INTRACELLULAR EXPRESSION OF THE MONOCLONAL ANTI-RAS ANTIBODY Y13-259 BLOCKS THE TRANSFORMING ACTIVITY OF RAS ONCOGENES

Microinjection of the anti-ras antibody Mab Y13-259 modifies ras funct ion and can induce temporary reversion of the transformed phenotype in mutant ras-transformed cells. Intracellular production of neutralizin g antibodies represents an approach to investigate the regulation of g ene function. The genes coding for the heavy and light chains of Mab Y 13-259 were isolated from a cDNA library. NIH3T3 cells transfected wit h heavy and light chain expression vectors produced functional anti-ra s antibody. The production of functional antibody did not require glyc osylation. To ensure that the antibody entered the cytoplasm and not t he secretory pathway, the hydrophobic leader sequences of both chains were removed and replaced with synthetic initiator sequences. The modi fied heavy chain gene was cloned under the control of the murine sarco ma virus long terminal repeat, and the light chain gene under the cont rol of the mouse mammary tumor virus long terminal repeat, which allow s the induction of light chain expression in the presence of dexametha sone. When both heavy and light chain genes were expressed in cells wi th activated ras (morphologically transformed) in the presence of dexa methasone, we observed phenotypic reversion to characteristics of nont ransformed cells. These experiments show that intracellular expression of antibodies can also be used as an alternative to analyze biologica l functions of a given protein.