Monoclonal Antibody Y13-259 Research Articles

M-Ras, a Widely Expressed 29-kD Homologue of p21 Ras: Expression of a Constitutively Active Mutant Results in Factor-Independent Growth of an Interleukin-3–Dependent Cell Line

M-Ras, a recently identified homologue of p21 Ras, is widely expressed, with levels of the 29-kD protein in spleen, thymus, and NIH 3T3 fibroblasts equaling or exceeding those of p21 Ras. A G22V mutant of M-Ras was constitutively active and its expression in an interleukin-3 (IL-3)-dependent mast cell/megakaryocyte cell line resulted in increased survival in the absence of IL-3, increased growth in IL-4, and, at high expression levels, in factor-independent growth. Expression of M-Ras G22V, however, had a negative effect on growth in the presence of IL-3, suggesting that M-Ras has both positive and negative effects on growth. Expression of M-Ras G22V in NIH-3T3 fibroblasts resulted in morphological transformation and growth to higher cell densities. M-Ras G22V induced activation of the c-fos promoter, and bound weakly to the Ras-binding domains of Raf-1 and RalGDS. Expression of a mutant of M-Ras G22V that was no longer membrane-bound partially inhibited (40%) activation of the c-fos promoter by N-Ras Q61K, suggesting that M-Ras shared some, but not all, of the effectors of N-Ras. An S27N mutant of M-Ras, like the analogous H-Ras S17N mutant, was a dominant inhibitor of activation of the c-fos promoter by constitutively active Src Y527F, suggesting that M-Ras and p21 Ras shared guanine nucleotide exchange factors and are likely to be activated in parallel. Moreover, M-Ras was recognized by the monoclonal anti-Ras antibody Y13-259, commonly used to study the function and activity of p21 Ras. Mammalian M-Ras and a Caenorhabditis elegans orthologue exhibit conserved structural features, and these are likely to mediate activation of distinctive signaling paths that function in parallel to those downstream of p21 Ras.

The Ras/Erk Pathway Induces Primitive Endoderm but Prevents Parietal Endoderm Differentiation of F9 Embryonal Carcinoma Cells

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

Activation of a Ca2+-dependent K+ current in mouse fibroblasts by lysophosphatidic acid requires a pertussis toxin-sensitive G protein and Ras.

Lysophosphatidic acid (LPA) is a bioactive lipid that acts through G protein-coupled plasma membrane receptors and mediates a wide range of cellular responses. Here we report that LPA activates a K+ current in NIH3T3 mouse fibroblasts that leads to membrane hyperpolarization. The activation occurs with an EC50 value of 1.7 nM LPA. The K+ current is Ca2+-dependent, voltage-independent, and completely blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin with IC50 values of 1.7, 16, and 62 nM, respectively. The underlying K+ channels possess a single channel conductance of 33 pS in symmetrical K+ solution. Pretreatment of cells with pertussis toxin (PTX), Clostridium sordellii lethal toxin, or a farnesyl protein transferase inhibitor reduced the K+ current amplitude in response to LPA to about 25% of the control value. Incubation of cells with the protein tyrosine kinase inhibitor genistein or microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 reduced it by more than 50%. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase A activator 8-bromo-cAMP had no effect. These results indicate that the K+ channel activation by LPA is mediated by a signal transduction pathway involving a PTX-sensitive G protein, a protein tyrosine kinase, and Ras. LPA is already known to activate Cl- channels in various cell types, thereby leading to membrane depolarization. In conjunction with our results that demonstrate LPA-induced membrane hyperpolarization by activation of K+ channels, LPA appears to be significantly involved in the regulation of the cellular membrane potential.

Ras activation in platelets after stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C.

Several reports have indicated that the small G-protein Ras is not present immunologically in platelets. However, here we report the identification of Ras in platelets by immunoprecipitation with the Ras-specific monoclonal antibodies Y13-259 or Y13-238, followed by Western blotting. The presence of Ras was not due to contamination of samples with erythrocytes or leucocytes. Immunofluorescence studies indicated that Ras was present in a peripheral rim pattern in fixed, permeabilized platelets, suggesting an intracellular, plasma membrane location. Activation of platelets with the thrombin receptor peptide42-50, the prostaglandin H2/thromboxane A2 mimetic U46619 or phorbol 12-myristate 13-acetate induced a rapid increase in GTP-bound, activated Ras. In each case, this increase was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide GF 109203X, suggesting that Ras is activated downstream of PKC in platelets. Thus the activation of Ras in platelets by agonists will now allow consideration of multiple potential Ras-dependent signal transduction pathways in platelet activation processes.

Structural basis of p21H-ras molecular switch inhibition by a neutralizing antibody

The ras oncogene product p21 functions as a molecular switch in the early section of the signal transduction pathway that is involved in cell growth and differentiation. When the protein is in its GTP-complexed form it is active in signal transduction, whereas it is inactive in its GDP-complexed form. The transforming activity of p21ras is neutralized by the mouse monoclonal antibody Y13-259, possibly by preventing GDP-GTP exchange. A molecular model of the variable fragment of Y13-259 has been derived using a knowledge-based prediction approach and computer-assisted modeling techniques. An analysis of this model while complexed with p21ras/(GDP) indicated that the two molecular switch regions are constrained by complex formation. Antibody binding inhibits GDP-GTP exchange through a mechanism of steric hindrance. Having identified necessary bound sites for inhibition, and explored their electrostatic properties, it should be possible to proceed with the design of antibody mimics as therapeutic agents in cancer control.

