Oncogenic Ras Proteins Research Articles

Evidence of enhancement of the ras oncogene protein product (p21) in a spectrum of human tumors.

Using a direct binding liquid competition radioimmunoassay, the amount of the ras oncogene protein product, p21, was quantitated in a variety of human tumors and adjacent apparently normal tissues. In 48 of 50 matched tumor and normal tissue biopsy specimens from 50 patients, more ras p21 was detected in the tumor than in its normal counterpart. Twenty-five of 28 breast tumors demonstrated more ras p21 than the average of the values obtained for fibroadenomas. Furthermore, in 17 of the 19 cases studied, over 20% more ras p21 was observed in breast carcinomas compared with their respective normal counterparts. More ras p21 was also demonstrated in the majority of tumors of the stomach, lung, colon and bladder compared with their respective adjacent normal tissues. Our data therefore indicate that ras p21 expression is quantitatively enhanced in many human tumors originating from several different tissue types.

Structural differences between a ras oncogene protein and the normal protein

One of the most commonly found transforming ras oncogenes in human tumours has a valine codon replacing the glycine codon at position 12 of the normal c-Ha-ras gene. To understand the structural reasons behind cell transformation arising from this single amino acid substitution, we have determined the crystal structure of the GDP-bound form of the mutant protein, p21(Val-12), encoded by this oncogene. We report here the overall structure of p21(Val-12) at 2.2 A resolution and compare it with the structure of the normal c-Ha-ras protein. One of the major differences is that the loop of the transforming ras protein that binds the beta-phosphate of the guanine nucleotide is enlarged. Such a change in the 'catalytic site' conformation could explain the reduced GTPase activity of the mutant, which keeps the protein in the GTP bound 'signal on' state for a prolonged period time, ultimately causing cell transformation.

Characterization and tumorigenicity of a butyrate-adapted T24 bladder cancer cell line.

We have adapted T24P, a tumorigenic subline of the T24 human bladder cancer cell line, to grow in 5 mM butyrate. In the presence of butyrate, the adapted cells (T24P/B) grow more slowly than the unadapted cells (T24P/C), have a lower saturation density, increased serum requirement for growth, loss of ability to form colonies when plated at low cell density, and decreased ouabain sensitivity. Morphologically, T24P/B cells in butyrate are large and flattened with increased cytoplasm. When T24P/B cells are grown without butyrate, the morphological changes, growth rate, plating efficiency, and ouabain sensitivity return to those of T24P/C. While the saturation density increases, it does not return to levels of T24P/C, and the size of colonies never reaches that of the T24P/C colonies. Both T24P/C and T24P/B are tumorigenic in nude mice, however, the T24P/B tumors differ grossly and microscopically from those produced by T24P/C in that they contain large cystic structures filled with clear fluid and lined by transitional cell epithelium with flattened surface layers. Although the transformed phenotype and tumorigenicity of T24P are modified by adaptation to growth in butyrate, no significant changes in ras oncogene RNA or protein expression were identified.

Serum Oncogene Proteins in Hazardous-Waste Workers

Oncogene protein detection may be a valuable molecular epidemiological marker of commitment to the carcinogenic pathway in worker populations exposed to carcinogens. This paper reports the use of serum immunoblotting with monoclonal antibodies to oncogene proteins to screen hazardous waste workers with potential carcinogen exposure who employ state-of-the-art work practices and personal protective equipment to minimize real exposures. Two individuals in this cohort were found to be trace positive for the ras oncogene protein in their serum, but this may have been due to their cigarette smoking. These results are contrasted to previous results in hazardous waste workers with real workplace carcinogen exposures.

Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates

The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5'-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.

Ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24- ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha- ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24- ras and c-Ha- ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.

Identification of microinjected cells using biotinylated antibodies and strep-avidin-conjugated horseradish peroxidase

Results from experiments using needle microinjection of cells are often compromised by an inability to readily demonstrate which cells within a population have been injected. The technique described here allows the unambiguous identification of cells that have been successfully microinjected. Sequential incubation of fixed cells with biotinylated anti-immunoglobulin antibodies, followed by horseradish peroxidase (HRP)-conjugated Strep-avidin and HRP substrate, provides a sensitive assay for identification of cells containing trace amounts of immunoglobulins. This allows direct correlation to the presence of injected molecules of effects on cell morphology, the ability to enter into DNA synthesis, or expression of specific genes. By a variety of criteria, nonspecific immunoglobulins do not adversely affect cellular processes when injected by themselves or in the presence of other proteins known to have biological effects when injected, such as cAMP-dependent protein kinase and the ras oncogene protein.

