A CYTOTOXIC RIBONUCLEASE - STUDY OF THE MECHANISM OF ONCONASE CYTOTOXICITY

Onconase, or P-30, is a protein initially purified from extracts of Ra na pipiens oocytes and early embryos based upon its anticancer activit y both in vitro and in vivo. It is a basic single-chain protein with a n apparent molecular mass of 12,000 daltons and is homologous to RNase A. In cultured 9L glioma cells, onconase inhibits protein synthesis w ith an IC50 of about 10(-7) M. The inhibition of protein synthesis cor relates with cell death determined by clonogenic assays. I-125-Labeled onconase binds to specific sites on cultured 9L glioma cells. Scatcha rd analysis of the binding data shows that onconase appears to bind to cells with two different affinities, one with a K(d) of 6.2 x 10(-8) and another of 2.5 x 10(-7) M. Each cell could bind about 3 x 10(5) mo lecules of onconase at each of the two affinity sites. The low affinit y K(d) is similar to the IC50 for onconase toxicity. Onconase also dem onstrates a saturability of cytotoxicity at a concentration that would saturate the low affinity binding site. Incubation at 4-degrees-C inc reased the binding of onconase to cells relative to 37-degrees-C bindi ng and also increased the sensitivity of cells to onconase toxicity, i ndicating that receptor binding may be an initial step in cell toxicit y. Onconase cytotoxicity can be blocked by metabolic inhibitors, NaN3 and 2-deoxyglucose, and cytotoxicity is potentiated 10-fold by monensi n. Ribonuclease activity appears necessary for onconase toxicity becau se alkylated onconase, which only retains 2% of the ribonuclease activ ity, was at least 100-fold less potent in inhibiting protein synthesis in cells. Onconase inhibition of protein synthesis in 9L cells coinci des with the degradation of cellular 28 S and 18 S rRNA. In contrast t o RNase A, onconase is resistant to two RNase inhibitors, placental ri bonuclease inhibitor and Inhibit-Ace(TM). Northern hybridization with placental ribonuclease inhibitor cDNA probe indicates that 9L glioma c ells contain endogenous placental ribonuclease inhibitor mRNA. Based o n these results, we propose that onconase toxicity results from oncona se binding to cell surface receptors, internalization to the cell cyto sol where it degrades ribosomal RNA, inhibiting protein synthesis and causing cell death.