Onconase (
Ranpirnase), a novel
ribonuclease isolated from Rana pipiens oocytes, was reported to suppress
cancer cell growth in vitro, reduce
tumor size in animals, and augment cytotoxicity of several chemotherapeutic agents. Since
onconase is currently in phase III clinical trials tested in treatment of
mesothelioma, much emphasis has been placed on the mechanism of its anti-
tumor activity. Previous studies have shown that
onconase-responsive cells become arrested at the G1/S checkpoint of the cell cycle and also undergo apoptosis. A proposed mechanism for these effects is that the enzymatic activity of
onconase targets cellular RNAs, in particular
tRNA, with an accompanying inhibition of
protein synthesis. In the present study, we have investigated the time- and dose-dependent effects of
onconase on growth of Jurkat SN
acute T-lymphocytic leukemia cells. Significant suppression of cell proliferation became evident after 72 and 96 h of treatment, and was most pronounced at the highest concentration (10 microg/ml; 8.3x10(-7) M) of
onconase. This reduction of cell proliferation, however, was not accompanied by measurable changes in distribution of cells at different phases of the cell cycle, but was paralleled by the induction of apoptosis, as assayed by flow cytometry, and with a modest decrease in the expression of a cell cycle regulatory
retinoblastoma protein (Rb). Further biochemical analysis revealed that growth suppression was closely coordinated with a down-regulation in the steady state and subcellular distribution of
NF-kappaB, a
transcription factor known to be functionally associated with cell survival. The reduction in expression of
NF-kappaB by
onconase appeared to coincide or even precede growth suppression, suggesting a causal relationship. To further test the hypothesis that cellular localization and expression of
NF-kappaB may be critical to cellular response to
onconase, we also studied the growth effects of
onconase in Jurkat-BalphaM cells, which, unlike the parent SN T cells, contain a stably transfected dominant-negative IkappaB gene. Growth suppression by
onconase in BalphaM cells was more pronounced and occurred earlier compared to SN cells, although still did not affect changes in cell cycle phase distribution. Contrary to expectation, however, diminution in
NF-kappaB expression by
onconase was even more pronounced in BalphaM cells, suggesting that this
transcription factor, while presumably prevented from dissociation from its inhibitory
protein IkappaB in these cells, is even more efficiently targeted for degradation by
onconase. These results implicate
NF-kappaB and its turnover as important determinants in the anti-proliferative/apoptotic effects of
onconase in
acute T-lymphocytic leukemia cells.