, hormonally dependent mammary tumors. Roughly 50% of the tumors have Ha-ras mutation, whereas 50% do not. The MNU-induced rat mammary tumor model was employed to examine the therapeutic efficacy of the farnesyltransferase inhibitor (FTI), R115777, and to examine the use of genomics in identifying susceptible tumors as well as identifying genes whose expression are modulated by FTI treatment. In animals bearing palpable mammary tumors (< 7 mm diameter), we performed a surgical biopsy, and 3 days following the biopsy, rats were treated with R115777 (50 mg/kg body wt/day) by gavage. Tumors with Ha-ras mutations underwent profound regression, with nearly 90% showing complete regressions within 4 weeks. In contrast, the non-Ha-ras mutation-bearing tumors yielded a more variable response, although roughly half of the non-Ha-ras mutation tumors underwent significant regression. These results show that although all tumors appear to respond to the FTI inhibitor the tumors with Ha-ras mutations were exquisitely sensitive. We employed a microarray approach to define potential targets and the mechanism of action of R115777 in Ha-ras mutant or wildtype tumors following treatment with FTI. In addition, we determined whether gene expression prior to FTI treatment can be used to differentiate highly sensitive tumors (Ha-ras mutant) and tumors with variable sensitivity (Ha-ras wildtype). Untreated or FTI-treated (4 days at 50 mg/kg body wt) tumors (Ha-ras mutant or wildtype) were examined using oligonucleotide arrays. A significant number of genes were differentially expressed in control rat mammary tumors with or without an activated Ha-ras mutation, suggesting that a microarray analysis might differentiate highly sensitive and variably sensitive tumors. Most of the genes whose expressions were modulated by FTI in tumors were independent of Ha-ras status and were presumably modulated by effects on farnesylation of proteins other than Ha-ras. However, treatment of Ha-ras-mutated mammary tumors with R155777 results in preferential modulation of genes involved in ras-MAP kinase signal transduction pathway and in decreased expression of many genes involved with cell proliferation. In contrast, several classes of genes are altered in rat mammary tumors without a mutated Ha-ras, suggesting that non-ras targets are involved. Ras pathway related genes, p53, WT1 and PCNA, were preferentially modulated in Ha-ras-mutated tumors, whereas modulation of genes in the G-protein pathway, various cytochrome p450s and RB1 are involved in Ha-ras wildtype tumors. Elucidation of gene expression changes in FTI-treated or control rat mammary adenocarcinomas will help in identifying potential pharmacodynamic markers of FTI treatment as well as potential molecular targets of R115777 and other FTIs.

Alkylating Agents↗