Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation

Purpose: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96-110 (PNC2) selectively blocks oncogenic (Val 12-contai ning) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferati on, we synthesized an inducible plasmid that expressed this peptide sequenc e. Our purpose was to test this expression system in oocytes to determine i f it was capable of causing selective inhibition of oncogenic ras-p21. Meth ods: We injected this plasmid and a plasmid expressing a control peptide in to oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). Result s: Microinjection of this plasmid into oocytes together with Val 12-p21 res ulted in complete inhibition of maturation in the presence of inducer. Anot her plasmid encoding the sequence for the unrelated control peptide, X13, w as unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasm id had no effect on the ability of insulin-activated normal cellular or wil d-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. Conclusi on: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p2 1 and may therefore be highly effective in blocking proliferation of ras-in duced cancer cells. Also, from the patterns of inhibition, by PNC2 and othe r ras- and raf-related peptides, of raf- and constitutively activated MEK-i nduced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.