Cytotoxic onconase and ribonuclease a chimeras: comparison andin vitrocharacterization

Publisher Website
Google Scholar
Abstract
Onconase, a protein isolated from fertilized Rana pipiens eggs, has antineoplastic properties both in vitro (Z. Darzynkiewicz, S. P. Carter, S. M. Mikulski, W. Ardelt, and K. Shogen, 1988, Cell Tissue Kinet. 21, 169–182) and in vivo (S. M. Mikulski, W. Ardelt, K. Shogen, E. H. Bernstein, and H. Menduke, 1990, J. Natl. Cancer Inst. 82, 151–153). The protein is a member of the pancreatic ribonuclease A superfamily and exhibits ribonuclease activity toward highly polymerized RNA (W. Ardelt, S. M. Mikulski, and K. Shogen, 1991, J. Biol. Chem. 266, 245–251). Previous work has demonstrated that bovine pancreatic ribonuclease A conjugated to transferrin (S. Rybak, S. Saxena, E. Ackerman, and R. Youle, 1991, J. Biol. Chem. 266, 21202–21207) or antibodies to the human transferrin receptor (D. Newton, O. Ilercil, D. Laske, E. Oldfield, S. Rybak, and R. Youle, 1992, J. Biol. Chem. 267, 169–182), acted like a class of reagents called immunotoxins to specifically inhibit protein synthesis in ligand-positive cells and prevent the growth of human glioblastoma tumors in vivo. To explore the properties of Onconase as an immunotoxin, this ribonuclease A homolog was conjugated to transferrin or an antibody (5E-9) to the human transferrin receptor and compared to bovine pancreatic ribonuclease A conjugates. The IC50's of both the Onconase and the ribonuclease A conjugates as inhibitors of protein synthesis in K562 human erythroleukemia cells were in the nano-molar range. Unconjugated Onconase also inhibited protein synthesis in K562 cells but was 20- to 100-fold less toxic on a molar basis than the Onconase conjugates. The time course profiles of Onconase and Onconase conjugates in affecting protein and RNA synthesis were very similar. Inhibition of protein synthesis was preceded by a decrease in RNA synthesis that subsequently increased relative to control values before it decreased again with time. Conjugated Onconase entered cells via a route mediated by transferrin or 5E-9 since excess of either ligand blocked toxicity of the conjugate but not the toxicity of unconjugated Onconase. Conjugated Onconase could also enter cells via the Onconase portion of the conjugate since it inhibited protein synthesis in cells that are not recognized by anti-human transferrin receptor antibody. ASPC-1 pancreatic carcinoma cells are essentially resistant to Onconase alone as measured by cell viability; however, when conjugated to antibody, Onconase did decrease the viability of ASPC-1 cells. This effect was markedly increased when the Onconase conjugate was combined with tamoxifen. These results demonstrate that the cytotoxic properties of a member of the ribonuclease A superfamily with inherent anti-tumor effects can be enhanced by conjugation to ligands capable of internalization. Therefore Onconase coupled to such ligands may have increased clinical relevance for some types of human cancer.