A cytotoxic ribonuclease. Study of the mechanism of onconase cytotoxicity.
Onconase, or P-30, is a protein initially purified from extracts of Rana pipiens oocytes and early embryos based upon its anticancer activity both in vitro and in vivo. It is a basic single-chain protein with an apparent molecular mass of 12,000 daltons and is homologous to RNase A. In cultured 9L glioma cells, onconase inhibits protein synthesis with an IC50 of about 10(-7) M. The inhibition of protein synthesis correlates with cell death determined by clonogenic assays. 125I-Labeled onconase binds to specific sites on cultured 9L glioma cells. Scatchard analysis of the binding data shows that onconase appears to bind to cells with two different affinities, one with a Kd of 6.2 x 10(-8) and another of 2.5 x 10(-7) M. Each cell could bind about 3 x 10(5) molecules of onconase at each of the two affinity sites. The low affinity Kd is similar to the IC50 for onconase toxicity. Onconase also demonstrates a saturability of cytotoxicity at a concentration that would saturate the low affinity binding site. Incubation at 4 degrees C increased the binding of onconase to cells relative to 37 degrees C binding and also increased the sensitivity of cells to onconase toxicity, indicating that receptor binding may be an initial step in cell toxicity. Onconase cytotoxicity can be blocked by metabolic inhibitors, NaN3 and 2-deoxyglucose, and cytotoxicity is potentiated 10-fold by monensin. Ribonuclease activity appears necessary for onconase toxicity because alkylated onconase, which only retains 2% of the ribonuclease activity, was at least 100-fold less potent in inhibiting protein synthesis in cells. Onconase inhibition of protein synthesis in 9L cells coincides with the degradation of cellular 28 S and 18 S rRNA. In contrast to RNase A, onconase is resistant to two RNase inhibitors, placental ribonuclease inhibitor and Inhibit-Ace. Northern hybridization with placental ribonuclease inhibitor cDNA probe indicates that 9L glioma cells contain endogenous placental ribonuclease inhibitor mRNA. Based on these results, we propose that onconase toxicity results from onconase binding to cell surface receptors, internalization to the cell cytosol where it degrades ribosomal RNA, inhibiting protein synthesis and causing cell death.
Wu
Y
Y
Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Mikulski
S M
SM
Ardelt
W
W
Rybak
S M
SM
Youle
R J
RJ
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000633:toxicity*; D001345:Autoradiography; D015152:Blotting, Northern; D020126:Brefeldin A; D002470:Cell Survival / Q000187:drug effects*; D003517:Cyclopentanes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004527:Egg Proteins / Q000378:metabolism* / Q000633:toxicity*; D004625:Embryo, Nonmammalian; D005910:Glioma; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009865:Oocytes; D010926:Placental Hormones / Q000096:biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011894:Rana pipiens; D012259:Ribonuclease, Pancreatic / Q000378:metabolism / Q000633:toxicity; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000633:toxicity*; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
D000970:Antineoplastic Agents; D003517:Cyclopentanes; D004527:Egg Proteins; D009363:Neoplasm Proteins; D010926:Placental Hormones; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C022052:placental ribonuclease inhibitor; D020126:Brefeldin A; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D007930:Leucine; C058703:ranpirnase