Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Two-dimensional polyacrylamide gel electrophoresis analysis of phosphorylated, membrane-localized ras p21 proteins

The ras p21 oncogene product migrates as a heterogeneous series of polypeptides as resolved by both one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). We have prepared polyclonal rat serum antibody to ras p21 and used this as well as monoclonal antibodies to immunoprecipitate forms of p21 synthesized in vivo in transformed NIH3T3 cells. Two-dimensional PAGE of p21 resolved two distinct groups of polypeptides, one acidic (pI 4.8-5.3) which we call the "A" forms, and one less acidic or more basic (pI 6.5-7.0) which we call the "B" forms. It is the membrane-localized, B forms of v-ras-Ha p21 that are predominantly phosphorylated in vivo.     

RAS enzyme-linked immunoblot assay discriminates p21 species: a technique to dissect gene family expression

The members of the RAS gene family of protooncogenes are of implied biological significance in oncogenesis. The precise role of these genes is unclear. One difficulty has been the inability to discriminate the individual p21 protein products of various ras genes in cell lines, de novo human tumors, and related normal tissues. In this report, specific proteins of the human c-Ha-ras-1, c-Ki-ras-2, and c-N-ras genes have been detected and discriminated by the differential use of various antisera recognizing these p21s. This enzyme-linked immunoblot assay utilizes a double antibody system in which monoclonal antibodies are initially used to immunoprecipitate the p21ras proteins. Immunoprecipitates are then subjected to one-dimensional Western blot analysis utilizing other antibodies raised against p21s, coupled with nonradiolabeled enzyme-linked colorimetric detection. By direct detection, the specific products of the three human ras genes can be discriminated. In addition, we describe the generation and characterization of a new anti-p21c-N-ras-specific antibody. The simultaneous expression into protein of multiple ras genes is unequivocally demonstrated in both homogeneous cell lines and heterogeneous human tissues. This new technique is also applicable for discrimination of the protein products of other gene families.     

Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays

Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family. We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products. Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines. One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21. Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin. These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product. The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material.     

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.     

Point-mutated p21ras couples a muscarinic receptor to calcium channels and polyphosphoinositide hydrolysis

A high-affinity muscarinic receptor is detectable both in normal 3T3 mouse fibroblasts and in their transformed counterpart obtained by transfection with the oncogene EJ/T24-H-ras. However, only the transformed cell line is responsive to muscarinic agonist carbamylcholine in terms of Ca2+ influx and polyphosphoinositide hydrolysis, whereas the normal cell line is unresponsive. Using a point-mutated p21ras protein and monoclonal antibodies anti-p21ras, we provide evidences that p21ras couples to receptor-operating calcium channels and to polyphosphoinositide hydrolysis a muscarinic receptor which is uncoupled in normal mouse fibroblasts.     

Intracellular immunization. Cloning and intracellular expression of a monoclonal antibody to the p21ras protein

Following the demonstration that intracellular expression of antibodies ('intracellular immunization') may be utilized to engineer new traits in mammalian cells, we undertook experiments to perturb the function of p21ras proteins, by engineering the intracellular expression of the anti-p21ras antibody Y13-259. The variable regions of this antibody have been cloned and, after verifying their antigen binding activity, expressed in general purpose vectors for the intracellular expression of antibodies. The results confirmed that the cloned antibody has been efficiently expressed both in the secretory and the intracellular forms. Thus, intracellular immunization of mammalian cells against p21ras, or any other antigen for which a monoclonal antibody is available, can now be performed.     

Expression of ras oncogene p21 during human fetal development as determined by monoclonal antibodies RAP-5, Y13-259, and DWP

In this report we describe the expression of the ras proto-oncogene p21 protein in various tissues during normal fetal development. Conventional, formalin fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging 9 to 42 weeks of gestation. Immunohistochemical localization of ras p21 was accomplished using the broadly reactive, mouse monoclonal antibodies RAP-5 and Y13-259. The monoclonal antibody DWP, which is specific for a mutated form of ras p21 having a valine/cysteine at amino acid position 12, was also used. Detectable expression of the p21 protein was seen at different time periods during fetal development depending on the tissue. The expression of ras p21 (as detected by RAP-5 and Y13-259) was noted in a wide range of cell types and tissues; intense immunostaining was noted in epithelial cells of the gastrointestinal tract, exocrine and endocrine pancreas, renal tubules and transitional urotheliem, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances ras p21 expression, when present, occurred during periods of rapid growth in given organ systems. However, some actively proliferating fetal tissues such as thymus and spleen, failed to express detectable ras p21 suggesting that factors other than cell cycle may influence its expression. No reactivity with DWP was noted in any of the tissues, suggesting that the mutated forms detected by this monoclonal antibody are not expressed during normal human embryogenesis. These data show that there is regulated expression, and broad distribution of this gene product in normal developing human fetal tissue.     

人原发性肝癌和肝癌细胞株的N-ras癌基因的产物p21

以原发肝癌及肝癌7402细胞株DNA转染NIH/3T3得到的转化细胞与材料,经~(35)S-甲硫氨酸参入,应用放射免疫吸附,聚丙烯酰胺凝胶电泳技术,发现原发性肝癌的转化细胞株中N-ras基因的表达产物p21明显高于非转化的细胞。7402细胞株和7402转化的细胞株可能存在有二个ras基因,一个是ras~N;另一个是ras~H。本文结果为证明N-ras是人肝癌的一种转化基因提供了证据。     

Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.     