The switch between two conformations of adenylate kinase

Crystalline adenylate kinase from porcine muscle cytosol can assume two interconvertible structures. Here, we report the refined structure of crystal form B at 3·3 Å resolution and compare it with crystal form A. Crystal forms A and B can be interconverted by protonation and deprotonation of His36, which is located deep in the active center cleft. The changes concern the molecular packing as well as the polypeptide chain conformation. On conversion from crystal form A to B, the N-terminal α-helix unwinds, the active center cleft opens to some extent and the nucleotide-binding glycine-rich loop 15–22 at the active center is detached from the bulk protein. This loop has counterparts in various important mononucleotide-binding proteins and is known to bind a phosphoryl group in adenylate kinase and in the oncogenic ras proteins. It is most likely involved in the phosphoryl transfer and the concomitant conformational changes. It is suggested that the two observed conformations are relevant for enzyme action in solution: they represent two of a series of three known snapshots depicting the enzyme during the substrate binding process.

Monoclonal Antibodies for Detecting Oncogenic Ras Proteins in Cancer Cells

Monoclonal Antibodies for Detecting Oncogenic Ras Proteins in Cancer Cells

Effects of ras-encoded proteins and platelet-derived growth factor on inositol phospholipid turnover in NRK cells.

The role of ras-encoded proteins and platelet-derived growth factor (PDGF) in inositol phospholipid metabolism has been studied. PDGF stimulates inositol phospholipid turnover in confluent normal rat kidney (NRK) cells and enhances hydrolysis of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in NRK cell membranes in the presence of guanosine 5'-[gamma-thio]triphosphate. The stimulatory effect of PDGF on phosphatidylinositol bisphosphate hydrolysis is not inhibited by pretreatment of NRK cells with pertussis toxin, implying that PDGF-stimulated phospholipase C activity of NRK cells is regulated by a pertussis toxin-insensitive guanine nucleotide-binding protein (G protein) that is different from Gi (inhibitory G protein) or Go (G protein of unknown function). When bacterially made human normal or oncogenic T24 ras protein is added to 32P-labeled NRK cell membranes in the presence of guanosine 5'-[gamma-thio]triphosphate, normal ras protein increases by 3-fold the formation of inositol trisphosphate, whereas T24 ras protein has no significant effect. In addition, normal ras protein and PDGF have additive effects on inositol trisphosphate production. Taken together, these data suggest that normal ras protein stimulates inositol phospholipid turnover in NRK cells by means of a pathway different from the PDGF-regulated one and that oncogenic ras protein is without significant stimulatory effect in this action.

Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

Requirement of One Functional RAS Gene and Inability of an Oncogenic ras Variant to Mediate the Glucose-Induced Cyclic AMP Signal in the Yeast Saccharomyces cerevisiae

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors

Microinjection of the activated ras oncogenic protein can induce the meiotic maturation of Xenopus laevis oocytes, a process that can also be triggered by progesterone or high concentrations of insulin. Cycloheximide and puromycin, well-known inhibitors of protein synthesis, block the maturation process induced by progesterone and insulin but do not affect the maturation caused by H- ras lys 12 protein microinjection. Theophylline, an inhibitor of cAMP phosphodiesterase that also affects oocyte protein synthesis, does cause a partial inhibition of ras protein-induced maturation. These findings indicate that ras protein acts on the oocyte maturation process at a point that is downstream of the protein synthesis requirement, a characteristic shared with the maturation promoting factor, an activity that appears in oocytes and mitotic cells at the onset of cell division.

