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 function and can induce temporary reversion of the transformed phenotype in mutant ras-transformed cells. Intracellular production of neutralizing antibodies represents an approach to investigate the regulation of gene function. The genes coding for the heavy and light chains of Mab Y13-259 were isolated from a cDNA library. NIH3T3 cells transfected with heavy and light chain expression vectors produced functional anti-ras antibody. The production of functional antibody did not require glycosylation. To ensure that the antibody entered the cytoplasm and not the secretory pathway, the hydrophobic leader sequences of both chains were removed and replaced with synthetic initiator sequences. The modified heavy chain gene was cloned under the control of the murine sarcoma virus long terminal repeat, and the light chain gene under the control of the mouse mammary tumor virus long terminal repeat, which allows the induction of light chain expression in the presence of dexamethasone. When both heavy and light chain genes were expressed in cells with activated ras (morphologically transformed) in the presence of dexamethasone, we observed phenotypic reversion to characteristics of nontransformed cells. These experiments show that intracellular expression of antibodies can also be used as an alternative to analyze biological functions of a given protein.
Montano
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National Cancer Institute, Frederick Cancer Research Facility, Maryland 21701, USA.
Jimenez
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A
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D011905:Genes, ras / Q000187:drug effects / Q000502:physiology*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D051379:Mice; D008845:Microinjections; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D021382:Protein Sorting Signals; D014415:Tunicamycin; D003907:Dexamethasone; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)