| Summary: | The small GTP binding proteins of the Ras superfamily have been implicated in
regulating cell growth and differentiation. Much of these data have been generated by the
microinjection of the neutralizing antibody to Ras, Y13.259. In studying the role of Ras in
mammalian cells I have chosen to attenuate Ras activity by the intracellular expression of a
neutralizing anti-Ras single chain Fv (scFv) derived from Y13.259. This approach has the
distinct advantage over microinjection in that it permits the study of the biochemical
consequence of abrogating Ras activity. The genes for the rat monoclonal, Y13-259, were
subcloned and engineered to produce a uni-valent monomeric protein which was shown to
recognize the same epitope on Ras (residues 60-76) as the parent antibody molecule. I
have successfully expressed scFv-Y13.259 in the SV-40 large-T transformed mouse
fibroblast line, SVT-2. Additionally, expression of scFv-Y13.259 did not decrease the
half-life of Ras, and the fact that 80-90% of the Ras could be detected complexed with
scFv-Y13.259 indicated that this simple method may be useful in uncovering functions of
Ras when a given site is masked by the scFv. Furthermore a myristylated version of scFv
Y13.259 that localizes to the plasma membrane caused a pronounced change in
morphology when expressed in SVT-2 cells.
When expressed in the B cell lymphoma line, WEHI-231, results showed that
blocking of the Switch II region of Ras by scFv-Y13.259 blocks proliferation and leads to
apoptosis. Given that the parental cells respond in the same manner to withdrawal of
serum, my results suggest that continued survival and proliferation of this tumor requires a
Ras dependant signal from serum. Cell cycle analysis of these scFv-Y13.259 expressing
WEHI-231 cells reveals that an apparent block at the G₁ to S phase boundary of the cell
cycle was the reason for their eventual demise.
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