The novel cyclic dinucleotide 3'-5' cyclic diguanylic acid binds to p21ras and enhances DNA synthesis but not cell replication in the Molt 4 cell line.

1. The effect of the novel, naturally occurring nucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.

Direct Measurement of the Binding of RAS to Neurofibromin Using a Scintillation Proximity Assay

Protein-protein interactions are of major importance in many cellular processes. When no enzymic activity is involved, assays for direct binding are required. One such example is the relatively weak interaction between oncogenic Pas and the GTPase-activating protein neurofibromin (NF1). The complex between the catalytic domain of NF1 and the GTP-form of oncogenic Pas protein dissociates rapidly; hence, equilibrium binding must be quantitated. Scintillation proximity assay (SPA) technology, a radioisotopic technique that requires no separation step, was used to characterize this interaction. Leu-61 Ras complexed with [3H]GTP was generated by nucleotide exchange in the presence of a GTP-regenerating system. A SPA signal was obtained when radiolabeled Pas was mixed with NF1 fused with glutathione S-transferase (GST), anti-GST, and protein A-coated SPA beads. This signal was abolished when any of the components were omitted and also by the addition of NaCl, which potently reduces the affinity of interaction between Pas and NF1. The neutralizing anti-Pas monoclonal antibody Y13-259 and the detergent n-dodecyl maltoside, a specific inhibitor of NF1 catalytic activity, both abolished the SPA signal from the NF1/Ras assay but neither affected a control SPA signal in which a [3H]GTP.GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology could be readily extended to the measurement of other protein-protein interactions and could form the basis for high-throughput screens for the discovery of novel therapeutic agents.

The Ras/Raf Signaling Pathway Is Required for Progression of Mouse Embryos Through the Two-Cell Stage

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Intracellular immunization with cytosolic recombinant antibodies.

We report the application of a strategy to inactivate cellular proteins in vertebrate cells based on the intracellular expression of immunoglobulin genes. We have selected, in this instance, the p21 protein, encoded by the ras proto-oncogene, as a target protein. The variable regions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody molecule or its single-chain Fv fragment (ScFv) derivative. In order to target the recombinant antibodies to the cytosol, their hydrophobic leader sequence for secretion was mutated or deleted. When these proteins are expressed in the cytosol of Xenopus laevis oocytes they colocalize with the endogenous p21ras protein in the cytoplasmic face of the oocyte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments block the ensuing meiotic maturation. Thus the intracellular expression of both whole antibodies and antibody domains can be used to block a biological function.

Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors.

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors.

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

Use of Living Columns to Select Specific Phage Antibodies

Here we demonstrate that it is possible to confront two recombinant microorganisms in order to select one using the other. We have shown that an epitope derived from p21ras expressed within the outer membrane protein, LamB, can be recognized both by the monoclonal antibody Y13-259, as well as the single chain Fv fragment derived from it. This specificity, which is maintained when the Y13-259 single chain Fv is expressed as a fusion protein with the phage fd gene 3 protein, has allowed us to use the living column of LamB-ras to purify Y13-259 phage from a background of non-binding phage, even at dilutions as high as 10 phage in 10(10) irrelevant phage.

Intracellular Expression of Anti-p21ras Single Chain Fv Fragments Inhibits Meiotic Maturation of Xenopus Oocytes

The recombinant variable regions of the monoclonal antibody Y13-259, directed against the p21ras protein, have been engineered for expression as intracellular single chain Fv fragments. The activity of the plasmid was confirmed by in vitro and in vivo translation of mRNA showing that the intracellularly expressed single chain fragments are stably and efficiently expressed as cytosolic proteins. The expression of the anti-p21ras single chain antibodies in the cytoplasm of Xenopus laevis oocytes leads to the inhibition of the insulin-induced meiotic maturation. This finding represents the first successful application of the strategy of intracellular antibodies to block a complex biological process in the cytosol of vertebrate cells.

