Characterization of monoclonal antibodies specific to the activated ras p21 with aspartic acid at position 13.

The ras proto-oncogenes encode membrane bound proteins (p21) which are structurally distinct from the proteins encoded by the activated transforming ras genes. These activated ras genes have been identified in various human tumors as well as their preneoplastic lesions such as colorectal tumors (20-40%), pancreatic carcinomas (95%), lung carcinomas (20-30%), myelodysplasia (40%) and acute myeloid leukemia (30%). The activation of ras p21 is due to amino acid substitutions at positions 12, 13 or 61 of the p21 protein. This report describes two monoclonal antibodies designated D129 and D146 raised against a synthetic peptide corresponding to amino acids 5-16 of ras p21 activated by the substitution of aspartic acid for glycine at position 13. D129 and D146 react specifically with the peptide with the aspartic acid substitution at position 13, but not with the peptide with valine at position 13 or the peptide containing the normal glycine at position 13. Western blot analysis demonstrates that D129 and D146 react specifically with p21 extracted from transformed NIH3T3 fibroblast lines containing aspartic acid at position 13. These studies also demonstrate that D146 is able to detect the activated p21 with aspartic acid at position 13 that is shed into the culture media. Studies demonstrate that MAb D146 specifically immunoprecipitates the cellular p21 with aspartic acid at position 13 from transformed NIH3T3 cells, whereas D129 cannot immunoprecipitate the activated p21. Using a sandwich ELISA format, D146 is able to detect the p21 with position 13 aspartic acid from cell extracts and culture fluids. The ability of D146 to function in the ELISA format raises the possibility that this assay maybe a quick and effective way of determining the presence of activated p21 with aspartic acid at position 13 in human fluids and tissues.

Activated Val-12 ras p21 in cell culture fluids and mouse plasma.

Activation of ras oncogenes has been associated with a variety of cancers as well as their precursor lesions. Ras proteins activated by substitutions at amino acid positions 12, 13 or 61 have not been identified in normal tissues and therefore their detection may have clinical value. In this study our objective was to determine whether activated ras proteins could be released into the extracellular environment. To test this hypothesis, we used ras-transformed NIH3T3 cells that express an activated p21 containing valine (Val-12 p21) at position 12 instead of the normal glycine (Gly-12 p21) and a monoclonal antibody (mAb) designated DWP that is specific for the activated Val-12 ras proteins. Culture fluids collected from NIH3T3 cells transformed by the activated Val-12 p21 were shown, using mAb DWP in a sandwich ELISA format, to contain the activated Val-12 p21. In contrast, culture fluids from non-Val-12-containing cells were unreactive with mAb DWP. PSV-LM-EJ cells which overexpress the activated Val-12 p21 were injected subcutaneously (SQ) into nude mice to produce tumors. At the time of gross tumor appearance (14-21 days after tumor cell inoculation), plasma was collected from the PSV-LM-EJ tumor-bearing mice as well as from a series of control mice. Employing mAb DWP as a detection reagent in the sandwich ELISA format, we were able to detect the Val-12 p21 in the plasma of the PSV-LM-EJ tumor-bearing mice. Activated Val-12 p21 was not present in the plasma of non-tumor-bearing mice, or in the plasma of mice bearing SQ tumors composed of non-Val-12 p21 ras-transformed cells. This report is the first description of an activated ras protein (Val-12 p21) in the plasma of tumor-bearing mice and demonstrates that the results of the Val-12 p21-specific ELISA could be validated with Western blot format.

Production and characterization of monoclonal antibodies to Ha-ras and N-ras p21.

The mammalian ras family consists of the Ha, Ki and N-ras genes that encode a series of 21,000 dalton proteins (p21). The three ras proteins participate in normal cell physiology and have been implicated in cellular transformation by either overexpression of the normal p21 or by mutation at positions 12, 13, or 61. To help understand the biological roles of the different ras proteins, we have generated monoclonal antibodies (Mabs) to the Ha-ras and N-ras p21. Mab Ha-770, raised to a Ha-specific synthetic peptide, reacts with Ha-ras recombinant p21 (r-p21) as well as cellular Ha-ras p21 by immunoprecipitation. Western blot and sandwich ELISA assays. Mab N-838, raised to an N-ras specific synthetic peptide, reacts with the N-ras recombinant p21 by immunoprecipitation, Western blot and sandwich ELISA assays. Mabs to the Ha-ras and N-ras p21 should be valuable reagents in assessing the individual roles of ras proteins in normal and neoplastic cells.

