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 killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.
Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, 21702, USA.
M R
MR
Smith
D L
DL
Newton
S M
SM
Mikulski
S M
SM
Rybak
D016475:3T3 Cells / Q000187:drug effects* / Q000201:enzymology; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004357:Drug Synergism; D004527:Egg Proteins / Q000633:toxicity; D005110:Extracellular Space / Q000201:enzymology; D007399:Interphase / Q000187:drug effects; D051379:Mice; D008845:Microinjections; D015689:Oncogene Protein p21(ras) / Q000502:physiology; D012259:Ribonuclease, Pancreatic / Q000633:toxicity; D012260:Ribonucleases / Q000633:toxicity*
D017304:Annexin A5; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004527:Egg Proteins; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D015689:Oncogene Protein p21(ras); C058703:ranpirnase