Dissecting the Mechanism

Select members of the bovine pancreatic noonuclease (RNase A) superfamily are toxic to tumor cells. RNase A. itself. is not normally cytotoxic. but Onconasee (ONC). an amphibian homologue. is a potent cytotoxin. This thesis examines the molecular basis of nDonucleasemediated cytotoxicity. The steps for ribonuclease cytotoxicity are: (1) cell-surface binding. (2) internalization. (3) translocation to the cytosol. and (4) degradation of cellular RNA. ONe and RNase A bind the surface of mammalian cells and are readily internalized in a dose-dependent manner. This internalization is mediated by acidic vesicles that are not dependent on the activity of dynamin. Internalized nDonucleases translocate to the cytosol from a pre-ER compartment. Sequence-specific lysosome-targeted degradation does not limit the toxicity of RNase A variants. Ribonucleases must evade the cytosolic ribonuclease inhibitor protein (RI) to be cytotoxic. A variant of G88R RNase A. K7 AlG88R RNase A. binds RI with lowered affinity and bas enhanced cytotoxicity. The role of RI as an intracellular modulator of nDonuclease cytotoxicity was investigated directly by varying its level within mammalian cells. RI limits the potency of toxic RNase A variants but has no effect on the cytotoxicity of ONe. indicating that ONe is not regulated by RI. The toxicity of ONe and RNase A variants correlates with the growth rate of cells. The biology of pancreatic nDonucleases and RI was explored by genetic methods. RI is a modular protein that has evolved rapidly by exon duplication. Pancreatic ribonuclease (Rib 1) and ribonuclease inhibitor (Rnh) genes are present in the mouse genome as a single