Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.
OBJECTIVE
We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21.


METHODS
We injected this plasmid and a plasmid expressing a control peptide into 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).


RESULTS
Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21.


CONCLUSIONS
We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.
Kovac
C
C
Department of Biology Chemistry, Long Island University, Brooklyn, NY 11201, USA.
Clie
L
L
Morin
J
J
Friedman
F K
FK
Robinson
R
R
Chung
D L
DL
Kanovsky
M
M
Flom
J
J
Brandt-Rauf
P W
PW
Yamaizumi
Z
Z
Michl
J
J
Pincus
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D007328:Insulin / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D008969:Molecular Sequence Data; D015689:Oncogene Protein p21(ras) / Q000037:antagonists & inhibitors*; D009865:Oocytes / Q000502:physiology*; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D014982:Xenopus laevis
D007328:Insulin; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D015689:Oncogene Protein p21(ras)