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.