Endowing human pancreatic ribonuclease with toxicity for cancer cells.
Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.
Leland
P A
PA
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Staniszewski
K E
KE
Kim
B M
BM
Raines
R T
RT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002455:Cell Division; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D006801:Humans; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D007700:Kinetics; D007938:Leukemia / Q000188:drug therapy; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity*; D012260:Ribonucleases / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D000970:Antineoplastic Agents; D018076:DNA, Complementary; D004220:Disulfides; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D003545:Cysteine; C058703:ranpirnase