Immunohistochemical evaluation of ras oncogene expression in pulmonary and pleural neoplasms

We undertook an immunohistochemical analysis of human bronchopulmonary epithelial neoplasms and pleural mesotheliomas using a monoclonal antibody which recognizes ras oncogene products (p21ras). The monoclonal antibody, RAP-5, recognizes both unaltered and certain mutated p21ras. Formalin fixed and paraffin embedded tissue samples of 187 lung epithelial tumors and 27 pleural mesotheliomas were investigated; normal and bronchiectatic lungs were similarly studied. Normal lung and pleural tissue did not immunostain except for occasional type II pneumocytes. Reactive type II pneumocytes adjacent to carcinomas and bronchiectasis immunostained consistently. Twenty four/34 (71%) squamous carcinomas immunostained. Only 8/50 (16%) adenocarcinomas immunostained focally and weakly whereas 19/24 (79%) bronchioloalveolar carcinomas immunostained. Eleven/18 (61%) large cell carcinomas immunostained with variable intensity. Eleven/13 (85%) carcinoids, 6/7 (85%) well differentiated neuroendocrine carcinomas, and 18/21 (86%) intermediate cell neuroendocrine carcinomas immunostained while none of 20 small cell neuroendocrine carcinomas immunostained. Only a few mesotheliomas were immunostained focally. Two/14 (14%) epithelial type and 1/9 (11%) biphasic type mesotheliomas immunostained weakly; none of 4 spindle cell mesotheliomas immunostained. We conclude that while at least occasional cases of most types of pulmonary epithelial neoplasms express p21ras, the frequency and intensity of the expression are distinctly greater in certain tumor types such as squamous, bronchioloalveolar, and neuroendocrine neoplasm except for the small cell type. Contrary to these lung epithelial neoplasms, most mesotheliomas did not immunostain for p21ras. Whether the enhanced p21ras expression may point to a different mechanism of transformation or may merely reflect differentiation features remains undetermined.     

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.     

Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals.

A family of normal vertebrate genes and oncogenes has been called the ras gene family. The name ras was assigned to this gene family based on the species of origin of the viral oncogenes of the rat-derived Harvey and Kirsten murine sarcoma viruses. There are now three known functional members of the ras gene family, and genes homologous to ras genes have been detected in the DNA of a wide variety of mammals and in Drosophila melanogaster. Prior experiments have detected proteins coded for by ras genes in a large number of normal cells, cell lines, and tumors. We report here the detection of ras-related proteins in D. melanogaster, a result predicted by the earlier detection of ras-related genes in the Drosophila genome. We also report for the first time the detection of ras-related proteins in a single-cell eucaryocyte, Saccharomyces cerevisiae. These proteins, approximately 30K in size, are recognized by both a monoclonal antibody which binds to the p21 coded for by mammalian ras genes and a polyclonal rat serum made by transplanting a v-Ha-ras-induced tumor in Osborne-Mendel rats. The p21 of v-Ha-ras and the 30K proteins from S. cerevisiae share methionine-labeled peptides as detected by two-dimensional tryptic peptide maps. The results indicate that S. cerevisiae synthesizes ras-related proteins. A genetic analysis of the function of these proteins for yeast cells may now be possible.     

Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.     

Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family.

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.     

Cellular ras activity and tumor cell proliferation

A monoclonal antibody able to specifically neutralize activity of the cellular proto-oncogene ras was microinjected into a variety of normal and tumor cell types in order to determine the requirement for c-ras activity during proliferation. Normal cells were always efficiently inhibited by the antibody, while most tumor cells continued to proliferate without inhibition following the injection. Tumor cells containing a mutant ras gene, however, exhibited an intermediate phenotype and were partially inhibited in proliferation by the injected anti-ras antibody. The mutant c-ras gene appears, therefore, to play a role in proliferation even of the mature tumor cell (although other gene products must also be involved). Mutations in most other tumor cells, however, fall into the class of oncogenes which promote proliferation independently of c-ras activity. In this way tumor cell proliferation is clearly distinguished from that of normal cells studied.     

Expression of ras-like proteins in embryonic and adult cells of Xenopus laevis

A polyclonal antibody raised against v-Ha-ras p21 was purified and its specificity was checked on Ha-ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one-dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full-grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two-dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modifications of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two-dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.     

Localization of pepstatin's inhibitory action during Fc-mediated antibody internalization: possible implications for antibody-mediated viral transmission

Antibody internalization via Fc receptors is an important cellular mechanism, possibly facilitating the entry of antigenic peptides or viral particles into cells when specific antibodies are present at the periphery. Using an experimental model of trophoblast cells, we have shown that anti-p21(ras) monoclonal antibodies can use IFN-gamma-induced surface Fcgamma receptors to enter the cell. This entry of anti-p21(ras) antibodies ultimately inhibits IFN-gamma-mediated class II antigen induction. Since there may be obvious and inevitable harmful aspects of this mechanism, during which Fc-mediated viral particle or autoantigen transport may occur, we concentrated efforts on defining a potent inhibitor able to eliminate such uptake. The results presented here show that the protease inhibitor pepstatin A efficiently inhibits Fcgamma receptor induction by IFN-gamma and also blocks the endocytic pathway followed by an antibody when it enters the cell at the level of early endosomal compartments. We thus postulate that the use of pepstatin A, because of its inhibition of autoantigen presentation or viral transmission, including that of HIV, may find important applications in therapeutic protocols.