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.     

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 of yeast adenylate cyclase by antibodies to ras p21.

Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.     

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.     

Reversal of transformed phenotype by monoclonal antibodies against Ha-ras p21 proteins

The transforming activities of p21 ras proteins have been determined by micro-injection of these proteins into NIH3T3 cells. In order to facilitate functional studies on the effect of ras proteins on malignant transformation and normal cellular growth, analysis has been made with three monoclonal antibodies (YA6-172, Y13-238 and Y13-259) as originally reported by Furth et al. (J virol 43 (1982) 294). Purified immunoglobulin of Y13-259 has the highest titer of binding to bacterially synthesized p21 ras proteins. Experimental analyses indicate that only Y13-259 antibody will neutralize the transforming activity of the co-injected bacterially synthesized ras protein and the neutralization effect was blocked by co-injection of excess ras protein. In addition, micro-injection of Y13-259 immunoglobulin into transformed NIH3T3 cells (obtained by DNA transfection of NIH3T3 cells with molecularly cloned ras gene) reversed their transformed phenotypes. These results indicate that both bacterially synthesized p21 ras proteins and the natural ras proteins produced in NIH3T3 cells were neutralized by Y13-259 antibody.     

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.     

A monoclonal antibody reactive with an activated ras protein expressing valine at position 12

Activated ras transforming genes have been described in a variety of neoplasms and encode 21,000-Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown to contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.     

Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.     

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.     

H-ras mutants lacking the epitope for the neutralizing monoclonal antibody Y13-259 show decreased biological activity and are deficient in GTPase-activating protein interaction.

We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Science 240:518-521, 1988). We showed that GAP had no effect on the in vitro GTPase activity of the deletion mutants of the normal p21 protein. Since similar deletions in mutants with activating lesions at position 12 or 59 or both showed decreased transforming activity, our results suggest that the recognition site for Y13-259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP and that this interaction is critical for biological activity of ras proteins.     

Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras proteins in the activation of phospholipase D.

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.     

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosis-promoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.     

A radioimmunocytological quantitative method for the rapid detection of ras oncogene p21 protein in mammalian cells

In order to provide a sensitive and quantitative detection method of ras p21 at the cytological level, the monoclonal antibody Y 13 259 and iodinated protein A were used to locate theras protein in various mammalian cell lines. The subsequent autoradiograph can be analysed by a computer-assisted system which showed in these reported experiments that the relative levels of p21 detected in these cells corresponded to results obtained earlier using conventional biochemical methods.To whom correspondence should be addressed.     

Application of automated image analysis to demonstrate the correlation between ras p21 expression and severity of gliomas

We found a direct correlation between increasing ras p21 protein immunopositivity and severity of human glioma using computer-assisted, digital-image processing to quantify the amount of p21 immunoreactive to the monoclonal antibody RAP-5. We determined that there was a significant difference in reactivity between glioblastoma multiformes and more-differentiated astrocytomas (experiment-wise error less than 0.05). This result confirmed the conclusions made on the same tumors using standard light microscopy and visual examination. Immunohistochemistry quantized by automated image analysis may be a useful adjunct to current histopathological strategies since it decreases assay subjectivity and variation.     

Regulation of a ras-related protein during development of Dictyostelium discoideum.

Recent work has shown that DNA sequences related to the mammalian ras proto-oncogenes are highly conserved in eucaryotic evolution. A monoclonal antibody (Y13-259) to mammalian p21ras specifically precipitated a 23,000-molecular-weight protein (p23) from lysates of Dictyostelium discoideum amoebae. Tryptic peptide analysis indicated that D. discoideum p23 was closely related in its primary structure to mammalian p21ras. p23 was apparently derived by post-translational modification of a 24,000-molecular-weight primary gene product. The amount of p23 was highest in growing amoebae, but declined markedly with the onset of differentiation such that by fruiting body formation there was less than 10% of the amoeboid level. The rate of p23 synthesis dropped rapidly during aggregation, rose transiently during pseudoplasmodial formation, and then declined during the terminal stages of differentiation. There was, therefore, a strong correlation between the expression of the ras-related protein p23 and cell proliferation of D. discoideum.     

The synthesis and degradation of ras-related gene products during growth and differentiation in Dictyostelium discoideum

A Dictyostelium discoideum protein with an Mr of 23,000 (p23dd-ras) is structurally related to the mammalian proto-oncogene ras-gene product, p21ras, and is specifically precipitated from cell-free extracts of D. discoideum by the Y13-259 monoclonal antibody against p21ras. p23dd-ras was degraded at rates that were very similar to those observed for total protein during both growth and differentiation, suggesting that the previously reported decline in p23dd-ras levels during differentiation is due to a change in the rate of synthesis rather than a change in the rate of degradation. p23dd-ras synthesis did not decrease immediately after the initiation of differentiation, but rather its rate of synthesis increased for the first 1-2 h, suggesting that p23dd-ras is not rapidly down-regulated in response to nutrient deprivation. There were differences in the extent of p23dd-ras turnover during the differentiation of the three tested strains, A-3, NC4, and V12-M2. The relative level of p23dd-ras dropped most rapidly in V12-M2, which may reflect the slightly faster differentiation process exhibited by this strain. In all three strains, very little p23dd-ras was present by the end of the differentiation process. A second protein with an Mr of 24,000 (p24dd-ras) was also immunoprecipitated using the Y13-259 antibody. The amount of p24dd-ras was small or undetectable in vegetative cells, but relatively larger amounts of p24dd-ras were synthesized in pseudoplasmodial cells. We found no evidence to suggest that p24dd-ras is a precursor of p23dd-ras.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.     

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms.

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.