RAS GENE ACTIVATION IN HUMAN SMALL INTESTINAL TUMORS

Expression of the ras oncoprotein in thirteen human small intestinal tumors was investigated employing an immunohistochemical technique. The level of ras p21 was analysed using the monoclonal antibody Y13-259 and the biotin-streptavidin-peroxidace-DAB technique. Nine out of thirteen tumors including one hyperplastic - metaplastic polyp, one adenomatous and papillary polyp of the duodenum, one adenomatous polyp, one leiomyoma, one carcinoid, one lymphoma, one angiosarcoma of the jejunum, one leiomyosarcoma and one metastatic adenocarcinoma of the colon, were found to ''press the ras p21 oncoprotein at elevated levels, as compared to adjacent normal tissue. Whereas in one Brunner's gland adenoma of the duodenum, one neurilemoma, one adenocarcinoma of the small intestine and one metastatic adenocarcinoma of the colon expression of ras p21 was not elevated. The detection of H-ras mutations in codon 12 and K-ras mutations in codons 12 and 13 was also examined using the polymerase chain reaction technique. Four out of the thirteen small intestinal tumors examined possessed mutations of the H-ras gene in codon 12. These included the following, tumors: one Brunner's gland adenoma of the duodenum, one lymphoma, one leiomyo-sarcoma and one metastatic adenocarcinoma of the colon. This is the first demonstration of ras mutations in small intestinal tumors. None of the tumors had mutations in K-ras codons 12 or 13 (Gly-->Asp) It is suggested that the ras p21 oncoprotein may be involved in the pathogenesis and H-ras mutations and be a molecular genetic marker in small intestinal tumors.

RAS P21 EXPRESSION IN NEUROBLASTOMAS AND GANGLIO-NEUROBLASTOMAS - CORRELATION WITH PATIENTS PROGNOSIS

Ras p21 expression was investigated in 28 cases of neuroblastomas and 12 cases of ganglio-neuroblastomas by the biotin-streptavidin immunoperoxidase method using the anti-ras p21 rat monoclonal antibody Y13-259. The results showed that 28% of neuroblastomas and 92% of ganglio-neuroblastomas demonstrated elevated ras p21 expression. However, in this series of patients 10 of 12 ganglio-neuroblastomas were staged as early disease (stage I or II) compared to 15 of 28 neuroblastomas, with similar staged disease. On analysing all of these tumours into early and late disease (i.e. stage I and II; and stage III and IV) a correlation was found between p21-ras overexpression and early disease (P<0.0001). As staging of neuroblastomas has been considered to be a prognostic indicator, it is argued that ras p21 over-expression maybe an indicator for a favourable prognosis in these patients.

Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.

The ras oncogene-encoded p21 protein is known to induce cell maturation of Xenopus laevis oocytes and malignant transformation of NIH 3T3 mouse fibroblasts. The pathways involved in oocytes and NIH 3T3 cells appear to be similar to one another. For example, in both cases, the ras p21-induced cellular events involve increased intracellular levels of the second messengers diacylglycerol and inositol phosphates, the former of which activates protein kinase C (PKC). To investigate the pathway of ras-induced oocyte maturation, we have explored the relationship between p21 protein and PKC. We show that the maturation signal from oncogenic p21 microinjected into Xenopus oocytes is completely blocked by the relatively specific PKC inhibitor CGP 41251, a staurosporine analogue that selectively inhibits PKC, but not by an inactive analogue of staurosporine, CGP 42700. Microinjection of purified PKC or of phorbol ester induces maturation of oocytes. PKC-induced maturation is inhibited by CGP 41251 but not by CGP 42700. Maturation induced by microinjected PKC is also not inhibited by two specific anti-p21 agents, the inactivating anti-p21 monoclonal antibody Y13-259 and the amino acid derivative azatyrosine. Both of these agents block p21-induced cell maturation. These results suggest that ras effects depend upon the action of PKC, whose activation is an event that occurs downstream of p21 in the maturation signal pathway.

Biochemical characterization of Artemia ras p21.

The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.

Ras p21 in breast tissue: associations with pathology and cellular localisation

Immunocytochemistry with monoclonal antibody Y13-259 demonstrated p21 ras in paraffin sections of breast tissue from 171 women: 85 with invasive breast carcinoma, 14 with non-invasive carcinoma and 72 with benign changes only. Many different tissue elements contributed to ras expression. Semiquantitative assessment showed that intensity of immunostaining in the normal epithelium of large ducts, small extralobular ducts and terminal duct lobular units (TDLU) was usually exceeded by that of myoepithelial cells. Vascular smooth muscle and apocrine epithelium also stained strongly, but the flat epithelial cells lining cysts did not express detectable p21 ras. There was a progressive increase from normal epithelium through epithelial hyperplasia of usual type and atypical hyperplasia to carcinoma in situ, without further increase in invasive carcinoma. Expression in carcinomas was inversely related to oestrogen receptor content but independent of the prognosis-associated variables of size, histological type, vascular invasion or lymph node metastasis.

Clinical significance of ras p21 overexpression for patients with an advanced colorectal cancer

The expression of ras oncogene product p21 was examined in 45 paraffin-embedded sections of primary advanced colorectal cancers, using the anti-v-H-ras p21 monoclonal antibody Y13-259. Fourteen of these specimens (31 percent) were stained positively. The incidence of lymphatic vessel invasion of cancer cells and lymph node metastasis correlated statistically with the overexpression of ras p21. The depth of invasion and incidence of liver metastasis in the p21-positive group were more prominent than in the p21-negative group. Statistically significant differences were evident in operative curability and clinical stage at initial surgery and in the long-term survival rate between these groups (P less than 0.05). We propose that ras p21 overexpression may serve as a marker to predict the prognosis of colorectal cancer.