Ras oncogene expression as a prognostic indicator in rectal adenocarcinoma

In order to investigate the value of ras oncogene expression as a prognostic indicator in rectal adenocarcinoma, we evaluated the level of ras gene protein product (p21) in the available material of 149 consecutive Dukes' B and C specimens resected at our institution between 1965 and 1981. Five year follow-up was available in all patients. Pathology slides and archival paraffin blocks were retrieved for confirmation of the original diagnosis, study of histopathological features, and measurement of p21 content. P21 titers were obtained using the RAP-5 monoclonal antibody in a semiquantitative immunohistochemical assay. Titer was expressed as the highest dilution of antibody giving definitive staining using the avidin-biotin peroxidase method. The analysis indicated that the group of tumors with high (⩾1:40,000) p21 titers had a worse 5-year survival (43.9 versus 64.3%, P < 0.02), higher incidence of distant metastases (51.8 versus 23.2%, P < 0.001), and more advanced Dukes' stage (53.7 versus 35.7% incidence of Dukes' C stage, P < 0.04) than tumors with low (< 1:40,000) titers. Multivariate analysis also demonstrated that the influence of the level of ras gene protein product on survival was independent of Dukes' stage. We conclude that detection of high levels of ras oncogene protein product indicates a group of tumors with a more aggressive behavior, characterized by a higher percentage of distant recurrences and worse prognosis. These findings suggest that measurement of p21 may become clinically important in identifying patients at high risk of recurrent disease.

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.

Dexamethasone-dependent inhibition of differentiation of C2 myoblasts bearing steroid-inducible N-ras oncogenes.

ras proteins are localized to the plasma membrane where they are postulated to interact with growth factor receptors and other proximal elements in intracellular cascades triggered by growth factors. The molecular events associated with terminal differentiation of certain skeletal myoblasts are inhibited by specific polypeptide growth factors and by constitutive expression of transforming ras oncogenes. To determine whether the inhibitory effects of ras on myogenic differentiation were reversible and to investigate whether muscle-specific genes remained susceptible to ras-dependent repression in terminally differentiated myotubes, the murine myoblast cell line, C2, was transfected with a plasmid containing a mutationally activated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat. Addition of dexamethasone to myoblasts bearing steroid-inducible ras oncogenes prevented myotube formation and induction of muscle creatine kinase and acetylcholine receptors. Inhibition of differentiation by dexamethasone occurred in a dose-dependent manner and was a titratable function of ras expression. In the presence of dexamethasone, myoblasts bearing steroid-inducible ras genes retained their dependence on exogenous growth factors to divide and exhibited contact inhibition of growth at confluent densities, indicating that the inhibitory effects of ras on differentiation were independent of cell proliferation. Removal of dexamethasone from N-ras-transfected myoblasts led to fusion and induction of muscle-specific gene products in a manner indistinguishable from control C2 cells. Examination of the effects of culture media conditioned by ras-transfected myoblasts on differentiation of normal C2 cells yielded no evidence for inhibition of differentiation via an autocrine mechanism. In contrast to the ability of N-ras to prevent up-regulation of muscle-specific gene products in myoblasts, induction of N-ras in terminally differentiated myotubes failed to extinguish muscle-specific gene expression. Together, these results suggest that oncogenic ras proteins reversibly activate an intracellular cascade that prevents establishment of the differentiated phenotype. The inability of ras to extinguish muscle-specific gene expression in terminally differentiated myotubes also suggests that ras may interfere with an early step in the pathway of myoblasts toward the differentiated state.

Clonal heterogeneity, experimental metastatic ability, and p21 expression in H-ras-transformed NIH 3T3 cells.

We examined individual clones of murine NIH 3T3 cells, transformed with the human bladder cancer (T24) H-ras oncogene, for p21 expression and for experimental metastatic ability in the immunodeficient chick embryo. We found that the clones were heterogeneous for both of these properties. In general, p21 expression was a good predictor of metastatic ability of the clones. Cells from poorly metastatic clones were passaged in the chick embryo metastasis assay to determine whether cells with increased metastatic ability could be selected. We found that the selected cells were more metastatic and that substantial increases in expression of p21 also accompanied this increase in metastatic ability. The relationship between p21 expression and metastatic ability appeared linear, with a high correlation coefficient (r = .85), suggesting that in this model system quantitative increases in metastatic properties can result from increased expression of the ras oncogene protein product p21.

Localization of phospholipase A2 in normal and ras-transformed cells.

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.

Crystallization of human c-H- ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H- ras, c-K- ras and N- ras). We report crystallization of several human c-H- ras oncogene proteins.

Three-Dimensional Structure of an Oncogene Protein: Catalytic Domain of Human c-H- ras P21

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.