Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially ki lled actively growing cells in both microinjection and cell culture experim ents. Moreover, agents that increased the number of cells in S phase such a s serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. C onversely, agents that decreased these proliferative pathways (dibutyryl cA MP and protein kinase A) correspondingly diminished Onconase cytotoxicity i n microinjection experiments, These results were also mimicked in cell cult ure experiments since log-phase v-ras-transformed NIH/3T3 cells were more s ensitive to Onconase (IC50 of 7 mu g/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 mu g/ml). Based on those data we postulated that Onconase-media ted cell death in NIH/3T3 cells was related to events occurring at two or m ore points in the cell cycle preferentially associated with late G(1)/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to micr oinjected RNase A than log phase cells and positive mediators of proliferat ive signal transduction did not enhance RNase A-mediated cytotoxicity. Take n together, these results demonstrate that these two RNases use different p athways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. P redictions formulated from these studies can be tested for relevance to RNa se actions in different target tumor cells. (C) 1999 Academic Press.