Generation of monoclonal antibodies specific for ras p21 Glu-12 oncoproteins: detection in carcinogen-induced mammary carcinomas.

ras genes have been shown to become oncogenes by single point mutations which result in amino acid substitutions that affect either their GTPase activity (positions 12, 13, 59, 61) or their affinity for GTP and GDP. Ras oncogenes and their corresponding proteins have been described in a variety of human cancers as well as in animal tumors induced by physical and chemical carcinogens. One of these animal tumor systems involves the induction of mammary carcinomas in rats by a single dose of N-nitroso-N-methylurea (NMU), a methylating carcinogen. These NMU-induced mammary carcinomas contain transforming H-ras genes activated by G----A transitions in the second nucleotide of their 12th codon, presumably a consequence of the pre-mutagenic lesions induced by NMU. These G----A mutations result in the replacement of the normal glycine in the 12th position of the ras p21 protein by a glutamic acid residue. In this study, we report the generation of monoclonal antibodies (Mab) reactive with oncogenic ras p21 proteins containing glutamic acid at position 12 (p21 Glu-12). Mab designated E184 specifically recognized activated ras p21 Glu-12 proteins but not normal p21 (Gly-12) or p21 proteins activated by other position 12 substitutions including arginine, aspartic acid, cysteine, valine or serine residues. Western blot analysis of NMU-induced mammary carcinomas demonstrated that Mab E184 recognized p21 proteins expressed in these rat tumors but not p21 present in normal tissues nor in other carcinogen-induced tumors known to carry H-ras oncogenes activated by mutations at position 61.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of anti-RAS p21 monoclonal antibodies.

Monoclonal antibodies (MAb) Ras 10 and Ras 11 were raised to an activated human Harvey-ras p21 and shown to react with recombinant p21 as well as p21 derived from human and rodent cells. Characterization studies by ELISA, immunoprecipitation and Western blot procedures demonstrated that MAb Ras 10 (IgG2a) and Ras 11 (IgG2b) react with normal p21, activated p21, and p21 from each of the Harvey, Kirsten and N-ras families. Studies illustrated that MAb Ras 10 and Ras 11 can also be used in flow cytometry and immunohistochemistry to specifically detect cellular p21. ELISA, immunoprecipitation and Western blot studies comparing rat anti-p21 MAb Y13-259 with Ras 10 and Ras 11 demonstrated that Ras 10 and Ras 11 had a greater sensitivity for ras protein detection than Y13-259. Collectively, these studies illustrate that MAb Ras 10 and Ras 11 can be applied to a variety of assay formats to detect ras proteins and, therefore, may be valuable tools in detecting and measuring of ras protein expression in normal, neoplastic and pre-neoplastic cells.

Mechanism of stimulation of prostaglandin synthesis by a factor from rheumatoid synovial tissue.

Rheumatoid synovial cell monolayers, with [1-14C]arachidonic acid ([1-14C]AA) incorporated into cell lipids, are stimulated by a factor (RSF) produced by explant cultures of rheumatoid synovial tissue to produce up to 50-fold increases in [1-14C]prostaglandin E2 and [1-14C]prostaglandin I2. In contrast, levels of free [1-14C]AA released from RSF-stimulated cells are generally lower than [1-14C]AA levels in cultures of untreated cells. These observations are inconsistent with a mechanism of prostaglandin stimulation consisting of an increase in phospholipase activity, because this mechanism would increase free AA levels as well as prostaglandins. A mechanism is proposed in which free AA is maintained at low steady-state levels by reacylation of free AA into phospholipids at a rate more rapid than its reaction with cyclooxygenase to form prostaglandins. In this mechanism, stimulation of the rate of the cyclooxygenase step by RSF accounts for increased prostaglandin synthesis as well as the decreased release of AA. On the basis of data previously reported by others, it is suggested that this mechanism may also be applicable to the stimulation of prostaglandin synthesis by several other agents. Preliminary characterization of the RSF indicates that it is a protein, and molecular seive chromatography indicates that its molecular weight is about 18,000. The production of RSF by rheumatoid synovial tissue is suppressed to undetectable levels by 1 microM dexamethasone.